CA2133956C - Dna sequencing method - Google Patents
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- CA2133956C CA2133956C CA002133956A CA2133956A CA2133956C CA 2133956 C CA2133956 C CA 2133956C CA 002133956 A CA002133956 A CA 002133956A CA 2133956 A CA2133956 A CA 2133956A CA 2133956 C CA2133956 C CA 2133956C
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
Abstract
There is disclosed a method for determining the sequence of a nucleic acid comprising the steps of: a) forming a single-stranded template comprising the nucleic acid to be sequenced; b) hybridizing a primer to the template to form a template/primer complex; c) extending the primer by the addition of a single labelled nucleotide; d) determining the type of the labelled nucleotide added onto the primer;
e) removing or neutralizing the label; and f) repeating steps (c) to (e) sequentially and recording the order of incorporation of labelled nucleotides. There is also disclosed apparatus for carrying out the method.
e) removing or neutralizing the label; and f) repeating steps (c) to (e) sequentially and recording the order of incorporation of labelled nucleotides. There is also disclosed apparatus for carrying out the method.
Description
WO 93/21340 ~ pCT/GB93/00848 DNA SeQUencinQ Method The present invention relates to a method for sequencing DNA. In particular, the present invention concerns a method for the automated sequencing of large fragments of DNA.
DNA sequence analysis has become one of the most important tools available to the molecular biologist. Current sequencing tEachnalogy allows sequence data to be obtained from virtually any DNA fragment. This has allowed not only the sequencing of entire genes and other genomic sequences but also the identification of the sequence of RNA
transcripts, by the sequencing of cDNA. Currently, emphasis is being placed on genomic sequencing in order to determine the DNA sequence of entire genomes. Ultimately, it is hoped that the sequence: of the human genome will be deciphered.
Traditional DNA sequencing techniques share three essential steps in their approaches to sequence determination.
Firstly, a multiplicity of DNA fragments are generated from a DNA species which it is intended to sequence. These fragments arE~ incomplete copies of the DNA species to be sequenced. The a.im, is to produce a ladder of DNA fragments, each a single: base longer than the previous one. This can be achieved by selective chemical degradation of multiple copies of the: DNA species to be sequenced, as in the Maxam and Gilbert method (A. Maxam and W. Gilbert, PNAS 74, p.560, 1977). Alternatively, the DNA species can be used as a template for a DNA polymerase to produce a number of incomplete cl.one~c, as in the Sanger method (F. Sanger, S.
Nicklen and A. Coulson, PNAS 74, p.5463, 1977). These fragments, which differ in respective length by a single base, are then separated on an apparatus which is capable of resolving single:-base differences in size. A thin polyacrylamid.e gel is invariably used in this process. The third and final step is the determination of the nature of the base at the end of each fragment. When ordered by the size of the fragments which they terminate, these bases 2 PCT/GB93/00&'' represent the sequence of the original DNA species.
Determination of the nature of each base is achieved by previously selecting the terminal base of each fragment. In the Sanger method, for example, dideoxy nucleoside triphosphates (ddNTPs) are used to selectively terminate growing DNA clones at an A, C, G or T residue. This means that four separate reactions need to be performed for each sequencing exercise, each in a separate tube using a different ddNTP. In one tube, therefore, each labelled fragment will terminate with an A residue, while in the next tube with a C residue, and so on. Separation of each group of fragments side-by-side on a polyacrylamide gel will show the sequence of the template by way of the relative size of the individual fragments.
In the Maxam and Gilbert method, on the other hand, the selectivity is achieved during the chemical degradation process. Chemicals are used which cleave DNA strands at A
only, C only, G and A or T and C. Use of limiting concentrations of such chemicals allows partial digestion of the DNA species. As in the Sanger method, four separate reactions must be performed and the products separated side-by-side on a polyacrylamide gel.
The disadvantages of these prior art methods are numerous.
They require a number of complex manipulations to be performed, in at least four tubes. They are susceptible to errors due to the formation of secondary structures in DNA, or other phenomena that prevent faithful replication of a DNA template in the Sanger method or which cause base-specificity to be lost by the chemical reactants of the Maxam and Gilbert method. The most serious problems, however, are caused by the requirement for the DNA fragments to be size-separated on a polyacrylamide gel. This process is time-consuming, uses large quantities of expensive chemicals, and severely limits the number of bases which can be sequenced in any single experiment, due to the limited WO 93/21340 ~ ~ ~ ~ ~ ~ ~ PCT/GB93/00848 resolution o:f the gel. Furthermore, reading the gels in order to extract the data is labour-intensive and slow.
A number of improvements have been effected to these sequencing mtathods in order to improve the efficiency and speed of DNA sequencing. Some of these improvements have related to the sequencing reaction itself. For example, improved polymerase enzymes have been introduced which lead to greater precision in the Sanger method, such as Sequenasem and Taquenase~. Improved reagents have not, however, significantly affected the speed of sequence data generation or significantly simplified the sequencing process.
In the interest of both speed and simplicity, a number of "Automated Sequencers" have been introduced in recent years (reviewed in 't. Hun~Capiller, R. Kaiser, B. Koop and L. Hood, Science, 254, p.59, 1991). These machines are not, however, truly automatic sequencers. They are merely automatic gel readers, which require the standard sequencing reactions to be carried out before samples are loaded onto the gel. They do provide a slight increase in speed, however, due to faster reading of the gels and collation of the data generated into computers for subsequent analysis.
Many automated sequencers exploit recent developments which have been made in labelling technology. Traditionally, radioactive labels in the form of 32P or 35S have been used to label each DNA :fragment. Recently, however, fluorophores have gained acceptance as labels. These dyes, attached either to the sequencing primer or to nucleotides, are excited to a ~'luorescent state on the polyacrylamide gel by a laser beam. An automated sequencer, therefore, can detect labelled fragments as they pass under a laser in a reading area. Use of dyes which fluoresce at different wavelengths allows individual labelling of A, G, C and T residues, which permits the producers of all four sequencing reactions to be run in a single lane of the gel.
WO 93/21340 PCT/GB93/0084' ~~~33~56 Even incorporating such refinements, however, automated sequencers can still produce no more than about 100kb of finished sequence per person per year. At this rate, it would take one person 73,000 years to sequence the human genome.
Clearly, if the aim of sequencing the human genome is to be achieved, current sequencing technology is entirely inadequate. In view of this, a few proposals have been made for alternative sequencing strategies which are not merely improvements of the old technology.
One such method, sequencing by hybridisation (SBH), relies on the mathematical demonstration that the sequence of a relatively short (say, 100kbp) fragment of DNA may be obtained by synthesising all possible N-mer oligonucleotides and determining which oligonucleotides hybridise to the fragment without a single mismatch (R. Drmanac, I. Labat, I.
Bruckner and R. Crkvenjakov, Genomics, 4, p.114, 1989; R.
Drmanac, Z. Stvanovic, R. Crkvenjakov, DNA Cell Biology, 9, p.527, 1990; W. Bains and G. Smith, J. Theor. Biol., 135, pp 303-307, 1988; K.R. Khrapko, et a1, FEBS lett., 256, pp.118-122, 1989; P.A. Pevzner, J. Biomolecular Structure and Dynamics, 7, pp.63-73, 1989; U. Maskos and E.M. Southern, Cold Spring Harbour Symposium on Genome Mapping and Sequencing, Abstracts, p.143, 1991). N can be 8, 9 or 10, such sizes being a compromise between the requirement for reasonable hybridisation parameters and manageable library sizes.
The technique can be automated by attaching the oligonucleotides in a known pattern on a two-dimensional grid. The fragment to be sequenced is subsequently hybridised to the oligonucleotides on the grid and the oligonucleotides to which the sequence has been hybridised are detected using a computerised detector. Determination of the sequence of the DNA is then a matter of computation.
WO 93/21340 r However, errors arise from the difficulty in determining the difference between perfect matches and single base-pair mismatches. Repetitive sequences, which occur quite 5 commonly in 'the Izuman genome, can also be a problem.
Another proposal. involves the fluorescent detection of single molecules (J. Jett et al., J. Biomol. Struct. Dyn., 7, p.301, 1989: D. Nguyen, et al., Anal. Chem., 56, p.348, 1987). In i:his method, a single, large DNA molecule is suspended in a flow stream using light pressure from a pair of laser beams. :Individual bases, each of which is labelled with a distinguishing f luorophore, are then cut from the end of the molecule and carried through a fluorescence detector by the flow stream.
Potentially, this method could allow the accurate sequencing of a large number of base pairs - several hundred - per second. However, feasibility of this method is not yet proven.
A third method is sequencing by scanning tunnelling microscopy (.3TM) (S. Lindsay, et al., Genet. Anal. Tech.
Appl., 8, p.E~, 1991: D. Allison et ai., Scanning Microsc., 4_, p.517, 1990: R. Driscoll et al., Nature, 346, p.294, 1990: M. Salmeron et al., J. Vac. Sci. Technol., 8_, p.635, 1990). This technique requires direct three-dimensional imaging of a 13NA molecule using STM. Although images of the individual bases can be obtained, interpretation of these images remains very difficult. The procedure is as yet unreliable arid the success rate is low.
A fourth method involves the detection of the pyrophosphate group released as a result of the polymerisation reaction which occurs when a nucleotide is added to a DNA primer in a primer extension reaction (E. D. Hyman, Anal. Biochem., 174, p. 423, 1988). This method attempts to detect the addition of single nucleotides to a primer using the WO 93/21340 PCT/GB93/00&
luciferase enzyme to produce a signal on the release of pyrophosphate. However, this method suffers a number of drawbacks, not least o.f which is that dATP is a substrate for luciferase and thus will always give a signal, whether it is incorporated into the chain or not. The added nucleotides are not labelled and no method is disclosed which will allow the use of labelled nucleotides.
In summary, therefore, each of the new approaches to DNA
sequencing described above, while solving some of the problems associated with traditional methods, introduces several problems of its own. In general, most of these methods are expensive and not currently feasible.
There is therefore a need for a sequencing method which allows the rapid, unambiguous sequencing of DNA at low cost.
The requirements for such a system are that:
1. it should not be based on gel resolution of differently-sized oligomers;
2. it should allow more rapid sequencing than present methods;
3. it should allow several DNA clones to be processed in parallel;
4. the cost of hardware should be reasonable;
5. it should cost less per base of sequence than current technology; and 6. it should be technically feasible at the present time.
The present invention provides such a sequencing system which comprises a method for the sequential addition of nucleotides to a primer on a DNA template.
According to a first aspect of the present invention, there is provided a method for determining the sequence of a nucleic acid comprising the steps of:
a) forming a single-stranded template comprising the nucleic acid to be sequenced;
b) hybridising a primer to the template to form a template/primer complex;
c) extending the primer by the addition of a single labelled nucleotide wherein said labelled nucleotide is not a chain elongation inhibitor;
d) determining the type of the labelled nucleotide added onto the primer;
e) removing or neutralising the label; and f) repeating steps (c) to (e) sequentially and recording the order of incorporation of labelled nucleotides.
In accordance with another aspect of the invention there is provided a process for sequencing a DNA fragment comprising the steps of: i) hybridising a capped primer containing a phosphorothioate nucleoside derivative to a template to form a template/primer complex; ii) adding a labelled deoxynucleoside triphosphate, wherein said labelled deoxynucleoside triphosphate is not a chain elongation inhibitor, together with heterogeneous chain terminators and a suitable polymerase to the template/primer complex; iii) removing excess reagents by washing; iv) measuring the amount of incorporated label; v) treating the templatelprimer complex with an exonuclease to remove the labelled deoxynucleoside triphosphate and the 7a chain terminators; vi) removing the exonuclease by washing;
vii) adding a phosphorothioate deoxynucleoside triphosphate corresponding to the labelled deoxynucleoside triphosphate added in Step ii) together with heterogeneous chain terminators; viii) removing excess reagents by washing; ix) treating the template/primer complex with an exonuclease to remove the chain terminators: x) removing the exonuclease by washing; and xi) repeating steps ii) to x), each time with a different deoxynucleoside triphosphate.
In accordance with yet another aspect of the invention there is provided a process for sequencing a DNA fragment comprising the steps of: i) hybridising a capped primer a containing phosphorothioate nucleoside derivative to a template to form a template/primer complex; ii) adding a labelled deoxynucleoside triphosphate, wherein said labelled deoxynucleoside triphosphate is not a chain elongation inhibitor, together with heterogeneous chain terminators and a suitable polymerase to the template/primer complex; iii) removing excess reagents by washing; iv) measuring the amount of incorporated label;
v) removing the labelled nucleotide and the chain terminators with an exonuclease; vi) removing the exonuclease by washing; vii) adding a phosphorothioate deoxynucleoside triphosphate together with heterogeneous chain elongation inhibitors not incorporated into the chain; viii) removing excess reagents by washing; and ix) repeating steps ii) to viii), each time with a different labelled deoxynucleoside triphosphate.
In accordance with still yet another aspect of the invention there is provided a process for sequencing a DNA
fragment comprising the steps of: i) hybridising a capped primer containing a phosphorothioate deoxynucleoside 7b derivative to a template to form a template/primer complex;
ii) adding a labelled deoxynucleoside triphosphate, wherein said labelled deoxynucleoside triphosphate is not a chain elongation inhibitor, together with heterogeneous chain elongation inhibitors not incorporated into the chain and a suitable polymerase to the template/primer complex;
iii) removing excess reagents by washing; iv) measuring the amount of incorporated label; v) repeating steps ii) to iv) until all four different labelled deoxynucleoside triphosphates in the presence of their corresponding heterogeneous chain elongation inhibitors not incorporated into the chain have been added; vi) removing all labelled nucleotides with exonuclease; vii) removing the exonuclease by washing; viii) adding the phosphorothioate deoxynucleoside triphosphate corresponding to the first labelled deoxynucleotide added to the reaction in step ii), together with heterogeneous chain elongation inhibitors not incorporated into the chain and a suitable polymerase to the template/primer complex; ix) removing excess reagents by washing; and x) repeating steps ix) and x) with the three remaining phosphorothioate deoxynucleoside triphosphates.
In accordance with still yet another aspect of the invention there is provided a process for sequencing a DNA
fragment comprising the steps of: i) hybridising a capped primer to a template to form a templatelprimer complex; ii) adding a fluorescent nucleoside triphosphate, wherein said fluorescent deoxynucleoside triphosphate is not a chain elongation inhibitor, together with three heterogeneous chain elongation inhibitors not incorporated into the chain and a suitable polymerase to the template/primer complex;
iii) removing excess reagents by washing; iv) measuring the amount of incorporated label; v) repeating steps ii) to iv) using all three different nucleoside triphosphates, each with a fluorescent label, in the presence of the respective heterogeneous chain elongation inhibitors not incorporated into the chain; and vi) destroying the fluorescent labels by bleaching with a laser or by a suitable chemical reaction, or removing the fluorescent labels by a chemical cleavage step.
In accordance with still yet another aspect of the invention there is provided a process for sequencing a DNA
fragment comprising steps of: i) hybridising a capped primer to a template to form a template/primer complex; ii) adding a nucleoside triphosphate labelled by attachment of a fluorescent dye group via a linker arm to the 3' moiety of the deoxyribose sugar thereon together with three non-labelled heterogeneous chain elongation inhibitors not incorporated into the claim and a polymerase, wherein said nucleoside triphosphate is not a chain elongation inhibitor; iii) removing the excess reagents by washing;
iv) measuring the amount of incorporated label; v) removing the fluorescent dye group by enzymatic cleavage; vi) removing excess reagents by washing; and vii) repeating steps ii) to vi), each time with a different labelled nucleoside triphosphate.
In accordance with still yet another aspect of the invention there is provided use of a DNA sequencing kit for carrying out a method or a process of the present invention, the DNA
sequencing kit comprising: i) a linker for attaching a DNA
template to a solid-phase matrix, the linker comprising a primer having a phosphorothioate deoxynucleoside residue at its 3' end; ii) chain elongation inhibitors; iii) fluorescently-labelled nucleoside triphosphates; iv) deoxynucleoside phosphorothioate triphosphates; v) a 5'-> 3' DNA polymerase; and vi) a 3'-> 5' exonuclease.
7d In accordance with still yet another aspect of the invention there is provided an automated sequencing machine adapted to sequence a nucleic acid essentially by executing 5 the steps of a method in accordance with the present invention, the machine comprising: i) a solid-phase support to which the template/primer complex is bound; ii) means for moving the solid-phase support and the bound template/primer complex, to expose the template/primer 10 complex to the necessary reagent and washing solutions, or means for sequentially pumping reagent and washing solutions over the bound template/primer complex; and iii) means for detecting the presence of a label.
In the method of the invention, a single-stranded template 15 is generated from a nucleic acid fragment which it is desired to sequence. Preferably, the nucleic acid is DNA.
Part of the sequence of this fragment may be known, so that a specific primer may be constructed and hybridised to the template. Alternatively, a linker may be ligated to a 20 fragment of unknown sequence in order to allow for hybridisation of a primer.
The template may be linear or circular. Preferably, the template is bound to a solid-phase support. For example, the template may be bound to a pin, a glass plate or a 25 sequencing chip. The provision of a solid phase template allows for the quick and efficient addition and removal of reagents, particularly if the process of the invention is automated. Additionally, many samples may be processed in parallel in the same vessel yet kept separate.
WO 93/21340 PCT/GB93/00&~
~z133956 Preferably, the template is attached to the solid support by means of a binding. linker. For example, one of the commercially available universal primers can be ligated to the 5' end of the template or incorporated easily to one of the ends of the templates by the polymerase chain reaction.
The binding linker may be attached to the solid support by means of a biotin/streptavidin coupling system. For example, the surface of the solid support may be derivatised by applying biotin followed by streptavidin. A biotinylated binding linker is then ligated to the template to bind it to the solid support or the biotinylated template generated by PCR is bound to the solid support.
In an alternative embodiment, an unligated binding linker is bound to the solid support by the biotin/streptavidin system. The template is then hybridised to the binding linker. The binding linker may be a separate binding linker, which is not the sequencing primer. Alternatively, the binding linker may also function as the sequencing primer.
Clearly, it is essential in the latter embodiment that the template should possess a region of complementarity with the binding linker bound to the support. Where the template is ligated to a linker, the complementarity may be provided by that linker. Alternatively, the binding linker may be complementary to a unique sequence within the template itself.
Preferably the solid support is derivatised using a mask so as to allow high resolution packaging of the templates) on the support. An array of template attachment areas can thereby be produced on a glass plate or sequencing chip, allowing parallel processing of a large number of different templates. Where pins are used as the solid support, a single pin is needed for each template. The single pins may WO 93/21340 ' PCT/GB93/00848 be grouped into arrays. It is envisaged that an array of 100 x 100 ping or attachment areas can be used, to allow the simultaneous processing. of 104 clones.
The primer is extended by a DNA polymerase in the presence of a single labelled nucleotide, either A, C, G or T.
Suitable DNA polymerases are, for example, Sequenase 2.0~, T4 DNA polymerase or the Klenow fragment of DNA polymerase 1 as well as heat--stable polymerases such as Taq polymerase (for example Taquenase~) and Vent polymerase.
In a manually operated procedure using a single template, the labelled nucleotides are used singly and sequentially in order to attempt t=o add that nucleotide to the primer. The nucleotide will add on to the primer when it is complementary to the next nucleotide in the template. It may take one, 'two, three or four steps before the appropriate labelled nucleotide is used. However, as soon as it is determined that a labelled nucleotide has been added onto the primer, step (e) can be performed.
In an automated procedure, especially where a large number of templates are being sequenced simultaneously, in step (c) all four labelled nucleotides are used sequentially and it is merely noted whi~~h of the labelled nucleotides is added, that is it is determined whether it is the f first, second, third or fourth labelled nucleotide which is added.
It has been found that nonspecific end-addition and misincorporation of nucleotides can lead to background problems when the incorporation step has been repeated a number of timsas. These side reactions are mainly due to the fact that a single nucleotide is present, instead of all four nucleoside triphosphates. In fact, it has been observed that: while it is possible to sequence certain templates by 'the sequential addition of single nucleotides in the absence' of the other three, significant problems have been encountered with other templates, particularly those 'z 13 3 9 5 6 PCT/GB93/00&
l0 templates containing multiple base repeats, due to non-specific incorporation of a nucleotide which is caused by the polymerase effectively jumping over a non-complementary base.
In order to ensure high accuracy -of operation during the primer extension step, it has been found advantageous to carry out step (c) in the presence of chain elongation inhibitors.
Chain elongation inhibitors are nucleotide analogues which either are chain terminators which prevent further addition by the polymerase of nucleotides to the 3' end of the chain by becoming incorporated into the chain themselves, or compete for incorporation without actually becoming incorporated. Preferably, the chain elongation inhibitors are dideoxy nucleotides. Where the chain elongation inhibitors are incorporated into the growing polynucleotide chain, it is essential that they be removed after incorporation of the labelled nucleotide has been detected, in order to allow the sequencing reaction to proceed using different labelled nucleotides. It has been found, as described below, that 3' to 5' exonucleases such as, for example, exonuclease III, are able to remove dideoxynucleotides. This finding allows the use of dideoxynucleotides as chain elongation inhibitors to promote the accuracy of the polymerase in the sequencing method of the invention. Accuracy of the polymerase is essential if 104 clones are to be processed simultaneously, since it is high polymerase accuracy which enables the sequencing reaction to be carried out on a single template instead of as four separate reactions.
Alternatively, the chain elongation inhibitors may be deoxynucleoside 5'-[a, Q-methylene) triphosphates. These compounds are not incorporated into the chain. Other nucleotide derivatives such as, for example, deoxynucleoside diphosphates or deoxynucleoside monophosphates may be used WO 93/21340 ~~ I 3 3 ~ 5 6 which are also nat incorporated into the chain.
It is furthermore envisaged that blocking groups on the 3' moiety of thsa deoxyribose group of the labelled nucleotide may be used to prevent nonspecific incorporation.
Preferably, l:herefore, the labelled nucleotide is labelled by attachment: of a f luorescent dye group to the 3' moiety of the deoxyribose group, and the label is removed by cleaving the fluorescent dye from the nucleotide to generate a 3' hydroxyl group. The fluorescent dye is preferably linked to the deoxyribose by a linker arm which is easily cleaved by chemical or enzymatic means.
Evidently, when nucleotide analogue chain elongation inhibitors a.re used, only the analogues which do not correspond to the labelled nucleotide should be added. Such analogues are referred to herein as heterogenous chain elongation inhibitors.
Label is ideally only incorporated into the template/primer complex if the labelled nucleotide added to the reaction is complementary to t:he nucleotide on the template adjacent the 3' end of the primer. The template is subsequently washed to remove any unincorporated label and the presence of any incorporated label determined. A radioactive label may be determined by counting or any other method known in the art, while fluorescent labels can be induced to fluoresce, for example by laser excitation.
It will be a~?parent that any label known in the art to be suitable for labelling nucleic acids may be used in the present invention. However, the use of fluorescent labels is currently preferred, due to the sensitivity of detection systems presently available for such labels which do not involve the use of radioactive substances.
Examples of flourescently-labelled nucleotides currently available include fluorescein-12-dUTP, fluorescein-15-dCTP, WO 93/21340 ~ 13 3 9 5 ~ pC'T/GB93/0084 fluorescein-15-dATP and flurescein-15-dITP. It has proved very difficult to synthesise a suitable fluroescent guanosine compound, so. an inosine compound is used in its place. Should a fluorescent guanosine compound become available, its use is envisaged in the present invention.
It has been found advantageous to use a mixture of unlabelled and labelled nucleo~~ides in the addition step.
When a fluorescent label is used, in order to produce all possible extension products on a template possessing a run of a particular nucleotide, the following ratios were found to be approximately optimal:
Fluorescein 15-dATP/dATP 500:1 -Fluarescein - 15-dITP/dGTP 500:1 Fluorescein - 12-dUTP/dTTP 15:1 Fluorescein - 12-dCTP/dCTP 15:1.
Preferably, therefore, the above ratios are used in connection with fluorescently - labelled nucleotides.
By repeating the incorporation and label detection steps until incorporation is detected, the nucleotide on the template adjacent the 3' end of the primer may be identified. Once this has been achieved, the label must be removed before repeating the process to discover the identity of the next nucleotide. Removal of the label may be effected by removal of the labelled nucleotide using a 3'-5' exonuclease and subsequent replacement with an unlabelled nucleotide. Alternatively, the labelling group can be removed from the nucleotide. In a further alternative, where the label is a fluorescent label, it is possible to neutralise the label by bleaching it with laser radiation.
If chain terminators or 3' blocking groups have been used, these should be removed before the next cycle can take place. Preferably, chain terminators are removed with a 3'-5' exonuclease. Preferably, exonuclease III is used. 3' blocking groups may be. removed by chemical or enzymatic cleavage of t:he blocking group from the nucleotide.
Where exonuc:lease III is used to remove the chain terminators, it is essential to prevent the exonuclease III
from chewing back along the growing chain to remove nucleotides which have alread; ~:aen incorporated, or even the primer itself" Preferab therefore, a nucleoside derivative which is resistant removal by exonucleases is used to replace the labelled nucleotides. Advantageously deoxynucleoside phosphorothioate triphosphates (dsNTPs) are used. Lik~awise, the primer preferably comprises a phosphorothioate nucleoside bases) at its 3' end which are incorporated during primer synthesis or an extra enzymatic capping step.
It is known that deoxynucleoside phosphorothioate derivatives resist digestion by exonuclease III (S. Labeit et al., DNA, 5_, p.173, 1986). This resistance is, however, not complete and conditions should be adjusted to ensure that excess digestion and removal of phosphorothioate bases does not occur.
For example, it .has been found that the pH of the exoIII
buffer used (50mM Tris/HC1, 5mM MgCl2) affects the extent of chewing back which occurs. Experiments carried out at pH
6.0, 7.0, 7.5., 8.0,. 8.5, 9.0 and 10.0 (37°C) reveal that pH
10.0 is the optimum with respect to the rate of reaction and specificity of exo:III. At this pH, the reaction was shown to go to completion in less than 1 minute with no detectable chewing back.
Once the label and terminators/blocking groups have been removed, the cycle is repeated to discover the identity of the next nucleotide.
_ ~13395fi In an alternative embodiment of the invention, steps (c) and (d) of the first aspect of the invention are repeated sequentially a plurality of times before removal or neutralisation of the label.
The number of times the steps (c) and (d) can be repeated depends on the sensitivity of the-apparatus used to detect when a labelled nucleotide has. been added onto the primer.
For instance, if each nucleotide is labelled with a different fluorescent label, the detection apparatus will need to be able to distinguish between each of the labels and will ideally be able to count the number of each type of fluorescent label. Alternatively, where each nucleotide is radioactively labelled or labelled with the same fluorescent dye, the apparatus will need to be able to count the total number of labels added to the primer.
As with the first embodiment of the invention, in a manual procedure using a single template, the labelled nucleotides are used singly and sequentially until a labelled nucleotide is added, whereupon the sequence is repeated. In an automated procedure all four labelled nucleotides are used sequentially and the apparatus is programmed to detect which nucleotides are added in what sequence to the primer.
Once the number of labels added has reached the resolving power of the detecting apparatus, removal or neutralisation of the label is carried out in a single step. Thus, the number of label removal steps is significantly reduced.
In this alternative embodiment, the steps (c) and (d) of the first aspect of the invention will preferably comprise:
i) adding a labelled nucleotide together with three heterogenous chain elongation inhibitors which are not incorporated into the chain, such as 5'-fa, p-methylene]
triphosphates;
13 ~ g ~ 6 PCT/GB93/00848 ii) removing excess reagents by washing;
iii) determining whether the label has been incorporated;
and iv) repeating steps (i) to (iii) using a different labelled nucleotide, either until a labelled nucleotide has been incorporated or until all four labelled nucleotides have been used.
This technique necessitates the use of a more sophisticated counter or label measuring device. Allowing for runs of repeated nucleotides, the label measuring device should be able to detect the presence of between four and sixteen labelled nucleotides accurately. For the measurement of long stretchE~s o:f repeated nucleotides, a device with a greater capacity may be required.
Scheme 1 According to a preferred aspect of the invention, a DNA
fragment is sequenced according to the following scheme:
1) a capped primer containing a phosphorothioate nucleoside derivative is hybridized to a template to form a template/primer complex;
2) a labESlled deoxynucleoside triphosphate (dNTP) together with heterogenous chain terminators and a suitable polymerase is added to the template/primer complex;
3) excess reagents are removed by washing;
4) the amount of incorporated label is measured;
5) the template/primer complex is treated with an exonuclease to remove the label and the dideoxynucleotides;
6) the exonuclease is removed by washing;
DNA sequence analysis has become one of the most important tools available to the molecular biologist. Current sequencing tEachnalogy allows sequence data to be obtained from virtually any DNA fragment. This has allowed not only the sequencing of entire genes and other genomic sequences but also the identification of the sequence of RNA
transcripts, by the sequencing of cDNA. Currently, emphasis is being placed on genomic sequencing in order to determine the DNA sequence of entire genomes. Ultimately, it is hoped that the sequence: of the human genome will be deciphered.
Traditional DNA sequencing techniques share three essential steps in their approaches to sequence determination.
Firstly, a multiplicity of DNA fragments are generated from a DNA species which it is intended to sequence. These fragments arE~ incomplete copies of the DNA species to be sequenced. The a.im, is to produce a ladder of DNA fragments, each a single: base longer than the previous one. This can be achieved by selective chemical degradation of multiple copies of the: DNA species to be sequenced, as in the Maxam and Gilbert method (A. Maxam and W. Gilbert, PNAS 74, p.560, 1977). Alternatively, the DNA species can be used as a template for a DNA polymerase to produce a number of incomplete cl.one~c, as in the Sanger method (F. Sanger, S.
Nicklen and A. Coulson, PNAS 74, p.5463, 1977). These fragments, which differ in respective length by a single base, are then separated on an apparatus which is capable of resolving single:-base differences in size. A thin polyacrylamid.e gel is invariably used in this process. The third and final step is the determination of the nature of the base at the end of each fragment. When ordered by the size of the fragments which they terminate, these bases 2 PCT/GB93/00&'' represent the sequence of the original DNA species.
Determination of the nature of each base is achieved by previously selecting the terminal base of each fragment. In the Sanger method, for example, dideoxy nucleoside triphosphates (ddNTPs) are used to selectively terminate growing DNA clones at an A, C, G or T residue. This means that four separate reactions need to be performed for each sequencing exercise, each in a separate tube using a different ddNTP. In one tube, therefore, each labelled fragment will terminate with an A residue, while in the next tube with a C residue, and so on. Separation of each group of fragments side-by-side on a polyacrylamide gel will show the sequence of the template by way of the relative size of the individual fragments.
In the Maxam and Gilbert method, on the other hand, the selectivity is achieved during the chemical degradation process. Chemicals are used which cleave DNA strands at A
only, C only, G and A or T and C. Use of limiting concentrations of such chemicals allows partial digestion of the DNA species. As in the Sanger method, four separate reactions must be performed and the products separated side-by-side on a polyacrylamide gel.
The disadvantages of these prior art methods are numerous.
They require a number of complex manipulations to be performed, in at least four tubes. They are susceptible to errors due to the formation of secondary structures in DNA, or other phenomena that prevent faithful replication of a DNA template in the Sanger method or which cause base-specificity to be lost by the chemical reactants of the Maxam and Gilbert method. The most serious problems, however, are caused by the requirement for the DNA fragments to be size-separated on a polyacrylamide gel. This process is time-consuming, uses large quantities of expensive chemicals, and severely limits the number of bases which can be sequenced in any single experiment, due to the limited WO 93/21340 ~ ~ ~ ~ ~ ~ ~ PCT/GB93/00848 resolution o:f the gel. Furthermore, reading the gels in order to extract the data is labour-intensive and slow.
A number of improvements have been effected to these sequencing mtathods in order to improve the efficiency and speed of DNA sequencing. Some of these improvements have related to the sequencing reaction itself. For example, improved polymerase enzymes have been introduced which lead to greater precision in the Sanger method, such as Sequenasem and Taquenase~. Improved reagents have not, however, significantly affected the speed of sequence data generation or significantly simplified the sequencing process.
In the interest of both speed and simplicity, a number of "Automated Sequencers" have been introduced in recent years (reviewed in 't. Hun~Capiller, R. Kaiser, B. Koop and L. Hood, Science, 254, p.59, 1991). These machines are not, however, truly automatic sequencers. They are merely automatic gel readers, which require the standard sequencing reactions to be carried out before samples are loaded onto the gel. They do provide a slight increase in speed, however, due to faster reading of the gels and collation of the data generated into computers for subsequent analysis.
Many automated sequencers exploit recent developments which have been made in labelling technology. Traditionally, radioactive labels in the form of 32P or 35S have been used to label each DNA :fragment. Recently, however, fluorophores have gained acceptance as labels. These dyes, attached either to the sequencing primer or to nucleotides, are excited to a ~'luorescent state on the polyacrylamide gel by a laser beam. An automated sequencer, therefore, can detect labelled fragments as they pass under a laser in a reading area. Use of dyes which fluoresce at different wavelengths allows individual labelling of A, G, C and T residues, which permits the producers of all four sequencing reactions to be run in a single lane of the gel.
WO 93/21340 PCT/GB93/0084' ~~~33~56 Even incorporating such refinements, however, automated sequencers can still produce no more than about 100kb of finished sequence per person per year. At this rate, it would take one person 73,000 years to sequence the human genome.
Clearly, if the aim of sequencing the human genome is to be achieved, current sequencing technology is entirely inadequate. In view of this, a few proposals have been made for alternative sequencing strategies which are not merely improvements of the old technology.
One such method, sequencing by hybridisation (SBH), relies on the mathematical demonstration that the sequence of a relatively short (say, 100kbp) fragment of DNA may be obtained by synthesising all possible N-mer oligonucleotides and determining which oligonucleotides hybridise to the fragment without a single mismatch (R. Drmanac, I. Labat, I.
Bruckner and R. Crkvenjakov, Genomics, 4, p.114, 1989; R.
Drmanac, Z. Stvanovic, R. Crkvenjakov, DNA Cell Biology, 9, p.527, 1990; W. Bains and G. Smith, J. Theor. Biol., 135, pp 303-307, 1988; K.R. Khrapko, et a1, FEBS lett., 256, pp.118-122, 1989; P.A. Pevzner, J. Biomolecular Structure and Dynamics, 7, pp.63-73, 1989; U. Maskos and E.M. Southern, Cold Spring Harbour Symposium on Genome Mapping and Sequencing, Abstracts, p.143, 1991). N can be 8, 9 or 10, such sizes being a compromise between the requirement for reasonable hybridisation parameters and manageable library sizes.
The technique can be automated by attaching the oligonucleotides in a known pattern on a two-dimensional grid. The fragment to be sequenced is subsequently hybridised to the oligonucleotides on the grid and the oligonucleotides to which the sequence has been hybridised are detected using a computerised detector. Determination of the sequence of the DNA is then a matter of computation.
WO 93/21340 r However, errors arise from the difficulty in determining the difference between perfect matches and single base-pair mismatches. Repetitive sequences, which occur quite 5 commonly in 'the Izuman genome, can also be a problem.
Another proposal. involves the fluorescent detection of single molecules (J. Jett et al., J. Biomol. Struct. Dyn., 7, p.301, 1989: D. Nguyen, et al., Anal. Chem., 56, p.348, 1987). In i:his method, a single, large DNA molecule is suspended in a flow stream using light pressure from a pair of laser beams. :Individual bases, each of which is labelled with a distinguishing f luorophore, are then cut from the end of the molecule and carried through a fluorescence detector by the flow stream.
Potentially, this method could allow the accurate sequencing of a large number of base pairs - several hundred - per second. However, feasibility of this method is not yet proven.
A third method is sequencing by scanning tunnelling microscopy (.3TM) (S. Lindsay, et al., Genet. Anal. Tech.
Appl., 8, p.E~, 1991: D. Allison et ai., Scanning Microsc., 4_, p.517, 1990: R. Driscoll et al., Nature, 346, p.294, 1990: M. Salmeron et al., J. Vac. Sci. Technol., 8_, p.635, 1990). This technique requires direct three-dimensional imaging of a 13NA molecule using STM. Although images of the individual bases can be obtained, interpretation of these images remains very difficult. The procedure is as yet unreliable arid the success rate is low.
A fourth method involves the detection of the pyrophosphate group released as a result of the polymerisation reaction which occurs when a nucleotide is added to a DNA primer in a primer extension reaction (E. D. Hyman, Anal. Biochem., 174, p. 423, 1988). This method attempts to detect the addition of single nucleotides to a primer using the WO 93/21340 PCT/GB93/00&
luciferase enzyme to produce a signal on the release of pyrophosphate. However, this method suffers a number of drawbacks, not least o.f which is that dATP is a substrate for luciferase and thus will always give a signal, whether it is incorporated into the chain or not. The added nucleotides are not labelled and no method is disclosed which will allow the use of labelled nucleotides.
In summary, therefore, each of the new approaches to DNA
sequencing described above, while solving some of the problems associated with traditional methods, introduces several problems of its own. In general, most of these methods are expensive and not currently feasible.
There is therefore a need for a sequencing method which allows the rapid, unambiguous sequencing of DNA at low cost.
The requirements for such a system are that:
1. it should not be based on gel resolution of differently-sized oligomers;
2. it should allow more rapid sequencing than present methods;
3. it should allow several DNA clones to be processed in parallel;
4. the cost of hardware should be reasonable;
5. it should cost less per base of sequence than current technology; and 6. it should be technically feasible at the present time.
The present invention provides such a sequencing system which comprises a method for the sequential addition of nucleotides to a primer on a DNA template.
According to a first aspect of the present invention, there is provided a method for determining the sequence of a nucleic acid comprising the steps of:
a) forming a single-stranded template comprising the nucleic acid to be sequenced;
b) hybridising a primer to the template to form a template/primer complex;
c) extending the primer by the addition of a single labelled nucleotide wherein said labelled nucleotide is not a chain elongation inhibitor;
d) determining the type of the labelled nucleotide added onto the primer;
e) removing or neutralising the label; and f) repeating steps (c) to (e) sequentially and recording the order of incorporation of labelled nucleotides.
In accordance with another aspect of the invention there is provided a process for sequencing a DNA fragment comprising the steps of: i) hybridising a capped primer containing a phosphorothioate nucleoside derivative to a template to form a template/primer complex; ii) adding a labelled deoxynucleoside triphosphate, wherein said labelled deoxynucleoside triphosphate is not a chain elongation inhibitor, together with heterogeneous chain terminators and a suitable polymerase to the template/primer complex; iii) removing excess reagents by washing; iv) measuring the amount of incorporated label; v) treating the templatelprimer complex with an exonuclease to remove the labelled deoxynucleoside triphosphate and the 7a chain terminators; vi) removing the exonuclease by washing;
vii) adding a phosphorothioate deoxynucleoside triphosphate corresponding to the labelled deoxynucleoside triphosphate added in Step ii) together with heterogeneous chain terminators; viii) removing excess reagents by washing; ix) treating the template/primer complex with an exonuclease to remove the chain terminators: x) removing the exonuclease by washing; and xi) repeating steps ii) to x), each time with a different deoxynucleoside triphosphate.
In accordance with yet another aspect of the invention there is provided a process for sequencing a DNA fragment comprising the steps of: i) hybridising a capped primer a containing phosphorothioate nucleoside derivative to a template to form a template/primer complex; ii) adding a labelled deoxynucleoside triphosphate, wherein said labelled deoxynucleoside triphosphate is not a chain elongation inhibitor, together with heterogeneous chain terminators and a suitable polymerase to the template/primer complex; iii) removing excess reagents by washing; iv) measuring the amount of incorporated label;
v) removing the labelled nucleotide and the chain terminators with an exonuclease; vi) removing the exonuclease by washing; vii) adding a phosphorothioate deoxynucleoside triphosphate together with heterogeneous chain elongation inhibitors not incorporated into the chain; viii) removing excess reagents by washing; and ix) repeating steps ii) to viii), each time with a different labelled deoxynucleoside triphosphate.
In accordance with still yet another aspect of the invention there is provided a process for sequencing a DNA
fragment comprising the steps of: i) hybridising a capped primer containing a phosphorothioate deoxynucleoside 7b derivative to a template to form a template/primer complex;
ii) adding a labelled deoxynucleoside triphosphate, wherein said labelled deoxynucleoside triphosphate is not a chain elongation inhibitor, together with heterogeneous chain elongation inhibitors not incorporated into the chain and a suitable polymerase to the template/primer complex;
iii) removing excess reagents by washing; iv) measuring the amount of incorporated label; v) repeating steps ii) to iv) until all four different labelled deoxynucleoside triphosphates in the presence of their corresponding heterogeneous chain elongation inhibitors not incorporated into the chain have been added; vi) removing all labelled nucleotides with exonuclease; vii) removing the exonuclease by washing; viii) adding the phosphorothioate deoxynucleoside triphosphate corresponding to the first labelled deoxynucleotide added to the reaction in step ii), together with heterogeneous chain elongation inhibitors not incorporated into the chain and a suitable polymerase to the template/primer complex; ix) removing excess reagents by washing; and x) repeating steps ix) and x) with the three remaining phosphorothioate deoxynucleoside triphosphates.
In accordance with still yet another aspect of the invention there is provided a process for sequencing a DNA
fragment comprising the steps of: i) hybridising a capped primer to a template to form a templatelprimer complex; ii) adding a fluorescent nucleoside triphosphate, wherein said fluorescent deoxynucleoside triphosphate is not a chain elongation inhibitor, together with three heterogeneous chain elongation inhibitors not incorporated into the chain and a suitable polymerase to the template/primer complex;
iii) removing excess reagents by washing; iv) measuring the amount of incorporated label; v) repeating steps ii) to iv) using all three different nucleoside triphosphates, each with a fluorescent label, in the presence of the respective heterogeneous chain elongation inhibitors not incorporated into the chain; and vi) destroying the fluorescent labels by bleaching with a laser or by a suitable chemical reaction, or removing the fluorescent labels by a chemical cleavage step.
In accordance with still yet another aspect of the invention there is provided a process for sequencing a DNA
fragment comprising steps of: i) hybridising a capped primer to a template to form a template/primer complex; ii) adding a nucleoside triphosphate labelled by attachment of a fluorescent dye group via a linker arm to the 3' moiety of the deoxyribose sugar thereon together with three non-labelled heterogeneous chain elongation inhibitors not incorporated into the claim and a polymerase, wherein said nucleoside triphosphate is not a chain elongation inhibitor; iii) removing the excess reagents by washing;
iv) measuring the amount of incorporated label; v) removing the fluorescent dye group by enzymatic cleavage; vi) removing excess reagents by washing; and vii) repeating steps ii) to vi), each time with a different labelled nucleoside triphosphate.
In accordance with still yet another aspect of the invention there is provided use of a DNA sequencing kit for carrying out a method or a process of the present invention, the DNA
sequencing kit comprising: i) a linker for attaching a DNA
template to a solid-phase matrix, the linker comprising a primer having a phosphorothioate deoxynucleoside residue at its 3' end; ii) chain elongation inhibitors; iii) fluorescently-labelled nucleoside triphosphates; iv) deoxynucleoside phosphorothioate triphosphates; v) a 5'-> 3' DNA polymerase; and vi) a 3'-> 5' exonuclease.
7d In accordance with still yet another aspect of the invention there is provided an automated sequencing machine adapted to sequence a nucleic acid essentially by executing 5 the steps of a method in accordance with the present invention, the machine comprising: i) a solid-phase support to which the template/primer complex is bound; ii) means for moving the solid-phase support and the bound template/primer complex, to expose the template/primer 10 complex to the necessary reagent and washing solutions, or means for sequentially pumping reagent and washing solutions over the bound template/primer complex; and iii) means for detecting the presence of a label.
In the method of the invention, a single-stranded template 15 is generated from a nucleic acid fragment which it is desired to sequence. Preferably, the nucleic acid is DNA.
Part of the sequence of this fragment may be known, so that a specific primer may be constructed and hybridised to the template. Alternatively, a linker may be ligated to a 20 fragment of unknown sequence in order to allow for hybridisation of a primer.
The template may be linear or circular. Preferably, the template is bound to a solid-phase support. For example, the template may be bound to a pin, a glass plate or a 25 sequencing chip. The provision of a solid phase template allows for the quick and efficient addition and removal of reagents, particularly if the process of the invention is automated. Additionally, many samples may be processed in parallel in the same vessel yet kept separate.
WO 93/21340 PCT/GB93/00&~
~z133956 Preferably, the template is attached to the solid support by means of a binding. linker. For example, one of the commercially available universal primers can be ligated to the 5' end of the template or incorporated easily to one of the ends of the templates by the polymerase chain reaction.
The binding linker may be attached to the solid support by means of a biotin/streptavidin coupling system. For example, the surface of the solid support may be derivatised by applying biotin followed by streptavidin. A biotinylated binding linker is then ligated to the template to bind it to the solid support or the biotinylated template generated by PCR is bound to the solid support.
In an alternative embodiment, an unligated binding linker is bound to the solid support by the biotin/streptavidin system. The template is then hybridised to the binding linker. The binding linker may be a separate binding linker, which is not the sequencing primer. Alternatively, the binding linker may also function as the sequencing primer.
Clearly, it is essential in the latter embodiment that the template should possess a region of complementarity with the binding linker bound to the support. Where the template is ligated to a linker, the complementarity may be provided by that linker. Alternatively, the binding linker may be complementary to a unique sequence within the template itself.
Preferably the solid support is derivatised using a mask so as to allow high resolution packaging of the templates) on the support. An array of template attachment areas can thereby be produced on a glass plate or sequencing chip, allowing parallel processing of a large number of different templates. Where pins are used as the solid support, a single pin is needed for each template. The single pins may WO 93/21340 ' PCT/GB93/00848 be grouped into arrays. It is envisaged that an array of 100 x 100 ping or attachment areas can be used, to allow the simultaneous processing. of 104 clones.
The primer is extended by a DNA polymerase in the presence of a single labelled nucleotide, either A, C, G or T.
Suitable DNA polymerases are, for example, Sequenase 2.0~, T4 DNA polymerase or the Klenow fragment of DNA polymerase 1 as well as heat--stable polymerases such as Taq polymerase (for example Taquenase~) and Vent polymerase.
In a manually operated procedure using a single template, the labelled nucleotides are used singly and sequentially in order to attempt t=o add that nucleotide to the primer. The nucleotide will add on to the primer when it is complementary to the next nucleotide in the template. It may take one, 'two, three or four steps before the appropriate labelled nucleotide is used. However, as soon as it is determined that a labelled nucleotide has been added onto the primer, step (e) can be performed.
In an automated procedure, especially where a large number of templates are being sequenced simultaneously, in step (c) all four labelled nucleotides are used sequentially and it is merely noted whi~~h of the labelled nucleotides is added, that is it is determined whether it is the f first, second, third or fourth labelled nucleotide which is added.
It has been found that nonspecific end-addition and misincorporation of nucleotides can lead to background problems when the incorporation step has been repeated a number of timsas. These side reactions are mainly due to the fact that a single nucleotide is present, instead of all four nucleoside triphosphates. In fact, it has been observed that: while it is possible to sequence certain templates by 'the sequential addition of single nucleotides in the absence' of the other three, significant problems have been encountered with other templates, particularly those 'z 13 3 9 5 6 PCT/GB93/00&
l0 templates containing multiple base repeats, due to non-specific incorporation of a nucleotide which is caused by the polymerase effectively jumping over a non-complementary base.
In order to ensure high accuracy -of operation during the primer extension step, it has been found advantageous to carry out step (c) in the presence of chain elongation inhibitors.
Chain elongation inhibitors are nucleotide analogues which either are chain terminators which prevent further addition by the polymerase of nucleotides to the 3' end of the chain by becoming incorporated into the chain themselves, or compete for incorporation without actually becoming incorporated. Preferably, the chain elongation inhibitors are dideoxy nucleotides. Where the chain elongation inhibitors are incorporated into the growing polynucleotide chain, it is essential that they be removed after incorporation of the labelled nucleotide has been detected, in order to allow the sequencing reaction to proceed using different labelled nucleotides. It has been found, as described below, that 3' to 5' exonucleases such as, for example, exonuclease III, are able to remove dideoxynucleotides. This finding allows the use of dideoxynucleotides as chain elongation inhibitors to promote the accuracy of the polymerase in the sequencing method of the invention. Accuracy of the polymerase is essential if 104 clones are to be processed simultaneously, since it is high polymerase accuracy which enables the sequencing reaction to be carried out on a single template instead of as four separate reactions.
Alternatively, the chain elongation inhibitors may be deoxynucleoside 5'-[a, Q-methylene) triphosphates. These compounds are not incorporated into the chain. Other nucleotide derivatives such as, for example, deoxynucleoside diphosphates or deoxynucleoside monophosphates may be used WO 93/21340 ~~ I 3 3 ~ 5 6 which are also nat incorporated into the chain.
It is furthermore envisaged that blocking groups on the 3' moiety of thsa deoxyribose group of the labelled nucleotide may be used to prevent nonspecific incorporation.
Preferably, l:herefore, the labelled nucleotide is labelled by attachment: of a f luorescent dye group to the 3' moiety of the deoxyribose group, and the label is removed by cleaving the fluorescent dye from the nucleotide to generate a 3' hydroxyl group. The fluorescent dye is preferably linked to the deoxyribose by a linker arm which is easily cleaved by chemical or enzymatic means.
Evidently, when nucleotide analogue chain elongation inhibitors a.re used, only the analogues which do not correspond to the labelled nucleotide should be added. Such analogues are referred to herein as heterogenous chain elongation inhibitors.
Label is ideally only incorporated into the template/primer complex if the labelled nucleotide added to the reaction is complementary to t:he nucleotide on the template adjacent the 3' end of the primer. The template is subsequently washed to remove any unincorporated label and the presence of any incorporated label determined. A radioactive label may be determined by counting or any other method known in the art, while fluorescent labels can be induced to fluoresce, for example by laser excitation.
It will be a~?parent that any label known in the art to be suitable for labelling nucleic acids may be used in the present invention. However, the use of fluorescent labels is currently preferred, due to the sensitivity of detection systems presently available for such labels which do not involve the use of radioactive substances.
Examples of flourescently-labelled nucleotides currently available include fluorescein-12-dUTP, fluorescein-15-dCTP, WO 93/21340 ~ 13 3 9 5 ~ pC'T/GB93/0084 fluorescein-15-dATP and flurescein-15-dITP. It has proved very difficult to synthesise a suitable fluroescent guanosine compound, so. an inosine compound is used in its place. Should a fluorescent guanosine compound become available, its use is envisaged in the present invention.
It has been found advantageous to use a mixture of unlabelled and labelled nucleo~~ides in the addition step.
When a fluorescent label is used, in order to produce all possible extension products on a template possessing a run of a particular nucleotide, the following ratios were found to be approximately optimal:
Fluorescein 15-dATP/dATP 500:1 -Fluarescein - 15-dITP/dGTP 500:1 Fluorescein - 12-dUTP/dTTP 15:1 Fluorescein - 12-dCTP/dCTP 15:1.
Preferably, therefore, the above ratios are used in connection with fluorescently - labelled nucleotides.
By repeating the incorporation and label detection steps until incorporation is detected, the nucleotide on the template adjacent the 3' end of the primer may be identified. Once this has been achieved, the label must be removed before repeating the process to discover the identity of the next nucleotide. Removal of the label may be effected by removal of the labelled nucleotide using a 3'-5' exonuclease and subsequent replacement with an unlabelled nucleotide. Alternatively, the labelling group can be removed from the nucleotide. In a further alternative, where the label is a fluorescent label, it is possible to neutralise the label by bleaching it with laser radiation.
If chain terminators or 3' blocking groups have been used, these should be removed before the next cycle can take place. Preferably, chain terminators are removed with a 3'-5' exonuclease. Preferably, exonuclease III is used. 3' blocking groups may be. removed by chemical or enzymatic cleavage of t:he blocking group from the nucleotide.
Where exonuc:lease III is used to remove the chain terminators, it is essential to prevent the exonuclease III
from chewing back along the growing chain to remove nucleotides which have alread; ~:aen incorporated, or even the primer itself" Preferab therefore, a nucleoside derivative which is resistant removal by exonucleases is used to replace the labelled nucleotides. Advantageously deoxynucleoside phosphorothioate triphosphates (dsNTPs) are used. Lik~awise, the primer preferably comprises a phosphorothioate nucleoside bases) at its 3' end which are incorporated during primer synthesis or an extra enzymatic capping step.
It is known that deoxynucleoside phosphorothioate derivatives resist digestion by exonuclease III (S. Labeit et al., DNA, 5_, p.173, 1986). This resistance is, however, not complete and conditions should be adjusted to ensure that excess digestion and removal of phosphorothioate bases does not occur.
For example, it .has been found that the pH of the exoIII
buffer used (50mM Tris/HC1, 5mM MgCl2) affects the extent of chewing back which occurs. Experiments carried out at pH
6.0, 7.0, 7.5., 8.0,. 8.5, 9.0 and 10.0 (37°C) reveal that pH
10.0 is the optimum with respect to the rate of reaction and specificity of exo:III. At this pH, the reaction was shown to go to completion in less than 1 minute with no detectable chewing back.
Once the label and terminators/blocking groups have been removed, the cycle is repeated to discover the identity of the next nucleotide.
_ ~13395fi In an alternative embodiment of the invention, steps (c) and (d) of the first aspect of the invention are repeated sequentially a plurality of times before removal or neutralisation of the label.
The number of times the steps (c) and (d) can be repeated depends on the sensitivity of the-apparatus used to detect when a labelled nucleotide has. been added onto the primer.
For instance, if each nucleotide is labelled with a different fluorescent label, the detection apparatus will need to be able to distinguish between each of the labels and will ideally be able to count the number of each type of fluorescent label. Alternatively, where each nucleotide is radioactively labelled or labelled with the same fluorescent dye, the apparatus will need to be able to count the total number of labels added to the primer.
As with the first embodiment of the invention, in a manual procedure using a single template, the labelled nucleotides are used singly and sequentially until a labelled nucleotide is added, whereupon the sequence is repeated. In an automated procedure all four labelled nucleotides are used sequentially and the apparatus is programmed to detect which nucleotides are added in what sequence to the primer.
Once the number of labels added has reached the resolving power of the detecting apparatus, removal or neutralisation of the label is carried out in a single step. Thus, the number of label removal steps is significantly reduced.
In this alternative embodiment, the steps (c) and (d) of the first aspect of the invention will preferably comprise:
i) adding a labelled nucleotide together with three heterogenous chain elongation inhibitors which are not incorporated into the chain, such as 5'-fa, p-methylene]
triphosphates;
13 ~ g ~ 6 PCT/GB93/00848 ii) removing excess reagents by washing;
iii) determining whether the label has been incorporated;
and iv) repeating steps (i) to (iii) using a different labelled nucleotide, either until a labelled nucleotide has been incorporated or until all four labelled nucleotides have been used.
This technique necessitates the use of a more sophisticated counter or label measuring device. Allowing for runs of repeated nucleotides, the label measuring device should be able to detect the presence of between four and sixteen labelled nucleotides accurately. For the measurement of long stretchE~s o:f repeated nucleotides, a device with a greater capacity may be required.
Scheme 1 According to a preferred aspect of the invention, a DNA
fragment is sequenced according to the following scheme:
1) a capped primer containing a phosphorothioate nucleoside derivative is hybridized to a template to form a template/primer complex;
2) a labESlled deoxynucleoside triphosphate (dNTP) together with heterogenous chain terminators and a suitable polymerase is added to the template/primer complex;
3) excess reagents are removed by washing;
4) the amount of incorporated label is measured;
5) the template/primer complex is treated with an exonuclease to remove the label and the dideoxynucleotides;
6) the exonuclease is removed by washing;
7) a phosphorothioate deoxynucleoside triphosphate corresponding to the labelled deoxynucleoside triphosphate added in Step 2 is added together with heterogenous chain terminators;
8) excess reagents are removed by washing;
9) the template/primer complex is treated with an exonuclease to remove the chain terminators;
10) the exonuclease is removed by washing; and 11) repeating step 2) to 10) four times, each time with a different labelled nucleotide, together with the appropriate heterogenous chain terminators.
For example, in Step 2 above the labelled nucleotide could be dATP. In this case, the heterogeneous chain terminators could be ddGTP, ddTTP and ddCTP. In step 7 phosphorothioate dATP would be added to replace the labelled dATP removed with the exonuclease in step 6. The cycle can then be repeated with another labelled nucleotide, for example dGTP, together with the heterogeneous dideoxynucleotides ddATP, ddTTP and ddCTP. This will cause label to be incorporated in all the chains propagating with G. This is followed in turn with labelled dTTP and labelled dCTP and continued again with dATP, dGTP, dTTP and dCTP and so on.
Scheme 2 According to a second preferred aspect of the invention, a DNA fragment is sequenced according to the follcwing scheme:
1) a capped primer containing a phosphorothioate nucleoside derivative is hybridized to a template to form a template/primer complex;
WO 93/21340 , ~ PCT/GB93/00848 2 ) a label:Led deoxynucleotide together with heterogeneous chain terminators and a suitable polymerase is added to the template/prim~~r complex;
3) excess reagents are removed by washing;
4) the amount of incorporated label is measured;
5) the lab~alled nucleotide and the chain terminators are removed with an exonuclease;
6) the exonuclease is removed by washing;
7) a phos;phorathioate deoxynucleotide together with heterogeneous chain elongation inhibitors not incorporated into the chain is added;
8) excess :reagents are removed by washing; and 9) steps 2 ) to 8) are repeated four times, each time with a different 1<ibelled nucleotide.
This scheme i,s essentially a sub-scheme of scheme 1. The main difference is that during the capping step 7, dideoxynucleoi=ides are replaced by the corresponding 5'-[a, ~-methylene] triphosphates derivatives. However, other chain elongation inhibitors like deoxynucloside diphosphate or deoxynucleoside monophosphate derivatives may also be used. Since i:hese derivatives cannot be incorporated into the growing polynuc:leotide chain there is no need to remove them. Hence, scheme 2 completely lacks the last exonuclease treatment step and t:he subsequent washing step of scheme 1.
Scheme 3 According to a third preferred aspect of the invention, a WO 93/21340 _ ~ ~~ ~ ~ ~ ~ ~ PGT/GB93/00&
DNA fragment is sequenced according to the following scheme:
1) a capped primer containing a phosphorothioate deoxynucleotide is hybridised to a template to form a template/primer complex;
2) a labelled nucleotide triphosphate together with heterogeneous chain elongation inhibitors not incorporated into the chain is added;
3) excess reagents are removed by washing;
4) the amount of incorporated label is measured;
5) steps 2 to 4, adding different labelled nucleotides in the presence of their corresponding heterogeneous chain elongation inhibitors not incorporated into the chain, are repeated until all four labelled nucleotides have been added;
6) all labelled nucleotides are removed with exonuclease;
7) the exonuclease is removed by washing;
8) the phosphorothioate deoxynucleotide corresponding to the first labelled deoxynucleotide added to the reaction in step 2 , is added together with heterogenous claim elongation inhibitors not incorporated into the chain and a suitable polymerase;
9) excess reagents are removed by washing; and 10) steps 8 and 9 are repeated with the three remaining phosphorothioate deoxynucleoside derivatives.
This scheme has the notable advantage of reducing overall number of exonuclease steps. All four labelled nucleotides are sequentially added to the chain and individually ~~_339~~
detected before all incorporated nucleotides are removed by a single exonuclease digestion step. The chase reactions are then carried out sequentially with the appropriate phosphorothioate nucleoside derivatives.
Scheme 4 In a fourth preferred aspect of the invention, a DNA
fragment is sequenced according to the following scheme:
1) a capped primer is hybridized to a template to form a template/primer complex;
2) a fluorescent nucleoside triphosphate, together with three heterogeneous chain elongation inhibitors not incorporated into the chain and a suitable polymerase, is added;
3) excess reagents are removed by washing;
4) the arnount of incorporated label is measured;
5) steps 2 to 4 are repeated using all three different nucleoside t.riphosphates, each with a fluorescent label, in the presen~~e of the respective heterogeneous chain elongation inhibitors not incorporated into the chain.
6 ) the f 7.uorescent labels are destroyed by bleaching with a laser or by a suitable chemical reaction, or the fluorescent labels are removed by a chemical cleavage step.
This scheme has the advantage that no enzymatic removal of incorporated label by way of an exonuclease reaction is required, nor is a chasing reaction with a phosphorothioate nucleotide derivative necessary. Instead, all incorporated fluorophore;s are chemically destroyed using either laser bleaching technology or suitable chemical reactions to destroy the: dysa or cleave the dye from the nucleotides.
WO 93/21340 _ '~ ~ ~ ~ ~ ~ ~ PCT/GB93/0084 Preferably, if the detector used permits quantitative measurement of incorporated label, the bleaching or cleaving step need only be carried out from time to time rather than after each successive addition.
Scheme 5 According to a fifth preferred aspect of the invention a DNA
fragment is sequenced according to the following scheme:
1) a capped primer is hybridized to a template;
2) a nucleoside triphosphate labelled by attachment of a fluorescent dye group via a linker arm to the 3' moiety of the deoxyribose sugar thereon is added;
3) excess reagents are removed by washing;
4) the amount of incorporated label is measured;
5) the fluorescent dye group is removed by enzymatic cleavage; and 6) excess reagents are removed by washing.
In Scheme 5, the nonspecific addition of labelled nucleotide is prevented by the 3' modification, so that the labelled nucleotide effectively acts as a chain terminator. Removal of the 3' blocking group is then all that is required to allow chain elongation to continue.
In a further aspect of the invention, there is provided a sequencing kit comprising at least three of the following:
i) a linker for attaching a DNA template to a solid-phase matrix, the linker comprising a primer having a deoxynucleoside phosphorothioate residue at its 3' end;
~133~~G
ii) chain elongation inhibitors;
iii) fluorescently-labelled nucleoside triphosphates;
iv) deoxynucleoside phosphorothioate triphosphates;
v) a 5'-> 3' ;DIVA polymerase;
vi) a 3'-> 5' exonuclease.
In addition, such a kit may comprise a solid support for carrying out the reaction, as well as biotinylated primers or linkers arid biot.in/streptavidin reagents for coupling the linker to th~~ solid support. The 3'-5' exonuclease may be exonuclease :CII. kurthermore, al::ernative chain elongation inhibitors, such as 3'-deoxyribose blocked labelled nucleotides, may be included. Preferably, the kit will comprise all of the components i-vi.
The invention also comprises an automatic sequencing machine capable of sequencing a nucleic acid essentially by executing the steps of a method according to the invention.
The machine is adapted either to move the solid support carrying the templates) into and out of all the necessary reagent and washing solutions, or to pump reagents and washing solutions over the solid support sequentially. The pin array type of support is better suited to the first procedure, winile glass plates and sequencing chips are more appropriate to the second.
Several washing steps are included between each reagent addition to minimise the carry-over of reagents.
The presence of label may be determined, in the case of a chip array, by passing the array over a fixed detector which records the level of label relative to the position of the array over the detector. In the case of a fixed glass plate WO 93/21340 , PCT/GB93/0084 X1.33956 or sequencing chip array, a radioactive or f luorescent image may be obtained by a fixed detector positioned above the array. Alternatively,.the glass plate or sequencing chip array and/or the detector may be movable. A two-dimensional image is produced by the detector and analysed by a computer.
Alternatively, optical fibres connected directly to a sequencing chip or to the pins in a pin array may be used to transmit data to a processor if used together with fluorescent labels.
The invention will now be described, for the purpose of illustration only, with reference to the following figures:
Figure 1 is a graph showing the correlation of emitted fluorescence to the number of nucleotides incorporated, using dUTP-12-fluorescein; and Figure 2 is as figure 1 except that dCTP-12-fluorescin is used.
EXAMPLES
Generation of template and binding to solid sut~port In this example an anchored single-stranded PCR product was used which was generated by known methods (T. Hultman et al., Nucleic Acids Res., 17, (1989), 4937-4946; D.S.C. Jones et al., DNA Sequence, 1 (1991), 279-283). Briefly, the template was generated by the polymerase chain reaction (PCR) using one biotinylated primer and one normal primer and the product subsequently bound to streptavidin coated magnetic beads. By treating the anchored double-stranded PCR product with alkali the non-anchored template strand is removed. All the steps were carried out as follows:
WO 93/21340 ~ I ~ 3 9 5 6 p~/GB93/00848 PCR was performed in 50 ~,1 using 0.5 ml test tubes. The following items were added: 30 ~cl water, 5 u1 of 10 x PCR
buffer (fetus) , 5 ~,1 of 2.5 mM dNTP's, 2.5 ~.1 of 10 ~M of the 5'-biot:Lnyl~ted universal reverse primer with the sequence: 5' Bio-AACAGCTATGACCATG 3', 2.5 ~1 of 10 ~M of the (-20) universal forward primer with the sequence: 5' GTAAAACGACGGCCAGT 3', 1 ~,1 of the Bluescript KS plasmid DNA
at the concentration 1 ng/~1, 0.5 ~cl (2.5 units) of native Taq polymerase (Cet:us) . After overlaying with light mineral oil the following cycles were performed: 95°C 90s, [95°C
30s, 55°C 60;s, 72°C 60s] x 35, 72°C 180s. All cycles were performed uaing the maximum heating and cooling rates possible with the Techne PHC-1 or PHC-2.
Binding the biotinylated PCR product with a length of approximately 250 by to the streptavidin-coated magnetic beads (Dynal) is accomplished by incubating 100 ~1 of beads under mineral oil .at room temperature for 5 min. The beads are sedimented using a strong magnet and the supernatant including the mineral oil is removed. Further traces of unused nucleotides, primers and buffers are removed by washing the heads with 100 ~:1 of water . The nonbiotinylated DNA strand i;s removed by incubating the beads with 50 ~1 of 0.15 M NaOH for 5 min. at room temperature. The beads are sedimented a.nd the supernatant is removed, followed by a further treatment with 50 u1 of 0.15M NaOH and three washings with 100 u1 of water. Finally the beads were resuspended in 10 ~1 of water.
Annealin4 of the seQUencin~primer to the anchored sink stranded DNA template To the 10 u1 resuspended beads with the anchored single-stranded DNA template (approximately 2 pmoles), 4 ~1 of 5 x Sequenase annealing buffer (200 mM Tris/HC1 pH 7.5 100 mM
MgCl2, 250 mM NaCl., USB) , and 4 ~cl (4 pmoles) of T7 primer with the sequence: 5' AATACGACTCACTATAG 3' are added. The ~.~3~95 6 PCT/GB93/0084;
' 24 mixture is heated for 3 min. at 65°C and then cooled on ice.
The template/primer complex is now ready for sequencing.
The following figure displays parts of its structure:
Complex 1: Polymer-streptavidin-biotin-5'-DNA-C-C-A-A-T-T-C-G-C-C-C-T-A-T-A-G-T-G-A-G-T-C-G-T-A-T-T-------3' 3'-G-A-T-A-T-C-A-C-T-C-A-G-C-A-T-A-A-------5' Capping of the primer with thionucleotides To the 18 u1 annealing mixture add 10 u1 of 100 uM dSGTP, dSCTP, ddATP, ddTTP, and 4 ~cl (5 units) of diluted sequenase 2.0 (USB), and incubate the mixture for 2 min. at room temperature. According to the complementary strand this adds the following five nucleotides sequentially to the primer: dSG, dSG, dSC, dSG, and ddA. The beads were sedimented using the magnet and the supernatant removed.
The beads were then washed two times with 50 u1 water.
Removing the dideox~nucleotide from the capped primer To the bead 10 ~1 (20 units) of an exonuclease solution in 50 mM Tris/HC1 pH 7.5, 5 mM MgCl2, 5 mM DTT were added and the mixture incubated 2 min. at 37°C. The reaction was stopped by sedimenting the beads with the magnet and removing the supernatant, followed by three washings with 50 ~1 of water. This step removed the dideoxy A-nucleotide from the 3'-end of the primer.
SEQUENCING BY SEQUENTIAL ADDITION OF SINGLE LABELLED
NUCLEOTIDES: FIRST COMPLETE CYCLE OF 9 STEPS
Scheme 1 Steps 2 and 3.
The beads (anchored template/primer complex 1) where resuspended in 13 ~.1 water. The following items were added:
5 ~.1 5 x sequenase buffer, 10 ~1 of a nucleotide mixture containing l0 ~,Ci of alpha-32P dATP of specific activity of ~~~~3~56 400 Ci/mmole, 4~,M cold dATP, 100 ~tM ddGTP, 100 ~,M ddTTP, 100 uM ddCTP, anc9 4 ~r,l of diluted sequenase 2Ø The mixture was incubated~,for 2 min_at 37°C and the reaction stopped by sedimenting the beads with the magnet and removing the 5 supernatant followed by three further washings with 50 ~1 of water. In this step, according to the complementary strand, two A-nucleotides and one dideoxy T-nucleotide were added to the 3'-end of the tapped primer.
10 Step 4 The label is counted with a hand counter Steps 5 and E.
The dideoxynucleotide and the labelled nucleotides were 15 removed by adding 10 ~1 (20 units) of an exonuclease III
solution in 50 mM Tris/HC1 pH 7.5, 5 mM MgCl2, 5 mM DTT and incubating the mixture for 2 min. at 37°C. The reaction was stopped by ,sedimenting the beads with the magnet and removing the supernatant, followed by three washings with 50 20 u1 of water. The removal of the label was checked by measuring ths~ mixture with the hand counter.
Steps 7 and ~.
In order to cap the primer, the beads were resuspended in 13 25 ~1 water. The following items were added: 5 ~1 5 x sequenase buffer, 10 u1 of a nucleotide mixture containing 100 ACM dSATP, 100 ~M ddGTP, 100 uM ddTTP, 100 ~N ddCTP, and 4 ~,1 of diluted sequenase 2Ø The mixture was incubated 2 min. at 37°C and the reaction stopped by sedimenting the beads with the magnet and removing the supernatant followed by three further washings with 50 ~,1 of water. In this step, two thiolat:ed A-nucleotides and one dideoxy T-nucleotide were added to the sequencing primer.
3 5 Steps 9 and 1.0 The dideoxy nucleotide was removed by adding 10 ~,1 (20 units) of the' above specified exonuclease III solution and incubating th.e mixture for 2 min. at 37°C. The reaction was WO 93/21340 PCT/GB93/00&
~~33g56 stopped by sedimenting the beads with the magnet and removing the supernatant, followed by three washings with 50 ~cl of water.
SEQUENCING BY SEQUENTIAL ADDITION OF SINGLE NUCLEOTIDES:
Scheme 1 Steps 2 and 3 The beads (anchored template/primer complex 1) were resuspended in 13 ~1 water. The following items were added:
5 u1 of 5 x sequenase buffer, 10 ~.1 of a nucleotide mixture containing 10 uCi of alpha-32P dTTP of specific activity of 400 Ci/mmol, 4 ACM cold dTTP, 100 ~,M ddGTP, 100 ~tM ddATP, 100 ~cM ddCTP, and 4 ~,1 of diluted sequenase 2Ø The mixture was incubated 2 min. at 37°C and the reaction stopped by sedimenting the beads with the magnet and removing the supernatant followed by three further washings with 50 ~,1 of water. In this step, according to the complementary strand, two T-nucleotides and one dideoxy G-nucleotide were added to the 3'-end of the capped primer.
Step 4 The label is counted with a hand counter.
Steps 5 and 6 The dideoxynucleotide and the labelled nucleotides were removed by adding 10 u1 (20 units) of the above specified exonuclease III solution and incubating the mixture for 2 min at 37°C. The reaction was stopped by sedimenting the beads with the magnet and removing the supernatant, followed by three washings with 50 u1 of water. The removal of the label was checked by measuring the mixture with the hand counter.
Steps 7 and 8 In order to cap the primer, the beads were resuspended in 13 ~cl water. The following items were added: 5 ~cl of 5 x sequenase buffer, 10 ~1 of a nucleotide mixture containing 13 3 9 5 G p~/GB93/00848 100 uM dSTTP, 100 ~.M ddGTP, 100 ~,M ddATP, 100 ~.M ddCTP, and 4 ~cl of diluted sequenase 2Ø The mixture was incubated 2 min. at 37°c, and the reaction stopped by sedimenting the beads with the magnet and removing the supernatant followed by three further washings with 50 ~cl of water. In this step, two thiolated T-nucleotides and one dideoxy G-nucleotide were added to the sequencing primer.
Steps 9 and 10 The dideoxy nucleotide was removed by adding 10 ~,1 (20 units) of th.e above specified exonuclease III solution and incubating the mixture for 2 min. at 37°C. The reaction was stopped by sedimenting the beads with the magnet and removing the supernatant, followed by three washings with 50 u1 of water.
This experiment was carried out in order to confirm that all the reactions described in example 1 yielded the correct elongation a.s well as degradation products. To prove this, the experiment described in example 1 was repeated using a 32P-labelled primer in combination with cold nucleotides.
The following modifications were made:
1. 4 pmo:les of a 5'-32P-labelled sequencing primer with the sequence 5' AATACGACTCACTATAG 3' was used in the annealing step;
2. In step 2 of the first cycle the labelled compound a-32P-dATP was omitted from the nucleotide mixture and the concentration of the cold dATP was increased to 100 ~,M;
3. In step 2 of the second cycle the labelled compound a-32-dTTP w~is omitted from the nucleotide mixture and the concentration of the cold dTTP was increased to 100 uM;
4. Step 4 in both cycles was not necessary;
5. After each enzymatic reaction and subsequent washing a 1/100 aliquot of the beads were removed and placed in a separate 0.'S ml test tube.
WO 93/21340 '~ ~ ~ ~ ~ ~ ~ PCT/GB93/0084 After performing all steps described in example 1, 5 ~,1 of 90% formamide dye mix were added to all the individual bead aliquots, the mixtures heated for 3 min. at 95°C, centrifuged at 13,OOOg for 5 seconds and cooled on ice. A
small aliquot (1 u1) of each sample was loaded into a individual well of a 20% polyacrylamide gel containing 7M
urea and electrophoresed at 700 Volts for 3 to 4 hours.
After electrophoresis the upper glass plate was removed and the exposed to a X-ray film for approximately 2 to 4 hrs.
The band pattern obtained was in full agreement with the predicted length of all primer elongation and degradation products.
Annealing and binding of the template/primer complex to solid sut~port In this example the biotinylated sequencing primer was f first annealed to the complementary region of a single-stranded M13 template and the complex subsequently bound via the 5' biotin moiety of the primer to the solid support (streptavidin beads). 2 ~g (1 pmole) of M13mp18 DNA was combined with 2 pmoles of 5' biotinylated (-20) universal forward primer with the sequence 5' GTAAAACGACGGCCAGT 3' in 40 mM Tris/HC1 pH 7.5, 20 mM MgCl2, 50 mM NaCl in a total of 10 ~1. The mixture was heated for 3 min. at 65°C and slowly cooled down to room temperature over a period of 10 min. 30 ~cl of streptavidin-coated magnetic beads (Dynal) were added 3 0 and the mixture incubated f or 5 minutes at room temperature .
The beads were sedimented, the supernatant removed, and the beads resuspended in 10 ~l of water.
Ca~pina of the primer with thionucleotides To the 18 ~cl annealing mixture add 10 ~1 of 100 ~M dsGTP, ddATP, ddTTP, ddCTP, and 4 ~1 (5 units) of diluted sequenase 2.0 (USB), and incubate the mixture for 2 min. at room temperature. According to the complementary strand this adds the following two nucleotides sequentially to the primer: d$G and ddA. The beads were sedimented using the magnet and t:he :>upernatant removed. The beads were then washed two times with 50 u1 water.
Removincr the dideoxvnucleotide from the capped primer To the bead 10 ~.;1 (20 units) of an exonuclease solution in 50 mM Tris-Hcl pH 7.5, 5 mM MgCl2, 5 mM DTT were added and the mixture incubated 2 min. at 37°C. The reaction was stopped by sedimenting the beads with the magnet and removing the supernatant, followed by three washings with 50 ~1 of water. This step removed the dideoxy A-nucleotide from the 3'-end of the primer.
SEQUENCING BY SEQUENTIAL ADDITION OF SINGLE LABELLED
NUCLEOTIDES: FIRST' COMPLETE CYCLE OF 9 STEPS
Scheme 1 Steps 2 and 3.
The beads (anchared template/primer complex 1) were resuspended :in 13 y1 water. The following items were added:
5 ~1 5 x seduenase buffer, 10 u1 of a nucleotide mixture containing 10 ~Ci of alpha-32P dATP of specific activity of 400 Ci-mmol, 4 ~,M cold dATP, 100 ~cM ddGTP, 100 ~M ddTTF, 100 ~M ddCTP, and 4 ~:l of diluted sequenase 2Ø The mi~:ture was incubated for. 2 min at 37°C and the reaction stoppe3 by sedimenting the beads with the magnet and removing the supernatant :Followed by three further washings with 50 u1 of water. In this step, according to the complementary strand, two A-nucleoi=ides and one dideoxy T-nucleotide were added to the 3' end of the capped primer.
Step 4 The label is counted with a hand counter.
Steps 5 and 6 WO 93/21340 PCT/GB93/00&
The dideoxynucleotide and the labelled nucleotides were removed by adding 10 ~1 (20 units) of an exonuclease III
solution in 50 mM Tris./HC1 pH 7.5 mM MgCl2, 5 mM DTT and incubating the mixture for 2 min. at 37°C. The reaction was 5 stopped by sedimenting the beads with the magnet and removing the supernatant, followed by three washings with 50 ~cl of water. The removal of the label was checked by measuring the mixture with the hand counter.
10 Steps 7 and 8 In order to cap the primer, the beads were resuspended in 13 ~cl water. The following items were added: 5 ~,1 5 x sequence buffer, 10 ~1 of a nucleotide mixture containing 100 ~M
dsATP, 100 ~M ddGTP, 100 ~M ddTTP, 100 uM ddCTP, and 4 ~C1 of 15 diluted sequenase 2Ø The mixture was incubated 2 min. at 37°C and the reaction stopped by sedimenting the beads with the magnet and removing the supernatant followed by three further washings with 50 u1 of water. In this step, two thiolated A-nucleotides and one dideoxy T-nucleotide were 20 added to the sequencing primer.
Steps 9 and 10 The dideoxy nucleotide was removed by adding 10 ~1 (20 units) of the above specified exonuclease III solution and 25 incubating the mixture f or 2 min . at 37 ° C. The reaction was stopped by sedimenting the beads with the magnet and removing the supernatant, followed by three washings with 50 ~.1 of water .
30 SEQUENCING BY SEQUENTIAL ADDITION OF SINGLE NUCLEOTIDES:
Scheme 1 Steps 2 and 3.
The beads (anchored template/primer complex 1) were resuspended in 13 ~1 water. The following items were added:
5 ~,1 of 5 x sequenase buffer, 10 ~,1 of a nucleotide mixture containing 10 ~Ci of alpha-32-P dTTP of specific activity of WO 93/21340 ~ ~ ~ ~ ~ ~ ~ PCT/GB93/00848 400 Ci/mmol, 4 ~M cold dTTP, 100 uM ddGTP, 100 ~.M ddATP, 100 uM ddCTP, anc3 4 ,u1 of diluted sequenase 2Ø The mixture was incubated 2 min. at 37°C and the reaction stopped by sedimenting the beads with the magnet and removing the supernatant followed by three further washings with 50 ~1 of water. In this step, according to the complementary strand, two T-nucleotides and one dideoxy C-nucleotide were added to the 3-end of the capped primer.
Steb 4 The label is counted with a hand counter.
Steps 5 and E~
The dideoxynucleotide and the labelled nucleotides were removed by adding 10 a 1 ( 2 0 units ) of the above specified exonuclease ::II solution and incubating the mixture for 2 min. at 37°C. The reaction was stopped by sedimenting the beads with the magnet and removing the supernatant, followed by three washings with 50 u1 of water. The removal of the label was checked by measuring the mixture with the hand counter.
Steps 7 and ~~
In order to cap the primer, the beads were resuspended in 13 u1 water. The following items were added: 5 u1 of 5 x sequenase buffer, :LO ~cl of a nucleotide mixture containing 100 ~M dSTTP, 100 ~M ddGTP, 100 ~M ddATP, 100 uM ddCTP, and 4 ~1 of diluted sequenase 2Ø The mixture was incubated 2 min. at 37°C and the reaction stopped by sedimenting the beads with the magnet and removing the supernatant followed by three further washings with 50 ~.1 of water. In this step, two thiolated T-nucleotides and one dideoxy C
nucleotide were added to the sequencing primer.
Steps 9 and 7.0 The dideoxy nucleotide was removed by adding 10 u1 (20 WO 93/21340 ~ ~ e~ ,~ ~ ~ ~ PCT/GB93/0084.°
units) of the above specified exonuclease III solution and incubating the mixture for 2 min. at 37°C. The reaction was stopped by sedimenting the beads with the magnet and removing the supernatant , f of lowed by three washings with 50 ~1 of water.
Template preparation, binding of the template to solid support, and annealing of the sequencing primer was performed as described in example 1, except that in the annealing step 4 u1 (4 pmoles) of radio-labelled T7 primer with the sequence: 32P-5'AATACGACTCACTATAG 3' are used.
Template/primer comt~lex 2:
Polymer-streptavidin-biotin-5'-C-C-A-A-T-T-C-G-C-C-C-T-A-T-A-G-T-G-A-G-T-C-G-T-A-T-T----3' 2 0 3'-G-A-T-A-T-C-A-C-T-C-A-G-C-A-T-A-A-32P-5' Capping of the primer with thionucleotides To the 18 ~1 annealing mixture add 10 ~1 of 100 uM deGTP, ddATP, ddTTP, and ddCTP and 4 ~1 (5 units) of diluted sequenase 2.0 (USB), and incubate the mixture for 2 min. at room temperature. According to the complementary strand this adds the following three nucleotides sequentially to the primer: dsG, dSG, ddC. The beads were sedimented using the magnet and the supernatant removed. The beads were then washed two times with 50 u1 water.
Removinct the dideoxynucleotide from the capped primer To the bead 10 u1 (20 units) of an exonuclease solution in 50 mM Tris/HC1 pH 7.5, 5 mM MgCl2, 5 mM DTT were added and the mixture incubated 2 min. at 37°C. The reaction was stopped by sedimenting the beads with the magnet and WO 93/21340 - r ~ ~ ~ ~ pCT/Gg93/00848 removing the supernatant, followed by three washings with 50 ~,1 of water. This step removed the dideoxy C-nucleotide from the 3'-end of the.primer.
FIRST SEQUENCING CYCLE (9 STEPS) Scheme 1 Steps 2 and 3 The beads (anchored template/primer complex 2) were resuspended :in 13. ~cl water. The following items were added:
5 u1 5 x sec~uenase buffer, 10 u1 of a nucleotide mixture containing 100 ul~t dCTP, 100 ~,M ddGTP, 100 ~,M ddATP, 100 ~M
ddTTP, and 4 u1 of diluted sequenase 2Ø The mixture was incubated for 2 m.rn. at 37 °C and the reaction stopped by sedimenting the beads with the magnet and removing the supernatant l:ollawed by three further washings with 50 ~1 of water. In this step, according to the complementary strand, one C-nucleol_ide a:nd one dideoxy G-nucleotide were added to the 3-end of the capped primer.
Step 4 This step is omitted because the label is located on the primer.
Steps 5 and ~5 The dideoxynucleotide and the labelled nucleotides were removed by adding 10 ~1 (20 units) of an exonuclease III
solution 50 mM Tris/HC1 pH 7.5 mM MgCl2, 5 mM DTT and incubating tree mixture for 2 min. at 37°C. The reaction was stopped by sedimenting the beads with the magnet and removing the supernatant , f of lowed by three washings with 50 ~1 of water.. The removal of the label was checked by measuring them mixture with the hand counter.
Steps 7 and F3 In order to cap the: primer, the beads were resuspended in 13 u1 water. The following items were added: 5 ~cl 5 x sequenase buffer, 10 ~,1 of a nucleotide mixture containing ~M dSCTP, 100 ~,M ddGTP, 100 ~M ddATP ~M ddTTP, and 4 u1 of diluted sequenase 2Ø The mixture was incubated 2 min.
5 at 37°C and the reaction stopg~d by sedimenting the beads with the magnet and removing the supernatant followed by three further washings with 50 ~,1 of water. In this step, one thiolated C-nucleotide and one dideoxy G-nucleotide were added to the sequencing primer.
Steps 9 AND 10 The dideoxy nucleotide was removed by adding 10 ~cl (20 units) of the above specified exonuclease III solution and incubating the mixture for 2 min. at 37°C. The reaction was stopped by sedimenting the beads with the magnet and removing the supernatant , followed by three washings with 50 ~,1 of water.
SECOND SEQUENCING CYCLE (9 STEPS) Scheme 1 Steps 2 and 3 The beads (anchored template/primer complex 2) were resuspended in 13 u1 water. The following items were added:
5 u1 5 x sequenase buffer, 10 ~,1 of a nucleotide mixture containing 100 uM dGTP, 100 ~M ddATP, 100 uM ddTTP, 100 ~M
ddCTP, and 4 u1 of diluted sequenase 2Ø The mixture was 3 0 incubated f or 2 min . at 37 ° C and the reaction stopped by sedimenting the beads with the magnet and removing the supernatant followed by three further washings with 50 u1 of water. In this step, according to the complementary strand, one G-nucleotide and one dideoxy A-nucleotide were added to the 3'-end of the capped primer.
Step 4 This step is omitted because the label is located on the ~y0 93/21340 _ ~ ~ ~ ~ 9 5 6 PCT/GB93/00848 primer.
Steps 5 and E: .
The dideoxyn~scleatide and the labelled nucleotides were 5 removed by adding 10 ~,1 (20 units) of an exonuclease III
solution in 50 mM Tris/HC1 pH 7.5, 5 mM MgClz, 5 mM DTT and incubating the mixture for 2 min. at 37°C. The reaction was stopped by :~edimenting the beads with the magnet and removing the :supernatant, followed by three washings with 50 10 ~,1 of water. The removal of the label was checked by measuring the mixture with the hand counter.
Steps 7 and 8, In order to cap the primer, the beads were resuspended in 13 15 u1 water. The following items were added: 5 u1 x sequenase buffer, l0 u1 of a nucleotide mixture containing 100 ~cM
dsGTP, 100 ACM ddATP, 100 uM ddTTP, 100 ~,M ddCTP, and 4 u1 of diluted sequenase 2Ø The mixture was incubated 2 min. at 37°C and the :reaction stopped by sedimenting the beads with 20 the magnet and removing the supernatant followed by three further washings with 50 ~,1 of water. In this step, one thiolated G-nucleotide and one dideoxy A-nucleotide were added to the sequencing primer.
25 Steps 9 and 10 The dideoxy nucleotide was removed by adding 10 u1 (20 units) of the above specified exonuclease III solution and incubating the mixture for 2 min at 37°C. The reaction was stopped by sedimenting the beads with the magnet and 30 removing the supernatant, followed by three washings with 50 ~c 1 of water .
THIRD SEQUENCING CfCLE (9 STEPS) 35 Scheme 1 Steps 2 and 3 The beads (anchored template/primer complex 2) were WO 93/21340 r PCT/GB93/0084p resuspended in 13 ~1 water. The following items were added:
~1 5 x sequenase buffer, 10 u1 of a nucleotide mixture containing 100 ~.M dATP, 100 ~M ddGTP, 100 ~,M ddTTP, 100 ACM
ddCTP, and 4 ~,1 of diluted s~quenase 2Ø The mixture was 5 incubated for 2 min at 37:°'C and the reaction stopped by sedimenting the beads with the magnet and removing the supernatant followed by three further washings with 50 ~.1 of water. In this step, according to the complementary strand, two A-nucleotides and one dideoxy T-nucleotide were added at the 3'-end of the capped primer.
Step 4 This step is omitted because the label is located on the primer.
SteQs 5 and 6 The dideoxynucleotide and the labelled nucleotides were removed by adding 10 ~,1 (20 units) of an exonuclease III
solution in 50 mM Tris/HC1 pH 7.5, 5 mM MgCl2, 5 mM DTT and incubating the mixture for 2 min at 37°C. The reaction was stopped by sedimenting the beads with the magnet and removing the supernatant, followed by three washings with 50 ~1 of water. The removal of the label was checked by measuring the mixture with the hand counter.
Steps 5 and 6 In order to cap the primer, the beads were resuspended in 13 ~cl water. The following items were added: 5 gel 5 x sequenase buffer, 10 ~1 of nucleotide mixture containing 100 ACM d$ATP, 100 uM ddGTP, 100 ~M ddTTP, 100 ~,M ddCTP, and 4 ~C1 of diluted sequenase 2Ø The mixture was incubated 2 min.
at 37°C and the reaction stopped by sedimenting the beads with the magnet and removing the supernatant followed by three further washings with 50 ~.1 of water. In this step, one thiolated A-nucleotide and one dideoxy T-nucleotide were added to the sequencing primer.
Steps 9 and 10 ~~ r~'39~6 The dideoxy nucleotide was removed by adding 10 ~,1 (20 units) of th~a abave specified exonuclease III solution and incubating the mixture ,for 2 min at 37°C. The reaction was stopped by sedimenting the beads with the magnet and removing the supernatant, followed by three washings with 50 u1 of water.
FOURTH SEQUE1JCING CYCLE (9 STEPS) Scheme 1 Steps 2 and :3 The beads (anchored template/primer complex 2) were resuspended in 13 ~,1 water. The following items were added:
5 ~1 5 x sec~uenase buffer, 10 u1 of a nucleotide mixture containing 10 ~cM dTTP, 100 uM ddGTP, 100 ~.M ddATP, 100 ACM
ddCTP, and 4 ~,l of diluted sequenase 2Ø The mixture was incubated fo:r 2 min. at 37°C and the reaction stopped by sedimenting the beads with the magnet and removing the supernatant follawed by three further washings with 50 ~cl of water. In this step, according to the complementary strand, two T-nucleotides and one dideoxy G-nucleotide were added to the 3'-end o:E the capper primer.
Step 4 This step i;s omitted because the label located on the primer.
Steps 5 and y The dideoxynucleotide and the labelled nucleotides were removed by adding 10 ~1 (20 units) of an exonuclease III
solution in ..°i0 mM Tris/HC1 pH 7.5, 5 mM MgCl2, 5 mM DTT and incubating the mixture for 2 min. at 37 °C. The reaction was stopped by sedimenting the beads with the magnet and removing the supernatant, followed by three washings with 50 u1 of water.. The removal of the label was checked by measuring the. mixture with the hand counter.
WO 93/21340 - '~ ~ ~ '~ 9 5 6 pCT/GB93/0084F
Stegs 7 and 8 In order to cap the primer, the beads were resuspended in 13 ~1 water. The following items were added: 5 ~1 5 x sequenase buffer, 10 ~1 of a nucleotide mixture containing 100 ACM dSTTP, 100 ~M ddGTP,'100 ~M ddATP, 100 uM ddCTP, and 4 ~1 of diluted sequenase 2Ø The mixture was incubated 2 min. at 37°C and the reaction stopped by sedimenting the beads with the magnet and removing the supernatant followed by three further washings with 50 ~1 of water. In this step, one thiolated T-nucleotides and one dideoxy G-nucleotide were added to the sequencing primer.
Steps 9 and 10 The dideoxy nucleotide was removed by adding 10 ~1 (20 units) of the above specified exonuclease III solution and incubating the mixture for 2 min. at 37°C. The reaction was stopped by sedimenting the beads with the magnet and removing the supernatant, followed by three washings with 50 ~,1 of water.
Fluorescein was used as a single tag attached to all four deoxynucleotides. In particular we used the following fluorescein-labelled deoxynucleoside triphosphates:
fluorescein-12-dUTP, fluorescein-15-dATP, fluorescein-15-dCTP, fluorescein-15-dITP.
GENERATION OF TEMPLATES
As a model template we used two single-stranded PCR products which were derived from the multicloning site of Bluescript II KS. Amplification of the Bluescript II KS vector DNA
using the biotinylated M13 (-21) forward primer and the nonbiotinylated M13 reverse primer yielded a PCR product which was anchored via the biotin moiety to streptavidin-coated beads as described in example 1. The nonbiotinylated ~y0 93/21340 ~ I 3 ~ ~ ~ 6 PCT/GB93/00848 (+) strand was removed by incubating the beads with 0.15 M
NaOH for 5 minutes followed by a wash with 0.15 M NaOH and three washes with water.. The template, comprising the (-) strand of tree multicloning site of the Bluescript II KS
vector, was named PCR template 1. Amplification of the Bluescript I:L KS vector using the biotinylated M13 reverse primer and t:he nonbiotinylated M13 (-21) forward primer yielded a PC;R product which was anchored via the biotin moiety to streptavidin-coated beads as described in example 1. The nonbiotinyl.ated (-) strand was removed by incubating the beads with 0.15 M NaOH for 5 minutes followed by a wash with 0.15 M NaOH and three washes with water. This template comprising t:he (~+) strand of the multicloning site of the Bluescript I:L KS vector was named PCR template 2.
SYNTHESIS OF' S'~-T'AMRA LABELLED SPECIFIC OLIGONUCLEOTIDE
PRIMERS
For each fluorescently-labelled nucleotide four different primers were designed using the Bluescript sequence of the PCR template 1 and 2. The primers were located in front of runs of a single nucleotide allowing incorporation of one, two, three, :Four,, or five nucleotides of the same kind.
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WO 93/21340 ~ ~ ~,~ ~ ~ ~ PCT/GB93/0084~' ANNEALING
In sixteen different annealing reactions, 2~,1 of water, 5~,1 of the appropriate single-stranded PCR template 1 or 2 (see tables), 2~1 of 5 x sequenase buffer and 1 u1 (0.5 pmol) of the appropriate TAMRA-labelled primer (see tables) were combined , heated at 65 ° C f or 3 minutes and then incubated on ice.
EXTENSION REACTIONS
In sixteen different extension reactions, to 6~,1 of each annealing mix, 2~1 of a nucleotide mixture (see tables) containing the appropriate unlabelled dNTPs (at 10 uM), the appropriate fluorescently-labelled dNTP (at 10 ACM), and the appropriate ddNTP (at 10~M) , and 2~,1 of diluted sequenase 2.0 were added and the mixture incubated at 37°C for 3 minutes. The reactions were stopped by adding 5~1 of 80%
formamide and heated for 3 min at 80°C followed by sedimenting the beads with a magnet and removing the supernatant.
DETECTION/IMAGING STEP (QUANTITATION) One ~.1 of each supernatant was measured using a SIT camera (model C2 400-08, Hamamatsu Photonics SA) mounted on a fluorescence microscope. The emitted fluorescence of the rhodamine dye TAMRA located at the 5'-end of the primer and the fluorescein dye introduced by nucleotide incorporation at the 3'-end of the primer was determined for each sample using appropriate filter systems. A control sample of 80%
formamide was also measured. The emitted fluorescence DI
fluorescein and DI rhodamine was recorded. The ratio of DI
fluorescein to QI rhodamine was used to normalise the data.
The results may be summarised as follows:
- Incorporation of up to five fluorescein-labelled pyrimidine nucleotides (fluorescein-12-U, fluorescein-15-C) ~13395~
-Quantitative measurements show a linear correlation between emitted fluorescence .and the number of incorporated fluorescein-:Labelled pyrimidine nucleotides. No quenching of fluorescence has been observed (see figures 1 and 2).
- Using the above. mentioned detection/imaging system from Hamamatsu Photonics we were able to detect as little as 108 molecules in a volume of approx. 1 n1 (concentration: 150 nM), allowincL, in principle, the use of up to 104 different templates on an array of 8 cm x 8 cm.
- Incorporation of up to two fluorescein-labelled purine nucleotides (fluorescein-15-A, fluorescein-15-I). Using the above detector system we were ab'; a to measure the difference between one a:nd two fluorescein-Labelled purine nucleotides.
Generation c>f template and binding to solid support, annealing of the sequencing primer to the anchored single stranded DN~~ template, capping of the primer with thionucleotides, and removing of the dideoxynucleotide from the capped primer were carried out as in example 1.
SEQUENCING B5.' SEQUENTIAL ADDITION OF SINGLE FLUORESCENTLY-LABELLED NUChEOTIDES: FIRST COMPLETE CYCLE OF 9 STEPS.
Scheme 1 steps 2 and =s The beads (anchored template/primer complex 1) were resuspended in 4 u1 water. The following items were added:
2 ~cl 5 x :~equenase buffer, 2~1 of a nucleotide mix containing 1C~~CM :Eluorescein-15-dATF (Boehringer Mannheim), 10 ~M ddGTP, 10 ~r,M ddTTP, 10 ~M ddCTP, and 2~,1 of diluted sequenase 2Ø The' mixture was incubated for 2 min at 37°C
and the reaction stopped by sedimenting the beads with the WO 93/21340 . PGT/GB93/0084~' '~1_3395~
magnet and removing the supernatant followed by three further washings with 50 u1 of water. In this step, according to the complementary strand, two fluorescein-15 A-nucleotides and one dideoxy T-nucleotide were added to the 3'- end of the capped primer..
step 4 The fluorescence was measured using a SIT camera (model C2 900-08, Hamamatsu Photonics SA) mounted on a fluorescence microscope.
steps 5 and 6 The dideoxynucleotide and the fluorescently-labelled nucleotides were removed by adding 10 ~cl (20 units) of an exonuclease-III solution in 50 mM Tris/HC1 Ph 7.5, 5 mM
MgCl2, 5 mM DTT and incubating the mixture for 2 min at 37°C. The reaction was stopped by sedimenting the beads with the magnet and removing the supernatant, followed by three washings with 50 ~,1 of water.
steps 7 and 8 In order to cap the primer, the beads were resuspended in 4 u1 water. The following items were added: 2 u1 5 x sequenase buffer, 2 ~1 of a nucleotide mixture containing 10 ACM dsATP, 10 ~,M ddGTP, 10 ~cM ddTTP, 10 ~,M ddCTP, and 2 ~1 of diluted sequenase 2Ø The mixture was incubated for 2 min at 37°C and the reaction stopped by sedimenting the beads with the magnet and removing the supernatant followed by three further washings with 50 ~,1 of water. In this step, two thiolated A-nucleotides and one dideoxy T-nucleotide were added to the sequencing primer.
steps 9 and l0 The dideoxy nucleotide was removed by adding 10 ~cl (20 units) of the above specified exonuclease-III solution and incubating the mixture for 2 min at 37°C. The reaction was stopped by sedimenting the beads with the magnet and removing the supernatant, followed by three washing with 50 u1 of water.
~'1.33~5~
WO 93/21340 PCf/GB93/00848 SEQUENCING BY SEQUENTIAL ADDITION OF SINGLE NUCLEOTIDES:
5 scheme 1 steps 2 and a The beads (anchored template/primer complex 1) were resuspended in 4 ~cl water. The following items were added:
2 ~1 of 5 x sequenase buffer, 2 ~,1 of a nucleotide mixture 10 containing l0 uM fluorescein-12-dUTP (Boehringer Mannheim), 10 uM ddGTP, 10 ~,M ddATP, 10 uM ddCTP, and 2 ~,1 of diluted sequenase 2Ø The mixture was incubated 2 min at 37°C and the reaction stopped by sedimenting the beads with the magnet and removing the supernatant followed by three 15 further washings with 50 ~1 of water. In this step, according to the: complementary strand, two fluorescein-labelled U-nucleotides and one dideoxy G-nucleotide were added to the 3'-end of the capped primer.
20 step 4 The fluorescence was measured using a SIT camera (model 400-08, Hamamatsu Photonics SA) mounted on a fluorescence microscope.
25 steps 5 and E>
The dideoxynucleotide and the labelled nucleotides were removed by adding 10 ~,1 (20 units) of the above specified exonuclease-7:II evolution and incubating the mixture for 2 min at 37°C. The reaction was stopped by sedimenting the 30 beads with the magnet and removing the supernatant, followed by three wasr~ings~ with 50 ~,1 of water.
steps 7 and Et In order to cap t:he primer, the beads were resuspended in 4 35 u1 water. The following items were added: 2 u1 of 5 x sequenase buffer, 2 u1 of a nucleotide mixture containing 10 ACM dSTTP, 10 ~cM ddGTP, 10 ~,M ddATP, 10 ACM ddCTP, and 2 ~1 of diluted seque~nase 2Ø The mixture was incubated for 2 min WO 93/21340 PCT/GB93/0084~
~,1'~39~~ 46 at 37°C and the reaction stopped by sedimenting the beads with the magnet and removing the supernatant followed by three further washings. with 50~c1 of water. In this step, two thiolated T-nucleotides and one dideoxy G-nucleotide were added to the sequencing primer.
steps 9 and 10 the dideoxy nucleotide was removed by adding 10 u1 (20 units) of the above specified exonuclease-III solution and incubating the mixture for 2 min at 37°C. The reaction was stopped by sedimenting the beads with the magnet and removing the supernatant, followed by three washings with 50 u1 of water.
Generation of template and binding to solid support, annealing of the sequencing primer to the anchored single-stranded DNA template, capping of the primer with thionucleotides, and removing of the dideoxynucleotide from the capped primer were carried out as in example 1.
SEQUENCING BY SEQUENTIAL ADDITION OF SINGLE FLUORESCENTLY-LABELLED NUCLEOTIDES: FIRST COMPLETE CYCLE OF 9 STEPS
scheme 1 steps 2 and 3 The beads (anchored template/primer complex 1) were resuspended in 4 ~1 water. The following items were added:
2 ~1 5 x sequenase buffer, 2 ~.1 of a nucleotide mix containing 500 uM fluorescein-15-dATP, 1.0 ~,M dATP, 10 ~M
ddGTP, 10 uM ddTTP, 10 ACM ddCTP, and 2 ~.1 of diluted sequenase 2Ø The mixture was incubated for 2 min at 37°C
and the reaction stopped by sedimenting the beads with the magnet and removing the supernatant followed by three further washings with 50 ~1 of water. In this step, as directed by the complementary strand, fluorescein-15-A-j ~ ~ ~'~ ~ ~ pCT/GB93/00848 nucleotides, A-nucleotides, and one dideoxy T-nucleotide were added to the 3'-end of the capped primer.
step 4 The fluorescence was measured using a SIT camera (model C2 400-08, Hama.matsu Photonics SA) mounted on a fluorescence microscope.
steps 5 and _6 The dideoxynucleotide, the deoxynucleotides and the fluorescently-labelled nucleotides were removed by adding 20 ~1 (20 units) of an exonuclease-III solution in 50 tnM
Tris/HCI pH 7.5,. 5 mM MgCl2 5 mM DTT and incubating the mixture for 2 min at 37°C. The reaction was stopped by sedimending the beads with the magnet and removing the supernatant, followed by three washings with 50 ~,1 of water.
steps 7 and 8 In order to ~~ap the primer, the beads were resuspended in 4 ~1 water. The following items were added: 2 ~cl 5 x sequenase buffer, 2 ~1 of a nucleotide mixture containing 10 ~,M dSATP, 10 ~M ddGTP, 10 ~M ddTTP, 10 uM ddCTP, and 2 ~1 of diluted sequenase 2Ø The mixture was incubated for 2 min at 37°C and the reaction stopped by sedimenting the beads with the magnet and removing the supernatant followed by the three further washings with 50 ~1 of water. In this step, two thiolate:d A~-nucleotides and one dideoxy T-nucleotide were added to the sequencing primer.
3 0 steps 9 and 10 The dideoxy nucleotide was removed by adding 10 ~l (20 units) of the above specified exonuclease-III solution and incubating the mixture for 2 min at 37°C. The reaction was stopped by sedimenting the beads with the magnet and removing the supernatant, followed by three washings with 50 ~cl of water.
SEQUENCING f3Y SEQUENTIAL ADDITION OF SINGLE NUCLEOTIDES:
~Z~.3395G
WO 93/21340 ' PCT/GB93/0084g scheme 1 steps 2 and 3 The beads (anchored template/primer complex 1) were resuspended in 4 ~1 water. The following items were added:
2 ~1 of 5 x sequenase buffer, 2 ~cl of a nucleotide mixture containing 15 ~,M fluoresce in-12-dUTP, 1.0 ~M dTTP, 10 ~,M
ddGTP, 10 ACM ddATP, 10 ACM ddCTP, and 2 ~,1 of diluted sequenase 2Ø The mixture was incubated for 2 min at 37°C
and the reaction stopped by sedimenting the beads with the magnet and removing the supernatant followed by three further washings with 50 u1 of water. In this step, as directed by the complementary strand, fluorescein-labelled U-nucleotides, T-nucleotides, and one dideoxy G-nucleotide were added to the 3'-end of the capped primer.
step 4 The fluorescence was measured using a SIT camera (model C2 400-08, Hamamatsu Photonics SA) mounted on a fluorescence microscope.
steps 5 and 6 The dideoxynucleotide and the labelled nucleotides were removed by adding 10 u1 (20 units) of the above specified exonuclease-III solution and incubating the mixture for 2 min at 37°C. The reaction was stopped by sedimenting the beads with the magnet and removing the supernatant, followed by three washings with 50 u1 of water.
steps 7 and 8 In order to cap the primer, the beads were resuspended in 4 u1 water. The following items were added: 2 dal of 5 x sequenase buffer, 2 ~1 of a nucleotide mixture containing 10 ~.M dSTTP, 10 ~cM ddGTP, 10 ~M ddATP, 10 ~,M ddCTP, and 2 ~,1 of diluted sequenase 2Ø The mixture was incubated 2 min at 37°C and the reaction stopped by sedimenting the beads with the magnet and removing the supernatant followed by three WO 93/21340 ~ ~ ~ ~ ~ ~ ~ PCT/GB93/00848 further washings with 50 ~,1 of water. In this step, two thiolated T--nucleotides and one dideoxy G-nucleotide were added to the sequencing primer.
steps 9 and 10 The dideoxy nucleotide was removed by adding 10 u1 (20 units) of th,e above specified exonuclease-III solution and incubating the mixture for 2 min at 37°C. The reaction was stopped by sedimenting the beads with the magnet and removing the supernatant, followed by three washings with 50 ~1 of water.
Template preparation, binding of the template to a solid support, and annealing the sequencing primer was performed as described in example 1.
Cap,~ing of the r~rimer with thionucleotides To the 18 u7. annealing mixture add 10 ~,1 of 100 uM dsGTP, ddATP, ddTTl?, and ddCTP and 4 ~cl (5 units) of diluted sequenase 2.0 (USES), and incubate the mixture for 2 min at room temperature. As directed by the complementary strand, this adds tree following three nucleotides sequentially to the primer: dSG, dsG, ddC. The beads were sedimented using the magnet and the supernatant removed. The beads were then washed two times with 50 ~,1 water.
Removing the dideaxynucleotide from the capped primer To the beads , 10 u:1 ( 2 0 units ) of an exonuclease solution in 50 mM Tris/EfCl pH 7.5, 5mM MgCl2, 5 mM DTT were added and the mixture incubated 2 min at 37°C. The reaction was the reaction stopped by sedimenting the beads with the magnet and remvoinc~ then supernatant, followed by three washings with 50 ~1 of water. This step removed the dideoxy C-nucleotide from the 3'-end of the primer.
WO 93/21340 PCT/GB93/0084~
~~~33956 FIRST SEQUENCING CYCLE (9 STEPS) scheme 1 steps 2 and 3 5 The beads (anchored template/primer complex 1) were resuspended in 4 ~1 of water. The following items were added: 2 u1 5 x sequenase buffer, 2 u1 of a nucleotide mixture containing 10 ~cM fluorescein-15-dCTP (Boehringer Mannheim), 10 ~M ddGTP, 10 ~,M ddATP, 10 ~M ddTTP, and 2 gel 10 of diluted sequenase 2Ø The mixture was incubated for 2 min at 37°C and stopped by sedimenting the beads with the magnet and removing the supernatant followed by three further washings with 50 ~,1 of water. In this step, as directed by the complementary strand, one fluorescein-15 labelled C-nucleotide and one dideoxy G-nucleotide were added to the 3'-end of the capped primer.
step 4 The fluorescence was measured using a SIT camera (model C2 20 400-08, Hamamatsu Photonics SA) mounted on a fluorescence microscope.
steps 5 and 6 The dideoxynucleotide and the fluorescein-labelled 25 nucleotides were removed by adding 10 ~1 (20 units) of an exonuclease-III solution in 50 mM Tris/HC1 pH 7.5, 5 mM
MgCl2, 5 mM DTT and incubating the mixture for 2 min at 37'C. The reaction was stopped by sedimenting the beads with the magnet and removing the supernatant, followed by 30 three washings with 50 ~.1 of water.
steps 7 and 8 In order to cap the primer, the beads were resuspended in 4 ~cl water. The following items were added: 2 ~,1 5 x 35 sequenase buffer, 2 ~.1 of a nucleotide mixture containing 10 ~,M dSCTP , 10 uM ddGTP , 10 ~cM ddATP , 10 ~.M ddTTP , and 2 a 1 of diluted sequenase 2Ø The mixture was incubated 2 min at 37°C and the reaction stopped by sedimenting the beads with the magnet and removing the supernatant followed by three further washings with 50u1 of water. In this step, one thiolated C-nulceotide~and one dideoxy G-nucleotide were added to the sequencing primer.
steps 9 and 7.0 The dideoxy nucleotide was removed by adding 10 ~1 (20 units) of ths: above specified exonuclease-III solution and incubating the mixture for 2 min at 37°C. The reaction was stopped by sedimenting the beads with the magnet and removing the supernatant, followed by three washings with 50 u1 of water.
SECOND SEQUErJCING CYCLE (9 STEPS) scheme 1 steps 2 and :3 The beads (anchored template/primer complex 1) were resuspended ~~n 4 ~,:L water . The following items were added:
2 ~1 5 x sequenase buffer, 2 ~1 of a nucleotide mixture containing 10 uM fluorescein-15-dITP (Boehringer Mannheim), 10 uM ddATP, 10 ~iM ddTTP, 10 ~cM ddCTP, and 2 ~1 of diluted sequenase 2Ø The mixture was incubated 2 min at 37°C and the reaction stopped by sedimenting the beads with the magnet and removing the supernatant followed by three further washings with 50 ~cl of water. In this step, as directed by the complementary strand, one fluorescein-labelled I-nucleotide and one dideoxy A-nucleotide were added to the 3'-end of the capped primer.
step 4 The fluorescence was measured using a SIT camera (model C2 400-08, Hamamatsu Photonics SA) mounted on a fluorescence microscope.
steps 5 and 6 The dideoxynucleotide and the labelled nucleotide were removed by adding 10 ~.1 (20 units) of an exonucleaseIII
WO 93/21340 c PCT/GB93/0084R
solution in 50 mM Tris/HC1 pH 7.5, 5 mM MgCl2, 5 mM DTT and incubating the mixture for 2 min at 37°C. The reaction was stopped by sedimenting the beads with the magnet and removing the supernartant, followed by three washings with 50 u1 of water.
steps 7 and 8 In order to cap the primer, the beads were resuspended in 4 ~cl water. The following items were added: 2 ~1 5 x sequenase buffer, 2 ~1 of a nucleotide mixture containing 10 ~M dSGTP, 10 ~,M ddATP, 10 ~cM ddTTP, 10 ~M ddCTP, and 2 ~C1 of diluted sequenase 2Ø The mixture was incubated for 2 min at 37°C and the reaction stopped by sedimenting the beads with the magnet and removing the supernatant followed by three further washings with 50 ~cl of water. In this step, one thiolated G-nucleotide and one dideoxy A-nucleotide were added to the sequencing primer.
steps 9 and 10 The dideoxy nucleotide was removed by adding 10 u1 (20 units) of the above specified exonuclease III solution and incubating the mixture for 2 min at 37°C. The reaction was stopped by sedimenting the beads with the magnet and removing the supernatant, followed by three washings with 50 ~cl of water.
THIRD SEQUENCING CYCLE (9 STEPS) scheme 1 steps 2.and 3 The beads (anchored template/primer complex 1) were resuspended in 4 ~1 water. The following items were added:
2 ~.1 5 x sequenase buffer, 2 u1 of a nucleotide mixture containing 10 ACM fluorescein-15-dATP, 10 ~M ddGTP, 10 uM
ddTTP, 10 uM ddCTP, and 2 ~1 of diluted sequenase 2Ø The mixture was incubated for 2 min at 37°C and the reaction stopped by sedimenting the beads with a magnet and removing the supernatant followed by three further washings with 50 ~1 of water. In this step, as directed by the complementary strand, two fluorescein-labelled A-nucleotides and one dideoxy T-nucleotide were added to the 3'-end of the capped primer.
step 4 The fluorescence was measured using a SIT camera (model C2 400-08, Hamamatsu Photonics SA) mounted on a fluorescence microscope.
steps 5 and 6 The dideoxynucleotide and the labelled nucleotides were removed by e~dding 10 ~cl (20 units) of an exonuclease-III
solution in 50 mM Tris/HC1 pH 7.5, 5 mM MgCl2, 5 mM DTT and incubating tlhe mixture for 2 min at 37°C . The reaction was stopped by sedimenting the beads with the magnet and removing the supernatant, followed by three washings wits ~0 ~cl of water.
steps 7 and 8 In order to cap th.e primer, the beads were resuspended in 4 ~cl water. The following items were added: 2 ~.1 5 x sequenase buffer, 2 al of a nucleotide mixture containing 10 uM deATP, 10 ~,M ddGTP, 10 ~M ddTTP, 10 ~M ddCTP, and 2 ~,1 of diluted sequenase 2Ø The mixture was incubated 2 min at 37°C and the: reaction stopped by sedimenting the beads with the magnet and removing the supernatant followed by three further washings with 50 ~.1 of water. In this step, two thiolated A--nucleotides and dideoxy T-nucleotide were added to the sequEancing primer.
steps 9 and 10 The dideoxy nucleotide was removed by adding 10 u1 (20 units) of tine above specified exonuclease III solution and incubating 'the mixture for 2 min at 37°C. The reaction was stopped by sedimenting the beads with the magnet and removing them supernatant, followed by three washings with 50 ~1 of water.
WO 93/21340 PCT/GB93/0084°
~z133g56 FOURTH SEQUENCING CYCLE (9 STEPS) scheme 1 steps 2 and 3 The beads (anchored template/primer complex 1) were resuspended in 4 ~1 water. The following items were added:
2 ~1 5 x sequenase buffer, 2 ~1 of a nucleotide mixture containing 10 uM fluorescein-12-dUTP, 10 uM ddGTP, 10 ~M
ddATP, 10 ~M ddCTP, and 2 ~1 of diluted sequenase 2Ø The mixture was incubated f or 2 min at 37 ° C and the reaction stopped by sedimenting the beads with the magnet and removing the supernatant followed by three further washings with 50 ~1 of water. In this step, as directed by the complementary strand, twofluorescein-labelled U-nucleotides and one dideoxy G-nucleotide were added to the 3'-end of the capped primer.
step 4 The fluorescence was measured using a SIT camera (model C2 400-08, Hamamatsu Photonics SA) mounted on a fluorescence microscope.
steps 5 and 6 The dideoxynucleotide and the labelled nucleotides were removed by adding 10 u1 (20 units) of an exonuclease-III
solution in 50 mM Tris/HC1 pH 7.5, 5 mM MgCl2, 5 mM DTT and incubating the mixture for 2 min at 37°C . The reaction was stopped by sedimenting the beads with the magnet and removing the supernatant, followed by three washings with 50 ~.1 of water. The removal of the label was checked by measuring the mixture with the hand counter.
3 5 stegs 7 and 8 In order to cap the primer, the beads were resuspended in 4 ~.1 water. The following items were added: 2 u1 5 x sequenase buffer, 2 u1 of a nucleotide mixture containing 10 uM dSTTP, 10 ~M ddGTP, 10 uM ddATP, 10 ~,M ddCTP, and 2 ~cl of diluted sequenase 2Ø The mixture was incubated for 2 min at 37°C and the reaction stopped by sedimenting the beads with the magnet and removing the supernatant followed by 5 three further washings with 50 ~1 of water. In this step, two thiolatEad T~-nucleotides and one dideoxy G-nucleotide were added t.o the sequencing primer.
steps 8 and 9 10 The dideoxy nucleotide was removed by adding 10 ~tl (20 units) of the ahove specified exonuclease-III solution and incubating the mixture for 2 min at 37°C. The reaction was stopped by sedimenting the beads with the magnet and remvoing the: supernatant, followed by three washings with 50 15 ~1 of water..
Template preparation, binding of the template to solid 20 support, and annealing of the sequencing primer was performed as described in example 1.
Capping of 'the primer with thionucleotides To the 18 u1 annealing mixture add 10 ~.1 of 100 ~M dBGTP, 25 ddATP, ddTTP, and ddCTP and 4 u1 (5 units) of diluted sequenase 2.0 (USB), and incubate the mixture for 2 min at room temperature. As directed by the complementary strand this adds the following three nucleotides sequentially to the primer: d$G, dSG, ddC. The beads were sedimented using 30 the magnet and the supernatant removed. The beads were then washed two times with 50 ~.1 water.
R_emovincr the dideoxynucleotide from the capped primer To the beads, 10 u1 (20 units) of an exonuclease solution in 35 50 mM Tris/HC1 pH 7.5, 5 mM MgCl2, 5 mM DTT were added and the mixture' incubated 2 min at 37°C. The reaction was stopped by sedimenting the beads with the magnet and removing the supernantant, followed by three washings with ~~133956 WO 93/21340 PCT/GB93/0084~
50 ~1 of water. This step removed the dideoxy C-nucleotide from the 3'-end of the primer.
FIRST SEQUENCING CYCLE (9 STEPS) scheme 1 steps 2 and 3 The beads (anchored template/primer complex 1) were resuspended in 4 gel water. The following items were added:
2 u1 5 x sequenase buffer, 2 u1 of a nucleotide mixture containing 15 uM fluorescein-15-dCTP, 1.0 uM dCTP, 10 uM
ddGTP, 10 uM ddATP, 10 uM ddTTP, and 2 ~cl of diluted sequenase 2Ø The mixture was incubated for 2 min at 37°C
and the reaction stopped by sedimenting the beads with the magnet and removing the supernatant followed by three further washings with 50 u1 of water.
step 4 The fluorescence was measured using a SIT camera (model C2 400-08, Hamamatsu Photonics SA) mounted on a fluorescence microscope.
steps 5 and 6 The dideoxynucleotide and the fluorescein-labelled nucleotides were removed by adding 10 ~1 (20 units) of an exonuclease-III solution in 50 mM Tris/HC1 pH 7.5, 5 mM
MgCl2, 5 mM DTT and incubating the mixture for 2 min at 37°C. The reaction was stopped by sedimenting the beads with the magnet and removing the supernatant, followed by three washings with 50 ~,1 of water.
steps 7 and 8 In order to cap the primer, the beads were resuspended in 4 ~,1 water. The following items were added: 2 ~1 5 x sequenase buffer, 2 ~,1 of a nucleotide mixture containing 10 ~M dSCTP, 10 ~M ddGTP, 10 ~M ddATP, 10 ~,M ddTTP, and 2 u1 of diluted sequenase 2Ø The mixture was incubated 2 min at 37°C and the reaction stopped by sedimenting the beads with ~1339~6 the magnet and removing the supernatant followed by three further washings with 50 ~1 of water. In this step, two thiolated C-nucleotides and one dideoxy G-nucleotide were added to the sequencing primer.
steps 9 and 10 The dideoxy nucleotide was removed by adding 10 ~,l (20 units) of th~~ above specified exonuclease III solution and incubating the mixture for 2 min at 37°C. The reaction was stopped by sedimenting the beads with the magnet and removing the supernatant, followed by three washings with 50 ~1 of water.
SECOND SEQUE;HCINr CYCLE (9 STEPS) scheme 1 steps 2 and 3 The beads (anchored template/primer complex 1) were resuspended in 4 ~cl water. The following items were added:
2 ~,1 5 x sequenase buffer, 2 u1 of a nucleotide mixture containing 500 ~,M fluorescein-15-dITP, 1.0 uM dGTP, 10 uM
cIATP, 10 uM ddTTP, 10 ~cM ddCTP, and 2 ~1 of diluted sequenase 2Ø The mixture was incubated for 2 min at 37°C
and the reaction stopped by sedimentin:~ the beads with the magnet and removing the supernatant followed by three further washings with 50 ~.1 of water.
stets 4 The fluorescence was measured using a SIT camera (model c2 400-08, Hamamatsu Photonics SA) mounted on a fluorescence microscope.
steps 5 and 6 The dideoxynucleotide and the labelled nucleotides were removed by adding 10 ~,1 (20 units) of an exonuclease-III
solution in 50 mM Tris/HC1 pH 7.5, 5 mM MgCl2, 5 mM DTT and incubating the mixture for 2 min at 37°C. The reaction was stopped by sedimenting the beads with the magnet and removing the supernatant, followed by three washings with 50 u1 of water.
WO 93/21340 ~~ ~ 3 3 g 5 6 PGT/GB93/00848 _steps 7 and 9 In order to cap the primer, the beads were resuspended in 4 u1 water. The following items were added: 2 u1 5 x sequenase buffer, 2 ~1 of.a nucleotide mixture containing 10 ~,M dSGTP, 10 uM ddATP, 10 ~M ddTTP, 10 ~M ddCTP, and 2 u1 of diluted sequenase 2Ø The mixture was incubated 2 min at 37°C and the reaction stopped by sedimenting the beads with the magnet and removing the supernatant followed by three further washings with 50 ~cl of water. In this step, one thiolated G-nucleotide and one dideoxy A-nucleotide were added to the sequencing primer.
step 9 and 10 The dideoxy nucleotide was removed by adding 10 u1 (20 units) of a specified exonuclease-III solution and incubating the mixture for 2 min at 37°C. The reaction was stopped by sedimenting the beads with the magnet and removing the supernatant, followed by three washings with 50 ~1 of water.
THIRD SEQUENCING CYCLE (9 STEPS) scheme 1 steps 2 and 3 The beads (anchored template/primer complex 1) were resuspended in 4 ~cl water. The following items were added:
2 u1 5 x sequenase buffer, 2 ~1 of a nucleotide mixture containing 500 ~.M fluorescein-15-dATP, 1 ~cM dATP, 10 ~M
ddGTP, 10 ~M ddTTP, 10 ~M ddCTP, and 2 ~C1 of diluted sequenase 2Ø The mixture was incubated for 2 min at 37°C
and the reaction stopped by sedimenting the beads with the magnet and removing the supernatant followed by three further washings with 50 ~1 of water.
step 4 The fluorescence was measured using a SIT camera (model C2 400-08, Hama:matsu Photonics SA) mounted on a fluorescence microscope.
steps 5 and 6 The dideoxyn.ucleotide and the labelled nucleotides were removed by adding 10 u1 (20 units) of an exonuclease-III
solution in X50 mM Tris/HC1 pH 7.5, 5mM MgCl2, 5 mM DTT and incubating tile mixture for 2 min at 37°C. The reaction was stopped by sedimenting the beads with the magnet and removing the supernatant, followed by three washings with 50 u1 of water.
steps 7 and .3 In order to c:ap the primer, the beads were resuspended in 4 ~:1 water. The following items were added: 2 u1 5 x sequenase buffer, 2 ~cl of a nucleotide mixture containing 10 ~M dSATP, 10 ~M ddGTP, 10 uM ddTTP, 10 uM ddCTP, and 2 ~1 of diluted s~equenase 2Ø The mixture was incubated 2 min at 37°C and the reaction stopped by sedimenting the beads with the magnet and removing the supernatant followed by three further washings with 50 ~1 of water. In this step, two thiolated A-nucleotides and one dideoxy T-nucleotide were added to the sequencing primer.
steps 9 and 10 The dideoxy nucleotide was removed by adding 10 ~,1 (20 units) of the above specified exonuclease-III solution and incubating t:he mixture for 2 min at 37°C. The reaction was stopped by sedimenting the beads with the magnet and 3 0 removing the supernatant, followed by three washings with 50 ~c 1 of water .
FOURTH SEQUENCING CYCLE (9 STEPS) scheme 1 steps 2 and 3 The beads (anc:hored template/primer complex 1) were resuspended in 4 u1 water. The following items were added:
2 u1 5 x sequenase buffer, 2 ~.1 of a nucleotide mixture WO 93/21340 PCT/GB93/0084'' ~~~~3~~6 containing 15 uM fluorescein-12-dUTP, 1.0 uM dTTP, 10 ~,M
ddGTP, 10 uM ddATP, 10 ACM ddCTP, and 2 u1 of diluted sequenase 2Ø The mixture was incubated for 2 min at 37°C
and the reaction stopped by sedimenting the beads with the 5 magnet and removing t2ie~ supernatant followed by three further washings with 50 ~1 of water.
step 4 The fluorescence was measured using a SIT camera (model C2 10 400-08, Hamamatsu Photonics SA) mounted on a fluorescence microscope.
steps 5 and 6 The dideoxynucleotide and the labelled nucleotides were 15 removed by adding 10 u1 (20 units) of an exonuclease-III
solution in 50 mM Tris/HC1 pH 7.5, 5 mM MgCl2, 5 mM DTT and incubating the mixture for 2 min at 37°C. The reaction was stopped by sedimenting the beads with the magnet and removing the supernatant, followed by three washings with 50 20 u1 of water. The removal of the label was checked by measuring the mixture with the hand counter.
steps 7 and 8 In order to cap the primer, the beads were resuspended in 4 25 ~1 water. The following items were added: 2 ~,1 5 x sequenase buffer, 2 ~1 of a nucleotide mixture containing 10 ACM dSTTP, 10 uM ddGTP, 10 uM ddATP, 10 uM ddCTP, and 2 ~1 of diluted sequenase 2Ø The mixture was incubated 2 min at 37°C and the reaction stopped by sedimenting the beads 30 with the magnet and removing the supernatant followed by three further washings with 50 ~cl of water. In this step, two thiolated T-nucleotides and one dideoxy G-nucleotide were added to the sequencing primer.
35 steps 8 and 9 The dideoxy nucleotide was removed by adding 10 ~1 (20 units) of the above specified exonucleaseIII solution and incubating the mixture for 2 min at 37°C. The reaction was ~.~ 33~~~~
WO 93/21340 - PCi'/GB93/00848 s=opped by sedimenting the beads with the magnet and removing the supernatant, followed by three washings with 50 ~,1 of water.
It will be understood that the invention is described above by way of example only, and that a variety of modifications will be apparent to those skilled in the art which fall within the scope of the appended claims.
For example, in Step 2 above the labelled nucleotide could be dATP. In this case, the heterogeneous chain terminators could be ddGTP, ddTTP and ddCTP. In step 7 phosphorothioate dATP would be added to replace the labelled dATP removed with the exonuclease in step 6. The cycle can then be repeated with another labelled nucleotide, for example dGTP, together with the heterogeneous dideoxynucleotides ddATP, ddTTP and ddCTP. This will cause label to be incorporated in all the chains propagating with G. This is followed in turn with labelled dTTP and labelled dCTP and continued again with dATP, dGTP, dTTP and dCTP and so on.
Scheme 2 According to a second preferred aspect of the invention, a DNA fragment is sequenced according to the follcwing scheme:
1) a capped primer containing a phosphorothioate nucleoside derivative is hybridized to a template to form a template/primer complex;
WO 93/21340 , ~ PCT/GB93/00848 2 ) a label:Led deoxynucleotide together with heterogeneous chain terminators and a suitable polymerase is added to the template/prim~~r complex;
3) excess reagents are removed by washing;
4) the amount of incorporated label is measured;
5) the lab~alled nucleotide and the chain terminators are removed with an exonuclease;
6) the exonuclease is removed by washing;
7) a phos;phorathioate deoxynucleotide together with heterogeneous chain elongation inhibitors not incorporated into the chain is added;
8) excess :reagents are removed by washing; and 9) steps 2 ) to 8) are repeated four times, each time with a different 1<ibelled nucleotide.
This scheme i,s essentially a sub-scheme of scheme 1. The main difference is that during the capping step 7, dideoxynucleoi=ides are replaced by the corresponding 5'-[a, ~-methylene] triphosphates derivatives. However, other chain elongation inhibitors like deoxynucloside diphosphate or deoxynucleoside monophosphate derivatives may also be used. Since i:hese derivatives cannot be incorporated into the growing polynuc:leotide chain there is no need to remove them. Hence, scheme 2 completely lacks the last exonuclease treatment step and t:he subsequent washing step of scheme 1.
Scheme 3 According to a third preferred aspect of the invention, a WO 93/21340 _ ~ ~~ ~ ~ ~ ~ ~ PGT/GB93/00&
DNA fragment is sequenced according to the following scheme:
1) a capped primer containing a phosphorothioate deoxynucleotide is hybridised to a template to form a template/primer complex;
2) a labelled nucleotide triphosphate together with heterogeneous chain elongation inhibitors not incorporated into the chain is added;
3) excess reagents are removed by washing;
4) the amount of incorporated label is measured;
5) steps 2 to 4, adding different labelled nucleotides in the presence of their corresponding heterogeneous chain elongation inhibitors not incorporated into the chain, are repeated until all four labelled nucleotides have been added;
6) all labelled nucleotides are removed with exonuclease;
7) the exonuclease is removed by washing;
8) the phosphorothioate deoxynucleotide corresponding to the first labelled deoxynucleotide added to the reaction in step 2 , is added together with heterogenous claim elongation inhibitors not incorporated into the chain and a suitable polymerase;
9) excess reagents are removed by washing; and 10) steps 8 and 9 are repeated with the three remaining phosphorothioate deoxynucleoside derivatives.
This scheme has the notable advantage of reducing overall number of exonuclease steps. All four labelled nucleotides are sequentially added to the chain and individually ~~_339~~
detected before all incorporated nucleotides are removed by a single exonuclease digestion step. The chase reactions are then carried out sequentially with the appropriate phosphorothioate nucleoside derivatives.
Scheme 4 In a fourth preferred aspect of the invention, a DNA
fragment is sequenced according to the following scheme:
1) a capped primer is hybridized to a template to form a template/primer complex;
2) a fluorescent nucleoside triphosphate, together with three heterogeneous chain elongation inhibitors not incorporated into the chain and a suitable polymerase, is added;
3) excess reagents are removed by washing;
4) the arnount of incorporated label is measured;
5) steps 2 to 4 are repeated using all three different nucleoside t.riphosphates, each with a fluorescent label, in the presen~~e of the respective heterogeneous chain elongation inhibitors not incorporated into the chain.
6 ) the f 7.uorescent labels are destroyed by bleaching with a laser or by a suitable chemical reaction, or the fluorescent labels are removed by a chemical cleavage step.
This scheme has the advantage that no enzymatic removal of incorporated label by way of an exonuclease reaction is required, nor is a chasing reaction with a phosphorothioate nucleotide derivative necessary. Instead, all incorporated fluorophore;s are chemically destroyed using either laser bleaching technology or suitable chemical reactions to destroy the: dysa or cleave the dye from the nucleotides.
WO 93/21340 _ '~ ~ ~ ~ ~ ~ ~ PCT/GB93/0084 Preferably, if the detector used permits quantitative measurement of incorporated label, the bleaching or cleaving step need only be carried out from time to time rather than after each successive addition.
Scheme 5 According to a fifth preferred aspect of the invention a DNA
fragment is sequenced according to the following scheme:
1) a capped primer is hybridized to a template;
2) a nucleoside triphosphate labelled by attachment of a fluorescent dye group via a linker arm to the 3' moiety of the deoxyribose sugar thereon is added;
3) excess reagents are removed by washing;
4) the amount of incorporated label is measured;
5) the fluorescent dye group is removed by enzymatic cleavage; and 6) excess reagents are removed by washing.
In Scheme 5, the nonspecific addition of labelled nucleotide is prevented by the 3' modification, so that the labelled nucleotide effectively acts as a chain terminator. Removal of the 3' blocking group is then all that is required to allow chain elongation to continue.
In a further aspect of the invention, there is provided a sequencing kit comprising at least three of the following:
i) a linker for attaching a DNA template to a solid-phase matrix, the linker comprising a primer having a deoxynucleoside phosphorothioate residue at its 3' end;
~133~~G
ii) chain elongation inhibitors;
iii) fluorescently-labelled nucleoside triphosphates;
iv) deoxynucleoside phosphorothioate triphosphates;
v) a 5'-> 3' ;DIVA polymerase;
vi) a 3'-> 5' exonuclease.
In addition, such a kit may comprise a solid support for carrying out the reaction, as well as biotinylated primers or linkers arid biot.in/streptavidin reagents for coupling the linker to th~~ solid support. The 3'-5' exonuclease may be exonuclease :CII. kurthermore, al::ernative chain elongation inhibitors, such as 3'-deoxyribose blocked labelled nucleotides, may be included. Preferably, the kit will comprise all of the components i-vi.
The invention also comprises an automatic sequencing machine capable of sequencing a nucleic acid essentially by executing the steps of a method according to the invention.
The machine is adapted either to move the solid support carrying the templates) into and out of all the necessary reagent and washing solutions, or to pump reagents and washing solutions over the solid support sequentially. The pin array type of support is better suited to the first procedure, winile glass plates and sequencing chips are more appropriate to the second.
Several washing steps are included between each reagent addition to minimise the carry-over of reagents.
The presence of label may be determined, in the case of a chip array, by passing the array over a fixed detector which records the level of label relative to the position of the array over the detector. In the case of a fixed glass plate WO 93/21340 , PCT/GB93/0084 X1.33956 or sequencing chip array, a radioactive or f luorescent image may be obtained by a fixed detector positioned above the array. Alternatively,.the glass plate or sequencing chip array and/or the detector may be movable. A two-dimensional image is produced by the detector and analysed by a computer.
Alternatively, optical fibres connected directly to a sequencing chip or to the pins in a pin array may be used to transmit data to a processor if used together with fluorescent labels.
The invention will now be described, for the purpose of illustration only, with reference to the following figures:
Figure 1 is a graph showing the correlation of emitted fluorescence to the number of nucleotides incorporated, using dUTP-12-fluorescein; and Figure 2 is as figure 1 except that dCTP-12-fluorescin is used.
EXAMPLES
Generation of template and binding to solid sut~port In this example an anchored single-stranded PCR product was used which was generated by known methods (T. Hultman et al., Nucleic Acids Res., 17, (1989), 4937-4946; D.S.C. Jones et al., DNA Sequence, 1 (1991), 279-283). Briefly, the template was generated by the polymerase chain reaction (PCR) using one biotinylated primer and one normal primer and the product subsequently bound to streptavidin coated magnetic beads. By treating the anchored double-stranded PCR product with alkali the non-anchored template strand is removed. All the steps were carried out as follows:
WO 93/21340 ~ I ~ 3 9 5 6 p~/GB93/00848 PCR was performed in 50 ~,1 using 0.5 ml test tubes. The following items were added: 30 ~cl water, 5 u1 of 10 x PCR
buffer (fetus) , 5 ~,1 of 2.5 mM dNTP's, 2.5 ~.1 of 10 ~M of the 5'-biot:Lnyl~ted universal reverse primer with the sequence: 5' Bio-AACAGCTATGACCATG 3', 2.5 ~1 of 10 ~M of the (-20) universal forward primer with the sequence: 5' GTAAAACGACGGCCAGT 3', 1 ~,1 of the Bluescript KS plasmid DNA
at the concentration 1 ng/~1, 0.5 ~cl (2.5 units) of native Taq polymerase (Cet:us) . After overlaying with light mineral oil the following cycles were performed: 95°C 90s, [95°C
30s, 55°C 60;s, 72°C 60s] x 35, 72°C 180s. All cycles were performed uaing the maximum heating and cooling rates possible with the Techne PHC-1 or PHC-2.
Binding the biotinylated PCR product with a length of approximately 250 by to the streptavidin-coated magnetic beads (Dynal) is accomplished by incubating 100 ~1 of beads under mineral oil .at room temperature for 5 min. The beads are sedimented using a strong magnet and the supernatant including the mineral oil is removed. Further traces of unused nucleotides, primers and buffers are removed by washing the heads with 100 ~:1 of water . The nonbiotinylated DNA strand i;s removed by incubating the beads with 50 ~1 of 0.15 M NaOH for 5 min. at room temperature. The beads are sedimented a.nd the supernatant is removed, followed by a further treatment with 50 u1 of 0.15M NaOH and three washings with 100 u1 of water. Finally the beads were resuspended in 10 ~1 of water.
Annealin4 of the seQUencin~primer to the anchored sink stranded DNA template To the 10 u1 resuspended beads with the anchored single-stranded DNA template (approximately 2 pmoles), 4 ~1 of 5 x Sequenase annealing buffer (200 mM Tris/HC1 pH 7.5 100 mM
MgCl2, 250 mM NaCl., USB) , and 4 ~cl (4 pmoles) of T7 primer with the sequence: 5' AATACGACTCACTATAG 3' are added. The ~.~3~95 6 PCT/GB93/0084;
' 24 mixture is heated for 3 min. at 65°C and then cooled on ice.
The template/primer complex is now ready for sequencing.
The following figure displays parts of its structure:
Complex 1: Polymer-streptavidin-biotin-5'-DNA-C-C-A-A-T-T-C-G-C-C-C-T-A-T-A-G-T-G-A-G-T-C-G-T-A-T-T-------3' 3'-G-A-T-A-T-C-A-C-T-C-A-G-C-A-T-A-A-------5' Capping of the primer with thionucleotides To the 18 u1 annealing mixture add 10 u1 of 100 uM dSGTP, dSCTP, ddATP, ddTTP, and 4 ~cl (5 units) of diluted sequenase 2.0 (USB), and incubate the mixture for 2 min. at room temperature. According to the complementary strand this adds the following five nucleotides sequentially to the primer: dSG, dSG, dSC, dSG, and ddA. The beads were sedimented using the magnet and the supernatant removed.
The beads were then washed two times with 50 u1 water.
Removing the dideox~nucleotide from the capped primer To the bead 10 ~1 (20 units) of an exonuclease solution in 50 mM Tris/HC1 pH 7.5, 5 mM MgCl2, 5 mM DTT were added and the mixture incubated 2 min. at 37°C. The reaction was stopped by sedimenting the beads with the magnet and removing the supernatant, followed by three washings with 50 ~1 of water. This step removed the dideoxy A-nucleotide from the 3'-end of the primer.
SEQUENCING BY SEQUENTIAL ADDITION OF SINGLE LABELLED
NUCLEOTIDES: FIRST COMPLETE CYCLE OF 9 STEPS
Scheme 1 Steps 2 and 3.
The beads (anchored template/primer complex 1) where resuspended in 13 ~.1 water. The following items were added:
5 ~.1 5 x sequenase buffer, 10 ~1 of a nucleotide mixture containing l0 ~,Ci of alpha-32P dATP of specific activity of ~~~~3~56 400 Ci/mmole, 4~,M cold dATP, 100 ~tM ddGTP, 100 ~,M ddTTP, 100 uM ddCTP, anc9 4 ~r,l of diluted sequenase 2Ø The mixture was incubated~,for 2 min_at 37°C and the reaction stopped by sedimenting the beads with the magnet and removing the 5 supernatant followed by three further washings with 50 ~1 of water. In this step, according to the complementary strand, two A-nucleotides and one dideoxy T-nucleotide were added to the 3'-end of the tapped primer.
10 Step 4 The label is counted with a hand counter Steps 5 and E.
The dideoxynucleotide and the labelled nucleotides were 15 removed by adding 10 ~1 (20 units) of an exonuclease III
solution in 50 mM Tris/HC1 pH 7.5, 5 mM MgCl2, 5 mM DTT and incubating the mixture for 2 min. at 37°C. The reaction was stopped by ,sedimenting the beads with the magnet and removing the supernatant, followed by three washings with 50 20 u1 of water. The removal of the label was checked by measuring ths~ mixture with the hand counter.
Steps 7 and ~.
In order to cap the primer, the beads were resuspended in 13 25 ~1 water. The following items were added: 5 ~1 5 x sequenase buffer, 10 u1 of a nucleotide mixture containing 100 ACM dSATP, 100 ~M ddGTP, 100 uM ddTTP, 100 ~N ddCTP, and 4 ~,1 of diluted sequenase 2Ø The mixture was incubated 2 min. at 37°C and the reaction stopped by sedimenting the beads with the magnet and removing the supernatant followed by three further washings with 50 ~,1 of water. In this step, two thiolat:ed A-nucleotides and one dideoxy T-nucleotide were added to the sequencing primer.
3 5 Steps 9 and 1.0 The dideoxy nucleotide was removed by adding 10 ~,1 (20 units) of the' above specified exonuclease III solution and incubating th.e mixture for 2 min. at 37°C. The reaction was WO 93/21340 PCT/GB93/00&
~~33g56 stopped by sedimenting the beads with the magnet and removing the supernatant, followed by three washings with 50 ~cl of water.
SEQUENCING BY SEQUENTIAL ADDITION OF SINGLE NUCLEOTIDES:
Scheme 1 Steps 2 and 3 The beads (anchored template/primer complex 1) were resuspended in 13 ~1 water. The following items were added:
5 u1 of 5 x sequenase buffer, 10 ~.1 of a nucleotide mixture containing 10 uCi of alpha-32P dTTP of specific activity of 400 Ci/mmol, 4 ACM cold dTTP, 100 ~,M ddGTP, 100 ~tM ddATP, 100 ~cM ddCTP, and 4 ~,1 of diluted sequenase 2Ø The mixture was incubated 2 min. at 37°C and the reaction stopped by sedimenting the beads with the magnet and removing the supernatant followed by three further washings with 50 ~,1 of water. In this step, according to the complementary strand, two T-nucleotides and one dideoxy G-nucleotide were added to the 3'-end of the capped primer.
Step 4 The label is counted with a hand counter.
Steps 5 and 6 The dideoxynucleotide and the labelled nucleotides were removed by adding 10 u1 (20 units) of the above specified exonuclease III solution and incubating the mixture for 2 min at 37°C. The reaction was stopped by sedimenting the beads with the magnet and removing the supernatant, followed by three washings with 50 u1 of water. The removal of the label was checked by measuring the mixture with the hand counter.
Steps 7 and 8 In order to cap the primer, the beads were resuspended in 13 ~cl water. The following items were added: 5 ~cl of 5 x sequenase buffer, 10 ~1 of a nucleotide mixture containing 13 3 9 5 G p~/GB93/00848 100 uM dSTTP, 100 ~.M ddGTP, 100 ~,M ddATP, 100 ~.M ddCTP, and 4 ~cl of diluted sequenase 2Ø The mixture was incubated 2 min. at 37°c, and the reaction stopped by sedimenting the beads with the magnet and removing the supernatant followed by three further washings with 50 ~cl of water. In this step, two thiolated T-nucleotides and one dideoxy G-nucleotide were added to the sequencing primer.
Steps 9 and 10 The dideoxy nucleotide was removed by adding 10 ~,1 (20 units) of th.e above specified exonuclease III solution and incubating the mixture for 2 min. at 37°C. The reaction was stopped by sedimenting the beads with the magnet and removing the supernatant, followed by three washings with 50 u1 of water.
This experiment was carried out in order to confirm that all the reactions described in example 1 yielded the correct elongation a.s well as degradation products. To prove this, the experiment described in example 1 was repeated using a 32P-labelled primer in combination with cold nucleotides.
The following modifications were made:
1. 4 pmo:les of a 5'-32P-labelled sequencing primer with the sequence 5' AATACGACTCACTATAG 3' was used in the annealing step;
2. In step 2 of the first cycle the labelled compound a-32P-dATP was omitted from the nucleotide mixture and the concentration of the cold dATP was increased to 100 ~,M;
3. In step 2 of the second cycle the labelled compound a-32-dTTP w~is omitted from the nucleotide mixture and the concentration of the cold dTTP was increased to 100 uM;
4. Step 4 in both cycles was not necessary;
5. After each enzymatic reaction and subsequent washing a 1/100 aliquot of the beads were removed and placed in a separate 0.'S ml test tube.
WO 93/21340 '~ ~ ~ ~ ~ ~ ~ PCT/GB93/0084 After performing all steps described in example 1, 5 ~,1 of 90% formamide dye mix were added to all the individual bead aliquots, the mixtures heated for 3 min. at 95°C, centrifuged at 13,OOOg for 5 seconds and cooled on ice. A
small aliquot (1 u1) of each sample was loaded into a individual well of a 20% polyacrylamide gel containing 7M
urea and electrophoresed at 700 Volts for 3 to 4 hours.
After electrophoresis the upper glass plate was removed and the exposed to a X-ray film for approximately 2 to 4 hrs.
The band pattern obtained was in full agreement with the predicted length of all primer elongation and degradation products.
Annealing and binding of the template/primer complex to solid sut~port In this example the biotinylated sequencing primer was f first annealed to the complementary region of a single-stranded M13 template and the complex subsequently bound via the 5' biotin moiety of the primer to the solid support (streptavidin beads). 2 ~g (1 pmole) of M13mp18 DNA was combined with 2 pmoles of 5' biotinylated (-20) universal forward primer with the sequence 5' GTAAAACGACGGCCAGT 3' in 40 mM Tris/HC1 pH 7.5, 20 mM MgCl2, 50 mM NaCl in a total of 10 ~1. The mixture was heated for 3 min. at 65°C and slowly cooled down to room temperature over a period of 10 min. 30 ~cl of streptavidin-coated magnetic beads (Dynal) were added 3 0 and the mixture incubated f or 5 minutes at room temperature .
The beads were sedimented, the supernatant removed, and the beads resuspended in 10 ~l of water.
Ca~pina of the primer with thionucleotides To the 18 ~cl annealing mixture add 10 ~1 of 100 ~M dsGTP, ddATP, ddTTP, ddCTP, and 4 ~1 (5 units) of diluted sequenase 2.0 (USB), and incubate the mixture for 2 min. at room temperature. According to the complementary strand this adds the following two nucleotides sequentially to the primer: d$G and ddA. The beads were sedimented using the magnet and t:he :>upernatant removed. The beads were then washed two times with 50 u1 water.
Removincr the dideoxvnucleotide from the capped primer To the bead 10 ~.;1 (20 units) of an exonuclease solution in 50 mM Tris-Hcl pH 7.5, 5 mM MgCl2, 5 mM DTT were added and the mixture incubated 2 min. at 37°C. The reaction was stopped by sedimenting the beads with the magnet and removing the supernatant, followed by three washings with 50 ~1 of water. This step removed the dideoxy A-nucleotide from the 3'-end of the primer.
SEQUENCING BY SEQUENTIAL ADDITION OF SINGLE LABELLED
NUCLEOTIDES: FIRST' COMPLETE CYCLE OF 9 STEPS
Scheme 1 Steps 2 and 3.
The beads (anchared template/primer complex 1) were resuspended :in 13 y1 water. The following items were added:
5 ~1 5 x seduenase buffer, 10 u1 of a nucleotide mixture containing 10 ~Ci of alpha-32P dATP of specific activity of 400 Ci-mmol, 4 ~,M cold dATP, 100 ~cM ddGTP, 100 ~M ddTTF, 100 ~M ddCTP, and 4 ~:l of diluted sequenase 2Ø The mi~:ture was incubated for. 2 min at 37°C and the reaction stoppe3 by sedimenting the beads with the magnet and removing the supernatant :Followed by three further washings with 50 u1 of water. In this step, according to the complementary strand, two A-nucleoi=ides and one dideoxy T-nucleotide were added to the 3' end of the capped primer.
Step 4 The label is counted with a hand counter.
Steps 5 and 6 WO 93/21340 PCT/GB93/00&
The dideoxynucleotide and the labelled nucleotides were removed by adding 10 ~1 (20 units) of an exonuclease III
solution in 50 mM Tris./HC1 pH 7.5 mM MgCl2, 5 mM DTT and incubating the mixture for 2 min. at 37°C. The reaction was 5 stopped by sedimenting the beads with the magnet and removing the supernatant, followed by three washings with 50 ~cl of water. The removal of the label was checked by measuring the mixture with the hand counter.
10 Steps 7 and 8 In order to cap the primer, the beads were resuspended in 13 ~cl water. The following items were added: 5 ~,1 5 x sequence buffer, 10 ~1 of a nucleotide mixture containing 100 ~M
dsATP, 100 ~M ddGTP, 100 ~M ddTTP, 100 uM ddCTP, and 4 ~C1 of 15 diluted sequenase 2Ø The mixture was incubated 2 min. at 37°C and the reaction stopped by sedimenting the beads with the magnet and removing the supernatant followed by three further washings with 50 u1 of water. In this step, two thiolated A-nucleotides and one dideoxy T-nucleotide were 20 added to the sequencing primer.
Steps 9 and 10 The dideoxy nucleotide was removed by adding 10 ~1 (20 units) of the above specified exonuclease III solution and 25 incubating the mixture f or 2 min . at 37 ° C. The reaction was stopped by sedimenting the beads with the magnet and removing the supernatant, followed by three washings with 50 ~.1 of water .
30 SEQUENCING BY SEQUENTIAL ADDITION OF SINGLE NUCLEOTIDES:
Scheme 1 Steps 2 and 3.
The beads (anchored template/primer complex 1) were resuspended in 13 ~1 water. The following items were added:
5 ~,1 of 5 x sequenase buffer, 10 ~,1 of a nucleotide mixture containing 10 ~Ci of alpha-32-P dTTP of specific activity of WO 93/21340 ~ ~ ~ ~ ~ ~ ~ PCT/GB93/00848 400 Ci/mmol, 4 ~M cold dTTP, 100 uM ddGTP, 100 ~.M ddATP, 100 uM ddCTP, anc3 4 ,u1 of diluted sequenase 2Ø The mixture was incubated 2 min. at 37°C and the reaction stopped by sedimenting the beads with the magnet and removing the supernatant followed by three further washings with 50 ~1 of water. In this step, according to the complementary strand, two T-nucleotides and one dideoxy C-nucleotide were added to the 3-end of the capped primer.
Steb 4 The label is counted with a hand counter.
Steps 5 and E~
The dideoxynucleotide and the labelled nucleotides were removed by adding 10 a 1 ( 2 0 units ) of the above specified exonuclease ::II solution and incubating the mixture for 2 min. at 37°C. The reaction was stopped by sedimenting the beads with the magnet and removing the supernatant, followed by three washings with 50 u1 of water. The removal of the label was checked by measuring the mixture with the hand counter.
Steps 7 and ~~
In order to cap the primer, the beads were resuspended in 13 u1 water. The following items were added: 5 u1 of 5 x sequenase buffer, :LO ~cl of a nucleotide mixture containing 100 ~M dSTTP, 100 ~M ddGTP, 100 ~M ddATP, 100 uM ddCTP, and 4 ~1 of diluted sequenase 2Ø The mixture was incubated 2 min. at 37°C and the reaction stopped by sedimenting the beads with the magnet and removing the supernatant followed by three further washings with 50 ~.1 of water. In this step, two thiolated T-nucleotides and one dideoxy C
nucleotide were added to the sequencing primer.
Steps 9 and 7.0 The dideoxy nucleotide was removed by adding 10 u1 (20 WO 93/21340 ~ ~ e~ ,~ ~ ~ ~ PCT/GB93/0084.°
units) of the above specified exonuclease III solution and incubating the mixture for 2 min. at 37°C. The reaction was stopped by sedimenting the beads with the magnet and removing the supernatant , f of lowed by three washings with 50 ~1 of water.
Template preparation, binding of the template to solid support, and annealing of the sequencing primer was performed as described in example 1, except that in the annealing step 4 u1 (4 pmoles) of radio-labelled T7 primer with the sequence: 32P-5'AATACGACTCACTATAG 3' are used.
Template/primer comt~lex 2:
Polymer-streptavidin-biotin-5'-C-C-A-A-T-T-C-G-C-C-C-T-A-T-A-G-T-G-A-G-T-C-G-T-A-T-T----3' 2 0 3'-G-A-T-A-T-C-A-C-T-C-A-G-C-A-T-A-A-32P-5' Capping of the primer with thionucleotides To the 18 ~1 annealing mixture add 10 ~1 of 100 uM deGTP, ddATP, ddTTP, and ddCTP and 4 ~1 (5 units) of diluted sequenase 2.0 (USB), and incubate the mixture for 2 min. at room temperature. According to the complementary strand this adds the following three nucleotides sequentially to the primer: dsG, dSG, ddC. The beads were sedimented using the magnet and the supernatant removed. The beads were then washed two times with 50 u1 water.
Removinct the dideoxynucleotide from the capped primer To the bead 10 u1 (20 units) of an exonuclease solution in 50 mM Tris/HC1 pH 7.5, 5 mM MgCl2, 5 mM DTT were added and the mixture incubated 2 min. at 37°C. The reaction was stopped by sedimenting the beads with the magnet and WO 93/21340 - r ~ ~ ~ ~ pCT/Gg93/00848 removing the supernatant, followed by three washings with 50 ~,1 of water. This step removed the dideoxy C-nucleotide from the 3'-end of the.primer.
FIRST SEQUENCING CYCLE (9 STEPS) Scheme 1 Steps 2 and 3 The beads (anchored template/primer complex 2) were resuspended :in 13. ~cl water. The following items were added:
5 u1 5 x sec~uenase buffer, 10 u1 of a nucleotide mixture containing 100 ul~t dCTP, 100 ~,M ddGTP, 100 ~,M ddATP, 100 ~M
ddTTP, and 4 u1 of diluted sequenase 2Ø The mixture was incubated for 2 m.rn. at 37 °C and the reaction stopped by sedimenting the beads with the magnet and removing the supernatant l:ollawed by three further washings with 50 ~1 of water. In this step, according to the complementary strand, one C-nucleol_ide a:nd one dideoxy G-nucleotide were added to the 3-end of the capped primer.
Step 4 This step is omitted because the label is located on the primer.
Steps 5 and ~5 The dideoxynucleotide and the labelled nucleotides were removed by adding 10 ~1 (20 units) of an exonuclease III
solution 50 mM Tris/HC1 pH 7.5 mM MgCl2, 5 mM DTT and incubating tree mixture for 2 min. at 37°C. The reaction was stopped by sedimenting the beads with the magnet and removing the supernatant , f of lowed by three washings with 50 ~1 of water.. The removal of the label was checked by measuring them mixture with the hand counter.
Steps 7 and F3 In order to cap the: primer, the beads were resuspended in 13 u1 water. The following items were added: 5 ~cl 5 x sequenase buffer, 10 ~,1 of a nucleotide mixture containing ~M dSCTP, 100 ~,M ddGTP, 100 ~M ddATP ~M ddTTP, and 4 u1 of diluted sequenase 2Ø The mixture was incubated 2 min.
5 at 37°C and the reaction stopg~d by sedimenting the beads with the magnet and removing the supernatant followed by three further washings with 50 ~,1 of water. In this step, one thiolated C-nucleotide and one dideoxy G-nucleotide were added to the sequencing primer.
Steps 9 AND 10 The dideoxy nucleotide was removed by adding 10 ~cl (20 units) of the above specified exonuclease III solution and incubating the mixture for 2 min. at 37°C. The reaction was stopped by sedimenting the beads with the magnet and removing the supernatant , followed by three washings with 50 ~,1 of water.
SECOND SEQUENCING CYCLE (9 STEPS) Scheme 1 Steps 2 and 3 The beads (anchored template/primer complex 2) were resuspended in 13 u1 water. The following items were added:
5 u1 5 x sequenase buffer, 10 ~,1 of a nucleotide mixture containing 100 uM dGTP, 100 ~M ddATP, 100 uM ddTTP, 100 ~M
ddCTP, and 4 u1 of diluted sequenase 2Ø The mixture was 3 0 incubated f or 2 min . at 37 ° C and the reaction stopped by sedimenting the beads with the magnet and removing the supernatant followed by three further washings with 50 u1 of water. In this step, according to the complementary strand, one G-nucleotide and one dideoxy A-nucleotide were added to the 3'-end of the capped primer.
Step 4 This step is omitted because the label is located on the ~y0 93/21340 _ ~ ~ ~ ~ 9 5 6 PCT/GB93/00848 primer.
Steps 5 and E: .
The dideoxyn~scleatide and the labelled nucleotides were 5 removed by adding 10 ~,1 (20 units) of an exonuclease III
solution in 50 mM Tris/HC1 pH 7.5, 5 mM MgClz, 5 mM DTT and incubating the mixture for 2 min. at 37°C. The reaction was stopped by :~edimenting the beads with the magnet and removing the :supernatant, followed by three washings with 50 10 ~,1 of water. The removal of the label was checked by measuring the mixture with the hand counter.
Steps 7 and 8, In order to cap the primer, the beads were resuspended in 13 15 u1 water. The following items were added: 5 u1 x sequenase buffer, l0 u1 of a nucleotide mixture containing 100 ~cM
dsGTP, 100 ACM ddATP, 100 uM ddTTP, 100 ~,M ddCTP, and 4 u1 of diluted sequenase 2Ø The mixture was incubated 2 min. at 37°C and the :reaction stopped by sedimenting the beads with 20 the magnet and removing the supernatant followed by three further washings with 50 ~,1 of water. In this step, one thiolated G-nucleotide and one dideoxy A-nucleotide were added to the sequencing primer.
25 Steps 9 and 10 The dideoxy nucleotide was removed by adding 10 u1 (20 units) of the above specified exonuclease III solution and incubating the mixture for 2 min at 37°C. The reaction was stopped by sedimenting the beads with the magnet and 30 removing the supernatant, followed by three washings with 50 ~c 1 of water .
THIRD SEQUENCING CfCLE (9 STEPS) 35 Scheme 1 Steps 2 and 3 The beads (anchored template/primer complex 2) were WO 93/21340 r PCT/GB93/0084p resuspended in 13 ~1 water. The following items were added:
~1 5 x sequenase buffer, 10 u1 of a nucleotide mixture containing 100 ~.M dATP, 100 ~M ddGTP, 100 ~,M ddTTP, 100 ACM
ddCTP, and 4 ~,1 of diluted s~quenase 2Ø The mixture was 5 incubated for 2 min at 37:°'C and the reaction stopped by sedimenting the beads with the magnet and removing the supernatant followed by three further washings with 50 ~.1 of water. In this step, according to the complementary strand, two A-nucleotides and one dideoxy T-nucleotide were added at the 3'-end of the capped primer.
Step 4 This step is omitted because the label is located on the primer.
SteQs 5 and 6 The dideoxynucleotide and the labelled nucleotides were removed by adding 10 ~,1 (20 units) of an exonuclease III
solution in 50 mM Tris/HC1 pH 7.5, 5 mM MgCl2, 5 mM DTT and incubating the mixture for 2 min at 37°C. The reaction was stopped by sedimenting the beads with the magnet and removing the supernatant, followed by three washings with 50 ~1 of water. The removal of the label was checked by measuring the mixture with the hand counter.
Steps 5 and 6 In order to cap the primer, the beads were resuspended in 13 ~cl water. The following items were added: 5 gel 5 x sequenase buffer, 10 ~1 of nucleotide mixture containing 100 ACM d$ATP, 100 uM ddGTP, 100 ~M ddTTP, 100 ~,M ddCTP, and 4 ~C1 of diluted sequenase 2Ø The mixture was incubated 2 min.
at 37°C and the reaction stopped by sedimenting the beads with the magnet and removing the supernatant followed by three further washings with 50 ~.1 of water. In this step, one thiolated A-nucleotide and one dideoxy T-nucleotide were added to the sequencing primer.
Steps 9 and 10 ~~ r~'39~6 The dideoxy nucleotide was removed by adding 10 ~,1 (20 units) of th~a abave specified exonuclease III solution and incubating the mixture ,for 2 min at 37°C. The reaction was stopped by sedimenting the beads with the magnet and removing the supernatant, followed by three washings with 50 u1 of water.
FOURTH SEQUE1JCING CYCLE (9 STEPS) Scheme 1 Steps 2 and :3 The beads (anchored template/primer complex 2) were resuspended in 13 ~,1 water. The following items were added:
5 ~1 5 x sec~uenase buffer, 10 u1 of a nucleotide mixture containing 10 ~cM dTTP, 100 uM ddGTP, 100 ~.M ddATP, 100 ACM
ddCTP, and 4 ~,l of diluted sequenase 2Ø The mixture was incubated fo:r 2 min. at 37°C and the reaction stopped by sedimenting the beads with the magnet and removing the supernatant follawed by three further washings with 50 ~cl of water. In this step, according to the complementary strand, two T-nucleotides and one dideoxy G-nucleotide were added to the 3'-end o:E the capper primer.
Step 4 This step i;s omitted because the label located on the primer.
Steps 5 and y The dideoxynucleotide and the labelled nucleotides were removed by adding 10 ~1 (20 units) of an exonuclease III
solution in ..°i0 mM Tris/HC1 pH 7.5, 5 mM MgCl2, 5 mM DTT and incubating the mixture for 2 min. at 37 °C. The reaction was stopped by sedimenting the beads with the magnet and removing the supernatant, followed by three washings with 50 u1 of water.. The removal of the label was checked by measuring the. mixture with the hand counter.
WO 93/21340 - '~ ~ ~ '~ 9 5 6 pCT/GB93/0084F
Stegs 7 and 8 In order to cap the primer, the beads were resuspended in 13 ~1 water. The following items were added: 5 ~1 5 x sequenase buffer, 10 ~1 of a nucleotide mixture containing 100 ACM dSTTP, 100 ~M ddGTP,'100 ~M ddATP, 100 uM ddCTP, and 4 ~1 of diluted sequenase 2Ø The mixture was incubated 2 min. at 37°C and the reaction stopped by sedimenting the beads with the magnet and removing the supernatant followed by three further washings with 50 ~1 of water. In this step, one thiolated T-nucleotides and one dideoxy G-nucleotide were added to the sequencing primer.
Steps 9 and 10 The dideoxy nucleotide was removed by adding 10 ~1 (20 units) of the above specified exonuclease III solution and incubating the mixture for 2 min. at 37°C. The reaction was stopped by sedimenting the beads with the magnet and removing the supernatant, followed by three washings with 50 ~,1 of water.
Fluorescein was used as a single tag attached to all four deoxynucleotides. In particular we used the following fluorescein-labelled deoxynucleoside triphosphates:
fluorescein-12-dUTP, fluorescein-15-dATP, fluorescein-15-dCTP, fluorescein-15-dITP.
GENERATION OF TEMPLATES
As a model template we used two single-stranded PCR products which were derived from the multicloning site of Bluescript II KS. Amplification of the Bluescript II KS vector DNA
using the biotinylated M13 (-21) forward primer and the nonbiotinylated M13 reverse primer yielded a PCR product which was anchored via the biotin moiety to streptavidin-coated beads as described in example 1. The nonbiotinylated ~y0 93/21340 ~ I 3 ~ ~ ~ 6 PCT/GB93/00848 (+) strand was removed by incubating the beads with 0.15 M
NaOH for 5 minutes followed by a wash with 0.15 M NaOH and three washes with water.. The template, comprising the (-) strand of tree multicloning site of the Bluescript II KS
vector, was named PCR template 1. Amplification of the Bluescript I:L KS vector using the biotinylated M13 reverse primer and t:he nonbiotinylated M13 (-21) forward primer yielded a PC;R product which was anchored via the biotin moiety to streptavidin-coated beads as described in example 1. The nonbiotinyl.ated (-) strand was removed by incubating the beads with 0.15 M NaOH for 5 minutes followed by a wash with 0.15 M NaOH and three washes with water. This template comprising t:he (~+) strand of the multicloning site of the Bluescript I:L KS vector was named PCR template 2.
SYNTHESIS OF' S'~-T'AMRA LABELLED SPECIFIC OLIGONUCLEOTIDE
PRIMERS
For each fluorescently-labelled nucleotide four different primers were designed using the Bluescript sequence of the PCR template 1 and 2. The primers were located in front of runs of a single nucleotide allowing incorporation of one, two, three, :Four,, or five nucleotides of the same kind.
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WO 93/21340 ~ ~ ~,~ ~ ~ ~ PCT/GB93/0084~' ANNEALING
In sixteen different annealing reactions, 2~,1 of water, 5~,1 of the appropriate single-stranded PCR template 1 or 2 (see tables), 2~1 of 5 x sequenase buffer and 1 u1 (0.5 pmol) of the appropriate TAMRA-labelled primer (see tables) were combined , heated at 65 ° C f or 3 minutes and then incubated on ice.
EXTENSION REACTIONS
In sixteen different extension reactions, to 6~,1 of each annealing mix, 2~1 of a nucleotide mixture (see tables) containing the appropriate unlabelled dNTPs (at 10 uM), the appropriate fluorescently-labelled dNTP (at 10 ACM), and the appropriate ddNTP (at 10~M) , and 2~,1 of diluted sequenase 2.0 were added and the mixture incubated at 37°C for 3 minutes. The reactions were stopped by adding 5~1 of 80%
formamide and heated for 3 min at 80°C followed by sedimenting the beads with a magnet and removing the supernatant.
DETECTION/IMAGING STEP (QUANTITATION) One ~.1 of each supernatant was measured using a SIT camera (model C2 400-08, Hamamatsu Photonics SA) mounted on a fluorescence microscope. The emitted fluorescence of the rhodamine dye TAMRA located at the 5'-end of the primer and the fluorescein dye introduced by nucleotide incorporation at the 3'-end of the primer was determined for each sample using appropriate filter systems. A control sample of 80%
formamide was also measured. The emitted fluorescence DI
fluorescein and DI rhodamine was recorded. The ratio of DI
fluorescein to QI rhodamine was used to normalise the data.
The results may be summarised as follows:
- Incorporation of up to five fluorescein-labelled pyrimidine nucleotides (fluorescein-12-U, fluorescein-15-C) ~13395~
-Quantitative measurements show a linear correlation between emitted fluorescence .and the number of incorporated fluorescein-:Labelled pyrimidine nucleotides. No quenching of fluorescence has been observed (see figures 1 and 2).
- Using the above. mentioned detection/imaging system from Hamamatsu Photonics we were able to detect as little as 108 molecules in a volume of approx. 1 n1 (concentration: 150 nM), allowincL, in principle, the use of up to 104 different templates on an array of 8 cm x 8 cm.
- Incorporation of up to two fluorescein-labelled purine nucleotides (fluorescein-15-A, fluorescein-15-I). Using the above detector system we were ab'; a to measure the difference between one a:nd two fluorescein-Labelled purine nucleotides.
Generation c>f template and binding to solid support, annealing of the sequencing primer to the anchored single stranded DN~~ template, capping of the primer with thionucleotides, and removing of the dideoxynucleotide from the capped primer were carried out as in example 1.
SEQUENCING B5.' SEQUENTIAL ADDITION OF SINGLE FLUORESCENTLY-LABELLED NUChEOTIDES: FIRST COMPLETE CYCLE OF 9 STEPS.
Scheme 1 steps 2 and =s The beads (anchored template/primer complex 1) were resuspended in 4 u1 water. The following items were added:
2 ~cl 5 x :~equenase buffer, 2~1 of a nucleotide mix containing 1C~~CM :Eluorescein-15-dATF (Boehringer Mannheim), 10 ~M ddGTP, 10 ~r,M ddTTP, 10 ~M ddCTP, and 2~,1 of diluted sequenase 2Ø The' mixture was incubated for 2 min at 37°C
and the reaction stopped by sedimenting the beads with the WO 93/21340 . PGT/GB93/0084~' '~1_3395~
magnet and removing the supernatant followed by three further washings with 50 u1 of water. In this step, according to the complementary strand, two fluorescein-15 A-nucleotides and one dideoxy T-nucleotide were added to the 3'- end of the capped primer..
step 4 The fluorescence was measured using a SIT camera (model C2 900-08, Hamamatsu Photonics SA) mounted on a fluorescence microscope.
steps 5 and 6 The dideoxynucleotide and the fluorescently-labelled nucleotides were removed by adding 10 ~cl (20 units) of an exonuclease-III solution in 50 mM Tris/HC1 Ph 7.5, 5 mM
MgCl2, 5 mM DTT and incubating the mixture for 2 min at 37°C. The reaction was stopped by sedimenting the beads with the magnet and removing the supernatant, followed by three washings with 50 ~,1 of water.
steps 7 and 8 In order to cap the primer, the beads were resuspended in 4 u1 water. The following items were added: 2 u1 5 x sequenase buffer, 2 ~1 of a nucleotide mixture containing 10 ACM dsATP, 10 ~,M ddGTP, 10 ~cM ddTTP, 10 ~,M ddCTP, and 2 ~1 of diluted sequenase 2Ø The mixture was incubated for 2 min at 37°C and the reaction stopped by sedimenting the beads with the magnet and removing the supernatant followed by three further washings with 50 ~,1 of water. In this step, two thiolated A-nucleotides and one dideoxy T-nucleotide were added to the sequencing primer.
steps 9 and l0 The dideoxy nucleotide was removed by adding 10 ~cl (20 units) of the above specified exonuclease-III solution and incubating the mixture for 2 min at 37°C. The reaction was stopped by sedimenting the beads with the magnet and removing the supernatant, followed by three washing with 50 u1 of water.
~'1.33~5~
WO 93/21340 PCf/GB93/00848 SEQUENCING BY SEQUENTIAL ADDITION OF SINGLE NUCLEOTIDES:
5 scheme 1 steps 2 and a The beads (anchored template/primer complex 1) were resuspended in 4 ~cl water. The following items were added:
2 ~1 of 5 x sequenase buffer, 2 ~,1 of a nucleotide mixture 10 containing l0 uM fluorescein-12-dUTP (Boehringer Mannheim), 10 uM ddGTP, 10 ~,M ddATP, 10 uM ddCTP, and 2 ~,1 of diluted sequenase 2Ø The mixture was incubated 2 min at 37°C and the reaction stopped by sedimenting the beads with the magnet and removing the supernatant followed by three 15 further washings with 50 ~1 of water. In this step, according to the: complementary strand, two fluorescein-labelled U-nucleotides and one dideoxy G-nucleotide were added to the 3'-end of the capped primer.
20 step 4 The fluorescence was measured using a SIT camera (model 400-08, Hamamatsu Photonics SA) mounted on a fluorescence microscope.
25 steps 5 and E>
The dideoxynucleotide and the labelled nucleotides were removed by adding 10 ~,1 (20 units) of the above specified exonuclease-7:II evolution and incubating the mixture for 2 min at 37°C. The reaction was stopped by sedimenting the 30 beads with the magnet and removing the supernatant, followed by three wasr~ings~ with 50 ~,1 of water.
steps 7 and Et In order to cap t:he primer, the beads were resuspended in 4 35 u1 water. The following items were added: 2 u1 of 5 x sequenase buffer, 2 u1 of a nucleotide mixture containing 10 ACM dSTTP, 10 ~cM ddGTP, 10 ~,M ddATP, 10 ACM ddCTP, and 2 ~1 of diluted seque~nase 2Ø The mixture was incubated for 2 min WO 93/21340 PCT/GB93/0084~
~,1'~39~~ 46 at 37°C and the reaction stopped by sedimenting the beads with the magnet and removing the supernatant followed by three further washings. with 50~c1 of water. In this step, two thiolated T-nucleotides and one dideoxy G-nucleotide were added to the sequencing primer.
steps 9 and 10 the dideoxy nucleotide was removed by adding 10 u1 (20 units) of the above specified exonuclease-III solution and incubating the mixture for 2 min at 37°C. The reaction was stopped by sedimenting the beads with the magnet and removing the supernatant, followed by three washings with 50 u1 of water.
Generation of template and binding to solid support, annealing of the sequencing primer to the anchored single-stranded DNA template, capping of the primer with thionucleotides, and removing of the dideoxynucleotide from the capped primer were carried out as in example 1.
SEQUENCING BY SEQUENTIAL ADDITION OF SINGLE FLUORESCENTLY-LABELLED NUCLEOTIDES: FIRST COMPLETE CYCLE OF 9 STEPS
scheme 1 steps 2 and 3 The beads (anchored template/primer complex 1) were resuspended in 4 ~1 water. The following items were added:
2 ~1 5 x sequenase buffer, 2 ~.1 of a nucleotide mix containing 500 uM fluorescein-15-dATP, 1.0 ~,M dATP, 10 ~M
ddGTP, 10 uM ddTTP, 10 ACM ddCTP, and 2 ~.1 of diluted sequenase 2Ø The mixture was incubated for 2 min at 37°C
and the reaction stopped by sedimenting the beads with the magnet and removing the supernatant followed by three further washings with 50 ~1 of water. In this step, as directed by the complementary strand, fluorescein-15-A-j ~ ~ ~'~ ~ ~ pCT/GB93/00848 nucleotides, A-nucleotides, and one dideoxy T-nucleotide were added to the 3'-end of the capped primer.
step 4 The fluorescence was measured using a SIT camera (model C2 400-08, Hama.matsu Photonics SA) mounted on a fluorescence microscope.
steps 5 and _6 The dideoxynucleotide, the deoxynucleotides and the fluorescently-labelled nucleotides were removed by adding 20 ~1 (20 units) of an exonuclease-III solution in 50 tnM
Tris/HCI pH 7.5,. 5 mM MgCl2 5 mM DTT and incubating the mixture for 2 min at 37°C. The reaction was stopped by sedimending the beads with the magnet and removing the supernatant, followed by three washings with 50 ~,1 of water.
steps 7 and 8 In order to ~~ap the primer, the beads were resuspended in 4 ~1 water. The following items were added: 2 ~cl 5 x sequenase buffer, 2 ~1 of a nucleotide mixture containing 10 ~,M dSATP, 10 ~M ddGTP, 10 ~M ddTTP, 10 uM ddCTP, and 2 ~1 of diluted sequenase 2Ø The mixture was incubated for 2 min at 37°C and the reaction stopped by sedimenting the beads with the magnet and removing the supernatant followed by the three further washings with 50 ~1 of water. In this step, two thiolate:d A~-nucleotides and one dideoxy T-nucleotide were added to the sequencing primer.
3 0 steps 9 and 10 The dideoxy nucleotide was removed by adding 10 ~l (20 units) of the above specified exonuclease-III solution and incubating the mixture for 2 min at 37°C. The reaction was stopped by sedimenting the beads with the magnet and removing the supernatant, followed by three washings with 50 ~cl of water.
SEQUENCING f3Y SEQUENTIAL ADDITION OF SINGLE NUCLEOTIDES:
~Z~.3395G
WO 93/21340 ' PCT/GB93/0084g scheme 1 steps 2 and 3 The beads (anchored template/primer complex 1) were resuspended in 4 ~1 water. The following items were added:
2 ~1 of 5 x sequenase buffer, 2 ~cl of a nucleotide mixture containing 15 ~,M fluoresce in-12-dUTP, 1.0 ~M dTTP, 10 ~,M
ddGTP, 10 ACM ddATP, 10 ACM ddCTP, and 2 ~,1 of diluted sequenase 2Ø The mixture was incubated for 2 min at 37°C
and the reaction stopped by sedimenting the beads with the magnet and removing the supernatant followed by three further washings with 50 u1 of water. In this step, as directed by the complementary strand, fluorescein-labelled U-nucleotides, T-nucleotides, and one dideoxy G-nucleotide were added to the 3'-end of the capped primer.
step 4 The fluorescence was measured using a SIT camera (model C2 400-08, Hamamatsu Photonics SA) mounted on a fluorescence microscope.
steps 5 and 6 The dideoxynucleotide and the labelled nucleotides were removed by adding 10 u1 (20 units) of the above specified exonuclease-III solution and incubating the mixture for 2 min at 37°C. The reaction was stopped by sedimenting the beads with the magnet and removing the supernatant, followed by three washings with 50 u1 of water.
steps 7 and 8 In order to cap the primer, the beads were resuspended in 4 u1 water. The following items were added: 2 dal of 5 x sequenase buffer, 2 ~1 of a nucleotide mixture containing 10 ~.M dSTTP, 10 ~cM ddGTP, 10 ~M ddATP, 10 ~,M ddCTP, and 2 ~,1 of diluted sequenase 2Ø The mixture was incubated 2 min at 37°C and the reaction stopped by sedimenting the beads with the magnet and removing the supernatant followed by three WO 93/21340 ~ ~ ~ ~ ~ ~ ~ PCT/GB93/00848 further washings with 50 ~,1 of water. In this step, two thiolated T--nucleotides and one dideoxy G-nucleotide were added to the sequencing primer.
steps 9 and 10 The dideoxy nucleotide was removed by adding 10 u1 (20 units) of th,e above specified exonuclease-III solution and incubating the mixture for 2 min at 37°C. The reaction was stopped by sedimenting the beads with the magnet and removing the supernatant, followed by three washings with 50 ~1 of water.
Template preparation, binding of the template to a solid support, and annealing the sequencing primer was performed as described in example 1.
Cap,~ing of the r~rimer with thionucleotides To the 18 u7. annealing mixture add 10 ~,1 of 100 uM dsGTP, ddATP, ddTTl?, and ddCTP and 4 ~cl (5 units) of diluted sequenase 2.0 (USES), and incubate the mixture for 2 min at room temperature. As directed by the complementary strand, this adds tree following three nucleotides sequentially to the primer: dSG, dsG, ddC. The beads were sedimented using the magnet and the supernatant removed. The beads were then washed two times with 50 ~,1 water.
Removing the dideaxynucleotide from the capped primer To the beads , 10 u:1 ( 2 0 units ) of an exonuclease solution in 50 mM Tris/EfCl pH 7.5, 5mM MgCl2, 5 mM DTT were added and the mixture incubated 2 min at 37°C. The reaction was the reaction stopped by sedimenting the beads with the magnet and remvoinc~ then supernatant, followed by three washings with 50 ~1 of water. This step removed the dideoxy C-nucleotide from the 3'-end of the primer.
WO 93/21340 PCT/GB93/0084~
~~~33956 FIRST SEQUENCING CYCLE (9 STEPS) scheme 1 steps 2 and 3 5 The beads (anchored template/primer complex 1) were resuspended in 4 ~1 of water. The following items were added: 2 u1 5 x sequenase buffer, 2 u1 of a nucleotide mixture containing 10 ~cM fluorescein-15-dCTP (Boehringer Mannheim), 10 ~M ddGTP, 10 ~,M ddATP, 10 ~M ddTTP, and 2 gel 10 of diluted sequenase 2Ø The mixture was incubated for 2 min at 37°C and stopped by sedimenting the beads with the magnet and removing the supernatant followed by three further washings with 50 ~,1 of water. In this step, as directed by the complementary strand, one fluorescein-15 labelled C-nucleotide and one dideoxy G-nucleotide were added to the 3'-end of the capped primer.
step 4 The fluorescence was measured using a SIT camera (model C2 20 400-08, Hamamatsu Photonics SA) mounted on a fluorescence microscope.
steps 5 and 6 The dideoxynucleotide and the fluorescein-labelled 25 nucleotides were removed by adding 10 ~1 (20 units) of an exonuclease-III solution in 50 mM Tris/HC1 pH 7.5, 5 mM
MgCl2, 5 mM DTT and incubating the mixture for 2 min at 37'C. The reaction was stopped by sedimenting the beads with the magnet and removing the supernatant, followed by 30 three washings with 50 ~.1 of water.
steps 7 and 8 In order to cap the primer, the beads were resuspended in 4 ~cl water. The following items were added: 2 ~,1 5 x 35 sequenase buffer, 2 ~.1 of a nucleotide mixture containing 10 ~,M dSCTP , 10 uM ddGTP , 10 ~cM ddATP , 10 ~.M ddTTP , and 2 a 1 of diluted sequenase 2Ø The mixture was incubated 2 min at 37°C and the reaction stopped by sedimenting the beads with the magnet and removing the supernatant followed by three further washings with 50u1 of water. In this step, one thiolated C-nulceotide~and one dideoxy G-nucleotide were added to the sequencing primer.
steps 9 and 7.0 The dideoxy nucleotide was removed by adding 10 ~1 (20 units) of ths: above specified exonuclease-III solution and incubating the mixture for 2 min at 37°C. The reaction was stopped by sedimenting the beads with the magnet and removing the supernatant, followed by three washings with 50 u1 of water.
SECOND SEQUErJCING CYCLE (9 STEPS) scheme 1 steps 2 and :3 The beads (anchored template/primer complex 1) were resuspended ~~n 4 ~,:L water . The following items were added:
2 ~1 5 x sequenase buffer, 2 ~1 of a nucleotide mixture containing 10 uM fluorescein-15-dITP (Boehringer Mannheim), 10 uM ddATP, 10 ~iM ddTTP, 10 ~cM ddCTP, and 2 ~1 of diluted sequenase 2Ø The mixture was incubated 2 min at 37°C and the reaction stopped by sedimenting the beads with the magnet and removing the supernatant followed by three further washings with 50 ~cl of water. In this step, as directed by the complementary strand, one fluorescein-labelled I-nucleotide and one dideoxy A-nucleotide were added to the 3'-end of the capped primer.
step 4 The fluorescence was measured using a SIT camera (model C2 400-08, Hamamatsu Photonics SA) mounted on a fluorescence microscope.
steps 5 and 6 The dideoxynucleotide and the labelled nucleotide were removed by adding 10 ~.1 (20 units) of an exonucleaseIII
WO 93/21340 c PCT/GB93/0084R
solution in 50 mM Tris/HC1 pH 7.5, 5 mM MgCl2, 5 mM DTT and incubating the mixture for 2 min at 37°C. The reaction was stopped by sedimenting the beads with the magnet and removing the supernartant, followed by three washings with 50 u1 of water.
steps 7 and 8 In order to cap the primer, the beads were resuspended in 4 ~cl water. The following items were added: 2 ~1 5 x sequenase buffer, 2 ~1 of a nucleotide mixture containing 10 ~M dSGTP, 10 ~,M ddATP, 10 ~cM ddTTP, 10 ~M ddCTP, and 2 ~C1 of diluted sequenase 2Ø The mixture was incubated for 2 min at 37°C and the reaction stopped by sedimenting the beads with the magnet and removing the supernatant followed by three further washings with 50 ~cl of water. In this step, one thiolated G-nucleotide and one dideoxy A-nucleotide were added to the sequencing primer.
steps 9 and 10 The dideoxy nucleotide was removed by adding 10 u1 (20 units) of the above specified exonuclease III solution and incubating the mixture for 2 min at 37°C. The reaction was stopped by sedimenting the beads with the magnet and removing the supernatant, followed by three washings with 50 ~cl of water.
THIRD SEQUENCING CYCLE (9 STEPS) scheme 1 steps 2.and 3 The beads (anchored template/primer complex 1) were resuspended in 4 ~1 water. The following items were added:
2 ~.1 5 x sequenase buffer, 2 u1 of a nucleotide mixture containing 10 ACM fluorescein-15-dATP, 10 ~M ddGTP, 10 uM
ddTTP, 10 uM ddCTP, and 2 ~1 of diluted sequenase 2Ø The mixture was incubated for 2 min at 37°C and the reaction stopped by sedimenting the beads with a magnet and removing the supernatant followed by three further washings with 50 ~1 of water. In this step, as directed by the complementary strand, two fluorescein-labelled A-nucleotides and one dideoxy T-nucleotide were added to the 3'-end of the capped primer.
step 4 The fluorescence was measured using a SIT camera (model C2 400-08, Hamamatsu Photonics SA) mounted on a fluorescence microscope.
steps 5 and 6 The dideoxynucleotide and the labelled nucleotides were removed by e~dding 10 ~cl (20 units) of an exonuclease-III
solution in 50 mM Tris/HC1 pH 7.5, 5 mM MgCl2, 5 mM DTT and incubating tlhe mixture for 2 min at 37°C . The reaction was stopped by sedimenting the beads with the magnet and removing the supernatant, followed by three washings wits ~0 ~cl of water.
steps 7 and 8 In order to cap th.e primer, the beads were resuspended in 4 ~cl water. The following items were added: 2 ~.1 5 x sequenase buffer, 2 al of a nucleotide mixture containing 10 uM deATP, 10 ~,M ddGTP, 10 ~M ddTTP, 10 ~M ddCTP, and 2 ~,1 of diluted sequenase 2Ø The mixture was incubated 2 min at 37°C and the: reaction stopped by sedimenting the beads with the magnet and removing the supernatant followed by three further washings with 50 ~.1 of water. In this step, two thiolated A--nucleotides and dideoxy T-nucleotide were added to the sequEancing primer.
steps 9 and 10 The dideoxy nucleotide was removed by adding 10 u1 (20 units) of tine above specified exonuclease III solution and incubating 'the mixture for 2 min at 37°C. The reaction was stopped by sedimenting the beads with the magnet and removing them supernatant, followed by three washings with 50 ~1 of water.
WO 93/21340 PCT/GB93/0084°
~z133g56 FOURTH SEQUENCING CYCLE (9 STEPS) scheme 1 steps 2 and 3 The beads (anchored template/primer complex 1) were resuspended in 4 ~1 water. The following items were added:
2 ~1 5 x sequenase buffer, 2 ~1 of a nucleotide mixture containing 10 uM fluorescein-12-dUTP, 10 uM ddGTP, 10 ~M
ddATP, 10 ~M ddCTP, and 2 ~1 of diluted sequenase 2Ø The mixture was incubated f or 2 min at 37 ° C and the reaction stopped by sedimenting the beads with the magnet and removing the supernatant followed by three further washings with 50 ~1 of water. In this step, as directed by the complementary strand, twofluorescein-labelled U-nucleotides and one dideoxy G-nucleotide were added to the 3'-end of the capped primer.
step 4 The fluorescence was measured using a SIT camera (model C2 400-08, Hamamatsu Photonics SA) mounted on a fluorescence microscope.
steps 5 and 6 The dideoxynucleotide and the labelled nucleotides were removed by adding 10 u1 (20 units) of an exonuclease-III
solution in 50 mM Tris/HC1 pH 7.5, 5 mM MgCl2, 5 mM DTT and incubating the mixture for 2 min at 37°C . The reaction was stopped by sedimenting the beads with the magnet and removing the supernatant, followed by three washings with 50 ~.1 of water. The removal of the label was checked by measuring the mixture with the hand counter.
3 5 stegs 7 and 8 In order to cap the primer, the beads were resuspended in 4 ~.1 water. The following items were added: 2 u1 5 x sequenase buffer, 2 u1 of a nucleotide mixture containing 10 uM dSTTP, 10 ~M ddGTP, 10 uM ddATP, 10 ~,M ddCTP, and 2 ~cl of diluted sequenase 2Ø The mixture was incubated for 2 min at 37°C and the reaction stopped by sedimenting the beads with the magnet and removing the supernatant followed by 5 three further washings with 50 ~1 of water. In this step, two thiolatEad T~-nucleotides and one dideoxy G-nucleotide were added t.o the sequencing primer.
steps 8 and 9 10 The dideoxy nucleotide was removed by adding 10 ~tl (20 units) of the ahove specified exonuclease-III solution and incubating the mixture for 2 min at 37°C. The reaction was stopped by sedimenting the beads with the magnet and remvoing the: supernatant, followed by three washings with 50 15 ~1 of water..
Template preparation, binding of the template to solid 20 support, and annealing of the sequencing primer was performed as described in example 1.
Capping of 'the primer with thionucleotides To the 18 u1 annealing mixture add 10 ~.1 of 100 ~M dBGTP, 25 ddATP, ddTTP, and ddCTP and 4 u1 (5 units) of diluted sequenase 2.0 (USB), and incubate the mixture for 2 min at room temperature. As directed by the complementary strand this adds the following three nucleotides sequentially to the primer: d$G, dSG, ddC. The beads were sedimented using 30 the magnet and the supernatant removed. The beads were then washed two times with 50 ~.1 water.
R_emovincr the dideoxynucleotide from the capped primer To the beads, 10 u1 (20 units) of an exonuclease solution in 35 50 mM Tris/HC1 pH 7.5, 5 mM MgCl2, 5 mM DTT were added and the mixture' incubated 2 min at 37°C. The reaction was stopped by sedimenting the beads with the magnet and removing the supernantant, followed by three washings with ~~133956 WO 93/21340 PCT/GB93/0084~
50 ~1 of water. This step removed the dideoxy C-nucleotide from the 3'-end of the primer.
FIRST SEQUENCING CYCLE (9 STEPS) scheme 1 steps 2 and 3 The beads (anchored template/primer complex 1) were resuspended in 4 gel water. The following items were added:
2 u1 5 x sequenase buffer, 2 u1 of a nucleotide mixture containing 15 uM fluorescein-15-dCTP, 1.0 uM dCTP, 10 uM
ddGTP, 10 uM ddATP, 10 uM ddTTP, and 2 ~cl of diluted sequenase 2Ø The mixture was incubated for 2 min at 37°C
and the reaction stopped by sedimenting the beads with the magnet and removing the supernatant followed by three further washings with 50 u1 of water.
step 4 The fluorescence was measured using a SIT camera (model C2 400-08, Hamamatsu Photonics SA) mounted on a fluorescence microscope.
steps 5 and 6 The dideoxynucleotide and the fluorescein-labelled nucleotides were removed by adding 10 ~1 (20 units) of an exonuclease-III solution in 50 mM Tris/HC1 pH 7.5, 5 mM
MgCl2, 5 mM DTT and incubating the mixture for 2 min at 37°C. The reaction was stopped by sedimenting the beads with the magnet and removing the supernatant, followed by three washings with 50 ~,1 of water.
steps 7 and 8 In order to cap the primer, the beads were resuspended in 4 ~,1 water. The following items were added: 2 ~1 5 x sequenase buffer, 2 ~,1 of a nucleotide mixture containing 10 ~M dSCTP, 10 ~M ddGTP, 10 ~M ddATP, 10 ~,M ddTTP, and 2 u1 of diluted sequenase 2Ø The mixture was incubated 2 min at 37°C and the reaction stopped by sedimenting the beads with ~1339~6 the magnet and removing the supernatant followed by three further washings with 50 ~1 of water. In this step, two thiolated C-nucleotides and one dideoxy G-nucleotide were added to the sequencing primer.
steps 9 and 10 The dideoxy nucleotide was removed by adding 10 ~,l (20 units) of th~~ above specified exonuclease III solution and incubating the mixture for 2 min at 37°C. The reaction was stopped by sedimenting the beads with the magnet and removing the supernatant, followed by three washings with 50 ~1 of water.
SECOND SEQUE;HCINr CYCLE (9 STEPS) scheme 1 steps 2 and 3 The beads (anchored template/primer complex 1) were resuspended in 4 ~cl water. The following items were added:
2 ~,1 5 x sequenase buffer, 2 u1 of a nucleotide mixture containing 500 ~,M fluorescein-15-dITP, 1.0 uM dGTP, 10 uM
cIATP, 10 uM ddTTP, 10 ~cM ddCTP, and 2 ~1 of diluted sequenase 2Ø The mixture was incubated for 2 min at 37°C
and the reaction stopped by sedimentin:~ the beads with the magnet and removing the supernatant followed by three further washings with 50 ~.1 of water.
stets 4 The fluorescence was measured using a SIT camera (model c2 400-08, Hamamatsu Photonics SA) mounted on a fluorescence microscope.
steps 5 and 6 The dideoxynucleotide and the labelled nucleotides were removed by adding 10 ~,1 (20 units) of an exonuclease-III
solution in 50 mM Tris/HC1 pH 7.5, 5 mM MgCl2, 5 mM DTT and incubating the mixture for 2 min at 37°C. The reaction was stopped by sedimenting the beads with the magnet and removing the supernatant, followed by three washings with 50 u1 of water.
WO 93/21340 ~~ ~ 3 3 g 5 6 PGT/GB93/00848 _steps 7 and 9 In order to cap the primer, the beads were resuspended in 4 u1 water. The following items were added: 2 u1 5 x sequenase buffer, 2 ~1 of.a nucleotide mixture containing 10 ~,M dSGTP, 10 uM ddATP, 10 ~M ddTTP, 10 ~M ddCTP, and 2 u1 of diluted sequenase 2Ø The mixture was incubated 2 min at 37°C and the reaction stopped by sedimenting the beads with the magnet and removing the supernatant followed by three further washings with 50 ~cl of water. In this step, one thiolated G-nucleotide and one dideoxy A-nucleotide were added to the sequencing primer.
step 9 and 10 The dideoxy nucleotide was removed by adding 10 u1 (20 units) of a specified exonuclease-III solution and incubating the mixture for 2 min at 37°C. The reaction was stopped by sedimenting the beads with the magnet and removing the supernatant, followed by three washings with 50 ~1 of water.
THIRD SEQUENCING CYCLE (9 STEPS) scheme 1 steps 2 and 3 The beads (anchored template/primer complex 1) were resuspended in 4 ~cl water. The following items were added:
2 u1 5 x sequenase buffer, 2 ~1 of a nucleotide mixture containing 500 ~.M fluorescein-15-dATP, 1 ~cM dATP, 10 ~M
ddGTP, 10 ~M ddTTP, 10 ~M ddCTP, and 2 ~C1 of diluted sequenase 2Ø The mixture was incubated for 2 min at 37°C
and the reaction stopped by sedimenting the beads with the magnet and removing the supernatant followed by three further washings with 50 ~1 of water.
step 4 The fluorescence was measured using a SIT camera (model C2 400-08, Hama:matsu Photonics SA) mounted on a fluorescence microscope.
steps 5 and 6 The dideoxyn.ucleotide and the labelled nucleotides were removed by adding 10 u1 (20 units) of an exonuclease-III
solution in X50 mM Tris/HC1 pH 7.5, 5mM MgCl2, 5 mM DTT and incubating tile mixture for 2 min at 37°C. The reaction was stopped by sedimenting the beads with the magnet and removing the supernatant, followed by three washings with 50 u1 of water.
steps 7 and .3 In order to c:ap the primer, the beads were resuspended in 4 ~:1 water. The following items were added: 2 u1 5 x sequenase buffer, 2 ~cl of a nucleotide mixture containing 10 ~M dSATP, 10 ~M ddGTP, 10 uM ddTTP, 10 uM ddCTP, and 2 ~1 of diluted s~equenase 2Ø The mixture was incubated 2 min at 37°C and the reaction stopped by sedimenting the beads with the magnet and removing the supernatant followed by three further washings with 50 ~1 of water. In this step, two thiolated A-nucleotides and one dideoxy T-nucleotide were added to the sequencing primer.
steps 9 and 10 The dideoxy nucleotide was removed by adding 10 ~,1 (20 units) of the above specified exonuclease-III solution and incubating t:he mixture for 2 min at 37°C. The reaction was stopped by sedimenting the beads with the magnet and 3 0 removing the supernatant, followed by three washings with 50 ~c 1 of water .
FOURTH SEQUENCING CYCLE (9 STEPS) scheme 1 steps 2 and 3 The beads (anc:hored template/primer complex 1) were resuspended in 4 u1 water. The following items were added:
2 u1 5 x sequenase buffer, 2 ~.1 of a nucleotide mixture WO 93/21340 PCT/GB93/0084'' ~~~~3~~6 containing 15 uM fluorescein-12-dUTP, 1.0 uM dTTP, 10 ~,M
ddGTP, 10 uM ddATP, 10 ACM ddCTP, and 2 u1 of diluted sequenase 2Ø The mixture was incubated for 2 min at 37°C
and the reaction stopped by sedimenting the beads with the 5 magnet and removing t2ie~ supernatant followed by three further washings with 50 ~1 of water.
step 4 The fluorescence was measured using a SIT camera (model C2 10 400-08, Hamamatsu Photonics SA) mounted on a fluorescence microscope.
steps 5 and 6 The dideoxynucleotide and the labelled nucleotides were 15 removed by adding 10 u1 (20 units) of an exonuclease-III
solution in 50 mM Tris/HC1 pH 7.5, 5 mM MgCl2, 5 mM DTT and incubating the mixture for 2 min at 37°C. The reaction was stopped by sedimenting the beads with the magnet and removing the supernatant, followed by three washings with 50 20 u1 of water. The removal of the label was checked by measuring the mixture with the hand counter.
steps 7 and 8 In order to cap the primer, the beads were resuspended in 4 25 ~1 water. The following items were added: 2 ~,1 5 x sequenase buffer, 2 ~1 of a nucleotide mixture containing 10 ACM dSTTP, 10 uM ddGTP, 10 uM ddATP, 10 uM ddCTP, and 2 ~1 of diluted sequenase 2Ø The mixture was incubated 2 min at 37°C and the reaction stopped by sedimenting the beads 30 with the magnet and removing the supernatant followed by three further washings with 50 ~cl of water. In this step, two thiolated T-nucleotides and one dideoxy G-nucleotide were added to the sequencing primer.
35 steps 8 and 9 The dideoxy nucleotide was removed by adding 10 ~1 (20 units) of the above specified exonucleaseIII solution and incubating the mixture for 2 min at 37°C. The reaction was ~.~ 33~~~~
WO 93/21340 - PCi'/GB93/00848 s=opped by sedimenting the beads with the magnet and removing the supernatant, followed by three washings with 50 ~,1 of water.
It will be understood that the invention is described above by way of example only, and that a variety of modifications will be apparent to those skilled in the art which fall within the scope of the appended claims.
Claims (19)
1. A method for determining the sequence of a nucleic acid comprising the steps of:
a) forming a single-stranded template comprising the nucleic acid to be sequenced;
b) hybridising a primer to the template to form a template/primer complex;
c) extending the primer by the addition of a single labelled nucleotide, wherein said labelled nucleotide is not a chain elongation inhibitor;
d) determining the type of the labelled nucleotide added onto the primer;
e) removing or neutralising the label; and f) repeating steps (c) to (e) sequentially and recording the order of incorporation of labelled nucleotides.
a) forming a single-stranded template comprising the nucleic acid to be sequenced;
b) hybridising a primer to the template to form a template/primer complex;
c) extending the primer by the addition of a single labelled nucleotide, wherein said labelled nucleotide is not a chain elongation inhibitor;
d) determining the type of the labelled nucleotide added onto the primer;
e) removing or neutralising the label; and f) repeating steps (c) to (e) sequentially and recording the order of incorporation of labelled nucleotides.
2. The method according to claim 1 wherein the template/primer complex is bound to a solid-phase support.
3. The method according to claim 1 or claim 2 wherein step (c) comprises the use of a mixture of both labelled and unlabelled nucleotides.
4. The method according to any one of claims 1 to 3 wherein the labelled nucleotide is added to the template/primer complex in the presence of chain elongation inhibitors.
5. The method according to claim 4 wherein the chain elongation inhibitors are chain terminators which are incorporated into the template/primer complex and step (e) further comprises removing the chain terminators.
6. The method according to claim 4 or claim 5 wherein the chain elongation inhibitor is a fluorescent dye group attached to the 3' moiety of the deoxyribose group of the labelled nucleotide, and step (e) comprises cleaving the fluorescent dye from the nucleotide to generate a 3' hydroxyl group.
7. The method according to claim 4 wherein the chain elongation inhibitors are not incorporated into the template/primer complex.
8. The method according to claim 7 wherein the chain elongation inhibitors are deoxynucleoside 5'-[.alpha., .beta.-methylene] triphosphates, deoxynucleoside diphosphates or deoxynucleoside monophosphates.
9. The method according to any one of claims 1 to 8 wherein the template/primer complex comprises a primer having a deoxynucleoside phosphorothioate base at its 3' end.
10. The method according to claim 9 wherein step (e) comprises:
i) removing the labelled nucleotide with an exonuclease; and ii) replacing the labelled nucleotide with a corresponding unlabelled phosphorothioate nucleoside derivative in the presence of chain elongation inhibitors.
i) removing the labelled nucleotide with an exonuclease; and ii) replacing the labelled nucleotide with a corresponding unlabelled phosphorothioate nucleoside derivative in the presence of chain elongation inhibitors.
11. The method according to any one of claims 1 to 10 wherein steps (c) and (d) are repeated sequentially a multiplicity of times before the removal or neutralisation of the label.
12. The method according to any one of claims 1 to 8 wherein the label is a fluorescent label and step (e) comprises neutralising the label by bleaching with laser radiation or by chemical means, or by dissociating the label from the labelled nucleotide.
13. A process for sequencing a DNA fragment comprising the steps of:
i) hybridising a capped primer containing a phosphorothioate nucleoside derivative to a template to form a template/primer complex;
ii) adding a labelled deoxynucleoside triphosphate, wherein said labelled deoxynucleoside triphosphate is not a chain elongation inhibitor, together with heterogeneous chain terminators and a suitable polymerase to the template/primer complex;
iii) removing excess reagents by washing;
iv) measuring the amount of incorporated label;
v) treating the template/primer complex with an exonuclease to remove the labelled deoxynucleoside triphosphate and the chain terminators;
vi) removing the exonuclease by washing;
vii) adding a phosphorothioate deoxynucleoside triphosphate corresponding to the labelled deoxynucleoside triphosphate added in Step ii) together with heterogeneous chain terminators;
viii) removing excess reagents by washing;
ix) treating the template/primer complex with an exonuclease to remove the chain terminators;
x) removing the exonuclease by washing; and xi) repeating steps ii) to x), each time with a different deoxynucleoside triphosphate.
i) hybridising a capped primer containing a phosphorothioate nucleoside derivative to a template to form a template/primer complex;
ii) adding a labelled deoxynucleoside triphosphate, wherein said labelled deoxynucleoside triphosphate is not a chain elongation inhibitor, together with heterogeneous chain terminators and a suitable polymerase to the template/primer complex;
iii) removing excess reagents by washing;
iv) measuring the amount of incorporated label;
v) treating the template/primer complex with an exonuclease to remove the labelled deoxynucleoside triphosphate and the chain terminators;
vi) removing the exonuclease by washing;
vii) adding a phosphorothioate deoxynucleoside triphosphate corresponding to the labelled deoxynucleoside triphosphate added in Step ii) together with heterogeneous chain terminators;
viii) removing excess reagents by washing;
ix) treating the template/primer complex with an exonuclease to remove the chain terminators;
x) removing the exonuclease by washing; and xi) repeating steps ii) to x), each time with a different deoxynucleoside triphosphate.
14. A process for sequencing a DNA fragment comprising the steps of:
i) hybridising a capped primer a containing phosphorothioate nucleoside derivative to a template to form a template/primer complex;
ii) adding a labelled deoxynucleoside triphosphate, wherein said labelled deoxynucleoside triphosphate is not a chain elongation inhibitor, together with heterogeneous chain terminators and a suitable polymerase to the template/primer complex;
iii) removing excess reagents by washing;
iv) measuring the amount of incorporated label;
v) removing the labelled nucleotide and the chain terminators with an exonuclease;
vi) removing the exonuclease by washing;
vii) adding a phosphorothioate deoxynucleoside triphosphate together with heterogeneous chain elongation inhibitors not incorporated into the chain;
viii) removing excess reagents by washing; and ix) repeating steps ii) to viii), each time with a different labelled deoxynucleoside triphosphate.
i) hybridising a capped primer a containing phosphorothioate nucleoside derivative to a template to form a template/primer complex;
ii) adding a labelled deoxynucleoside triphosphate, wherein said labelled deoxynucleoside triphosphate is not a chain elongation inhibitor, together with heterogeneous chain terminators and a suitable polymerase to the template/primer complex;
iii) removing excess reagents by washing;
iv) measuring the amount of incorporated label;
v) removing the labelled nucleotide and the chain terminators with an exonuclease;
vi) removing the exonuclease by washing;
vii) adding a phosphorothioate deoxynucleoside triphosphate together with heterogeneous chain elongation inhibitors not incorporated into the chain;
viii) removing excess reagents by washing; and ix) repeating steps ii) to viii), each time with a different labelled deoxynucleoside triphosphate.
15. A process for sequencing a DNA fragment comprising the steps of:
i) hybridising a capped primer containing a phosphorothioate deoxynucleoside derivative to a template to form a template/primer complex;
ii) adding a labelled deoxynucleoside triphosphate, wherein said labelled deoxynucleoside triphosphate is not a chain elongation inhibitor, together with heterogeneous chain elongation inhibitors not incorporated into the chain and a suitable polymerase to the template/primer complex;
iii) removing excess reagents by washing;
iv) measuring the amount of incorporated label;
v) repeating steps ii) to iv) until all four different labelled deoxynucleoside triphosphates in the presence of their corresponding heterogeneous chain elongation inhibitors not incorporated into the chain have been added;
vi) removing all labelled nucleotides with exonuclease;
vii) removing the exonuclease by washing;
viii) adding the phosphorothioate deoxynucleoside triphosphate corresponding to the first labelled deoxynucleotide added to the reaction in step ii), together with heterogeneous chain elongation inhibitors not incorporated into the chain and a suitable polymerase to the template/primer complex;
ix) removing excess reagents by washing; and x) repeating steps ix) and x) with the three remaining phosphorothioate deoxynucleoside triphosphates.
i) hybridising a capped primer containing a phosphorothioate deoxynucleoside derivative to a template to form a template/primer complex;
ii) adding a labelled deoxynucleoside triphosphate, wherein said labelled deoxynucleoside triphosphate is not a chain elongation inhibitor, together with heterogeneous chain elongation inhibitors not incorporated into the chain and a suitable polymerase to the template/primer complex;
iii) removing excess reagents by washing;
iv) measuring the amount of incorporated label;
v) repeating steps ii) to iv) until all four different labelled deoxynucleoside triphosphates in the presence of their corresponding heterogeneous chain elongation inhibitors not incorporated into the chain have been added;
vi) removing all labelled nucleotides with exonuclease;
vii) removing the exonuclease by washing;
viii) adding the phosphorothioate deoxynucleoside triphosphate corresponding to the first labelled deoxynucleotide added to the reaction in step ii), together with heterogeneous chain elongation inhibitors not incorporated into the chain and a suitable polymerase to the template/primer complex;
ix) removing excess reagents by washing; and x) repeating steps ix) and x) with the three remaining phosphorothioate deoxynucleoside triphosphates.
16. A process for sequencing a DNA fragment comprising the steps of:
i) hybridising a capped primer to a template to form a template/primer complex;
ii) adding a fluorescent nucleoside triphosphate, wherein said fluorescent deoxynucleoside triphosphate is not a chain elongation inhibitor, together with three heterogeneous chain elongation inhibitors not incorporated into the chain and a suitable polymerase to the template/primer complex;
iii) removing excess reagents by washing;
iv) measuring the amount of incorporated label;
v) repeating steps ii) to iv) using all three different nucleoside triphosphates, each with a fluorescent label, in the presence of the respective heterogeneous chain elongation inhibitors not incorporated into the chain; and vi) destroying the fluorescent labels by bleaching with a laser or by a suitable chemical reaction, or removing the fluorescent labels by a chemical cleavage step.
i) hybridising a capped primer to a template to form a template/primer complex;
ii) adding a fluorescent nucleoside triphosphate, wherein said fluorescent deoxynucleoside triphosphate is not a chain elongation inhibitor, together with three heterogeneous chain elongation inhibitors not incorporated into the chain and a suitable polymerase to the template/primer complex;
iii) removing excess reagents by washing;
iv) measuring the amount of incorporated label;
v) repeating steps ii) to iv) using all three different nucleoside triphosphates, each with a fluorescent label, in the presence of the respective heterogeneous chain elongation inhibitors not incorporated into the chain; and vi) destroying the fluorescent labels by bleaching with a laser or by a suitable chemical reaction, or removing the fluorescent labels by a chemical cleavage step.
17. A process for sequencing a DNA fragment comprising steps of:
i) hybridising a capped primer to a template to form a template/primer complex;
ii) adding a nucleoside triphosphate labelled by attachment of a fluorescent dye group via a linker arm to the 3' moiety of the deoxyribose sugar thereon together with three non-labelled heterogeneous chain elongation inhibitors not incorporated into the chain and a polymerase, wherein said nucleoside triphosphate is not, a chain elongation inhibitor;
iii) removing the excess reagents by washing;
iv) measuring the amount of incorporated label;
v) removing the fluorescent dye group by enzymatic cleavage;
vi) removing excess reagents by washing; and vii) repeating steps ii) to vi), each time with a different labelled nucleoside triphosphate.
i) hybridising a capped primer to a template to form a template/primer complex;
ii) adding a nucleoside triphosphate labelled by attachment of a fluorescent dye group via a linker arm to the 3' moiety of the deoxyribose sugar thereon together with three non-labelled heterogeneous chain elongation inhibitors not incorporated into the chain and a polymerase, wherein said nucleoside triphosphate is not, a chain elongation inhibitor;
iii) removing the excess reagents by washing;
iv) measuring the amount of incorporated label;
v) removing the fluorescent dye group by enzymatic cleavage;
vi) removing excess reagents by washing; and vii) repeating steps ii) to vi), each time with a different labelled nucleoside triphosphate.
18. Use of a DNA sequencing kit for carrying out the method of any one of claims 1 to 12 or the process of any one of claims 13 to 17, the DNA sequencing kit comprising:
i) a linker for attaching a DNA template to a solid-phase matrix, the linker comprising a primer having a phosphorothioate deoxynucleoside residue at its 3' end;
ii) chain elongation inhibitors;
iii) fluorescently-labelled nucleoside triphosphates;
iv) deoxynucleoside phosphorothioate triphosphates;
v) a 5'- > 3' DNA polymerase; and vi) a 3'- > 5' exonuclease.
i) a linker for attaching a DNA template to a solid-phase matrix, the linker comprising a primer having a phosphorothioate deoxynucleoside residue at its 3' end;
ii) chain elongation inhibitors;
iii) fluorescently-labelled nucleoside triphosphates;
iv) deoxynucleoside phosphorothioate triphosphates;
v) a 5'- > 3' DNA polymerase; and vi) a 3'- > 5' exonuclease.
19. An automated sequencing machine adapted to sequence a nucleic acid essentially by executing the steps of a method according to any one of claims 1 to 15, the machine comprising:
i) a solid-phase support to which the template/primer complex is bound;
ii) means for moving the solid-phase support and the bound template/primer complex, to expose the template/primer complex to the necessary reagent and washing solutions, or means for sequentially pumping reagent and washing solutions over the bound template/primer complex; and iii) means for detecting the presence of a label.
i) a solid-phase support to which the template/primer complex is bound;
ii) means for moving the solid-phase support and the bound template/primer complex, to expose the template/primer complex to the necessary reagent and washing solutions, or means for sequentially pumping reagent and washing solutions over the bound template/primer complex; and iii) means for detecting the presence of a label.
Applications Claiming Priority (3)
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|---|---|---|---|
| GB9208733.7 | 1992-04-22 | ||
| GB929208733A GB9208733D0 (en) | 1992-04-22 | 1992-04-22 | Dna sequencing method |
| PCT/GB1993/000848 WO1993021340A1 (en) | 1992-04-22 | 1993-04-22 | Dna sequencing method |
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| Publication Number | Publication Date |
|---|---|
| CA2133956A1 CA2133956A1 (en) | 1993-10-28 |
| CA2133956C true CA2133956C (en) | 2007-02-20 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002133956A Expired - Lifetime CA2133956C (en) | 1992-04-22 | 1993-04-22 | Dna sequencing method |
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|---|---|
| US (1) | US6087095A (en) |
| EP (1) | EP0640146B1 (en) |
| JP (1) | JPH07507681A (en) |
| AT (1) | ATE159766T1 (en) |
| AU (1) | AU4020893A (en) |
| CA (1) | CA2133956C (en) |
| DE (1) | DE69314951T2 (en) |
| ES (1) | ES2110604T3 (en) |
| GB (1) | GB9208733D0 (en) |
| WO (1) | WO1993021340A1 (en) |
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| AU2674092A (en) * | 1991-09-09 | 1993-04-05 | Baylor College Of Medicine | Method and device for rapid dna or rna sequencing determination by a base addition sequencing scheme |
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| GB9208733D0 (en) | 1992-06-10 |
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