CA1340851C - Dna sequencing - Google Patents

Abstract

A method for sequencing a strand of DNA, including the steps of: providing the strand of DNA;
annealing the strand with a primer able to hybridize to the strand to give an annealed mixture; incubating the mixture with a deoxyribonucleoside triphosphate, a DNA
polymerase, and a chain terminating agent under conditions in which the polymerase causes the primer to be elongated to form a series of DNA products differing in length of the elongated primer, each DNA product having a chain terminating agent at its elongated end;
the number of each DNA product being approximately the same for substantially all DNA products differing in length from 1 to 20 bases.

Description

1~408~~
DNA SEQUENCING
Back round of the Invention This invention relates to DNA sequencing and in particular to automated methods for DNA sequencing.
DNA sequencing is generally carried out by the method of Sanger et al. (Proc. Nat. Acad. Sci. USA
74:5463, 1977) and involves enzymatic synthesis of single strands of DNA. from a single stranded DNA
template and a primer. Referring to Fig. 1, four separate syntheses are carried out. A single stranded l0 template is provided along with a primer which hybridizes to the template. The primer is elongated using a DNA polymerase, and each reaction terminated at a specific base (guan.ine, G, adenine, A, thymine, T, or cytosine, C) via the incorporation of an appropriate chain terminating agent, for example, a dideoxynucleotide. Enzymes currently used for this method of sequencing include: the large fragment of Escherichia coli DNA polymerase I ("Klenow" fragment), reverse transcriptase, Taq polymerase, and a modified 2o form of bacteriophage~ T7 DNA polymerase.
Still referring to Fig. 1, the four DNA
synthesis reactions result in formation of four series of DNA products, each product having one defined terminus and one variable terminus. The defined terminus starts with the primer molecule. The variable terminus ends with a chain terminating agent specific 1~~~~'~v for the nucleotide base (either G, A, T, or C) at which the synthesis reaction terminated. The four different series of products are each separated on the basis of their molecular weight, in four separate lanes in a high resolution polyacrylamide gel, to form four series of bands, with each band on the gel corresponding sequentially to a specific nucleotide in the DNA
sequence. Thus, the relative positions of the bands identify the positions in the DNA sequence of each given nucleotide base. Generally, the DNA products are l0 labelled so that the bands produced are readily detected. As shown in Fig. 1, the intensity of the bands is generally non-uniform, within a single lane, because band intensity is directly related to the total number or concentration of DNA products of the same molecular weight in a specific lane, and this number varies from one product to another even when they are of approximately t:he same molecular weight and even when they contain the same chain terminating agent.
Using the above methodology, automated systems for DNA sequence analysis have been developed. One instrument, manvufactured by EG&G, uses a 32P-label and a DNA polymeras~e, and the resulting DNA products separated by gel electrophoresis. Toneguzzo et al., 6 Biotechniques 460, 1988. A 32P-detector at the bottom of the gel scans for radioactivity as it passes through the bottom of the gel. Four synthesis reactions are required for ea~~h template to be sequenced, as well as four lanes on each gel, a separate lane being used for products terminated by each specific chain terminating agent, as shown for example in Fig. 1.
Kanbara et al., 6 Biotechnology 816, 1988, have replaced the 32~P-labelled primer, described above, with a fluorescent-labelled primer. The resulting 13~~~~1 fluorescently labelled products are excited with a laser at the bottom of the gel and the fluorescence detected with a CRT monitor. This procedure also requires four synthesis reactions and four lanes on the gel for each template to be sequenced.
Applied Biosystems manufactures an instrument in which four different primers are used, each labelled with a different fluorescent marker. Smith et al., 13 Nuc. Acid. Res. 2399, 1985; and 321 Nature 674, 1986.
Each primer is used in a separate reaction containing one of four did~eoxynucleotides. After the four reactions have :been carried out they are combined together and ruin in a single lane on a gel. A laser at the bottom of t:he gel is used to detect fluorescent products after they have been permeated or electrophoresed through the gel. This system requires four separate annealing reactions and four separate synthesis reactions for each template, but only a single lane on the gel. Computer analysis of the sequence is made easier by :having all four bands in a single lane.
DuPont provides an instrument in which a different fluorescent marker is attached to each of four dideoxynucleoside triphosphates. Prober et al., 238 Science 336, 1987. A single annealing step, a single polymerase reaction (containing each of the four labelled dideoxynucleosides triphosphates) and a single lane in the sequencing gel are required. The four different fluorescent markers in the DNA products are detected separately as they are electrophoresed through the gel.
Englert et al., U.S. Patent 4,707,237 (1987), describes a multichannel electrophoresis apparatus having a detection means, disposed substantially across the whole width. of the gel, which can sense labelled DNA
products as they migrate past the detector means in four separate lanes, and identifies the channel or lane in which the sample is l.acated. Preferably, radioisotopic labels are used..
Inherent to procedures currently used for DNA
sequence analysis is the necessity to separate either radioactively or fluorescently-labelled DNA products by a gel permeation procedure such as polyacrylamide or other gel electrophoresis, and then detect their locations relative to one another along the axis of permeation or movemerut through the gel. The accuracy of this procedure is determined in part by the uniformity of the signal in bands which have permeated approximately the same distance through the gel.
Differences or variations in signal intensities between nearby bands create :>everal problems. First, they decrease the sensitivity of the method, which is limited by the ability to detect the bands containing the weakest signal:. Second, they create difficulties in determining whether a band with a weak signal is a true signal due to t;he incorporation of a chain terminating agent, or an artifact: due to a pause site in the DNA, where the polyaterase has dissociated. Third, they decrease the ac:curac~~ in determining the DNA sequence between closel~~ spacs~d bands since the strong signal of one band may mask thE~ weak signal of its neighbor.
Summary of the Invention All of the l:oregoing problems are overcome in the present invention, where approximately the same amounts of DNA producers of similar molecular weights are produced in a :>equencing reaction, and thus nearby bands in the sequencing ge:L, in the same lane, are of approximately t:he sarne intensity.

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The ability to produce nearby bands of approximately t'.he same intensity is useful since it permits the results of any sequencing reaction to be read more easily and with greater certainty. Further, since the DNA products from a sequencing reaction with a specific chain terminating agent form bands which are of approximately t:he same intensity as that of nearby bands, band intensity itself provides a specific label for the series of bands so formed. The number of DNA
products of approximately the same molecular weight produced by a given chain terminating agent varies depending upon the concentration of the chain terminating agent. Thus, by using a different concentration of each of the four chain terminating agents for the synthesis, the DNA products incorporating one chain terminating agent are distinguished from DNA
products of approximately the same molecular weight incorporating other chain terminating agents in that they differ in number or amount; consequently, the bands of DNA products can b~e identified as to chain terminating agent simply by their intensity as compared to the intensities of nearby bands. As a result, two or more series of DNA products, each series having a different chain terminating agent, can be subjected to gel permeation in a single lane and identified, i.e., distinguished from each other, by the intensity of each band as compared to t:he intensity of nearby bands.
Moreover, the syntheses of DNA products incorporating different chain terminating agents need not be carried out separately, in separate containers, but may all be carried out simultaneously in a single reaction vessel, and the same label, e.g., radioisotopic, fluorescent, etc. can, if desired, be used for all chain terminating agents instead of a different label for each, thus simplifying the procedure.
It should bs~ noted, however, that there is a gradual decrease in intensity of all bands of DNA
products as they permeate through the gel, those that have travelled the shortest distance displaying less intensity than those which have travelled the farthest distance. Neve~rless, the relative intensity of each band as compared to nearby bands at any location along the axis of permeation remains approximately the same throughout. This conservation of relative intensity throughout the extent: of permeation makes possible the present invention.
By "nearby bands" is meant those in the same lane within about 20--30mm either ahead of or behind the band in question, measured along the axis of permeation. In genei:al, the nearby bands include DNA
products differing from the one in question by no more than 20 bases (i.e., with a mass differing by no more than about 6, OtlO dali~ons ) .
In general, the invention features a DNA
polymerase for use in DNA sequencing reactions, which, in a sequencing reaction, causes DNA products of slightly different molecular weight to be produced in approximately Equal numbers. Thus, when such DNA
products are separated in a gel matrix they form bands, with nearby bands being of approximately the same intensity. Without being bound to any particular theory, the in~rentor~s regard this uniformity in intensity as bE~ing due to the polymerase not discriminating between normal nucleoside triphosphates and chain term:inatin~~ agents, such as dideoxynucleoside triphosphates.

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In a first aspect, the invention features a method for sequencing a strand of DNA, including the steps of: providing the strand of DNA; annealing the strand with a primer able to hybridize to the strand to give an annealed mixture; incubating the annealed mixture with a deoxyribonucleoside triphosphate, a DNA
polymerase, and a first chain terminating agent under conditions in which the polymerase causes the primer to be elongated to form a first series of first DNA
products differing in. length of the elongated primer, each first DNA product having a chain terminating agent at its elongated end; the number of each first DNA
product being approximately the same for substantially all DNA products differing in length from 1 to 20 bases. Preferably, the method further includes the steps of: separating the first DNA products by gel permeation according to molecular weight to form a first series of bands, eacr~ first series band representing a first DNA product of a given molecular weight, wherein the intensity of each nearby first series band is approximately the same for substantially all first series bands; a.nd det:ermining the position of each first band.
By "su.bstant:ially all" is meant that at least 9 out of 10 (or 19 out of 20) nearby bands have approximately the same intensity. That is, only occasional bands will. have a different intensity. This different intensity z:esults from artifacts. One example of such an artifact is the compression of two or more DNA products of different molecular weight within one band. The result of two such compressions are shown in Fig. 2 where the art:ifactual bands are marked with an asterisk. By approximately the same is meant that band 1'~~0~~~.
_8_ intensity varies by at most 2 fold, most preferably at most 1.2 fold. By gel permeation is meant to include existing polyacrylamide gels used for DNA sequencing, and any other mechanism for separating DNA products according to their molecular weight.
5~-~ In one embodiment, production of nearby bands of approximately the same intensity is achieved by incubating a DNA polymerase in a solution containing manganese or iron ions.
In one preferred embodiment, the method further includes the steps of providing a second chain terminating agent in the annealed mixture at a concentration different from the first chain terminating agent, wherein the DNA polymerase causes production of a second series of second DNA products, each second DNA
product having the sE~cond chain terminating agent at its elongated end, the number of each second DNA product being approximately t;he same for substantially all DNA
products differing in length from 1 to 20 bases, wherein the number of substantially all the first and all the second DNA products differing in length from 1 to 20 bases is distirnctly different. Most preferably, the second series of second DNA products form a second series of band:. when separated by gel permeation according to molecul<ir weight, wherein the intensity of substantially ~~11 nearby second series bands is approximately t:he sarne, and the intensity of substantially all bands of the first series is distinctly and distinguishably different from the intensity of each nearby band of the second series, and the method further includes the step of determining the position and intensity of each band, the intensity being representative of a ,particular band series.

i~~gi By distinctly different is meant that a band of one series can be distinguished from a nearby band (i.e., a band with a length differing from 1 to 20 bases) in the other series. That is, a machine which measures the number of DNA products of a specific molecular weight can distinguish the two series of DNA
products from each other.
In another ~~referred embodiment, the method includes providing two other chain terminating agents wherein the polymerase causes production of a second and third series of second and third DNA products, the number of each second and third DNA products being approximately the same for substantially all DNA
products differing in length from 1 to 20 bases, wherein the number of substantially all the first, all the second and all the third DNA products differing in length from 1 to 20 bases is distinctly different. Most preferably, each second and third series of the second and third DNA products form a different series of second and third band;, when separated by gel permeation according to molecul<~r weight, wherein the intensity of substantially all nearby second series bands is approximately t:he same, the intensity of substantially all nearby third series bands is approximately the same, and wherein thEa intensity of substantially all nearby bands of different sE~ries is distinctly different; and the method furt;her includes the steps of determining the position and intensity of each band, the intensity being representative of a particular band series.
In yet: another preferred embodiment, the method includes providing in the annealed mixture four different deox5~ribon~icleoside triphosphates and four different chain terminating agents, wherein the DNA

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- i0 -polymerase cau:>es production of second, third and fourth series of second, third and fourth DNA products, the number of each second, third and fourth DNA products being approximately i~he same for substantially all DNA
products differing in length from 1 to 20 bases, wherein the number of substantially all the first, all the second, all thE~ third and all the fourth DNA products differing in lE~ngth :E'rom 1 to 20 bases is distinctly different. Mo:~t preferably, each second, third and fourth series produce series of second, third and fourth l0 bands, when separated by gel permation according to molecular weight, wherein the intensity of substantially all nearby second series bands, or substantially all nearby third sE~ries hands, or substantially all nearby fourth series hands is approximately the same, and wherein the ini:ensity of substantially all nearby bands in a different series is distinctly different; most preferably, thE~ method further includes the steps of determining thE~ position and intensity of each band, the intensity being rep resentative of a particular band 2o ser ies .
In other preferred embodiments, the annealed mixture is provided with a manganese or iron ion, wherein the ion causes the polymerase to be non-discriminatory for a chain terminating agent; the DNA products a:re separated according to molecular weight in less than four lanes of a gel; the intensity of each band is measured by a gel reading apparatus; the DNA
polymerase is ~~hosen from a T7-type DNA polymerase, the large fragment of E. coli DNA polymerase I, and Taq polymerase; and the chain terminating agent is a dideoxynucleoside triphosphate.

..

In related aspects, the invention features a method for sequencing a :strand of DNA, including the steps of either (a) providing a DI;fA polymerise, and incubating the polymerise and the strand of DNA in a solution including an ion of manganese or iron and a chain terminating agent; or (b) providing a DNA polymerise which is substantially non-discriminating for a chain terminating agent.
The invention also features a method for sequencing a strand of DNA, comprising the steps of:
providing a L)NA po:lymerase, and incubating said po:lymerase and said strand of DNA in a solution comprising a m<~nganese ion and a chain terminating agent wherein said ion :is provided at a concentration effective for causing production of approximately equal numbers of molecules of these DNA products which differ in size from each other by no more than 2r) bases.
In another related aspect, the invention features a method for producing a 1)NA polymerise for DNA sequencing, including the step of mixing the DNA polymerise in a solution including a manganese o:r iron ion.
In another aspect, the invention features a solution including a T7-type DNA polymerise, or a Taq polymerise, and a manganese or iron ion. Preferably the ion is at a concentration from 0.005 to 100 millimolar.
In another aspect, the invention features a kit for sequencing DNA having a DNA polymerise, a chain terminating agent, and a compound comprising a manganese or iron ion.
In preferred embodiments, the polymerise is a T7-type 13~~~~i DNA polymerase, the large fragment of E- coli DNA polymerase I, or Taq polymerase; the c:hairs terminating agent is a dideoxynucleotide; and t:he kit further includes a deoxyribonucleoside triphosphate.
The invention provides a method for sequencing DNA
comprising:
providing a DNA po7Lymerase in a reaction medium in which said DNA polymeras:e is substantially non-discriminating for a chain terminating agent,, and incubating se~.id DN~~ in said reaction medium with said DNA
polymerase, a primer, four deoxyribonucleoside triphosphates and a chain terminating agent to elongate said primer to form a series of DNA products differing in the length of the elongated primer and having said chain terminating agent at the elongated end, the number of mole<~ules of each said DNA product being approximately the same j=or substantially all. those DNA products differing in size by no more than 20 bases.
The invention also provides a method for sequencing a strand of DNA, comprising the steps of:
providing said strand hybridized with a primer able to hybridize to said strand, to give a hybridized mixture, and incubating said hybridized mixture with four deoxyribonucleosic~e trihhosphates, a DNA polymerase, and a plurality of chain terminating agents in a reaction medium in which said polymerase r.~~uses said primer to be elongated to form a series of DNA products differing in the length of the elongated primer, each I~NA product having a first said chain terminating agent at its elongated end, wherein the number of 13~0~~
12a molecules of said DNA products is approximately the same for substantially all DNA products differing in length by no more than 20 bases.
The invention also provides a method for sequencing a strand of DNA, co~ripris:ing the steps of:
providing a DNA po:lymerase, and incubating said po7Lymerase and said strand of DNA in a solution comprising an iron ion and a chain terminating agent wherein said ion i.s provided at a concentration effective for causing production of approximately equal numbers of molecules of DNA products differing in size by no more than 20 bases.
The invention further provides a method for sequencing a strand of DNA, comprising the steps of:
providing said str<~nd hybridized with a primer able to hybridize to said strand to give an hybridized mixture, incubating said hybridized mixture with four deoxyribonucleos:ide triphosphates, a. DNA polymerase, and a plurality of different ~~hain terminating agents each capable of terminating the DD1A synthesis at a different nucleotide base, to cause said primer to be elongated to form a series of DNA
products differing in length with each DNA product having a chain terminating agent at its elongated end, the amount of each said chain terminating ;gent being distinctly different from that of each other chain terminating agent, and said incubation causing the total amount of said series terminating with one chain terminating agent to be distinctly different from the total amount of s<~id series terminating with each different chain terminating agent, and 12b ~.34~~~~!
separating said series according to length, whereby the DNA products of approximately the same length containing one chain terminating agent are distinctly different in amount from those containing any other chain terminating agent.
In another aspect, the invention features a method for automated sequencing of DNA, including providing a polymerase which :is substantially non-discriminating for a chain terminating agent and causes production of a series of DNA products difff:ring in molecular weight and terminating with the same chain terminating agent, wherein the DNA
products produce :~ubsta:ntially all nearby bands of approximately the same intensity.
By subsi~antially non-discriminating is meant that chain terminating agents are incorporated uniformly along the length of the DNA, regardless of the DNA sequence. By approximately the same is meant that the intensity differs by at most two- to three-fold.
In anoi~her aspect, the invention features an automated DNA sequencing apparatus having a reactor for providing at leasi~ two series of DNA products formed from a single primer and a DNA strand, each DNA product of a series differing in mole~~ular 'weight and having a chain terminating agent at one end; delivering means for providing said DNA
products to; separating means for separating the DNA products to form a series of bands, the intensity of substantially all nearby bands in a series being approximately the same, and the intensity of subsl~antially all nearby bands in a different ~.3~~~i 12c series being diff~:rent, band reading means positionable near said separating m~aans for determining the position and intensity of each band after separating said band reading means being operahly linked to; computing means for determining the D1JA sequence of the DNA strand directly from the position and :intensity of the bands.
In preferred embodiments, the reactor includes a manganese or iron ion, and a T7-type DNA polymerase.
In another aspect, the invention features a solution or kit including ~~ pyro;phosphatase, a DNA polymerase, and a chain terminating agent or dITP; and a method for DNA
sequencing, including providing pyrophosphatase in the sequencing reaction. Inclusion of pyrophosphatase in a sequencing reaction reduces the level of pyrophosphate and improves the uniformity of band intensity of nearby bands.
The invention further provides an automated DNA
sequencing apparal~us comprising:
a reactor comprising reagents which provide a series of DNA products formed from a primer and a DNA strand, wherein said reactor comprises a manganese ion, delivery means for providing said DNA products to a separator :Eor separating said DNA products along one axis of the separ~~tor to form a series of bands, a band reader positionable near said separator for determining the position and intensity of each said band along said axis after said separating, said band reader being operably linked t~~

12d a computer that determines the DNA sequence of said DNA
strand solely from said position and said intensity of said bands along said axis a:nd not from the wavelength of emission of light from any label that may be present in the separating means.
The invention additionally provides an automated DNA
sequencing apparai~us, comprising:
a separator :Eor separating DNA products along one axis of the separator, said products being formed from a primer and a DNA strand in the presence of a manganese ion, to form a series of bands, a band reader positionable near said separator for determining the position and intensity of each said band along said axis after s~~id separating, said band reader being operably linked to;
a computer that determines the DNA sequence of said DNA
strand solely from both said position and intensity of said bands along said .axis and not from the wavelength of emission of light from any label that may be present in the separating means. Preferably the separator consists essentially of a single lane containing four separate series of DNA products with each of the :DNA products labelled with the same label.
The invention also provides an automated DNA
sequencing apparatus comprising a computer that determines the DNA sequence of a DNA strand solely from the position and intensity of bands formed from DNA products separated along one axis of a separator and not from the wavelength of 13~O~~i 12e emission of light from any label that may be present in the separator, said D1JA products being produced by a DNA
sequencing method from said DNA strand.
The invention further provides an automated DNA
sequencing apparai:us comprising a computer means that compute a DNA sequence so:Lely f=rom intensity and position data obtained form at :Least 'two series of DNA products labelled with an identical label and produced by a sequencing reaction performed in the presence of manganese ions after separating l0 said DNA products in the same lane of a separator to form a series of bands acid not from the wavelength of emission of light from the label that is present in the separator.
In any of the above aspects, the manganese or iron ion may be provide=d in vthe presence of a chelate, such as citrate or isocitrate. Such chelates are I

13~~0~~~

thought to provide a more controlled level of the desired ion in a DNA sequencing reaction.
In a final aspect, the invention features a T7 DNA polymerase D Lys 118-Arg 145, and DNA encoding this polymerase. This polymerase has no detectable exonuclease activity.
We have found conditions under which DNA
polymerases can be modified to change their ability to incorporate a chain terminating agent at the elongating terminus of a primer DNA in the presence of a DNA
l0 template. This ability allows DNA sequencing to be performed with lower concentrations of chain terminating agents, thus greatly lowering the costs of a DNA
sequencing reaction. Further, we have found that DNA
polymerases having this ability produce nearby bands in a sequencing gel which are of approximately uniform intensity. That is, the polymerase is no longer discriminating, to ar.~y great extent, between incorporating chain germinating agents and normal deoxynucleoside triphosphates. We have shown that at least three polymerases can be modified in this way, including a modified T7 DNA polymerase, the large fragment of E, coli DNA polymerase I, and Taq polymerase. Other polymerases having homology to these polymerases will also work in the invention.
Another advantage of this invention is that the concentration of any given chain terminating agent to be used in a sequencing reaction is readily calculated, since band intE~nsity is directly related to the concentration of any chain terminating agent and is the same for each such agent.
The modified polymerases of this invention are particularly u:;eful in DNA sequencing reactions since only a single :;equenc:ing reaction containing all four ~.3~0~~~

chain terminating agents at four different concentrations is necessary. Thus, less than four different sequencing reactions can be used for any particular DNA template.
Other features and advantages of the invention will be apparer.~t from the following description of the preferred embodiments thereof, and from the claims.
Description of the Preferred Embodiments The drawings will first briefly be described.
Drawings l0 Fig. 7. is a schematic representation of DNA
sequencing by t:he met=hod of Sanger et al., supra.
Figs. 2-7 are graphical representations of relative band i.ntens:ities of six different sequencing gels scanned bh an Applied Biosystems Model 370A DNA
Sequencing System, each from a single gel lane containing a sEaquenc:ing reaction mixture resulting from using a genetically rnodified T7 DNA polymerase in the presence of various mixtures of manganese or~magnesium and various di~leoxynucleosides.
In each of these Figures, the DNA sequenced was mGPl-2 (encoding T7 IzNA polymerase, Tabor et al., Proc.
Nat. Acad. Sci~~ USA, 84:4767, 1987), and the primer was the fam primer of Applied Biosystems. In each case the unprocessed (r<iw) output for the fam primer is shown.
The start and End of each output are indicated. In addition, the ~~ositi~~ns of the sequences are shown, with respect to their corresponding position in wild type T7 DNA. (Dunn et al., ,J. Mol. Biol. 8:452, 1983) The points on each graph marked by an asterisk represent regions of com~~ressi~on, where at least two DNA products of different molecular weight migrate at the same position on thc~ gel. Compressions are generally described by Tabor et al., Proc. Nat. Acad. Sci. USA, 84:4767, 1987.

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Fig. 8 is a graph showing the optimum concentration of magnesium and manganese for DNA
polymerase activity for a genetically modified T7 DNA
polymerase in vthe presence and absence of 4.0 mM
isocitrate.
Fig. ~~ is a graph showing the effect of different concentrations of isocitrate in the presence of 10 mM magne~;ium or manganese on DNA polymerase activity for a genetically modified T7 DNA polymerase.
Fig. 10 is a schematic map of pGPS-8, a plasmid l0 that encodes f~~r a genetically modified T7 DNA
polymerase lacking amino acids Lys 118 through Arg 145, that lacks exo:nuclease activity.
Fig. 11 is a diagrammatic representation of an automatic sequencing apparatus of this invention.
DNA Polymerase DNA polymerases useful in this invention include those belonging to a class of homologous polymerases including T7-type DNA polymerases (such as T7, T3, ~I, III, H, W31, gh-1, Y, A1122, or SP6), the large fragment of E. coli DNA polymerase I and Taq polymerase. By homologous polymerases is meant an enzyme that discriminates against dideoxynucleoside triphosphates compared to deoxynucleoside triphosphates in the presence of magnesium; however, when magnesium is replaced by manganese the discrimination against dideoxynucleoside triphosphates is reduced. These polymerases are used. in a DNA sequencing reaction under conditions in which they produce nearby bands of approximately uniform intensity (with about a 1.5- to 2.0-fold variation in intensity) when the DNA products of the sequencing reaction are run in a gel. By nearby is meant to include bands representing DNA products of molecular weight differing by as much as 6000, i.e., 20 131~~~~.

bases. The actual value of this intensity will decrease along the length of the gel, as described below and shown in the Figures. Band intensity reflects the number of DNA products within a certain band. Labels, such as fluorophores or radioisotopes, are used to produce a readily detectable band of intensity reflective of the number of such DNA products. Thus, in this invention, nearby bands derived from one sequencing reaction with one chain terminating agent have approximately the sane number of DNA products and thus a uniform band intensity. The sequencing conditions include incubation of the polymerase in the presence of specific divalent or trivalent cations such as manganese (II and III), ferrous and ferric ions; monovalent and divalent cation.s which have no detectable effect, or are detrimental to DNA synthesis, include: K, Na, Ba, Be, Ca, Ce, Cr, Co, Cu, rfi, Si and Zn. The anion is unimportant, for example, chloride, acetate, and sulfate are suitable. Under these conditions the requirement for chain terminating agents, such as dideoxynucleosides, i.s lessened by almost 1000-fold for enzymes such as, large fragment of E. coli DNA polymerase I and Taq polymerase, and by about 10-fold for a modified T7 pol.ymerase. A chelator may also be provided in this solution in order to help regulate the concentration of avaulable divalent metal ions. For example, citrate or :~socitrate may be provided. These chelates are thought to maintain the level of, for example, free manganese ions at a concentration of between 10 and 100 uM over a wide range of manganese concentrations. Than is, the chelator acts as a buffer.
The DDIA pol~nnerases of this invention do not discriminate significantly between dideoxynucleoside analogs and deoxynuc:ieosides along the length of the DNA
template. That: is, :in the presence of manganese or iron these polymera:;es arE~ unable to discriminate between a nucleotide that; has a 3' hydroxyl group versus one that does not (i.e., has t;wo hydrogens at the 3' position of the ribose). However, these polymerases do discriminate against modifications at other positions on the nucleosides, even in the presence of manganese or iron.
For example, tree pol5nnerases do discriminate against some dideoxynuc;leoside analogs which have fluorescent groups attachect compared to deoxynucleosides. However, the polymerase do not: discriminate to a different extent at neighboring, or nE~arby nucleotides, on the basis of the presence on absence of the modification to the dideoxynucleoside. ".Chus, while they discriminate strongly against these analogs, requiring higher concentrations for a DNA sequencing reaction compared to unmodified didE~oxynuc:leosides, the intensity of nearby bands will still be uniform. For example, there is a 10 fold discrimination against dideoxy ITP (ddITP), compared to dideoxy GTP (ddGTP), in the presence of Mn.
However, all ttie bands produced in a sequencing reaction are of equal intensify with ddITP since there is no differential d»scrim:ination along the length of the DNA
template.
Thus, the polymerases of this invention provide a uniform efficiency of incorporation of chain terminating agE~nts, even if they discriminate against overall incorporation.
Chain terminating agents useful in this invention include dideoxynucleosides having a 2', 3' dideoxy structure. ether agents useful in the invention are those able to specifically terminate a DNA
sequencing reacaion at a specific base, and are not discriminated against by the polymerase under the above conditions.

In order to determine whether any particular DNA polymerase, in combination with any particular chain terminating agE~nt, or other component of a sequencing reaction mixture, is useful in this invention, a standard sequencing reaction is performed, as described below and shown in the drawings, and the extent of band formation, and the uniformity of nearby bands in a sequencing gel, reviewed. If the polymerase reaction does not extend the primer by at least 20 bases, it is not suitable under tree conditions used. Adjacent band l0 uniformity within a t:wo-fold or less range is useful in this invention, preferably the uniformity is within a 1.0-1.5 fold range. Similarly, determination of optimum cation concentration,, or of other potential cations useful in the invention, is determined by use of this sequencing reacaion under various conditions. For example, cations are tested in ranges from 0.005-100 mM. An examplE~ of such an experiment follows:
DNA s5~nthes:is is measured using a 17-mer primer of sequence 5'--GTAAAACGACGGCCAGT-3' (New England Biolabs catalog number 1211) that has been labeled with 32P at its 5' end and annealed to single-stranded mGPl-2 DNA.
Tabor et al., 3?roc. Nat. Acad. Sci. USA 84:4767 (1987) and Tabor et a:L., J. Biol. Chem. 262:16212 (1987). Any other template is equally useful in this reaction. This primer-templatES is used in a reaction that contains a DNA polymerase in the presence of a range of concentrations of a metal ion. Reactions are carried out in the pre;aence of a given concentration of all 4 deoxynucleotides (dN'TPS, 20-200 uM), and over a range of concentrations of one dideoxynucleotide (ddNTP, in this example, ddGTP from 10-500 uM). The DNA products are then analy:aed by polyacrylamide gel electrophoresis, 1~~~~~~1 where DNA synthesis is detected as extensions of the primer producing bands, representing extensions of various molecular weights, in the gel.
In a specific example, each reaction mixture (10u1) contained 0.1 ~.g 32P-primer-template, 40 mM
Tris-HC1 pH 7.5, 5 mM dithiothreitol (DTT) 5uM to 20mM
metal ion, 10 to 500 uM 4dNTPs, 1 to 500 uM ddNTPs, and 2 units of a DNA polymerase. Incubation was at 37°C
for 15 min. Th,e reaction was stopped by addition of 10u1 of 90% formamide, 50 mM EDTA, and 0.1%
l0 bromophenol blue.
The resulting samples were heated at 75°C for two minutes imcr~ediats~ly prior to loading onto a polyacrylamide gel (8% acrylamide, 0.3% bisacrylamide) in 7M urea, 100 mM Tris-borate, pH 8.9. Electrophoresis was at 2000 volts for 2 hours. The gel was fixed in 50%
methanol, 10% acetic acid for 30 min., dried, and exposed, for au.toradi.ography. Band intensity in each lane of the resulting film was determined by~scanning each lane with a densitometer. The densitometer used was a double-beam recording instrument, model MkIIIC
(Joyce, Loebl s: Co., Ltd., Gateshead-on-tyre, II, England). Any suitable densitometer instrument for scanning gels will also work. Alternatively, the uniformity of t:he resulting bands can be determined by scanning the DrfA products as they are electrophoresed within the gel.
The agility to incorporate a given ddNTP
compared to thE~ corresponding dNTP for any one enzyme is measured as the ratio of ddNTP to dNTP necessary to allow DNA synthesis that terminates in a ffixed range, detected as producing bands of no greater than a fixed molecular weight. That is, the bands produced in the reaction end within <i specified range in the sequencing gel. Thus, if one enzyme discriminates 1000-fold greater against a given ddNTP compared to another enzyme, a 1000-fold higher ratio of ddNTP to dNTP will be necessary to obtain bands terminating at the corresponding sites i.n the same range of the gel.
Manganese (Mn) Following is a series of examples of the use of a modified T7 hNA polymerise or the large fragment of E.
coli DNA polyme~rase I: in DNA sequencing reactions with Mn present in the sequencing buffer. These examples are not limiting to this invention and are given simply to provide those skilled in the art with guidelines for use of DNA polymera.ses of this invention. As described above, those skilled in the art can readily determine other conditior,.s under which DNA polymerises of this invention can be produced that will give the properties described here with respect to uniformity of chain terminating ags~nt incorporation and use in a sequencing reaction.
The s~>ecific: modified T7 DNA polymerise used in the following examples was genetically modified to have no detectable e~xonuc7.ease activity. This genetically modified DNA polymerise is termed ~Lys 118-Arg 145 (~28) since thE~ amino acid region from Lys 118 through Arg 145 in T7 DNA po~'~ymerase is deleted. The gene encoding this F>olymei:ase was constructed in a plasmid I pGPS-8 as a variant of the plasmid pGPS-5 that is described in Tabor et: al., U.S. Patent No. 4,795,699.
Referring to Fig. 10, pGPS-8 includes pACYC177 resected at BamHI and HincII sites, T7 DNA from bases 5667 to 6166 containing ~l.lA and ~1.1H, and T7 DNA
bases 14,306 to 16,869 containing gene 5 with ~~~~8~

modifications shown in Fig. 10. pGPS-8 was constructed by first synthesizing the 34 mer, 5'CCGGCAAGTTGCC;CGGGA2'GCTCGAGGAGCAGGG 3'. This oligonucleotide~ was used as a primer for DNA synthesis on the single-stranded DNA of M13 mGPS-2, that contains an insert that encodes T7 gene 5, and is described in Tabor et al., Id. DNA synthesis and mutant selection was performed a.s described in Tabor et al. Id. After construction of the desired mutation in mGPS-2, the appropriate region of T7 gene 5 that contains the 84 by deletion was inserted into pGPS-5 by isolating an EcoRI
to H~aI fragment containing T7 DNA from positions 14,306 to 15,610, including the region including the 84 by deletion, and l.igating it into the comparable region of pGPS-5. The derivative pGPS-8 was confirmed to contain the deletion bh the presence of the SmaI and XhoI sites that are created by t:he mutagenesis, and by DNA sequence analysis of the region containing the 84 by deletion.
pGPS-8 was transformE~d into the strain K38/pTrx-3, to create the strain K38/pTrx-3/pGPS-8. Induction of K38/pTrx-3/pGP°.i-8, and purification of the genetically altered T7 DNA polymerase, was carried out using the same procedure as th~it described for the analogous strain K38/pTr~~-3/pGP5-5 in Tabor et al., Id. Since this polymerasE~ has no detectable exonuclease activity chemical modification, as described by Tabor et al., Id., is not ne<:essar~~ before its use in a DNA sequencing reaction. The genetically modified T7 DNA polymerase used in the examples below was a preparation with an activity of 1000 units/ml.
Example 1: DNi~ Sec~uencin Reaction Usin Manganese Standard DN;~ sequencing reaction methodology is used for sequencing :DNA in the presence of Mn. For T7 ~.3~~~35~.

DNA polymerase the general sequencing steps are described in detail i.n Tabor et al., Id. Briefly, the steps and conditions are as follows:

A. Annealing F:eaction In ths~ annealing reaction the following solution was preparect:

DNA to be sequs~nced (e. g., mGPl-2 DNA) in 10 mM Tris-HC1 pH7.5,0.1 mM EDTA, 2ug/7u1 7ul 5X SeqBuf (200 mM Tri.s-HC1 pH7.5, 5mM MnCl2, 2 250 mM NaCl) Primer (New England Biolabs-l7mer, Cat #1210 1 0.5 pm/ui) 1o ul This solution Gras heated at 65C 2 min, and slow cooled to room temperature.

B. Labeling rE~actlOIl In thEa labeling reaction the following solution was prepared:

Annealing reaction mixture 10 ul Dithiothreitol 0.1 M 1 f35S]dATP, New England Nuclear NEG-034H 1 dTTP, dCTP, dG~'P 1.5 uM each 2 Genetically mmodified T7 DNA polymerase, 1 unit/ul (~Lys118-Arg 145, as described above) 2 16 ul This was incubcited at room temperature for 5 min.

1~~~~~~.

C. Termination Reaction In thE~ termination reactions, four reaction mixtures were prepared as follows:
G A T C
5X SeqBuf 0.6 0.6 0.6 0.6 ul 4dNTPs (3mM) 0.3 0.3 0.3 0.3 ul H20 1.9 1.9 1.9 1.9 ul ddGTP 0.2 mM (tid=dideoxy) 0.2 ul ddATP 0.2 mM 0.2 ul ddTTP 0.2 mM 0.2 ul ddCTP 0.2 mM 0.2 ml 3 3 3 3 ul The termination mixtures were incubated at 37C for 2 min, and then :3 girl aliquots of the completed labeling reaction added to each termination mixture. The resulting solui:ion was incubated at 37C for 5 min.

The tE~rmina~tion reactions were stopped with 5 ul of 90% formamide, 20 mM EDTA, 0.2%

bromophenol-blue, xylene-cyanol, pH 8Ø The resulting samples were heated at 75C for two minutes, loaded onto a polyacrylamide gel (8% acrylamide, 0.3% bisacrylamide) in 7M urea, 100 mM Tris borate pH 8.9, and electrophoresed at 2000 volts for 2 hours. The gel was fixed in 50% methanol, 10% acetic acid for 30 min, dried and used to ex~~ose film by autoradiography.

The e:!~posed gel was developed, and the intensity of radioactive bands in each lane was determined by ,scanning each lane with a densitometer (Joyce, Loebl ~~ Co., Ltd., model number MkIIIC).

When 'the same sample is run in the presence of magnesium in place of manganese, the underlined bases in the following triplets are 2-5 fold more intense than ~.3t~(~~~~.

adjacent bases whenever these triplets appear: TCT, AAG, GCA, CCT. However, in the example just described, bands corresponding t:o every base in all the triplets just shown havE~ the Name intensity, differing by at most 20% from one another"
Example 2: Sec e~ ncinq reaction using manganese, 2X ddGTP and 1X ddCTP to differentiate between G and C by relative band nrar~c; t; ac .
t In this example, only one vessel yeas used to perform a sequE~ncing reaction to determine the sequence of two types of: bases (namely C and G) in a DNA
template. The steps were as follows:
In thE~ annealing reaction the following solution was prepared:
mGPl-2 DNA (2.7 mM in 10 mM
Tris-HC1 pH 7.'.i, 0.1 mM EDTA) 8.6 ul 5X SeqBuf 4 Primer (ABI fans primer, 0.4 pm/~rl) 2 H20 5.4 20 ul This solution was heated at 65°C 2 min, and slow cooled to room temperature. The fam primer is labelled with a fluorescent label which can be detected as it passes through a sequEancing gel, using the ABI Model 370 A DNA
Sequencing Sysi~em.
In thcs extension reaction the following solution was prepared.
Annealing reaci~ion mixture 20 ul Dithiothreitol 0.1 M 1 4 dNTP 3 mM 3 ddGTP 30 uM 3 ddCTP 30 uM 1.5 28.5 ul This solution eras inc:ubated at 37°C for 2 min, 1.5 ~1 of genetically modified T7 DNA polymerase (~28), 1 unit/~1, added, and t:he solution incubated at 37°C, for 10 min. Tree reacaion was stopped by adding 5 ul of 100 mM EDTA, pH 8.Ø
The rEasulting fragments were precipitated as follows: 3.5 ul. 3M sodium acetate, and 100 ul 100%
ethanol was added. After incubation on ice for 10 min, the mixture was centrifuged for 30 min at "°C in a microcentrifugEa. ThEa pellet was washed with 500 ~1 70% ethanol, and centrifuged again for 5 min. The supernatant was decanted, and the pellet dried by centrifuging under vacuum for several minutes. The sample was then resuspended in 5 ul 90% formamide, 50 mM EDTA pH 8.0,, heated at 75°C for two minutes, and loaded onto an ABI Model 370A DNA Sequencing System.
The instrument was r~sn and the unprocessed (raw) data was collected as described in the User's Manual for the model 370A insi~rument (Preliminary version, March 1987, Sections 3, 4 and 5). Unprocessed (raw) output for only the fam primer is shown.
The output from this reaction is shown in Fig.
4. Each G is :represented by a tall peak and each C by a short one. Th~ss, the sequence of G's and C's in the DNA
is determinable from the peak height. Thus, from one sequencing rea~~tion, with only one label used for all DNA products, a DNA sequence of G's and C's can be determined. Peak height becomes reduced along the length of a gel since products of higher molecular weight are present in lower amounts. However, the difference between a nearby G and C remains about 2 fold along the gel, while that of a pair of nearby G's or a pair of nearby C's is approximately uniform (varying about 1.1- to 1.4-fold), correcting for the decrease in ~,'~~~~~1.

intensity for each additional position along the sequence. For example, in Fig. 4 the signal decreases 2 fold for a given series of bands over a period of approximately 60 basE~s. Thus there is a 1.16% decrease inherent at each additional position along the template in this examples (since Chi is 1.0116 for Chi60=2).
It is important to distinguish between bands of different inter.~sity clue to different efficiency of chain termination within nearby bands, and two ox more bands migrating together during electrophoresis. The latter event, called a~ compression, is an artifact of gel electrophoresis., and not the DNA sequencing reaction itself, and is not eliminated by using manganese. One example of such a compression is marked by an asterisk (*) in Fig. 4. If one knows that such a compression represents the co-migration of two DNA products, as the one noted in Fi.g. 4, then that band is an accurate marker of a band of 2X intensity.
The precise sequence in a region of compression cannot be determined. In order to determine this sequence, it is necessary either 1) to determine the sequence in thE~ reverse orientation, 2) run the sequencing gel under stronger denaturing conditions, i.e., higher tE~mperat;ure, or by the addition of 50%
formamide, or :3) use a nucleotide analog, e.g., dITP or deazaGTP, in place o:E dGTP. Compressions are due to the formation of sizable hairpins in the DNA under the conditions of del electrophoresis; incorporation of these nucleotide analogs destabilize most of these hairpins.
Compressions due to hairpin structures can be of virtually any length, depending on the extent and strength of the hairvpin. Thus, with Mn all nearby bands have an approximately equal numbers of DNA products of ~.'~~ob~3.

the same molecular weight, but do not necessarily have a similar band intensity due to compressions.
Referring to Fig. 2, the above method was used with just ddGT>:~ in the presence of manganese at a 1 mM
final concentration. Each band on the resulting gel is represented in Fig. :>. as a peak. The intensity of a band is reflect:ed by the height of each peak. With manganese, nearby band intensity and thus peak height is approximately uniform along the gel, differing between nearby bands bh less than 5 or 10%.
In cor~trast,~ the output shown in Fig. 3 represents the same experiment run in the presence of magnesium instEaad of manganese. Here, nearby band intensity and thus peak height varies as much as 10 fold.
Referring to Fig. 5, with all four dideoxy nucleotides at equal concentrations (0.75 uM final concentration for each ddNTP, as in Example 2) in a sequencing reaction :in the presence of manganese, nearby bands and corresponding peaks are approximately uniform, varying by no more than about 1.5-fold again decreasing in absolute intensity for DNA products of higher molecular weigrit. In contrast, in the presence of magnesium and all four dideoxy nucleotides at equal concentration, as shown in Fig. 6, nearby band intensity varies greatly.
By varying the concentration of each ddNTP in a sequencing reacaion the complete nucleotide sequence of a strand of DN~~ can be determined. An example of such a procedure is shown in Fig. 7, where the concentration of each ddNTP differs b!t 30% intervals: ddGTP (4.5 uM
2.2x), ddATP (3.0 ~M, 1.7x), ddTTP (2 uM, 1.3x) and ddCTP (1.4 uM, 1.0x). The DNA sequence determined from this graph is shown below the second line in the Figure. Only ti mistakes (shown by a 'v') were made 1'3~0~~1 compared to the actual DNA sequence. These mistakes can be eliminated by using greater ratios of each ddNTP
(e. g., lxddCTP, 2xdd'TTP, 4xddATP and 8xddGTP).
Similarly, by measuring peak areas, rather than peak height, the results are more accurate. Computer programs to measure such peak areas are readily written for existing DrIA sequencing machines.
Referring to Fig. 8, the optimum concentration of manganese in a sequencing reaction is 1 mM, in the absence of a chelatoz: such as citrate or isocitrate, compared to 20 mM for. magnesium. When 40 mM isocitrate is present in t:he reaction, the polymerase activity in the presence of: manganese is stimulated 4-fold, and the optimum manganE~se concentration is 5 to 20 mM.
Referring to Fig. 9, at 10 mM manganese concentration, the optimal isocitrat:e concentration is 40 mM, resulting in a 4-fold st~.mulat:ion of polymerase activity. At to mM magnesium concentration, any amount of isocitrate has an inhibitory Effect on polymerase. These results were obtained by pei:forming polymerase reactions, as described below, in the presence of various chelator and ion concentrations. Specifically, reactions (200 ul) contained 40 mN4 Tris-HCl, pH7.5, 5 mM dithiothreitol, 0.5 mM denaturE~d calf thymus DNA, 0.3 mM dGTP, dATP, dCTP and [3H]d'.CTP (20 cpm/pm), 50 ug/ml BSA, and the indicated concE3ntrat:ions of MgCl2, MnCl2 or sodium isocitrate. Reactions were begun by the addition of 0.1 unit of genetic: ally modified T7 DNA polymerase (~Lys118-Argl4l5). Incubation was 37°C for 30 min.
Reactions were stopped by the addition of 3 ml of 1N HC1 and 0.1 M sodincn pyrophosphate, and the acid insoluble radioactivity Haas determined. One unit of DNA
polymerase catalyzes the incorporation of 10 nmoles of total nucleotide into an acid-insoluble form in 30 min.

1~~~:'~_~

under the conditions of the assay. (Tabor et al. J.
Biol. Chem. 262, 16212, (1987)).
Pyrophosphatase When chemically modified T7 DNA polymerase is used for DNA se~~uencing, specific fragments disappear upon prolonged incubation (Tabor and Richardson, Proc.
Nat. Acad. Sci. USA 84:4767, 1987). We refer to the sites where this occurs as "holes" since this process creates a space in the sequencing gel. The holes occur more frequently when dITP is used in place of dGTP.
The de~~radation of specific fragments is an obvious problem in reading DNA sequencing gels. The absence of a fragment is either missed completely when the sequence is read, resulting in a deletion in the determined sequence, or else a hole is observed that can only be interpreted as an unknown base at that position.
The current solution to this problem is to keep the reaction tunes short. This is unsatisfactory for two reasons. First, it makes running the reactions technically more difficult, since one is forced to work very rapidly in order to terminate the reactions soon after they are :begun. More importantly, some bands are extremely sensitive to this degradation, and disappear even after very short reactions times.
We have constructed a genetically altered form of T7 DNA polym~erase (~28, described above) that has no detectable level of exonuclease activity (<10 7 the level of the wild-type enzyme, or >10,000 times lower than the chemically modified T7 DNA polymerase). We expected that, since the holes appear with prolonged incubation, theyy were presumably due to exonuclease activity, and thus would not occur when this genetically modified form of T7 DNA polymerase was used. However, the radioactive fragments mentioned above still ~.'~r~ ~~:~.

disappear at the same rate when either chemically or genetically modified T7 DNA polymerase is used.
We have determined that this loss of specific bands is due to pyrophosphorolysis activity of the polymerase. This activity is not due to the exonuclease activity of DNA. polymerase, but rather to the reversal of the polymerase activity: in the presence of pyrophosphate (PPi), the polymerase will add PPi to the terminal nucleotide that is located at the 3' terminus of the chain, in this case releasing a dideoxynucleoside 5'-triphosphate. See generally Deutscher et al. J.
Biol. Chem. 244:3019, 1969; and Kornberg, DNA
Replication pp. 125-126, published by Freeman & Co., SF. This reaction ha.s the effect of removing the block at the 3' terminus, permitting synthesis to extend further along the template. PPi normally accumulates in a DNA synthesis reaction mixture, since it is a product of the polymerization; reaction. The site of pyrophosphorolysis is DNA sequence dependent, and thus the holes described above are produced only at specific sites.
To overcome this problem, the pyrophosphorolysis rE~action must be inhibited. One way to inhibit pyrophosphorolysis is to break down the pyrophosphate as it is generated in the polymerase reaction, by adding the enzyme pyrophosphatase. Other solutions include altering the pyrophosphate by other enzymatic reactions, or preventing the pyrophosphorolysis reaction by the addition of an analog that inhibits this activity of the DNA polymerase. We have found that the addition of even trace amounts of this enzyme (on.e thousandth the molar ratio of DNA
polymerase molecules) to the sequencing reactions completely sta~~ilize:: the specific class of fragments mentioned above and eliminates production of holes. In the presence of both the genetically altered form of T7 DNA polymerase (~28) and pyrophosphatase, all bands are stable upon even prolonged incubation (up to 2 hours).
For automated sequencing, using differential band intensity, it is critical that the intensity of every band is determined entirely by the ratio of ddNTP

to dNTP. Pyrophosphorolysis will create ambiguities by diminishing the intensity of some bands. Thus, addition of pyrophosphatase is particularly useful in this sequencing procedure.

Pyrophosphatase should be added whenever chemically or genetically modified T7 DNA polymerase or other polymerases are used for sequencing, at at least an amount sufficient to catalyze the hydrolysis of the PPi formed at a rate that will prevent the accumulation of PPi to a level that will lead to pyrophosphorolysis.

This is particularly true when dITP is used in place of dGTP, in which case the appearance of holes due to pyrophosphorolysis reaction occurs to a greater extent.

Example 3: Protocol using pyrophosphatase in sequencing reactions In this example, a normal sequencing protocol was followed. The only modification was that yeast inorganic pyrophosphatase was used. The source of the pyrophosatase is not important, however in this example we used Sigma yeast inorganic pyrophosphatase catalog number I-4503, without further purification, or further purified on an FPLC mono Q column, and Worthington's y east inorganic pyrophosphatase without further purification. The pyrophosphatase was added to modified T7 DNA polymerase prior to adding the polymerase to the labeling reaction. 'Typically, 2 units (0.25 ug) of ~i'~G~~-YJ't~rr~

1'~~~~~:

polymerase were used per sequencing reaction set, and 0.001 units of ;Yeast inorganic pyrophosphatase (4 ng).
A wide range of pyrophosphatase activity will work successfully: ~D.O1 ng to 1 ~g of yeast pyrophosphatase per sequencing reaction have bee n tested with success.

For example, in the annealing reaction the following solution was prepared:

mGPl-2 DNA (in 10 mM Tris-HC1 pH7.5,0.1 mM EDTA) 7u1 5X SeqBuf 2 Primer (New England Biolabs-l7mer, 0.5 pm/ul Cat #1211) 1 10 ul This solution was heated at 65C 2 min, and slow cooled to room temperature.

In the labeling reaction the following solution was prepared:

Annealing reaction mixture 10 girl Dithiothreitol 0.1 M 1 35S dATP, New England Nuclear NEG-034H 1 3 dNTP (1.5 uM each dTTP, dCTP, 3 uM dITP) 2 Enzyme mixture (see below) 2 16 ul Enzyme mixture:
Genetically modified T7 DNA polymerase, D Lys118-Argl45 1 unit/ul Yeast inorganic pyrophosphatase 0.01 units ul in 20 :mM Tris-HC1 pH 7.5, 10 mM f3-mercaptoethanol, 50 ug/:ml bovine serum albumin This solution was incubated at room temperature for 5 min.
In the termination reactions the following four reaction mixtures were prepared:

G A T C

5X SeqBuf ( see .above ) 0 . 6 0 . 6 0 . 6 0 . 6 lr 4dNTPs (3mM each dATP, dTTP, dCTP, and 6 mM dITP) 0.3 0.3 0.3 0.3 ul H20 1.9 1.9 1.9 1.9 ul ddGTP 0.03 mM 0.2 ul ddATP 0.2 mM 0.2 ul ddTTP 0.2 mM 0.2 ul ddCTP 0.2 mM 0.2 ml 3 3 3 3 ul These termination mixtures were incubated at 37C

for 2 min, and :3 ul aliquots of the labeling reaction added to each termination mixture. The resulting solutions were incubated at 37C for 60 min.

Each tercnination reaction was stopped with 5 ul of 90% formamide, 20 mM EDTA, 0.2% bromophenol-blue, xy 1 ene-cyano 1, 1?H 8 . 0 .

The resu:Lting samples were heated at 75C for two minutes, loaded onto a polyacrylamide gel (8%

polyacrylamide, 0.3% ~bisacrylamide) in 7M urea, 100 mM

Tris-borate, pH 8.9, and electrophoresed at 2000 volts for 2 hours. The gel was fixed in 50% methanol, and 10%

acetic acid for 30 mi:n, dried, and used to expose film by autoradiogral?hy.

Apparatus Referring to Fig. 11, apparatus 100, suitable for automated DNA sc=quencing, includes a reactor 102 including the above described reagents 104, for example, DNA polymerase, manganese or iron ions, chelators and pyrophosphatase. The apparatus is also provided with a gel box 106, fo:r separating DNA products according to their molecular weights, and a gel reading means 108 for detecting the DISA products as they pass through the gel (shown by dashed arrows 107). Further, a computing means 110 is provided to calculate the intensity of bands of DNA products, and the position of the bands relative to one another. If the DNA products are run in one lane, then the computer means is able to compute the DNA sequence from the band intensity and position.
Standard computer programs are used to perform this Y, function.
Other embodiments are within the following claims.

Claims (43)

1. A method for sequencing a strand of DNA comprising the steps of:
providing said strand of DNA, annealing said strand with a primer able to hybridize to said strand, to give an annealed mixture, and incubating said annealed mixture with a deoxyribonucleoside triphosphate, a DNA polymerase, and a first chain terminating agent, characterized in that said incubation is carried out in the presence of divalent or trivalent manganese or iron cations and under conditions in which said polymerase causes said primer to be elongated to form a first series of first DNA products differing in the length of the elongated primer, each said first DNA product having a said chain terminating agent at its elongated end, the quantity of each said first DNA product being approximately the same for substantially all DNA products differing in length from 1 to 20 bases.

2. The method of claim 1 further including the steps of:
separating said first DNA products by gel permeation according to molecular weight to form a first series of bands, each said first series band representing a said first DNA
product of a particular molecular weight, wherein the intensity of each nearby first series band is approximately the same for substantially all said first series bands, and determining the position of each said first series band.

3. The method of claim 1 further characterized by providing one to three different chain terminating agents in said annealed mixture, each at a concentration different from that of every other chain terminating agent, wherein said DNA
polymerase causes production of one to three additional series of additional DNA products, each DNA product of each additional series having one of said additional chain terminating agents at its elongated end, the quantity of each of said additional DNA product having the same chain terminating agent being approximately the same for all lengths within a range of 20 bases and being distinctly different from the quantity of all DNA products having a different chain terminating agent and being in a different series but having approximately the same molecular weight.

4. The method of claim 3 further including the steps of:
separating all said DNA products by gel permeation according to molecular weight to form a total of two to four series of bands respectively, each said band of a series representing a DNA product having the same chain terminating agent as every other band in said series and being of a particular molecular weight, wherein the intensity of each 36a nearby band within the came series is approximately the same and is distinctly different from the intensity of each nearby band of a different series, and determining the position and intensity of each band of each series.

5. The method of claim 1, 2 or 3 further characterized in that said annealed mixture is provided with a manganese or iron ion, wherein said ion causes said polymerise to be non-discriminatory for said chain terminating agents.

6. The method of claim 1, 2 or 3 further characterized in that said DNA polymerase is chosen from T7-type DNA polymerase, the large fragment of E. coli DNA polymerise I, and Taq polymerase.

7. The method of claim 1, 2 or 3 further characterized in that said chain terminating agent is a dideoxynucleoside triphosphate.

8. The method of claim 1, 2 or 3 further characterized in that said annealed mixture is provided with a chelator.

9. The method of claim 1, 2 or 3 wherein said annealed mixture is provided with a manganese or iron ion, wherein said ion causes said polymerise to be non-discriminatory for said chain terminating agents and said DNA polymerise is chosen from T7-type DNA polymerase, the larger fragment of E. coli DNA polymerise I, and Taq polymerase.

10. The method of claim 1, 2 or 3 wherein said annealed mixture is provided with a manganese or iron ion, wherein said ion causes said polymerase to be non-discriminatory for said chain terminating agents, said DNA polymerise is chosen from T7-type DNA polymerase, the large fragment of E. coli DNA polymerase I, and Taq polymerise, said chain terminating agent is dideoxy-nucleoside triphosphate and said annealed mixture is provided with a chelator.

11. A kit for use in sequencing DNA comprising a supply of a DNA polymerase and a chain terminating agent, and a compound comprising a manganese or iron ion.

12. The kit of claim 11 characterized in that said polymerase is a T7-type DNA polymerase, Klenow fragment, or Taq polymerase and said chain terminating agent is a dideoxynucleoside triphosphate.

13. The kit of claim 11 characterized in that it contains a pyrophosphatase.

14. A method for sequencing a strand of DNA, comprising the steps of:
providing said strand hybridized with a primer able to hybridize to said strand, to give an hybridized mixture, incubating said hybridized mixture with four deoxyribonucleoside triphosphates, a DNA polymerase, and a first chain terminating agent, wherein said DNA polymerase causes said primer to be elongated to form a first series of first DNA
products differing in the length of the elongated primer, each said first DNA product having a said chain terminating agent at its elongated end, the number of molecules of each said first DNA products being approximately the same for substantially all DNA products differing in length by no more than 20 bases, and providing a second chain terminating agent in said hybridized mixture at a concentration different from said first chain terminating agent, wherein said DNA polymerase causes production of a second series of second DNA products differing in the length of the elongated primer, each said second DNA product having said second. chain terminating agent at its elongated end, the number of molecules of each said second DNA products being approximately the same for substantially all second DNA products differing in length from each other by from 1 to 20 bases, and being distinctly different from the number of molecules of all said first DNA products having a length differing by no more than 20 bases from that of said second DNA products.

15. The method of claim 14 further including the steps of:
separating said first and second DNA products by gel permeation according to molecular weight to form a first and a second series of bands respectively, each said first or second series band representing a said first or second DNA product, respectively, of a given molecular weight, wherein the intensity of each nearby first series band is approximately the same for substantially all. said first series bands, and wherein the intensity of substantial.l.y all nearby second series bands is approximately the same, and wherein the intensity of substantially all bands of the first series is distinctly different from the intensity of each nearby band of the second series, and determining the position and intensity of each said first and second series band.

16. The method of claim 14 further comprising the step of:
providing a third chain terminating agent in said hybridized mixture at a concentration different from said first and second chain terminating agents, wherein said polymerase causes production of a third series of third DNA products differing in the length of the elongated primer, each said third DNA product having said third chain terminating agent at its elongated end, the number of molecules of each said third DNA
product being approximately the same for substantially all said third DNA products which differ in length by no more than 20 bases, wherein the numbers of molecules of substantially all said first, all said second and all said third DNA products are distinctly different foam the numbers of molecules of all those DNA products of a different series which are of lengths differing from them by no more than 20 bases.

17. The method of claim 16 further including the steps of:
separating said first, second and third DNA products by gel permeation according to molecular weight to form a first, second and third series of bands, respectively, each said first, second or third series band representing a said first, second or third DNA product, respectively, of a given molecular weight wherein the intensity of each nearby first series band is approximately the same for substantially all said first series bands, wherein the intensity of substantially all nearby second series bands is approximately the same, and the intensity of substantially all nearby third series bands is approximately the same, and wherein the intensity of substantially all nearby bands of different series is distinctly different, and determining the position and intensity of each said band.

18. The method of claim 16 further comprising the step of:
providing in said hybridized mixture a fourth chain terminating agent at a concentration different from said first, second and third chain germinating agents, wherein said DNA
polymerase causes production of a fourth series of fourth DNA
products differing in the length of the elongated primer, each said fourth DNA product having said fourth chain terminating agent at its elongated end, the number of molecules of each said fourth DNA product being approximately the same or substantially all fourths DNA products differing in length by no more than 20 bases, wherein the number of molecules of substantially all said first, all said second, all said third, and all said fourth series of DNA products is distinctly different from all the number of molecules of those DNA products of a different series which differ in length from them by no more than 20 bases.

19. The method of claim 18, further including the steps of:
separating said first, second, third and fourth DNA
products by gel permeation according to molecular weight to form a first, second, third and fourth series of bands respectively, each said first, second, third and fourth series band representing a said first, second, third or fourth DNA product of a given molecular weight, wherein each said second, third and fourth series produce second, third and fourth bands, wherein the intensity of substantially all nearby first series bands, or substantially all nearby second series bands, or substantially all nearby third series bands, or substantially all nearby fourth series bands is approximately the same, and wherein the intensity of substantially all nearby bands in a different series is distinctly different, and determining the position and intensity of each said band.

20. The method of any one of claims 14 to 19 wherein said hybridized mixture is provided with a manganese or iron ion, wherein said ion causes said polymerase to be non-discriminatory for a said chain terminating agent.

21. The method of claim 19 wherein said DNA products are separated according to molecular weight in less than four lanes of a gel.

22. The method of claim 21 wherein the intensity of each band is measured by a gel reading apparatus.

23. The method of any one of claims 14 to 19 wherein each said chain terminating agent is a dideoxynucleoside triphosphate.

24. The method of claim 20, wherein said hybridized mixture is provided with a chelator.

25. The method of claim 24, said chelator being citrate or isocitrate.

26. A method for sequencing a strand of DNA, comprising the steps of:
providing a DNA polymerase, and incubating said polymerase and said strand of DNA in a solution comprising a manganese ion and a chain terminating agent wherein said ion is provided at a concentration effective for causing production of approximately equal numbers of molecules of these DNA products which differ in size from each other by no more than 20 bases.

27. A kit for sequencing DNA comprising:
a first container comprising a DNA polymerase, a second container comprising a chain terminating agent, and a compound comprising a manganese ion.

28. A kit for sequencing DNA comprising:
a first, container comprising a DNA polymerase, a second container comprising a chain terminating agent, and a compound comprising an iron ion.

29. The kit of claim 27 or 28 wherein said polymerase is a T7-type DNA polymerase, suitable for DNA sequencing, Klenow, or Taq polymerase.

30. The kit of claim 26, 27 or 28 wherein each said chain terminating agent is a dideoxynucleoside triphosphate.

31. The kit of claim 29 further comprising a deoxyribonucleoside triphosphate.

32. The kit of claim 27 further comprising a chelator.

33. The kit of claim 32 said chelator being citrate or isocitrate.

34. A method for sequencing DNA comprising:
providing a DNA polymerase in a reaction medium in which said DNA polymerase is substantially non-discriminating for a chain terminating agent, and incubating said DNA in said reaction medium with said DNA
polymerase, a primer, four deoxyribonucleoside triphosphates and a chain terminating agent to elongate said primer to form a series of DNA products differing in the length of the elongated primer and having said chain terminating agent at the elongated end, the number of molecules of each said DNA product being approximately the same for substantially all those DNA products differing in size by no more than 20 bases.

35. The method of claim 34 wherein said DNA polymerase is a T7-type DNA polymerase provided in a solution comprising manganese ions.

36. The method of claim 20 wherein said manganese ions are at a concentration between 5 µM and 20 mM.

37. The method of claim 26 or 34 wherein said manganese ions are at a concent rat ion between 5 µM and 20 mM.

38. A method for sequencing a strand of DNA, comprising the steps of:
providing said strand hybridized with a primer able to hybridize to said strand, to give a hybridized mixture, and incubating raid hybridized mixture with four deoxyribonucleoside triphosphates, a DNA polymerase, and a plurality of chain terminating agents in a reaction medium in which said polymerase causes said primer to be elongated to form a series of DNA products differing in the length of the elongated primer, each DNA product having a first said chain terminating agent at its elongated end, wherein the number of molecules of said DNA products is approximately the same for substantially all DNA products differing in length by no more than 20 bases.

39. The method of claim 38, wherein said reaction medium comprises manganese ions.

40. The method of claim 39, wherein said manganese ions are at a concentration between 5 µM and 20 mM.

41. The method of claim 39, wherein said reaction medium comprises a chelator and manganese ions at a concentration between 0.005 and 100 mM.

42. The method of claim 26 wherein said solution comprises a chelator and said manganese ion is at a concentration between 0.005 and 100 mM.

43. The method of claim 26 wherein said manganese ion is at a concentration between 5µM and 20 mM.
43. The method of any one of claims 14 to 19, wherein said hybridized mixture is provided with a manganese ion, wherein said ion causes said polymerase to be non-discriminating for a said chain terminating agent.
45. The method of any one of claims 14 to 19, wherein said DNA polymerase is selected from the group consisting of the large fragment of E. coli DNA polymerase I and Taq polymerase.
46. The method of any one of claims 14 to 19, wherein said DNA polymerase is a T7-type DNA polymerase.
47. A method for sequencing a strand of DNA, comprising the steps of:
providing a DNA polymerase, and incubating said 46a polymerase and said strand of DNA in a solution comprising a magnesium ion and a chain terminating agent wherein said ion is provided at a concentration effective for causing production of approximately equal numbers of molecules of DNA products differing in size by no more than 20 bases.
48. A method for sequencing a strand of DNA, comprising the steps of:
providing said strand hybridized with a primer able to hybridize to said strand to give an hybridized mixture, incubating said hybridized mixture with four deoxyribonucleoside triphosphates, a DNA polymerase, and a plurality of different chain terminating agents each capable of terminating the DNA synthesis at a different nucleotide base, to cause said primer to be elongated to form a series of DNA
products differing in length with each DNA product having a chain terminating agent at its elongated end, the amount of each said chain terminating agent being distinctly different from that of each other chain terminating agent, and said incubation causing the total amount of said series terminating with one chain terminating agent to be distinctly different from the total amount of said series terminating with each different chain terminating agent, and separating said series according to length, whereby the DNA
products of approximately the same length containing one chain terminating agent are distinctly different in amount from those containing any other chain terminating agent.

49. An automated DNA sequencing apparatus comprising:
a reactor comprising reagents which provide at least two series of DNA products formed from a single primer and a DNA
strand, each said DNA product of a said series differing in molecular weight and having a chain terminating agent at one end, delivering means for providing said DNA products to a separating means for separating said DNA products along one axis of a separator to form a series of bands, the intensity of substantially all nearby bands in a series being approximately the same, and the intensity of substantially all nearby bands in any one series being different from those of other series, band reading means positionable near said separating means for determining the position and intensity of each said band after said separating along said one axis, said band reading means being operably linked to a computing means that determines the DNA sequence of said DNA strand solely from said position and intensity of said bands along said axis and not from the wavelength of emission of light from any label that may be present in the separating means.
50. The apparatus of claim 49 wherein said reagent comprises a manganese or iron ion.
51. The apparatus of claim 49 wherein said reagent comprises T7-type polymerase and a manganese or iron ion.

52. The apparatus of claim 49, 50 or 51, said reagent further comprising a chelator.
53. The apparatus of claim 52, said chelator being citrate or isocitrate.
54. An automated DNA sequencing apparatus comprising:
a reactor comprising reagents which provide a series of DNA products formed from a primer and a DNA strand, wherein said reactor comprises a manganese ion, a connector for providing said DNA products to a separator for separating said DNA products along one axis of the separator to form a series of bands, a band reader positionable near said separator for determining the position and intensity of each said band along said axis after said separating, said band reader being operably linked to a computer that determines the DNA sequence of said DNA strand solely from said position and said intensity of said bands along said axis and not from the wavelength of emission of light from any label that may be present in the separating means.
55. The apparatus of claim 54 wherein said reagent comprises a T7-type DNA polymerase.
56. The apparatus of claim 54 or 55 wherein said reagent further comprises a chelator.

57. The apparatus of claim 56 wherein said chelator is citrate or isocitrate.
58. An automated DNA sequencing apparatus, comprising:
a separator for separating DNA products along one axis of the separator, said products being formed from a primer and a DNA strand in the presence of a manganese ion, to form a series of bands, a band reader positionable near said separator for determining the position and intensity of each said band along said axis after said separating, said band reader being operably linked to a computer that determines the DNA sequence of said DNA strand solely from both said position and intensity of said bands along said axis and not from the wavelength of emission of light from any label that may be present in the separating means.
59. The apparatus of claim 58 wherein said separator consists essentially of a single lane containing four separate series of DNA products with each said DNA product being labelled with the name label.
60. An automated DNA sequencing apparatus comprising a computer that determines the DNA sequence of a DNA strand solely from the position and intensity of bands formed from DNA products separated along one axis of a separator and not from the wavelength of emission of light from any label that may be present in the separator, said DNA products being produced by a DNA sequencing method from said DNA strand.

61. An automated DNA sequencing apparatus comprising a computer means that compute a DNA sequence solely from intensity and position data obtained from at least twa series of DNA
products labelled with an identical label and produced by a sequencing reaction performed in the presence of manganese ions after separating said DNA products in the same lane of a separator to form a series of bands and not from the wavelength of emission of light from the label that is present in the separator.