The present invention relates to a method for the direct, exponential amplification and sequencing of DNA molecules as well as the use of the method. The direct, exponential amplification and sequencing of DNA molecules is referred to as “DEXAS” in the following.
DNA sequence determination as developed by Sanger et al. ((1977) Proc. Natl. Acad Sci. USA 74, 5463-5467) is usually carried out with a T7 DNA polymerase (Tabor S. and Richardson, C. C. (1989) Proc. Natl. Acad. Sci. USA 86, 4076-4080). This method requires relatively large amounts of a purified, single-stranded DNA template. Recently cycle sequencing has been developed (Murray, V. (1989) Nucleic Acids Res. 17, 8889). This method does not require a single-stranded template and allows the sequence reaction to be initiated with relatively small amounts of template. However, the template DNA has to be purified to almost complete homogeneity and is usually prepared by means of cloning in plasmids (Bolivar, F. et al., (1977) Gene 2, 95-113) and subsequent plasmid purification (Birnboim, H. C. and Doly, J. (1979) Nucleic Acids Res. 7, 1513-1523) or by means of PCR amplification (Mullis, K. B. and Faloona, F. A. (1987) Methods Enzymol. 155, 335-350). Only one primer is used in both of the methods described above.
In one embodiment of the cycle sequencing which is referred to as “coupled amplification and sequencing” or “CAS” Ruano and Kidd ((1991) Proc. Natl. Acad. Sci. USA 88, 2815-2819; U.S. Pat. No. 5,427,911) have shown that one can use a two-step protocol to generate sequences from DNA templates. In the first step 15 PCR cycles are carried out with Taq
DNA polymerase in the absence of dideoxynucleotides in order to prepare an adequate amount of sequencing template. In a second step in which dideoxynucleotides and a labelled primer are added, CAS produces the sequence as well as the additional amplification of the target sequence. Two primers are used in both steps of the method.
Taq DNA polymerase, that is used in coupled DNA sequencing reactions strongly discriminates against ddNTPs and preferably incorporates dNTPs if it is furnished with a mixture of ddNTPs as well as dNTPs. In addition it incorporates each ddNTP, i.e. ddATP, ddCTP, ddGTP, ddTTP, with a strongly varying efficiency. Hence the optimization of the CAS process requires careful titration of the dideoxynucleotides.
Furthermore since coupled amplification and sequencing depends on the amount of the initial DNA, the distance between the two primers and the concentrations and the ratios of the ddNTPs and dNTPs relative to one another and to each other, the optimization of coupled amplification and sequencing reactions (CAS) requires that the reaction conditions are individually optimized for a particular DNA fragment.
All the methods described above require an interruption between the first step of exponential amplification of the template DNA and the second step for the synthesis of truncated DNA molecules and also require the individual optimization of a given DNA fragment which can be tedious and time-consuming and can lead to errors especially when sequencing a large number of different DNA molecules or when processing large amounts of samples in a hospital or laboratory or when sequencing rare samples for forensic or archaeological studies.
For this reason it would be advantageous to have available a method for sequencing nucleic acids which simultaneously potentiates the exponential amplification of molecules of full length and of molecules of truncated length in the reaction which leads to a reduction of the required amount of starting nucleic acid molecules and does not require an interruption of the exponential amplification step and of the sequencing step so that the whole reaction can be carried out more rapidly and with fewer manipulations.
SUMMARY OF THE INVENTION
The object of the present invention is to provide an improved, rapid and reliable method for sequencing DNA molecules, preferably genomic DNA.
A further object of the present invention is to provide a direct method for nucleic acid sequencing which simultaneously increases the exponential amplification of molecules of full length as well as of molecules of truncated length in the reaction which leads to a reduction of the initial amount of nucleic acid molecules that are required for the cycling reaction.
A further object of the present invention is to provide an improved, rapid and reliable method for sequencing DNA molecules, preferably genomic DNA that can be carried out in a single step in a single container.
A further object of the present invention is to provide an application according to the invention for sequence determination in medical diagnostics, forensics and population genetics.
Further objects of the invention are obvious to a person skilled in the art from the description.
In contrast to the above-described “CAS” method a DNA polymerase is used as the thermostable DNA polymerase which, compared to wild-type Taq DNA polymerase, has a reduced discrimination against the four ddNTPs in the buffer and under the conditions that are used for the thermocycling. More preferably a DNA polymerase is used which carries a “Tabor-Richardson” mutation or a functional derivative thereof which also has no 5′-3′exonuclease activity such as e.g. AmplitaqFS™ (Taq DNA polymerase (-exo5′-3′)(F667Y), Tabor and Richardson (1995), loc. cit.), Taquenase™ (Taq DNA polymerase Δ235 (-exo5′-3′) (F667Y), Tabor and Richardson (1995), loc. cit.) and Thermo Sequenase™ (Taq DNA polymerase (-exo5′-3′) (F667Y), Tabor and Richardson (1995), loc. cit.) as well as mixtures thereof or other DNA polymerases and mixtures thereof which are thermostable can also be used in the method of the present invention.
Surprisingly the use of a DNA polymerase which, in comparison to wild-type Taq DNA polymerase, has a reduced discrimination against the four ddNTPs, enables the simultaneous and exponential synthesis of truncated as well as of full fragments from the start of the cycling reaction. Hence the present invention concerns a method for the direct sequencing of a nucleic acid molecule from a complex mixture of nucleic acids, such as e.g. total genomic human DNA, containing a reaction buffer, deoxynucleotides or derivatives thereof and a dideoxynucleotide or another terminating nucleotide and a thermostable polymerase which has a reduced discrimination against ddNTPs in comparison to wild-type Taq DNA polymerase. Within the sense of the present invention direct sequencing means that the nucleic acid fragment to be sequenced is simultaneously amplified and sequenced in one step without interrupting the reaction and without prior amplification of the nucleic acid fragment to be sequenced by the known methods and in such a manner that an unequivocal sequence ladder is readable.
The principle of DEXAS is that the initial and subsequent cycle sequencing reaction is carried out with two primers, a first primer, and a second primer which lies on the strand complementary to the first, which are preferably present in a non-equimolar ratio and serve to simultaneously produce adequate template molecules of full length as well as truncated molecules which contribute to the sequencing of the DNA molecule. Four reactions are prepared, one for the determination of each base, so that each reaction contains two primers preferably in a non-equimolar ratio to one another of which either one is labelled and the other is unlabelled or both are differently labelled. The said non-equimolar ratio between the first primer and the second primer enables the simultaneous and exponential synthesis of the truncated as well as of the full fragments from the start of the cycling reaction. Furthermore each reaction contains from the start the DNA template to be sequenced as well as a buffer solution, thermostable DNA polymerase, thermostable pyrophosphatase (optionally), the four deoxynucleotides or derivatives thereof and a dideoxynucleotide or a terminating nucleotide e.g. 3-aminonucleotide or 3′-ester-derivatized nucleotides.
Thereafter cycles for denaturing and extension are carried out so that in each of these cycles two types of extension products are formed from each primer. Each primer functions such that it initiates extension products which are long enough to reach the other primer position. Simultaneously products are initiated by each primer which, due to the incorporation of a dideoxynucleotide, are terminated before the other primer position is reached. The former said products (products of full length) serve in the following cycles as a template for the production of further DNA strands of full length and are also used as templates for extensions that contribute to the sequence reaction, and the latter products (truncated products) accumulate during the cycles and contribute to the sequence ladder that is generated. Hence DEXAS results in the simultaneous exponential production of a sequencing template and a sequence ladder in a single tube without having to interrupt the thermocycling reaction.
Therefore the use of the present invention enables the DNA sequence of multicopy and single-copy regions of DNA to be determined in a single step.
Hence the present invention for the first time provides a method which enables the nucleic acid to be sequenced to be simultaneously amplified and sequenced from a complex mixture of nucleic acids, such as e.g. total genomic human DNA, without prior amplification by the known methods, in one step i.e. without interrupting the reaction and such that an unequivocal sequence ladder is readable wherein at least one thermostable DNA polymerase, a nucleic acid molecule, a first primer, a second primer, a reaction buffer, deoxynucleotides or derivatives thereof and at least one dideoxynucleotide or another terminating nucleotide is present in the initial reaction mixture.
Furthermore the aforementioned object and goals of the present invention are achieved by the provision of a method for sequencing DNA molecules in which truncated DNA molecules as well as DNA molecules of full length are simultaneously and exponentially synthesized between two positions on the said DNA molecule in a thermocycling reaction which initially contains a DNA molecule, a first primer, a second primer, a reaction buffer, a thermostable DNA polymerase, thermostable pyrophosphatase (optionally), deoxynucleotides or derivatives thereof, and a dideoxynucleotide or another terminating nucleotide thereof wherein the initial ratio of the said primers in the said thermocycling reaction is not equal to 1.
In a preferred embodiment of the method of the invention the ratio of the said primers to one another is about 2:1 to about 3:1, most preferably 2:1.
In a further preferred embodiment of the method of the invention the said primers have such a length that the signal-to-noise ratio between the specific truncated DNA molecules and the unspecific DNA molecules is large enough not to substantially prevent the reading of the sequence. The said primers preferably have a length of at least 25 nucleotides.
Primers can be synthesized by means of methods known in the state of the art. For example primers can be synthesized using known methods which do not significantly change the stability or function of the said primers during the nucleic acid sequencing method of the present invention.
Furthermore the PNA-DNA hybrid oligonucleotides (see Finn, P. J. et al., N. A. R. 24, 3357-3363 (1996), Koch, T. et al., Tetrahedron Letters, 36, 6933-6936 (1995), Stetsenko, D. A, et al., Tetrahedron Letters 37, 3571-3574 (1996), Bergmann, F. et al., Tetrahedron Letters 36, 6823-6826 (1995) and Will, D. W. et al., Tetrahedron 51, 12069-12082 (1995)) are also regarded as primers for the method according to the invention.
In a further preferred embodiment of the invention the said first primer is labelled. Moreover it is preferable that the said first primer and second primer are labelled differently. Any suitable agents or methods known in the state of the art can be used as single or differential labelling agents and methods, provided that they do not significantly change the stability or function of the said primer in the DNA sequencing method of the present invention. For example single and differential labels can be selected from the group which comprises those enzymes such as β-galactosidase, alkaline phosphatase and peroxidase, enzyme substrates, coenzymes, dyes, chromophores, fluorescent, chemiluminescent and bioluminescent labels such as FITC, Cy5, Cy5.5, Cy7, Texas-red and IRD40 (Chen et al., (1993), J. Chromatog. A 652: 355-360 and Kambara et al. (1992), Electrophoresis 13: 542-546) ligands or haptens such as e.g. biotin and radioactive isotopes such as 3H, 35S, 32P, 125I and 14C.
The method according to the invention can also be carried out as a “hot start” method. In this case it is ensured that the activity of the polymerase or polymerases only starts at an increased temperature in order to suppress a polymerization on unspecifically hybridized primers at lower temperatures. One possibility is that the thermocycling reaction additionally contains a polymerase-inhibiting agent. Polymerase antibodies are for example available commercially which only denature at higher temperatures and thus release enzyme activity of the polymerase. However, polymerases modified by genetic engineering that are present in an inactive form at lower temperatures would also be conceivable.
DEXAS is relatively insensitive to various buffers and various deoxynucleotides and dideoxynucleotide concentrations and can be carried out using various thermostable DNA polymerases.
The number of thermocycles can be from about 18 to about 50 cycles depending on the amount of template DNA and its purity.
Buffer components which can be used can include Tris-HCl at a pH of about 9.0 to 9.5 and at a concentration of about 10 to 30 mM, ammonium sulfate at a concentration of about 10 to 20 mM preferably 15 mM, MgCl2 at a concentration of about 3.5 to 5.5 mM optionally about 0.05 mM mercaptoethanol, about 0.28% Tween20® and/or about 0.02% Nonidet 40® but, however, are not limited to these.
Deoxynucleotides may be selected from dGTP, dATP, dTTP and dCTP but are not limited to these. According to the invention it is additionally also possible to use derivatives of deoxynucleotides which are defined as those deoxynucleotides which are able to be incorporated by a thermostable DNA polymerase into growing DNA molecules that are synthesized in the thermocycling reaction. Such derivatives can include thionucleotides, 7-deaza-2′-dGTP, 7-deaza-2′-dATP as well as deoxyinosine triphosphate that can also be used as a substitute deoxynucleotide for dATP, dGTP, dTTP or dCTP, but are not limited to these. The aforementioned deoxynucleotides and derivatives thereof are preferably used at a concentration between about 300 μM and 2 mM.
Dideoxynucleotides can be selected from ddGTP, ddATP, ddTTP and ddCTP but, however, are not limited to these. According to the invention it is also additionally possible to use derivatives of dideoxynucleotides which are defined as those dideoxynucleotides that are able to be incorporated by a thermostable DNA polymerase into growing DNA molecules that are synthesized in a thermo-cycling reaction. In addition it is also possible to use other terminating nucleotides e.g. 3′-amino nucleotide or 3′-ester-derivatized nucleotides. Preferred concentrations of ddNTPs are between about 1 and 5 μM.
In the method according to the invention the preferred ratio of dNTPs to ddNTPs (dNTPs:ddNTPs) is between 100:1 and 1000:1 preferably between 300:1 and 600:1.
In a further preferred embodiment of the method of the invention the said method is carried out at a temperature at which the signal-to-noise ratio between the specific truncated DNA molecules and the unspecific DNA molecules is large enough not to substantially impede reading of the sequence. It is less important to optimize the annealing temperature. In the case of human single-copy DNA sequences the highest possible annealing temperature drastically reduces the background. In this case the annealing and synthesis steps of the thermocycling reaction are preferably carried out at a minimum temperature of 62° C., more preferably at 66° C. and most preferably at at least about 68° C.
The template of the DNA molecule to be sequenced is preferably present as a total genomic DNA molecule which does not have to be cloned or purified, but this may be the case. In one embodiment of the invention the genomic DNA has a length of more than or equal to 2 kb. Other forms of DNA that can be used as templates include cloned or uncloned mitochondrial DNA, partially purified or unpurified DNA such as e.g. plasmid DNA of bacterial colonies. DEXAS functions well with about 250 ng template DNA for the determination of mitochondrial DNA sequences and about 1 μg template DNA for determining single-copy DNA sequences such as e.g. total genomic DNA, but it also functions with smaller amounts of mitochondrial or genomic DNA. The method according to the invention can also be used for the direct sequencing of unpurified single-stranded or double-stranded DNA from bacteriophages. DEXAS is in addition relatively independent of the base composition of the template.
In a preferred embodiment the method according to the invention is furthermore characterized in that each thermocycling reaction to determine the position of A, G, C and T in the said DNA molecule is carried out in a single step, in a single container, vessel or tube.
Suitable sources of nucleic acid molecules in the method according to the invention are body fluids such as sperm, urine, blood or fractions of these, hairs, an individual cell, cells or fractions thereof, hard tissue such as bones or soft tissue or fractions thereof and cell cultures or fractions thereof.
The present invention also serves for the application of the method according to the invention for the determination of a nucleotide sequence of a given nucleic acid molecule e.g. for sequencing Shotgun libraries with two labels for large-scale genome projects and in medical diagnostics, forensics and population genetics. The method of the present invention can be used to detect genetic mutations or polymorphisms, to identify the origin of the sequenced nucleic acid or to detect the presence of foreign or infectious agents in a sample.
The present invention relates to all combinations of all procedures of the above methods.
After preparation the sequencing reactions can be loaded directly onto a sequencing gel such as e.g. after addition of a commonly used application buffer (e.g. formamide which contains 20 mM EDTA (pH 7.4) and 6 mg/ml dextran blue) and denaturation (e.g. for 4 minutes at 96° C.). The sequence ladder can be read according to known methods. The method of the invention is well suited for automation. Since the two primers in the reaction are provided with different labels which can for example be detected with two different wavelengths, the method of the present invention enables the simultaneous sequencing of both strands of a template and the detection of both reactions in one or several gel lanes. In general many DEXAS reactions that are carried out using different dyes can be carried out simultaneously in the same tube and applied to a sequencing instrument that is equipped with several lasers or be detected by other methods such as e.g. autoradiography.
A further subject matter of the present invention is a kit for the direct sequencing of a nucleic acid molecule from a complex mixture of nucleic acids, such as e.g. total genomic human DNA, containing a reaction buffer, deoxynucleotides or derivatives thereof and a dideoxynucleotide or a further terminating nucleotide and a thermostable polymerase which has a reduced discrimination against ddNTPs compared to wild-type Taq DNA polymerase. Within the sense of the present invention direct sequencing means that the nucleic acid fragment to be sequenced is simultaneously amplified and sequenced, without prior amplification of the nucleic acid fragment to be sequenced by the known methods, in a single step without interrupting the reaction and such that an unequivocal sequence ladder can be read.
A further subject matter of the present invention is a kit for the direct sequencing of a nucleic acid molecule of a complex mixture of nucleic acids containing a reaction buffer, deoxynucleotides or derivatives thereof and a dideoxynucleotide or another terminating nucleotide, a thermostable polymerase and two primers whose ratio is larger than 1. The kit particularly preferably contains a thermostable polymerase which has a reduced discrimination against ddNTPs in comparison to the wild-type Taq DNA polymerase.