CA1335263C - T7 dna polymerase - Google Patents

Abstract

A method for producing blunt-ended double stranded DNA
from a linear DNA molecule having a single stranded region, wherein the 3' end of said molecule is double stranded and has no 3' protruding termini, comprising incubating said DNA molecule with a processive DNA polymerase essentially free from naturally occurring exonuclease activity. Also provided is a method for in vitro mutagenesis of a cloned DNA fragment comprising providing a primer and a template, said primer having contiguous bases able to base-pair with contiguous bases of said template except at least one base which is unable to base-pair with said template, and extending said primer with a processive DNA polymerase having less than 500 units of exonuclease activity per mg of polymerase.

Description

~ 1 1 3 3 52 6 3 60724-20g4F
This application is a divisional application of application No. 556,390 filed on January 13th, 1988.
This invention relates to a method for producing blunt-ended double stranded DNA from a linear DNA molecule having a single stranded region, wherein the 3' end of said molecule is double stranded and has no 3' protruding termini, comprising incubating said DNA molecule with a processive DNA polymerase essentially free from naturally occurring exonuclease activity.
The invention relates to DNA polymerases suitable for DNA sequencing.
DNA sequencing involves the generation of four populations of single stranded DNA fragments having one defined terminus and one variable terminus. The variable terminus always terminates at a specific given nucleotide base (either guanine (G), adenine (A), thymine (T), or cytosine (C)). The four different sets of fragments are each separated on the basis of their length, on a high resolution polyacrylamide gel; each band on the gel corresponds colinearly to a specific nucleotide in the DNA sequence r thus identifying the positions in the sequence of the given nucleotide base.
Generally there are two methods of DNA sequencing. One method (Maxam and Gilbert sequencing) involves the chemical degradation of isolated DNA fragments, each labeled with a single radiolabel at its defined terminus, each reaction yielding a limited cleavage specifically at one or more of the four bases (G, A, T or C). The other method (dideoxy sequencing) involves the enzymatic synthesis of a DNA strand. Four '~' - 2 - l 3 3 5 ~ 6 3 60724-209E
sQparate syntheses are run, each reaction being caused to terminate at a spscific base ~G, A, T or C) via incorporation of the appropriate chain terminating dideoxynucleotide. The latter method is pref~rred since the DNA fragments are uniformly labelled ~instead of end labell~d) and thu~ the larger DNA fragments contain increasingly more radioactivity. Further, 35S-labelled nucleotides can be used in place of 3aP-labelled nucleotide~, resulting in sharper definition; and the reaction products are simpla to interpret since each lane corresponds only to eithQr G, A, T or C. Tha enzyme used for most dideoxy seguencing i8 the Escherichia coli DNA-polymerase I large fragment ("Xlenow"~. Another ~olymerase used is AMV reverse transcriptase.
Su~ary of the Invention The invention of the parent application features a method for determining tho nuclsotide base sequence of a DNA
molecule, comprising annealing the DNA molecule with a primer molecule able to hybridise to the DNA molacula:
incubating separate portions of the annealed mixture in at least four vessels with four different deoxynucleo~ide tripho~phates, a proces6ive DNA
polymerase wh~rein the polymerasQ remain~ bound to a DNA
moleculs for at least S00 base~ before dissociating in an en~iro~msnt~l condition normally used in the exten~ion r~action of a DNA seguencing reaction, the polym ~a~e ha~ing le~s than 500 unit~ of sYo~clease ac~i~ity po~ mg of polymera~e, and one of four DNA
synthe~i~ terminating agent3 which terminata DNA
synthe~is at a ~pecific nucleotide base. The agent terminate- at a different specific nuclaotide base in ~ach of the four ves~als. The DNA products of the incubating reaction are separated according to their cize so that at least a part of the nucleotidQ base seguence of the DNA molecule can be determinad.

_ 3 _ 1 335263 In preferred embodiments the polymerase remains bound to the DNA molecule for at least 1000 bases before dissociating; the polymerase is substantially the same as one in cells infected with a T7-type phage (i.e., phage in which the DNA polymerase requires host ~hioredoxin as a subunit; for example, the T7-type phage is T7, T3, ~ II, H, W31, gh-l, Y, A1122, or SP6, S~udier, 9s Virology 70, 1979); the polymerase is non-discriminating for dideoxy nucleotide analogs; the polymerase i8 modified to havo less than 50 units of exonuclease activity per mg of polymerase, more preferably less than 1 unit, even more preferably less ~han 0.1 unit, and most preferably has no detectable exonuclease activity; the polymerasQ is able to utilize primers of as short as 10 bases or preferibly as short as 4 bases; the primer comprises four to forty nucleotide bases, and is single stranded DNA or RNA; the annealing step comprises heating the DNA molecule and the primer to abovQ 65C, preferably from 65C to 100C, and allowing the heated mixture to cool to below 65C, preferably to 0C to 30C; the incubating step comprises a pulse and a chase step, wherein the pulse step comprises mixing the annealed mixture with all four different deoxynucleoside triphosphates and a processive DNA polymerase, wherein at least one of the deo~y~ucleoside triphosphates is labelled; most - pr0ferably the pulse step performed under conditions in which the polymerase does not exhibit its processivity and is for 30 seconds to 20 minutes at 0C to 20C or where at least one of the nucleotide triphosphates is limiting; and the chase step comprises a~ing one of the chain terminating agents to four separate aliquots of the mixture after the pulse step; preferably the chase ~ 60724-2094F

step i3 for 1 to 60 minutes at 30C to 50C; the terminating agent is a dideoxynucleotide, or a limiting level of one deoxynucleoside triphosphate; one of the four deoxynucleotide~ is dITP or deazaguanosine;
labelled primers ara used so that no puls~ step is required, preferably the label is radioactive or fluorescent; and th~ polymerase is unable to exhibit it~
procsssi~ity in a seeond environmental condition normally used in the pulse reaction of a DNA sequencing reaction.
Inventions of divisional applications feature a) a method for produeing blunt ended double-stranded DNA
moleeulQs from a linQar DNA moleeule having no 3' protruding termini, using a proeessive DNA polymerase lS free from ~Yo~uelease aetivity; b) a method of amplifieation of a-DNA sequenee comprising annealing a first and seeond primer to opposite strand~ of a touble stranded DNA sequence and incubating the annealed mixture with a proCQs~ive DNA polymerase having less than 500 unit~ of sYo~elease aetivity per mg of polymerase, preferably le~s than 1 unit, wherein the irst and second primers anneal to opposite strants of the DNA sequenee; in prefQrred embodimants the primers have their 3' end~ direeted toward eaeh other; and the 2s me~hod further eompri~ea, after the incubation step, denaturing th- r-~ulting DNA, annealing the first and second ~rimer- to the resulting DNA and ineubating the annealed misture with tho polymerase; preferably the eyele of denaturing, annealing and ineub~ting is repeat~d from 10 to ~0 time~; e) a method for in vitro mutagen!e~i- of eloned DNA fragment~, eompri~ing providing a eloned fragment and synthe~izing a DNA
s~rand using a proeessi~e DNA polymerase ha~ing less than 1 unit of eYon~elease aetivity per mg of polymerase; d) a method of produeing aeti~e T7-type DNA
polymerase from eloned DNA fragment~ under th~ eontrol ~ 5 1 335263 of non-leaky promoters (see below) in the same cell comprising inducing expression of the genes only when the cells are in logarithmic growth phase, or stationary phase, and isolating the polymerase from the cell;
preferably the cloned fragments are under the control of a promoter requiring T7 RNA polymerase for expression;
e) a gene encoding a T7-type DNA polymerase, the gene being genetically modified to reduce the activity of naturally occurring exonuclease activity: most preferably a histidine (His) residue is modified, even more preferably His-123 of gene 5; f) the product of the gene encoding genetically modified polymerase; g) a method of purifying T7 DNA polymerase from cells comprising a vector from which the polymerase i8 expressed, comprising the steps of lysing the cells, and passing the polymerase over an ion-exchange column, over a DE52 DEAE column, a phosphocellulose column, and a hydroxyapatite column; preferably prior to the passing step the method comprises precipitating the polymerase with ammonium sulfate: the method further comprises the step of passing the polymerase over a Sephadex*DEAE A50 column; and the ion-exchange column is a DE52 DEAE
column; h) a method of inactivating exonuclease activity in a DNA polymerase solution comprising incubating the zs solution in a vessel containing oxygen, a reducing agent and a transition metal; i) a kit for DNA sequencing, comprising a processive DNA polymerase, defined as above, having les~ than S00 units of exonuclease activity per mg of polymerase, wherein the polymerase is able to exhibit its processivity in a first environmental condition, and preferably unable to exhibit its processivity in a second environmental condition, and a reagent necessary for the sequencing, *Trademark ~ 1 3 3 5 2 6 3 60724-209~

selected from a chain terminating agent, and dITP; j) a method for labelling the 3' end of a DNA fraqment compri 8 ing incubating the DNA fragment with a processive DNA polymerase having less than 500 units of exonuclease activity per mg of polymerase, and a labelled deoxynucleotide; X) a method for in vitro mutagenQsis of a cloned DNA fragment comprising providing a primer and a template, the primer and the template having a speciic mismatched base, and extending the primer with a procQssive DNA polymerase; and 1) a method for in vitro mutagenesis of a cloned DNA fragment comprising providing the cloned fragment and synthesizing a DNA
s~rand using a processive D~A polymerase, having less than 50 unit~ of exonuclease activity, under conditions which cause misincorporation of a nucleotide base.
The inventions provide a DNA polymerase which is procassive, non-discriminating, and can utilize short primers. Further, the polymerase has no associated exonuclease activity. These are ideal proparties for the above describ~d methods, and in particular for DN~
sequencing reactions, since the background level of radioactivity in the polyacylamide gels is negligible, there are few or no artifactual bands, and the bands are sharp -- making the DNA saquence easy to read. Further, such a polymera~Q allow~ novel mQthods of sequencing long DNA frag~nent~, as i3 d~scribed in detail below.
Other featurer and advantage~ of the inventions will be apparent from the following description of the . preferrQd ambodim~nt~ thQreof and from the claims.

Description of the Preferred Embodiments The drawings will first briefly be described.
Drawinqs Figs. 1-3 are d~agrammatic representations of the vectors pTrx-2, mGPl-l, and pGP5-5 respectively;
Fig. 4 is a graphical representation of the selective oxidation of T7 DNA polymerase:
Fig. 5 is a graphieal representation of the ability of modified T7 polymerase to synthesize DNA in the presance of etheno-dAT2; and lo Fig. 6 i5 a diagrammatic representation of the enzymatie amplifieation of genomie DNA u~ing modifiad T7 DNA polymerase.
Fig. 7, 3 and 9 are the nucleotide sequences of pTrx-2, a part of pGPS-5 and mGPl-2 respectively.
Fig. 10 i5 a diagrammatie representation of pGP5-6.
DNA PolYm~rase In gsneral the DNA polymerase is processive, ha~ no associated eYon~elease aetivity, does not diseriminata against nucleotide analog incorporation, and ean utiliz~ small oligonueleotides (such as tetramers, heY~msrs and oetamers) as sp~eific primQrs. Thase p~op~rties will now be discussed in detail.
2s Procas~i~ity By proe~-~ivity i8 meant that the DNA
polymera~ able to eontinuously ineorporate many nucleotide- w ing the same primer-template without dissoeiating fro~ the template, under conditions normalIy us~d for DNA sequeneing sxtension reaetions.
The dagree of proees~ivity varie3 with different ~olymerasQs: some ineorporate only a few base~ before dissoeiating (e.g. Klenow (about 15 basQs), T~ DNA

polymerase (about 10 bases), T5 DNA polymerase ~about 180 bases) and reverse transcriptase (about 200 bases) (Das et al. J. Biol. Chem. 254:1227 1979; Bambara et al., J. Biol. Chem 2S3:413, 1978) while others, such as those of the present invention, will remain bound for at least 500 bases and preferably at least 1,000 bases under suitable environmental conditions. Sueh environmental conditions include having adequate supplie~ of all four deoxynueleoside triphosphates and an ineubation temperature from 10C-50C. Proeessivity is greatly enhA"eed in the presenee of E. coli single stranded bindi~g (ssb), protein.
With proc2ssive enzymes termination of a sequeneing reaetion will oceur only at those bases which have ineorporated a chain terminating agent, sueh as a dideoxynucleotide. If the DNA polymerase is non-processive, then artifactual bands will arise during sequencing reactions, at positions corresponding to the nucleotide where the polymerase dissoeiated. Freguent dissoeiation ereates a baekground of bands at ineorreet pcsitions and obseures the true DNA sequenee. This problem i8 partially eorreeted by ineubating the reaetion mixture for a long time (30-60 min) with a high coneentration of substrates, whieh "ehase" the artifaetual band~ up to a high moleeular waight at the top of the gel, away from the region where the DNA
sequenee i~ read. This i8 not an ideal solution sines a non-proee~sive DNA polymerase has a high probability of dissoeiating from the template at regions of eompaet seeondary strueturQ, or hairpins. Reinitiation of primer elongation at these sites is ineffieient and the usual result is the formation of bands at the same position for all four nueleotides, thus obseuring the DNA sQquenee.

Analoq discrimination The DNA polymerases do not discriminate significantly between dideoxy-nucleotide analogs and normal nucleotides. That is the chance of incorporation of an analog is approximately the same as that of a normal nucleotide or at least incorporates the analog with at least 1/10 the efficiency that of a normal analog.
The polymerases also do not discriminate significantly against some other analogs. This is important since, in addition to the four normal deoxynucleotide triphosphates (dGTP, dATP, dTTP and dCTP), sequencing reactions require the incorporation of other types of nucleotide derivatives such as 2 radioactively-or fluorescently-labelled nucleotide triphosphates, usually for labelin~ the synthesized strands with 35S, 32p, or other chemical agents. When a DNA polymerase does not discriminate against analogs the same probability will exi.st for the incorporation of an analog as for a normal nucleotide. For labelled nucleotide triphosphates this is important in order to efficiently label the synthesized DNA strands using a minimum of radioactivity.
Further, lower levels of analogs are required with such enzymes, making the sequencing reaction cheaper than with a discriminating enzyme.
~ iscriminating polymerases show a different extent of discrimination when they are polymerizing in a processive mode versus when stalled, struggling to synthesize through a secondary structure impediment. At such impediments there will be variability in the intensity of different radioactive bands on the gel, which may obscure the sequence.

9a ~ 335~63 60724-2094F
Exonuclease Activity The DNA polymerase has less than 50%, preferably less than 1%, and most preferably less than 0.1%, of the normal or naturally associated level of exonuclease activity (amount of activity per polymerase .

molecule). 8y normal or naturally associated level is meant the eYon~el6asQ aetivity of unmodified T7-type polymerase. Normally the assoeiated activity is about 5,000 units of eYo~l~clease activity per mg of polymerase, measured as described below by a modification of the procedure of Chase et al. ~249 J. Biol. Chem. 4545, 197~). F~o~uelQasQs inerease the fidelity of DNA
synthesis by exeising any newly synthesized bases whieh are ineorreetly basepaired to the template. Sueh assoeiated e~n~elease aetivities are detrimental to the quality of DNA sequeneing reaetions. They raise the mi ~ l required eoncentration of nueleotide preeursors which must be added to the reaetion sinee, when the nueleotide eoncentration falls, the polymerasa aetivity slows to a rate eomparable with the eYon~elease~aetivity, resulting in no net DNA synthesis, or even degradation of the synthesized DN~.
More importantly, assoeiated eYo~uelease aetivity will cause a DNA polymerase to idle at regions in the template with seeondary strueture ;mre~;ments. When a polymerase approaches such a strueture its rate of synthesis deerease3 as it struggles to pass. An assoeiated exonuelease will exeise the newly synthesized DNA when the polymerase stalls. As a eonsequence numerous cyeles of synthesi~ and exeision will oeeur. This may result in the polymerase eventually synthesizing past the - hairpin (with no detriment to the guality ofithe sequeneing reaetion); or the polymerase may dissoeiate from the synthe~ized strand (resulting in an artifaetual band at the same position in all four sequeneing reactions): or, a chain terminating agent may be incorporated at a high freguency and produce a wide variability in the intensity of different fragments in a sequeneing gel. This happens beeauæe the frequeney of incorporation of a chain terminating agent at any given site increases with the number of opportunities the polymerase has to incorporate the chain terminating nucleotide, and so the DNA
polymerase will incorporate a chain-terminating agent at a much higher frequency at sites of idling than at other sites.
An ideal sequencing reaction will produce bands of uniform intensity throughout the gel. This is essential for obtaining the optimal exposure of the X-ray film for every radioactive fragment. If there is variable intensity of radioactive bands, then fainter bands have a chance of going undetected. To obtain uniform radioactive intensity of all fragments, the DNA po~ymerase should spend the same interval of time at each position on the DNA~ showing no preference for either the addition or removal of nucleotides at any given site. This occurs if the DNA polymerase lacks any associated exonuclease, so that it will have only one opportunity to incorporate a chain terminating nucleotide at each position along the template.
Short Primers The DNA polymerase is able to utilize primers of 10 bases or less, as well as longer ones, most preferably of 4-20 bases. The ability to utilize short primers offers a number of important advantages to DNA sequencing. The shorter primers are cheaper to buy and easier to synthesize than the usual 15-20-mer primérs. They also ànneal faster to complementary sites on a DNA
template, thus making the sequencing reaction faster. Further, the ability to utilize small (e.g., six or seven base) oligonucleotide primers for DNA sequencing permits strategies not otherwise possible for sequencing long DNA fragments. For 12 ~ 3 3 5 2 6 3 60724-2094F
example, a kit containing 80 random hexamers could be generated, none of which are complementary to any sites in the cloning vector. Statistically, one of the 80 hexamers sequences will occur an average of every 50 bases along the DNA fragment to be sequenced. The determination of a sequence of 3000 bases would require only five sequencing cycles. First, a "universal" primer (e.g., New England Biolabs #1211 , sequence 5' GTAAAACGACGGCCAGT

3') would be used to sequence about 600 bases at one end of the insert. Using the results from this sequencing reaction, a new primer would be picked from the kit homologous to a region near the end of the determined sequence. In the second cycle, the sequence of the next 600 bases would be determined using this primer. Repetition of this process five times would determine the complete sequence of the 3000 bases, without necessitating any subcloning, and without the chemical synthesis of any new oligonucleotide primers. The use of such short primers may be enhanced by including gene 2.5 and 4 protein of T7 in the sequencing reaction.
DNA polymerases having the above properties include modified T7-type polymerases. That is the DNA polymerases requires host thioredoxin as a sub-unit, and they are substantially identical to a modified T7 DNA polymerase or to equivalent enzymes isolated from related phage, such as T3, ~I, ~II, H, W31, gh-1, Y, A1122 and SP6. Each of these enzymes can be modified to have properties similar to those of the modified T7 enzyme. It is possible to isolate the enzyme from phage infected cells directly, but preferably the enzyme is isolated from cells *Trademark 13 60724-20s4F
which overproduce it. By substantially identical is meant that the enzyme may have amino acid substitutions which do not affect the overall properties of the enzyme. One example of a particularly desirable amino acid substitution is one in which the natural enzyme is modified to remove any exonuclease activity.
This modification may be performed at the genetic or chemical level (see below~.
Cloning T7 Polymerase We shall describe the cloning, overproduction, purification, modification and use of T7 DNA polymerase. This processive enzyme consists of two polypeptides tightly comple~ed in a one to one stoichiometry. One is the phage T7-encoded gene 5 protein of 84,000 daltons (Modrich et al. 150 J. Biol. Chem. 5515, 1975), the other is the E. coli encoded thioredoxin, of 12,000 daltons (Tabor et al., J. Biol. Chem. 262:16, 216, 1987). The thioredoxin is an acce~sory protein and attaches the gene 5 protein (the non-processive actual DNA polymerase) to the primer template. The natural DNA polymerase has a very active 3' to 5' exonuclease associated with it. This activity makes the polymerase useless for DNA sequencing and must be inactivated or modified before the polymerase can be used. This is readily performed, as ~escribed below, either chemically, by local oxidation of the exonuclease domain, or genetically, by modifying the coding region of the polymerase gene encoding this activity.
pTrx-2 In order to clone the trxA (thioredoxin) gene of E. coli wild type E. coli DNA was partially cleaved with Sau3A and the t 335263 13a 6072~-209 fragments ligated to BamHI-cleaved T7 DNA isolated from .strain T7 ST~ tTabor et al., in Thioredoxin and Glutaredoxin Systems:
Structure and Function (Holmgren et al., eds) pp. 285-300, Ra~en ~ress, NY: and Tabor et al., suPra). The ligated DNA
was transfected into E. coli trxA cells, the mixture plated onto trxA cells, and the resulting T7 plaques picked. Since T7 cannot grow without an active E. coli trxA gene only those phages containing the trxA gene could form plaques. The cloned trxA genes were located on a 470 base pair HincII fragment.
In order to overproduce thioreodoxin a plasmid, lo pTrx-2, was as constructed. Briefly, the 470 base pair HincII fragment cont~ining the trxA gene was isolated by standard procedure (Maniatis et al., Cloning: A
Laboratory Manual, Cold Spring Harbor ~abs., Cold Spring -Harbor, N.Y.), and ligated to a deri~ative of pBR322 containing a Ptac promoter (pta~c-12, Amann et al., 25 Gene 167, 1983). Referring to Fig. 2, ptac-12, containing B-lactamase and Col El origin, was cut with PvuII, to yi~ld a fragment of 2290 bp, which was then ligated to two tandem copies of trxA (HincII fragment) using commercially available linkers (SmaI-BamHI
polylinker), to form pTrx-2. The complete nucleotide seguence of pTrx-2 is shown in Figure 7. Thioredoxin production i~ now under the control of the tac promoter, and thus can be specifically induced, e.g. by IPTG
tisopropyl B-D-thiogalactoside).
PGP5-S and mGPl-2 Some gen~ products ~of T7 are lethal when expressed in E. coli. An expression system was de~eloped to facilitate cloning and expression of, lethal gQnes, bas~d on the inducible expression of T7 RNA polymerase. Gene 5 protein is lethal in some E.
coli strains and an example of such a system is describ~d by Tabor et al. 82 Proc. Nat. Acad. Sci. 1074 = == =--(1985) where T7 gene 5 was placed under the control of the ~10 promoter, and is only expressed when T7 RNA
polymerase is preseht in the cell.
Briefly, pGP5-5 (Fig. 3) was constructed by standard procedur~s using synthetic BamHI linkers to join T7 fragment from 14306 (NdeI) to 16869 (AhaIII), containing gene 5, to the 560 bp fragment of T7 from 5667 (HincII) to 6166 (Fnu4Hl) containing both the ~l.lA and ~1.18 promoters, which are recognized by T7 RNA polymerase, and the 3kb BamHI-HincII fragment of pACYC177 (Chang et al., 134 J. Bacteriol. 11~1, 1978).
The nucleotide seguence of the T7 inserts and linkers in shown in Fig. 8. In this plasmid gene 5 is only expressed when T7 RNA polymerase i8 provided in the cell.
Referring to Fig. ~3, T7 RNA polymerase is provided on phage vector mGP1-2. This is similar to pGPl-2 (Tabor et al., id.) except that the fragmen~ of T7 from 3133 (HaeIII) to 5840 (HinfI), containing T7 RNA
polymerase wa~ ligated, using linkers (B~lII and SalI
respectively), to BamHI-SalI cut M13 mp8, placing the polymerase gene under control of the lac promoter. The complete nucleotide se~uenee of mGPl-2 is shown in Fig. 9.
Since pGP5-5 and pTrx-2 have different origins of replication (respe~ivQly a PlSA and a ColEl origin) they can bo tranformed i~to one cell simultaneously.
pTrx-2 exprQ3ses large ~uantities of thiore~ovin in the presence of IPTG. mGPl-2 can coexist in the same cell as thesQ two pla~mids and be used to regulate expression of T7-DNA polymera~e from pGPS-5, simply by causing production of T7-RNA polymQrase by inducing the lac promoter with, e.g., IPTG.
.

Overproduction of T7 DNA polymerase There are several potential strategies for overproducing and reconstituting the two gene products of trxA and gene S. The same cell strains and plasmids can be utilized for all the strategies. In the preferred strategy thQ two genes are co-overexpressed in the sama cell. (This i8 because gene 5 is susceptible to proteases until thioredoxin is bound to it.) As described in detail below, one procedure is to place the two genQs separately on each of two compatible plasmids in the same cell. Alternatively, the two genes could be placed in tandem on the same plasmid. It is important that the T7-gene 5 is placed under the control of a non-leaky inducible promoter, such as ~l.lA, ~l.lB
and ~10 of T7, as the synthesis of even small quantities of the two polypeptides together is toxic in most E. coli cells. By non-leaky is meant that less than 500 molecules of the gene product are produced, per cell generation time, from the gene when the promoter, controlling the gene's expression, is not activated.
Preferably the T7 RNA polymerase expression system is used although other expression systems which utilize inducible promoters could also be used. A leaky promoter, e.g., plac, allows more than 500 molecules of protein to be synthe~ized, even when not in~Ge~, thus cells containing lethal genes under the control of such - a promoter gro~ poorly and are not suitable in this invention. rt i8 Of" course possible to produce these products in cells whare thay are not lethal, for example, the plac promoter is suitable in such cells.
In a sscond strategy each gene can be cloned and overexpressed separately. Using this strategy, the cells containing the individually overproduced polypeptides are combined prior to preparing the extracts, at which point the two polypeptides form an active T7 DNA polymerase.
Example 1: Production of T7 DNA polymerase E. coli strain 71.18 ~Me5sing et al., Proc.
Nat. Acad. Sci. 74:3642, 1977) is used for preparing stocks of mGPl-2. 71.18 is stored in 50% glycerol at -80~C. and is streaked on a standard mlnimal media agar plate. A single colony is grown overnight in 25 ml standard M9 media at 37C, and a single plaque of mGPl-2 is obtained by titering the stock using fre~hly prepared 71.18 cells. The plague is used to inoculate 10 ml 2X
L3 ~2~ Bacto-Tryptone, 1~ yea8t extract, 0.5~ NaCl, 8mM
NaOH) containing JM103 grown to an A590-0.5. This culture will provide the phage stock for preparing a large culture of m~Pl-2. After 3-12 hours, the 10 ml culture is centrifuged, and the supernatant used to infect the large (2L) culture. For the large culture, 4 X 500 ml 2X LD is inoculated with 4 X 5 ml 71.18 cells grown in M9, and i~ shaken at 37C. When the large culture of cells has grown to an A590-1.0 (approximately three hours), they are inoculated with 10 ml of supernatant containing the starter lysate of mGPl-2. The infected cells are then grown overnight at 37C. The next day, the cells are rc...~ved by centrifugation, and the supernatant is raady to use for induction of ~3a~pGP5-5/pTrx-2 (see below). The supernatant can ~be ~tored at ~C or approximately six month~, at a titer -5 X 1011 ~/ml. At this titer, 1 L of phage will infect 12 liter~ of cells at an A590-5 with a multiplicity of infection of 15. If the titer is low, the m~Pl-2 phage can be concentrated from the supernatant by dis~olving NaCl (60 gm/liter) and PE~-6000 (6S gm/liter) in the supernatant, allowing the *Trademark mixture to settle at 0C for 1-72 hours, and then centrifuging (7000 rpm for 20 min). The precipitate, which contains the mGPl-2 phage, is resuspended in approximately l/20th of the original volume of M9 media.
K38/pGP5-5/pTrx-2 is the E. coli strain ~genotype HfrC (~)) containing the two compatible plasmidæ pGPS-5 and pTrx-2. pGPS-S plasmid has a P15A
origin of replication and expresses the kanamycin (Rm) resistance gene. pTrx-2 has a ColEI origin of replication and expresses the ampicillin (Ap) resistance gene. The plasmids are introduced into K38 by standard procedures, selecting KmR and ApR respectively. The cells K38/pGPS-S/pTrx-2 are stored in 50~ glycerol at lS -80C. Prior to use they are streaked on a plate containing 50~g/ml ampicillin and kanamycin, grown at 37C overnight, and a single colony grown in 10 ml LB
media containing 50~g/ml ampicillin and kanamycin, at 37C for 4-6 hours. The 10 ml cell culture is used to inoculate 500 ml of LB media containing 50~g/ml ampicillin and kanamycin and sh~ken at 37C overnight.
The following day, the S00 ml culture is used to inoculate 12 liters of 2X LB-KPO4 media (2%
Bacto-Tryptone, 1% yeast extract, 0.5% NaCl, 20 mM
KPO4, 0.2% dextrose, and 0.2% casamino acids, pH 7.4), and grown wit~ aeration in a fermentor at 37C. When the cells reach an ~590~5~0 (i.e. logarithmic or stationary phase cells), they are infected with mGPl-2 at a multiplicity of infection of 10, and IPTG is added (final concsntration 0.5m~). The IPTG induces production of thiore~oYin and the T7 RNA polymerase in mGPl-2, and th~ncs induces production of the cloned DNA

polymerase. The cells are grown for an a~ditional 2.s hours with stirring and aQration~ and then harvested.
The cell pellet i8 resuspended in 1.5 L 10% sucrose/20 mM Tris-HCl, pH 8.0/25 mM EDTA and re-spun. Finally, the cell pellet is resuspended in 200 ml 10% sucrose/20 mM Tris-HCl, pH 8/1.0 mM EDTA, and frozen in liquid N2. From 12 liters of induced cells 70 gm of cell paste are obtained containing approximately 700 mg gene 5 protein and 100 mg thiore~oYi~.
lo K38/pTrx-2 (K38 eontaining pTrx-2 alone) ovQrproduees thiore~oYin, and it is addQd as a "boostQr"
to extraets of K38/pGPS-5/pTrx-2 to insure that thiore~oY~n i8 in excQss over gene 5 protein at tha outset of tha purifieation. The K38/pTrx-2 eells are stored in 50% glyeerol at -80C. Prior to use they are streaked on a plate containing 50 ~g/ml ampicillin, grown at 37~C for 2~ hours, and a single colony grown at 37C overnight in 25 ml LB media containing 50 ~g/ml ampieillin. The 25 ml culture is used to inoeulate 2 L
of 2X LB media and sh~ken at 37C. When tha cells reach an A590-3.0, the ptac promoter, and thus thioredoxin produetion, is indueed by the addition of IPTG (final eoneentration 0.5 mM). The eells are grown with shaXing for an additional 12-16 hours at 37C, harvQsted, resuspsn~s~ in 600 ml 10% sucrose/20 mM Tris-HCl, pH
8.0/25 ~M EDTA, and rQ-spun. Finally, the cells are resusr~n~s~ in ~0 ml 10% sucrose/20 mM Tris-HCl, pH
8/0.5 m~ EDTA, and frozen in liquid N2. From 2L of cell~ 16 gm of cell pa~t~ arQ obtain~d containing 150 mg of thiore~oY~n.
Assays for the polym~rase involve the use of single-stranded ealf thymus DNA (6mM) as a substrate.
This i8 prepared imms~iatQly prior to us~ by .

denaturation of double-stranded calf thymus DNA with 50 m~ NaOH at 20OC for 15 min., followed by neutralization with HCl. Any purified DNA can be used as a template for the polymerase assay, although preferably it will have a length greater than 1,000 bases.
The standard T7 DNA polymerase assay used is a modification of the procedure described by Grippo et al.
(246 J. 3iol. Chem. 6867, 1971). The standard reaction mix (200 ~1 final volume) contains 40 mM Tris/HCl pH
7.5, 10 mM MgC12, 5 mM dithiothreitol, 100 nmol alkali-denatured calf thymus DNA, 0.3 mM dGTP, dATP, dCTP and ~3H]dTTP (20 cpm/pm), 50 ~g/ml ~SA, and varying amounts of T7 DNA polymerasQ. Incubation i8 at 37C (10C-45C) for 30 min (5 min-60 min). The reaction is ~topped by the addition of 3 ml of cold (0C) 1 N HCl-O.l M pyrophosphate. Acid-insoluble radioactivity is determined by the procedure of Hinkle at al. (250 J. 3iol. Chem. 5523, 1974). The DNA is precipitatad on ice for 15 min (5 min-12 hr), then precipitated onto glass-fiber filters by filtration.
The filters are washed five time~ with 4 ml of cold (0C) 0.1M HCl-0.lM pyrophosphate, and tWiCQ with cold (0C) 90% ethanol. After drying, the radioactivity on the filters is cou~ted using a non-agueous scintillation fluor.
one unit of polymerase activity catalyzes the inco~rporation of 10 nmol of total nucleotide into an acid-soluble for~ in 30 min at 37C, under the conditions given above. Native T7 DNA polymerase and modified T7 DNA polymerase (see below) have the same specific polymerase activity ~ 20%, which ranges between 5,000-20,000 units/mg for native and 5,000-50,000 units!mg for modified polymerase) depen~ng upon the preparation, using the standard assay conditions stated above.

T7 DNA polymerase is purified from the above extracts by precipitation and chromatography techniques. An example of such a purification follows.
An extract of frozen cells (200 ml K38/pGPS-5/pTrx-2 and 40 ml K38/pTrx-2) are thawed at 0C overnight. The cells are combined, and 5 ml of lysozyme ~15 mg/ml) and 10 ml of NaCl (5M) are added.
After 45 min at 0C, the cells are placed in a 37qC
water bath until thair temperature reaches 20C. The cells are then frozen in liquid N2. An additional 50 lo ml of NaCl (SM) i8 added, and the cells are thawed in a 37C water bath. After thawing, the cells are gently mixed at O~C for 60 min. The lysate is centrifuged for one hr at 35,000 rpm in a Beckman 45Ti rotor. The supernatant (250 ml) is fraction I. It contains approximately 700 mg gene 5 protein and 250 mg of thioredoxin (a 2:1 ratio ~hioredoxin to gene 5 protein).
90 gm of ammonium sulphate is dissolved in fraction I (250 ml) and stirred for 60 min. The suspension is allowed to sit for 60 min, and the resulting precipitate collected by centrifugation at 8000 rpm for 60 min. The precipitate is redissolved in 300 ml of 20 mM Tris-HCl pH 7.5/5 mM
2-mercaptoethanol/0.1 mM EDTA/10% glycerol (~uffer A).
Thi~ is fraction II.
. A column of Whatman*DE52 DEAE ( 12.6 cm2 x 18 ;cm) is prepared and washed with Buffer A. Fraction II
iB dialyzed overnight against two changes of 1 L of ~uffer A each until the conductivity of Fraction II has a conductivity equal to that of Buffer A containing 100 mM NaCl. Dialyzed Fraction II is applied to the column at a flow rate of 100 ml/hr, and washed with 400 ml of 3uffer A containing 100 mM NaCl. Proteins are eluted *Trademark ` 1 335263 with a 3.5 L gradient rom 100 to 400 mM NaCl in 3uffer A at a flow rate of 60 ml/hr. Fractions containing T7 DNA polymerase, which elutes at 200 mM NaCl, are pooled. This is fraction III ~lgO ml).
A column of Whatman Pll~phosphocellulose (12.6 cm2 x 12 cm) iY prepared and washed with 20 mM KPO4 pH 7.4/5 mM 2-mercaptoethanol/0.1 mM EDTA/10 ~ glycerol (Buffer ~). Fraction III is diluted 2-fold ~380 ml) with Buffer B, then applied to the column at a flow rate of 60 ml/hr, and washed with 200 ml of Buffer B
containing lOOmM XCl. Proteins are eluted with a 1.8 L
gradient from 100 to 400 mM KCl in Buffer B at a flow rat~ of 60 ml/hr. Fraction8 containing T7 DNA
polymerase, which eluteY at 300 mM KCl, are pooled.
Thi~ is fraction IV (370 ml).
A column of DEAE-Sephade ~A-50 (4.9 cm2 x 15 cm) is prepared and washed with 20 mM Tris-HCl 7.0/0.1 mM dithiothreitol/0.1 mM EDTA/10~ glycerol ~Buffer C).
Fraction rv is dialyzed against two changes of 1 L
8ufer C to a final conductivity equal to that of Buffer C containln~ 100 mM NaCl. Dialyzed fraction IV is applied to the column at a flow rate o 40 ml/hr, and washed with 150 ml of Buffer C containing 100 mM NaCl.
Proteins are eluted with a 1 L gradient from 100 to 300 mM NaCl in Buffer C at a flow rate of 40 ml/hr.
Fractions containing T7 DNA polymerase, which elutes at 210 mM NaCl, are pooled. This is fraction V (120 ml).
A column of BioRad HTP hydroxylapatite (4.9 cm2 x lS cm) i8 prepared and washed with 20 mM KPO4, pH 1.4/10 mM 2-mercaptoethanol/2 mM Na citrate/10%
glycerol (Buffer D). Fraction V is dialyzed againYt two change~ of 500 ml Buffer D each. Dialyzed fraction V is applied to the column at a flow ratQ of 30 ml/hr, and .
*Trademark washed with 100 ml of Buffer D. Proteins arQ eluted with a 900 ml gradient from 0 to 180 mM KPO4, pH 7.4 in Buffer D at a flow rate of 30 ml/hr. Fractions eontaining T7 DNA polymerase, which elutes at 50 mM
KPO4, are pooled. This is fraction VI (130 ml). It eontains 270 mg of homogeneous T7 DNA polymerase.
Fraction ~I is dialyzed versus 20 mM KPO4 pH
7.4/0.1 mM dithiothreitol/0.1 mM EDTA/50% glycQrol.
This is concentrated fraction VI (~65 ml, 4 mg/ml), lo and is stored at -20C.
The isolated T7 polymerase has eYo~llelease acti~ity assoeiated with it. As statQd above this must be inactivated. An example of inactivation by chemieal modification follows.
~15 Concentrated fraction VI is dialyzed overnight against 20 mM KPO4 pH 7.4/0.1 mM dithiothreitol/10%
glycerol to remove the EDTA present in the storage buffer. After dialysis, the concentration is adjusted to 2 mg/ml with 20 mM KPO4 pH 7.4/0.1 mM
dithiothreitol/10~ glycerol, and 30 ml (2mg/ml)-aliquots are placed in 50 ml polypropylene tubes. (At 2 mg/ml, the molar eoneentration of T7 DNA polymerase is 22 ~M.) Dithiothr~itol (DTT) and ferrous ammonium sulfate (Fe(NH4)2~SO4)26H2O) are prepared fresh immadiately before use, and added to a 30 ml . aliquot of T7 DNA polymerase, to eoneentrations of 5 mM
DTT (0.6 ml of a 250 mM stoek) and 20~M
Fe(~H4)2(SO~)26H2O (0.6 ml of a 1 mM stoek).
During modifieation the molar eoncentrations of T7 DNA
polymQrase and iron are eaeh approximately 20 ~M, while DTT is in 2SOX molar exeess.

; The modification is carried out at 0C under a saturated OA~Ye-- atmosphQre as follows. The reaction mixture is plaeed on ice within a dessicator, the d~ssieator is purged of air by evaeuation and subsequently filled with 100% oxygen. This cycle is repeated three times. The reaetion can be performed in air (20% oxygen), but oceurs at one third the ratQ.
The time course of loss of exonuelease activity is shown in Fig. 4. 3H-labaled double-stranded DNA (6 cpm/pmol) was prepared from bactQriophage T7 as d~seribed by Richardson (15 J. Molee. Biol. 49, 1966).
3H-labeled singlQ-stranded T7 DNA was prepared imm~diately prior to U8Q by denaturation of double-stranded 3H-labeled T7 DNA with 50 mM NaOH at 20C for 15 min, followed by neutralization with HC1.
The standard exonueleasQ assay used is a modification of the procedure deseribed by Chase et al. (su~ra). The standard reaction mixture (100 ~1 final volume) eontained 40 mM Tris/HCl pH 7.5, 10 mM MgC12, 10 mM
dithiothreitol, 60 nmol H-labeled single-stranded T7 DNA (6 epm/pm), and varying amounts of T7 DNA
polymerasa. 3H-labelQd double-stranded T7 DNA ean also be used as a substrate. Also, any uniformly radioaetively labeled DNA, single- or double-strandQd, ean be used for th~ assay. Also, 3' end lab~led single-or double-stranded DNA ean be used for the assay. After - ineubation at 37-C for lS min, the reaetion is stopped by the addition of 30 ~1 of BSA (10mg/ml) and 25 ~1 of TCA ~100% w/v). The assay ean be run at 10C-45C
for 1-60 min. The DNA is preeipitated on iee for 15 min (1 min - 12 hr), then centrifuged at 12,000 g for 30 min (5 min - 3 hr). 100 ~1 of the supernatant is used to determinQ the aeid-soluble radioaetivity by adding it to ~ 1 335263 400 ~1 water and 5 ml of aqueous scintillation cocktail.
One unit of exonuclease activity catalyzes the acid solubilization of 10 nmol of total nucleotide in 30 min under the conditions of thQ assay. Native T7 DNA
polymerase has a spQcific exonuclease activity of 5000 units/mg, using the standard assay conditions stated above. The specific eYon~clease activity of the modified T7 DNA polymerase depends upon the extent of chemical modification, but ideally is at least lO-lOO-fold lower than that of native T7 D~A polymerase, or 500 to 50 or le~s units/mg using the standard asæay conditions stated above. When double stranded substrate i3 used the exonuclease activity i8 about 7-fold higher.
Under the conditions outlined, the eYontlclease activity decays exponentially, with a half-life of decay of eight hours. Once per day the reaction vessel is mixed to distribute the soluble o~en, otherwise the reaction will proceed more rapidly at the surface where the concentration of oxygen is higher. Once per day 2.5 ~M DTT (O.3 ml of a fresh 250 mM stock to a 30 ml reaction) is added to replenish the oxidized DTT.
After eight hours, the eYo~clease activity of T7 DNA polymerase has been reduced 50%, with negligible loæs of polymerase activity. The 50% 1088 may be tha result of the complete inactivation of eYonuclease activity of half the polymerase molecules, rather than a general reduction of the rate of exonuclQase activity in all the molecules. Thus, after an eight hour reaction all the molecules have normal polymerase activity, half the molecules have normal exonuclease activity, while the other half have <0.1~ of their-origina} eYonuclease activity.

- 26 - l 335263 When 50% of the molecules are modified (an eight hour reaction), the enzyme is suitable, although suboptimal, for DNA sequencing. For more optimum quality of DNA sequencing, the reaction is allowed to proceed to greater than 99% modification (having less than 50 units of s~on~lclease activity), which requires four days.
After four days, the reaction mixture is dialyzed against 2 changes of 250 ml of 20 mM KPO4 pH 7.4/0.1 mM
dithiothreitol/0.1 mM EDTA/50% glycerol to remove the iron. The modified T7 DNA polymerase (-4 mg/ml) is stored at -20C.
The reaction mechanism for chemical modification of T7 DNA polymerase depends upon reactive oxy~e~ species generated by the presence of reduced transition metals such as Fe2+ and o~yge--. A possible reaction mechanism for the generation of hydroxyl radicals is outlined below:

~l) Fe2+ + 2 ~ Fe3 + i (2) 2 O~ + 2 H ~ H2O2 + 2 ( 3 ) FQ2 + H2O2 ~ Fe3 + OH' + OH

In equation l, oxidation of the reduced metal ion yields superoxide radical, 2 The superoxide radical can undergo a dismutation reaction, producing hydrogen peroxide (equation 2). Finally, hydrogen peroxide can rsact with reduced metal ions to form hydroxyl radicals, OH (the Fenton reaction, equation 3). The oxidized metal ion is recycled to the reduced form by r~dueing agents such as dithiothreitol (DTT).
These reaetive oxygen speeies probably inactivate protein~ by irreversibly chemically altering specific amino acid residues. Such damage is observed in SDS-PAGE of fragments of gene 5 produced by CNBr or trypsin. Some fragments disappear, high molecular weight cross linking oeeurs, and some fragments are broken into two smaller fragments.
As prQviously mentioned, osygen, a reducing agent (e.g. DTT, 2-mereaptoathanol) and a transition metal ~e.g. iron) are essential elements of the modification reaetion. The reaetion oeeurs in air, but is stimulated thre~-fold by use of 100~ o~yya... The rsaction will occu~ slowly in the absence of added transition metals due to thQ presence of traee quantities of transition metals (1-2~M) in most buffer preparations.
As e~yec~ed, inhibitors of the modification reaetion include anaerobic conditions (e.g., N2) and mstal chelators ~e.g. EDTA, citrate, nitrilotriacetate). In addition, the enzymes catalase and superoxide dismutase may inhibit the reaction, consistent with the essential role of reactive oxygen 2s spaeies in the generation of modified T7 DNA polymerase.
As an al~ernative proeedure, it is possible to - genetieally mutate the T7 gene 5 to specifically inaetivate the eYsn~elease domain of the protein. The T7 gene S protein purified from sueh mutants is ideal for use in DNA seguencing without the need to chemically inactivate the eYonuelease by oxidation and without the secondary damage that inevitably occurs to the protein during chemical modification.
Genetically modified T7 DNA polymerase can be isolated by r~ndo~ly mutagenizing the gene 5 and then 1 ~3~263 screening for those mutants that have lost exonuclease activity, without loss of polymQrase activity.
Mutagenesis is performed as follows. Single-stranded DNA containing gene S (e.g., cloned in pEMBL-8, a plasmid containing an origin for single stranded DNA
replication) under the control of a T7 RNA polymerase promoter is prepared by standard procedure, and treated with two different chemical mutagens: hydrazine, which will mutate C's and T's, and formic acid, which will mutate G's and A's. Myers et al. 229 Science 242, 1985. The DNA is mutagenized at a dose which results in a~ average of one base being altered per plasmid molecule. The single-stranded mutagenized plasmids are ~hen primed with a universal 17-mer primer (see above), and used as templates to synthesizQ the op~osite strands. The synthesized strands contain rA~domly incorporated bases at positions corresponding to the mutated bases in the templates. The double-stranded mutagenized DNA is then used to transform the strain K3~/pGPl-2, which is strain K38 containing the plasmid pGPl-2 (Tabor et al., su~ra). Upon heat induction this strain eYpresses T7 RNA polymerase. The transformed cells are plated a~ 30C, with approximately 200 colonies per plata.
Screening for cells having T7 DNA polymerase lacking eY~n~lclQafi~ activity ic based upon the following f~nd~ng. The 3' to 5' ~Yonuclease of DNA polymerases serves a proofreading function. When bases are mi~incorporated, the eYo~ clease will remove the newly incorporated base which is recognized as "abnormal".
This i8 the case for the analog of dATP, etheno-dATP, which is readily incorporated by T7 DNA polymerase in place of dATP. However, in the presence of the 3' to 5' exonuclease of T7 DNA polymerase, it is excised as rapidly as it is incorporated, resulting in no net DNA
synthesis. As shown in figure 6, using the alt~rnating copolymer poly d~AT) as a template, native T7 DNA
polymerase catalyzes extensive DNA synthesis only in the presence of dATP, and not etheno-dATP. In contrast, modified T7 DNA polymerase, because of its lack of an associated eYon~lcleasQ, stably incorporates etheno-dATP
into DNA at a rat~ comparable to dATP. Thus, using poly d~AT) as a template, and dTTP and etheno-dATP as precursors, native T7 DNA polymQrase is unable to synthesizQ DNA from this template, while T7 DNA
polymerase which has lost its eYo~clease activity will be able to use thi~ template to synthesize D~A.
The proeedure for lysing and screening large number of eolonies is deseribed in Raetz t72 Proe. Nat.
Acsd. Sei. 2274, 1975). 3riefly, the K38/pGPl-2 cells transformed with the mutagenized gene 5-containing plasmids are transferred from the petri dish, where they are present at approximately 200 colonies per plate, to a piece of filter paper ("replica plating"). The filter paper dises are then plaeed at 42C for 60 min to induee the T7 RNA polymerasQ, which in turn expresses the gen~
5 protein. Thioredoxin is eonstitutively produeed from the chromosomal gane. Lysozyme is added to the filter paper to lyse the cells. After a freeze thaw step to ensure CQll ly8i8, the filter paper dises are ineubated with poly d(AT), t~32P]dTTP and etheno-dATP at 37C
for 60 min. The filter paper dises are then washed with aeid to remove the unineorporated t32P]dATP. DNA will preeipitate on the filter paper in aeid, while nucleotides will be soluble. The washed filter paper is then used to expose X-ray film. Colonies whieh have indueed an active T7 DNA polymerase which is deficient - 30 _ 1 3 3 52 6 3 in its eYo~uclease will have incorporated acid-insoluble 32p, and will be visible by autoradiography. Colonies expressing native T7 DNA polymerase, or expressing a T7 DNA polymerase defective in polymerase activity, will not appear on the autoradiograph.
ColoniQs which appear positive are recovered from the master petri dish containing the original colonies. Cells containing each potQntial positive clone will be induced on a larger scale (one liter) and T7 DNA polymerase purified from each preparation to ascertain the levels of exonuclease associated with each mutant. Those low in exonuclease are appropriate for D~A seguencing.
Directed mutagenesis may also be used to isolate genetic mutants in the eYqn~lclease domain of the T7 gene 5 protein. The following is an example of this procedure.
T7 DNA polymerase with reduced exonuclease activity (modified T7 DNA polymerase) can also be distinguished from native T7 DNA polymerase by its ability to synthesize through regions of secondary structure. Thus, with modified DNA polymerase, DNA
synthesis from a labeled primer on a template having secondary structure will result in significantly longer extensions, compared to llnmq~ified or native DNA
polymerase. This assay provides a basis for screening for th~ convQrsion of small percentagQs of DNA
polymerase molecules to a modified form.
The above assay was used to screen for altered T7 DNA polymerase after treatment with a number of chemical reagents. Three rQactions resultQd in conversion of the enzyme to a modified form. The first is treatment with iron and a reducing agent, as - 31 - 1 3~52~3 describ~d above. The other two involve treatment of the enzyme with photooxidizing dyes, Rose Bengal and methylene blue, in the presence of light. The dyes must be titrated carefully, and even under optimum conditions the specificity of inactivation of exonuclease activity over polymerase activity is low, compared to thQ high specificity of the iron-induced oxidation. Since these dyes are ~uite specific for modification of histidine residues, this re~ult strongly implicates histidine residues as an essential species in the exonuclease active site.
There are 23 histidine residues in T7 gene 5 protein. Eight of these residues lie in the amino half -of the protein, in the region wher~, based on the homology with ths large fragment of E. coli DNA
polymerase I, the exonuclease domain may be located (Ollis et al. Nature 313, 818. 1984). As described below, seven of the eight histidine residues were mutated individually by synthesis of appropriate oligonucleotides, which were then incorporated into gene 5. These correspond to mutants 1, and 6-10 in table 1.
The mutations WQre constructed by first cloning the T7 gene 5 from p¢P5-3 ~Tabor et al., J. Biol. Chem.
282, 1987) into the SmaI and HindIII sites of the vector 2s M13 mpl8, to give mGPS-2. (The vector used and the source of gene 5 ar0 not critical in this procedure.) - Single-stranded mGPS-2 DNA was prepared from a strain that incorporates deoxyuracil in place of deoxythymidine (Kun~el, Proc. Natl. Acad. Sci. USA 82, 488, 1985).
Thi~ procedure provides a strong selection for survival of only the synthesized strand (that containing the mutation) when transfected into wild-type E.coli, since the strand containing uracil will be preferentially degraded.

Mutant oligonucleotides, 15-20 bases in length, were synthesized by standard procedures. Each oligonucleotide was annealed to the template, extended using native T7 DNA polymerase, and ligated using T4 DNA
ligase. Covalently closed circular molecules were isolated by agarose gel electrophoresis, run in the presence of 0.5~g/ml ethidium bromide. The resulting pu~ified molecule~ were then used to transform E. coli 71.18. DNA from the resulting plagues was isolated and lo th0 relevant region seguenced to confirm each mutation.
The following summarizes the oligonucleotides used to generate genQtic mutants in the gene 5 eYonuclease. The mutations created are ~nderlined.
Amino acid and base pair numbers are taken from Dunn et al., 166 J. Molec. Biol. 477, 1983. The relevant wild type seguences of the region of gene 5 mutated are also shown.
Wild type sequence:
1~9 (aa~ --- . 122 123 Leu Leu Arg Sor Gly ~y~ leu Pro Gly lys Arg Phe Gly Ser ~is Ala leu Glu CTT CTG CGT TCC GGC AAG TTG CCC GGA AAA CGC TTT G&G TCT cac GCT TTG GAG
14677 (~7 bp) _ . .

.

t~l ` at~ H~ 123 ~ Ser 123 Primer u ed: S' CGC TTT GG~ TC~ ~C GCT TTG 3' Mutant ~equenco:

leu leu Arg Ser Gly lys Leu Pro Gly Lys Arg Phe Gly Ser ~ Ala leu Glu c$'r CTG CGT TCC GGC AAG TTG CCC GGA AAA CGC TTT GG~ TC~ ~,C GCT TTG GAG

~at~o~ 2: Deletlon o~ Ser 122 and Hi 123 Pr~er u.~ed: 5' GGA AAA CGC TTT GG~, GC~ TTG GAG &CG 3 ' 6 ba~e deletion Mutant ~equence:

Leu Leu Arg Ser Gly Ly~ Leu Pro Gly Ly~ Arg Phc Gly ~ Ala Leu Glu CTT CTG CGT TCC GGC AAG TTG CCC GGA AAA CGC TTT GG ----- ------ GCC TTG GAG

. ~ 33 ~ 1 335263 M~t~tion 3: Ser 122, Hi~ 123 -~Ala 122, Glu 123 Primer u.~ed: 5' CGC TTT GGG GCT ~A_ GCT TTG G 3' Mutant qequence:

Leu Leu Arg Ser Gly Lys Leu Pro Gly Lys Ar~ Phe Gly ala ~L~ Ala Leu Glu CTT CTG CGT TCC GGC AAG TTG CCC GGA AAA CGC TTT GGG QCT 9Ag GCT TTG GAG

Mutation 4: Lys 118, Arg 119 -~ Glu 118, Glu 119 Primer used: 5' 5' G CCC GGG GAA ~a~ TTT GGG TCT CAC GC 3' Mutant sequence:

Leu Leu Arg Ser Gly Ly.~ Leu Pro Gly ~L~ ~ Phe Gly Ser ~is Ala Leu Glu CTT CTG CGT TCC GGC AAG TTG CCC GG_ ~AA 9a~ TTT GGG TCT CAC GCT TTG GAG

~UtAtlQn 5:. Arg 111, Ser 112, Lya 114 -~ Glu 111, Ala 112, Glu 114 Primsr uqed : 5' G GGT CTT CTG 9aa _CC GGC GAG TTG CCC GG 3' Mutant qequence:

Leu Leu ~ 7a Gly ~L~ Leu Pro Gly Lys Arg Phe Gly Ser ~is Ala Leu Glu CTT CTG ~8a ~CC GGC _AG TTG CCC GGA AAA CGC TTT GGG TCT CAC GCT TTG GAG

T' t~tiQn 6: Hil 59, Hia 62 -~ Ser 59, Ser 62 PrlMer uaed: S' ATT GTG TTC ~CC AAC GGa 5~C AAG TAT GAC G 3' Wild-type aequence:
aa: 55 59 62 . Leu Ile Val Phe Hia Asn Gly ~is Ly~ Tyr Asp Val CTT ATT GTG TTC CAC AAC GGT CAC AAG TAT GAC GTT
T7 bp: 14515 Mutant se~uen~e:

Leu Ile Val Phe ~ Asn Gly S-~ Ly-~ Tyr Asp Val CTT ATT GTG TTC ~C AAC GG~ 5CC AAG TAT GAC GTT

.

. ~ - 34 ~ l 335263 . .

Mutatlo~ 7: Hi~ 82 -~ Ser 82 Primer u.~ed: S' GAG TTC 5CC CTT CCT CG 3' wild-eype ~equence:

aa: 77 82 Leu Asn Arg Glu Phe ~is Leu Pro Arg Glu Asn TTG AAC CGA GAG TTC CAC CTT CCT CGT GAG AAC
T7 bp: 14581 Mutant sequence: 82 Leu Asn Arg Glu Phe 59~ Leu Pro Arg Glu Asn TTG AAC CGA GAG TTC 5~C -TT CCT CGT GAG AAC

~ut~tio~ 8: Arg 96, Hi~ 99 ~Leu 96, Ser 99 Primcr u.~ed: 5' C5~ TTG ATT 5CT TCC AAC CTC 3' Wild-type aequenc~:

aa: 93 96 99 Val Leu Ser Arg LQU Ile ~ia Ser Asn Leu Lys Asp Thr Asp GTG TTG TCA CGT TTG ATT CAT TCC AAC CTC AAG GAC ACC GAT
T7 bp: 14629 Mutant ~equence:

Val Leu Ser ~ Leu Ile ~ Ser Asn Leu Lys Asp Thr Asp GTG TTG TCA C5~ TTG ATT 5CT TCC AAC CTC AAG GAC ACC GAT

~tatiQ~ 9: Hi.~ 190 -~ Ser 190 erimer u~ed: S' CT GAC AAA 5CT TAC TTC CCT 3' Wild-type -e~lu~ ~r~:

Leu Leu S-r ASp Ly~ ~'~ Tyr Phe Pro Pro Glu CTA CTC TCT GAC AAA CAT TAC TTC CCT CCT GAG
T7 bp: 14905 Mu~ant ~equence:

LQU Leu Ser Asp Ly~ Tyr Phe Pro Pro Glu _ 35 _ 1 335 2 63 ~t2t~0n 10: Hi-~ 218 -~Ser 218 Pri3er U-~ed: 5' GAC ATT GAA ~T CGT GCT GC 3' Wi1d-tYPe SeqUenCe:
aa: 214 218 Val A~p Ile Glu ~ Arg Ala Ala Trp Leu Leu GTT GAC ATT GAA CAT CGT GCT GCA TGG CTG CTC
T7 bP: 149g2 MUtant ~eqUenCe: 218 Val A~p Ile Glu ~C~ Arg Ala Ala Trp Lau leu GTT GAC ATT GAA ~CT CGT GCT GCA TGG CTG ~TC

M'?t~O~ : De1eeiOn Of am1nO aCid 118 tO 123 Pr~3~r USed: 5' C GGC AAG TTG CCC GG~ GCT TTG GAG GCG TGG G ~' 18 ba~e deletiOn W~d-qpe s~
146~7 (T~ bP~TCC GGC AAG TTG CCC GGY LAYA Arg Phe Gly Ser ~i~ Al 126 MUtant ~
LeU LeU Arg Ser Gly Ly~ LeU Pro Gly~ 6 anino acida) ~ Al Leu Glu CTT CTG CGT TCC GGC AAG TTG CCC GG~ hases)-------- ~GCT TTG GAG

~t~t~^~ 12: ~S 123 ~G1U 123 Pri~er USed: 5' GGG TCT 9A~ GCT TTG G 3' MUtant ~e~U~n~

Leu Leu Arq Ser Gly Lys Leu Pro Gly Ly~ Arg Phe Gly Ser ~L~ Ala Leu Glu CTT CTG CGT TCC GGC AAG TTG CCC GGA AAA CGC TTT GGG TCT gAQ GCT TTG GAG

M~t~tiQ~ 13~ g 131, Lys 136. Lys 140, Lys 144, A~g 14S 3 Glu 131, Glu 136, Glu 140, Glu 144, Glu 145) Pr~m~r u.~d: S' GGT $AT 93G ~3C GGC GAG ATG 9AG GGT GAA TAC 9AA GAC GAC TTT 9aG -~A ATG
CTT GAA G 3' Wild-type 5~v< '1"
129 (aa) 131 136 140 144 145 Gly Tyr Arg ~u Gly Glu ~et ~ys Gly Glu Tyr ~y~ A~p A~p Phe ry~ Met reU Glu Glu GGT TAS CGC TTA GGC GAG ATG AAG GGT GAA TAC AAA GAC GAC TTT AAG CGT ATG CTT GAA G
14737 (T1 bp~

Mu~ant s~v - ~
129 (aa) 131 136 140 144 145 Gly Tyr ~L~ L u Gly Glu ~ee g~ Gly Glu Tyr ~LU Asp A~p Ph~ Met Leu Glu GluGGT TAT 9~ 9~ GGC GAG ATG ÇAG GGT GAA TA_ 9AA GAC GAC TTT ~AG 9a~ ATG CS$ GAA G
14737 (T7 bp) ~ 335263 - 36a -Each mutant gene 5 protein was produced by infection of the mutant phage iAto K38~pGPl-2, as follows. The cells were grown at 30C to an A59031Ø The temperature was shifted to 4~C for 30 min., to induce T7 RNA polymerase. IPTG was added to O.S mM, and a lysate of each phage was added at a moi~l0. Infected cells wera grown at 37C for 90 min.
The cells were then harvested and extracts prepared by standard procedures for T7 gene 5 protein.
Extracts were partially purified by passage over a phosphocellulose and DEAE A-50 colum~, and assayed by measuring the polymerase and exonuclease acti~ities dir~ctly, as described abo~e. The results are shown in Table l.

Table l SUMMARY OF EXONUC~ASE AND POLYMERASE
A~L1VI~LI~S OF T7 GENE 5 MUTANTS
~x~n~elease Polymerase Uutant activitY, % activity, %

[Wild-~ype] tlOO]a tlO03b Mu~ant 1 (~s 123 ~ Serl23) 10-25 >90 Muta~t 2 (A Ser 122, His 12.3) 0.2-0.4 >90 Mutant 3 (Ser 122,His 123 ) Ala 122,Glu 123) <2 >90 ~ 37 ~ 1 335263 Table 1 SUMMARY OF EXONUCLEASE AND POLYMERASE

Exonuclease Polymerase ~utant actiyity~ ~ activity, ~u~nt4 (Lysll8,A~g119-~G1ull8,G1u119) ~30 >90 Mutant5 (A~g111,S~ 112, Lys 114 -~
G1u111,A1al12,G1ul14) ~75 ~90 Muunt6 ~H~59,EGs62-~S~ 59, Ser 62)~75 ~90 ~uEnt7 ~s82-~ Ser 82) ~75 ~go MuEnt8 (A~g 96, H~s 99 -~ Leu 96,S~ 99) ~75 ~go Mutant 9 ~s 190 ) Scr 190) ~75 ~go ~utant 10 (H~s218 )Scr218) ~75 ~go Mu~nt11 (~ Lysl18,A~gll9,Phc12Q
Gly121,S~ 122,E~s123) <0.02 ~90 M~nt12 (H~123 ~G1u123) d0 ~90 Mutant 13 (A~g131,Lys 136, Lys 140, Lys 144, A~g145-~
Glu 131, Glu 136, G1u140,G1u144,G1u 145) c30 ~90 a. ~xonuclease activity was measured on single stranded t3H~T7 DNA. 100% P~on~clease activity corresponds tO 5, 000 uni.s/mg, b. Polymerase activity was measured using single-stranded calf thymus DNA. 100% polymerase activity corresponds to 8,000 units/mg.

Of the seven histidines tested, only one (His 1~3: mutant 1) ha~ the enzymatic activities characteristic of modified T7 DNA polymerase. T7 gene 5 protein was purified from this mutant using DEAE-cellulose, phosphocellulose, DEAE-SeF~a~eY and hydroYylapatite chromatography. While the polymerase activity was nearly normal (>90% the level of the native enzyme), the eYo~uclease activity was reduced 4 to 10-fold.
A variant of this mutant was constructed in which both His 123 and Ser 122 were deleted. The gene 5 protein purified from this mutant has a 200-500 fold lower eYonuclease activity, again with retention of >90% -of the polymerase activity.
These data strongly suggest~that His 123 lies in the active site of the exonuclease domain of T7 gene 5 protein. Furthermore, it is likely that the His 123 is in fact the residue being modified by the oxidation involving iron, G~ygen and a reducing agent, since such oxidation has been shown to modify histidine residues in other proteins (Levine, J. Biol. Chem. 258: 11823, 1983; and Hodgson et al. Biochemistry 14: 5294, 197S).
The level of residual exonuclease in mutant 11 is comparable to the levels obtainable by chemical modification.
Although mutations at His. residues are described, mutation~ at nearby sites or even at distant sites may also produce mutant enzymes suitable in this invention, e.g., lys and arg (mutants 4 and 15).
Similarly, although mutations in some His residues have little effect on eYonllclease activity that does not necessarily indicate that mutations near these residues will not affect eYo~uclease activity.

Mutations which are especially effective include those having d~letions of 2 or more amino acids, preferably 6-8, for example, near the His-123 region. Other mutations should reduce exonuclease activity further, or completely.
As an example of the use of these mutant strains the following is illustrative. A pGP5-6 (mutation ll)-containing strain has been deposited with the ATCC (see below). The strain is grown as described above and induced as described in Taber et al. J. Biol.
Chem. 262:16212 (1987). K38/pTrx-2 cells may be added to increase the yield of genetically modified T7 DNA
polymerase .
The above noted deposited strain also contains plasmid pGPl-2 which expresses T7 RNA polymerase. This plasmid is described in Tabor et al., Proc. Nat. Acad.
Sci. USA 82:1074, 1985 and was deposited with the ATCC
on March 22, 1985 and assigned the number 40,175.
Referring to Fig. 10, pGP5-6 includes the following ses-~ts:

1. EcoRI-SacI-SmaI-BamHI polylinker seguence from M13 mplO (21bp).

2. T7 bp 14309 to 16747, that contains the T7 gene 5, with the following modifications:
T7 bp 1~703 is chan~ed from an A to a G, creating a SmaI site.
T7 bp 1430~ to 14321 inclusive are deleted (18 bp).

3. SalI-PstI-HindIII polylinker sequence from M13 mp 10 (15 bp) 4. pBR322 bp 29 (HindIII site) to pBR322 bp 375 (BamHI
site).

~ 335263 S. T7 bp 22855 to T7 bp 22927, that contains the T7 RNA
Polymerase promoter ~10, with BamHl linkers inserted at each end (82 bp).
!

6. p3R322 bp 375 (BamHI site) to pBR322 bp 4361 (EcoRI
site).

DNA Sequencin~ Usin~ Modified T7-type DNA PolYmerase DNA synthesis reactions using modified T7-type D~A polymerase result in chain-terminated fragments of uniform radioacti~e intensity, throughout the range of several bases to thousands of bases in length. There is virtually no background due to terminations at sites independsnt of chain terminating agent incorporation (i.e. at pause sites or secondary structure impe~iments).
Seguencing reactions using modified T7-type DNA
polymerase consis~ of a pulse and chase. By pulse is meant that a short labelled DNA fragment is synthesized;
by chase is meant that the short fragment is lengthene~
until a chain terminating agent is incorporated. The rationale for each step differs from conventional DNA
sequencing reactions. In the pulse, the reaction is incubated at 0C-37C for 0.5-4 min in the presence of high levels of three nucleotide triphosphates (e.g., dGTP, dCTP and dTTP) and limiting levels of one other - labelled, carrier-free, nucleotide triphosphate, e.g., - t35S] dATP. Under these~conditions the modified polymerase is unable to exhibit its processive character, and a population of radioactive fragments will be synthesized ranging in size from a few bases to several hlmdred bases. The purpose of the pulse is to radioactively label each primer, incorporating maximal radioactivity while using minim~l levels of radioactive .

nucleotides. In this example, two conditions in the pulsQ reaction (low temperature, e.g., from 0-20C, and limiting levels of dATP, e.g., from 0.1~M to l~M) prevent the modified T7-type DNA polymerase from exhibiting its proCQssi~e character. Other essential environmental components of the mixture will have similar effects, e.g., limiting more than one nucleotide triphosphate or increasing the ionic strength of the reaction. If the primQr is already labelled (e.g., by kinasing) no pulsa step is required.
In the chasQ, the reaction is incubated at 45C
for 1-30 min in the prQsence of high levels (50-S00~M) of all four deoxynucleo6ide triphosphates and limiting levels ~l-50~M) of any one of the four chain terminating agent~, e.g., didQoxynucleosidQ
triphosphates, such that DNA synthesis is terminated after an average of 50-600 bases. The purpose of the chase is to extend each radioactively labelQd primer under conditions of processivQ DNA synthQsis, terminating each extension exclusively at correct sites in four separate reactions using each of the four dideoxynuclQoside triphosphates. Two conditions of the chase (high temparature, e.g., from 30-50C) and high levels (above S0~M) of all four deo~y-,ucleoside triphosphate~) allow the modified T7-type DNA polymerasQ
to exhibit its processive character for tens of thousands of basQs; thu~ the sa~mQ polymerase moleculQ will synthesiza from the primer-template until a dideo~ cleotide is incorporated. At a chasQ
tempQratura of 45C synthesis occurs at >700 nuclQotides/sQc. Thus, for seguencing reactions the chase is complete in 1Q88 than a second. ssb increasQs processivity, for example, when using dITP, or when using low tQmpQraturQs or high ionic strength, or low levels of triphosphates throughout the sequencing reaction.

Either ~a35S]dATP,[a32P]dATP or fluorescently labelled nucleotides can be used in the DNA
sequencing reactions with modified T7-typQ DNA
polymerase. If the fluorescent analog is at the 5' end of the primer, then no pulse step is required.
Two components determine the average extensions of the synthesis reactions. First is the length of time of the pulse reac~ion. sincQ the pulse is done in the absence of chain terminating agQnts~ the longer the pulse reaction time, the longer the primer extensions. At 0C
the polymerase extensions average lO nucleotides/sec.
Second is the ratio of deoxyribonucleoside triphosphates to chain terminating agents in the chase reaction. A
modified T7-type DNA polymerase does not discriminate against the incorpo~ration of these analogs, thus the a~erage length of extension in the chase is four times the ratio of the deoxynucleoside triphosphate concentration to the chain terminating agent concentration in the chase reaction. Thus, in order to shorten the average size of the extensions, the pulse time i8 shortened, e.g., to 30 sec. and/or the ratio of chain terminating agent to deG~y-,ucleoside triphosphate concentration is raised in the chase reaction. This can b~ done either by raising the concentration of the chain 2s terminating agent or lowering the concentration of deoxynucleoside triphosphate. To increase the average l~ngth of the extQnsion3, the pulse time is increased, e.g., to 3-~ min; and/or the concentration of chain terminating agent is lowered (e.g., from 20~M to 2~M) in the chase reaction.
Example 2: DNA se~uencin~ usina modified T7 DNA
polymerase The following is an example of a sequencing protocol using didQoxy nucleotides as terminating agents.

9~1 of single-stranded M13 DNA (mGPl-2, prepared by standard procedures) at 0.7 mM concentration is mixed with 1 ~1 of complementary sequencing primer (standard universal 17-mQr, O.S pmole primer / ~1) and 2.5 ~1 5X annealing buffer (200 mM Tris-HCl, pH 7.S, 50 mM MgC12) heated to 6SC for 3 min, and slow cooled to room temperature over 30 min. In the pulse reaction, 12.5 ~1 of the above annealed mix was mixed with 1 ~1 dithiothreitol 0.1 M, 2 ~1 of 3 dNTPs (dGTP, dCTP, dTTP) 3 mM each (P.~ Biochemicals, in TE), 2.S
~1 t~35S]dATP, (1500 Ci/mmol, New England Nuclear) and 1 ~1 of modified T7 DNA polymerase dascribed in Example 1 (0.~ mg/ml, 2500 units/ml, i.e. 0.4 ~g, 2.5 units) and incuba~ed at 0C, for 2 min, after vortexing and centrifuging in a microfuge for 1 sec. The time of incubation can vary from 30 sec to 20 min and temperature can vary from 0C to 37C. Longer times are used for determining sequences distant from the primer.
4.5 ~1 aliquots of the above pulse reaction are added to each of four tubes containing the chase mixes, preheated ~o 4SC. The four tubes, labeled G, A, T, C, each contain trace amounts of either dideoxy (dd) G, A, T, or C (P-L Biochemicals). The specific chase solutions are given below. Each tube contains 1.5 ~1 dATP lmM, 0.5 ~1 SX annealing buffer (200 mM Tris-HCl, pH 7.5, SOmM~MgC12), and 1.O ~1 ddNTP 100 ~M
~wherQ ddNTP corr~onds to ddG,A,T or C in the respective tubes). Each chase reaction is incubated at 45C (or 30C-50C) for 10 min, and then 6 ~1 of stop solution (90~ formamide, lOmM EDTA, 0.1% xylenecyanol) is added to each tube, and the tube placed on ice. The chase times can vary from 1-30 min.

The sequencing reactions are run on standard, 6% polyacrylamide sequencing gel in 7M urea, at 30 Watts for 6 hours. Prior to running on a gel the reactions are heated to 75C for 2 min. The gel is fixed in 10%
acetic acid, 10% methanol, dried on a gel dryer, and exposed to Kodak OMl high-contrast autoradiography film overnight.
Example 3: DNA sequencinq usina limitinq concentrations of dNTPs In this examplQ DNA sequenee analysis of mGPl-2 D~A is performed using limiting levels of all four deoxyribon~leleosid~ triphosphatQ3 in the pulsQ
reaetion. This method has a n~mher of advantagQs over the protoeol in example 2. First, the pulse reaetion runs to co~pletion, whereas in the previous protoeol it was necessary to interrupt a time eourse. As a eonsequenee the reaetions are easier to run. Seeond, with this method it is easier to control the extent of the elongations in the pulse, and so the efficiency of labeling of sequenees near thQ primer (the first 50 baSQS) i8 inereasEd approximately 10-fold.
7 ~1 of 0.75 mM single-stranded Ml3 DNA
(mGPl-2) was mixed with 1~1 of eomplementary sQqueneing primer (17-mer, 0.5 pmole primer/~l) and 2 ~l 5X ann~aling buffer (200 mM Tris-HCl pH 7.5, 50 mM MgC12~ 2SO mM ~aCl) heated at 65C for 2 min, and slowly e~ooled to room temperature over 30 min. In the puls~ reaetion 10 ~l of the above annealed mix was mixed with 1 ~1 dithiothreitol 0.1 M, 2 ~1 of 3 dNTPs ~dGTP, dCTP, dTTP) 1.5 ~M eaeh, O.5 ~l t35S]dATP, (alO~M) (about lO~M, 1500 Ci/mmol, NHW England Nuelear) and 2 ~l modified T7 DNA
polymerase (0.1 mg/ml, lOOO units/ml, i.e., 0.2 ~g, 2 units) and ineubated at 37C for 5 min. (The temperature and time of incubation can be varied from 20C-45C and 1-60 min., respectively.) 3.5 ~1 aliquots of the above pulse reaction were added to each of four tubes containing the chasa S mi.xes, which were preheated to 37C. The four tubes, labeled G, A, T, C, each contain trace amounts of either di.deoxy G, A, T, C. The specific chase solutions are gi.ven below. Each tube contains 0.5 ~l 5X annealing buffer (200 mM Tris-HCl pH 7.5, 50 mM MgC12, 250 mM
NaCl), l ~1 4dNTPs (dGTP, dATP, dTTP, dCTP) 200 ~M
each, and 1.0 ~l ddNTP 20 ~M. Each chase reaction is incubated at 37C for 5 min (or 20C-45C and 1-60 min res~ac~ively), and then 4 ~1 of a stop solution (95% formamide, 20 mM EDTA, 0.05% xylene-cyanol) added to each tube, and the tube placed on ice prior to running on a standard polyacrylamide sequencing gel as described above.
Example 4: RePlacement of dGTP with dITP for DNA
se~uencin~
In order to sequence through regions of compression in DNA, i.e., regions having compact secondary structure, it is common to use dITP (Mills et al., 76 Proc. Natl. Acad. Sci. 2232, 1979) or deazaguanosine triphosphate (deaza GTP, Mizusawa et al., 14 Nuc. Acid Ras. 1319, 1986). We have found that both ana~Qgs function wall with T7-type polymerases, espe;cially with dITP in the presence of ssb. Preferably these reactions are performQd with the above described genetically modified T7 polymerase, or the chase reaction is for 1-2 min., and/or at 20C to reduce exsnuclease degradation.
Modified T7 DNA polymerase efficiently utilizes dITP or deaza-GTP in place of dGTP. dITP is substituted for dGTP in both the pulse and chase mixes at a concentration two to five times that at which dGTP is used. In tha ddG chase mix ddGTP is still used (not ddITP).
The chasa reactions using dITP are sensitive to the residual low levels (about 0.01 units) of eYonllclease activity. To avoid this problem, the chase reaction times should not exceed 5 min when dITP is used. It is recommended that the four dITP reactions be run in conjunetion with, rather than to the exclusion of, the four reactions using dGTP. If both dGTP and dITP are routinely used, the number of raquired mixes ean be ~inimized by: (1) Leaving dGTP and dITP out of the ehase mixes, whieh means that the four chase mixes can be used for both dGTP and dITP chasQ rQaetions. (2) -Adding a high eoneentration of dGTP or dITP (2~1 at 0.5 mM and 1-2.S mM respeetively) to the appropriate pulse mix. The two pulse mixes then each eontain a low concentration of dCTP,dTTP and ~a35S]dATP, and a high concentration of either dGTP or dITP. This modifieation does not usually adversely effeet the quality of the sequencing reactions, and reduees the required number of pulse and chase mixes to run reactions using both dGTP and dITP to six.
The sequencing reaction is as for example 3, except that two o the pulse mixes contain a) 3 dNTP mix 2s for dGTP: l.S ~M dCTP,dTTP, and 1 mM dGTP and b) 3 ~dNTP mix for dITP: 1.5 ~M dCTP,dTTP, and 2 mM dITP.
In the chase reaction dGTP is re...oved from the chase mixes (i.e. the cha~e mixe~ contain 30 ~M dATP,dTTP
and dCTP, and one of the four didQoxynuclQotides at 8 ~M), and the chase time using dITP does not exceed 5 min.

- 47 _ 1 33 5263 Deposits Strains R38/pGP5-5/pTrx-2, K38/pTrx-2 and M13 mGPl-2 have been deposited with the ATCC and assigned numbers 67,287, 67,286, and 40,303 respectively. These deposits were made on January 13, 1987. Strain K38/pGPl-2/pGP5-6 was deposited with the ATCC. On December 4, 1987, and assigned the number 67571.
Applicants' and their assignees acknowledge their responsibility to replace these cultures should they die before the end of the term of a patent issued hereon, 5 years after the last request for a culture, or 30 years, whichever i5 the longer, and its responsibility to notify the depository of the issuance of such a patent, at which time the deposits will be ~ made irrevocably available to the public. Until that time the deposits will be made irrevocably available to the Commissioner of Patents under the terms of 37 CFR
Section 1-14 and 35 USC Section 112.
Other Embod;ments Other embodiments are within the following claims.
~ther uses of the modified DNA polymerases of this invention, which take advantage of their processivity, and lac~ of eY~nl~clease activity, include the direct enzymatic amplification of genomic DNA
25~ se~uences. Thi~ has been described, for other polymerase~, by Saiki et al., 230 Science 1350, 1985;
and Scharf, 233 Science 1076, 1986.
Referring to Fig. 6, enzymatic amplification of a specific DNA region entails the use of two primers which anneal to opposite strands of a double stranded DNA sequence in the region of interest, with their 3' ends directed toward one another (see dark arrows). The actual procedure involves multiple (10-40, preferably 16-20) cycles of denaturation, annealing, and DNA

~ ~ 335263 synthe~is. Using this procedure it is possible to amplify a specific region of human genomic DNA over 200,000 times. As ~ result the specific gene fragment represents about one part in five, rather than the initial one part in a million. This greatly facilitates both the cloning and the direct analysis of genomic DNA. For diagnostic uses, it can speed up the analysis from several weeXs to 1-2 days.
Unlika Rlenow fragment, where the amplification process i8 limited to fragments under two hundred bases in length, modified T7-type DNA polymerases should (preferably in conjuction with E. coli DNA bin~ing protein, or s3b, to prevent "snapback formation of single stranded DNA) permit the amplification of DNA
fragmQnts thousands of bases in length.
The modified T7-type DNA polymerases are also suitable in standard reaction mixtures: for a) filling in 5' protruding termini of DNA fragments generated by restriction enzyme cleavage; in order to, for example, produce blunt-ended double stranded DNA from a linear D~A molecule having a single stranded region with no 3' protruding termini; b) for labeling the 3' termini of restriction fragments, for mapping mRNA start sites by Sl nuclease analysis, or sequencing DNA using the Maxam and Gilbert chemical modification procedure; and c) for in vitro mutago~esis of cloned DNA fragments. For ~xample, a ch~mically synthesized primer which contains specific mismatched bases i~ hybridized to a DNA
template, and then extended by the modified T7-type DNA
polymerase. In thls way the mutation becomes pQrmanently incorporated into the synthesized strand.
It is advantageous for the polymerase to synthesize from the primer through the entire length of the DNA. This ~ 49 ~ 1 3 3 52 6 3 is most efficiently done using a processive DNA
polymerasQ. Alternatively mutagenesis is performed by misincorporation during DNA synthesis (see above). This application is used to mutagenize specific regions of cloned DNA fragments. It is important that the enzyme used lack eYonl~clease activity. By standard reaction mixture is meant a buffered solution containing the polymerase and any necessary deoxynucleosides, or other compounds.

Claims (40)

1. A method for producing blunt-ended double stranded DNA
from a linear DNA molecule having a single stranded region, wherein the 3' end of said molecule is double stranded and has no 3' protruding termini, comprising incubating said DNA molecule with a processive DNA
polymerase essentially free from naturally occurring exonuclease activity.

2. A method for producing blunt-ended double stranded DNA
from a linear DNA molecule having a single stranded region, wherein the 3' end of said molecule is double stranded and has no

3' protruding termini, comprising incubating said DNA molecule with a T7-type DNA
polymerase essentially free from naturally occurring exonuclease activity.

3. The method of claim 2, wherein said T7-type DNA
polymerase is T7 DNA polymerase.

4. A method for labelling the 3' end of a DNA fragment comprising incubating said DNA fragment with a processive DNA
polymerase having less than 500 units of exonuclease activity per mg of polymerase and a labelled deoxynucleotide.

5. A method for labelling the 3' end of a DNA fragment comprising incubating said DNA fragment with a T7-type DNA
polymerase having less than 500 units of exonuclease activity per mg of polymerase and a labelled deoxynucleotide.

6. A method for in vitro mutagenesis of a cloned DNA
fragment comprising providing a primer and a template, said primer having contiguous bases able to base-pair with contiguous bases of said template except at least one base which is unable to base-pair with said template, and extending said primer with a processive DNA polymerase having less than 500 units of exonuclease activity per mg of polymerase.

7. A method for in vitro mutagenesis of a cloned DNA
fragment comprising providing said cloned fragment and a processive DNA polymerase, contacting said cloned fragment with said polymerase under conditions suitable for synthesizing a DNA
strand from said fragment, wherein said processive DNA polymerase has less than 500 units of exonuclease activity per mg of polymerase, and wherein said conditions cause formation of said DNA strand by incorporation of a plurality of contiguous bases able to base-pair with said fragment and incorporation of a nucleotide base unable to base-pair with said fragment.

8. A method for in vitro mutagenesis of a cloned DNA
fragment comprising providing a primer and a template, said primer having contiguous bases able to base-pair with contiguous bases of said template except at least one base which is unable to base-pair with said template, and extending said primer with a T7-type DNA polymerase having less than 500 units of exonuclease activity per mg of polymerase.

9. A method for in vitro mutagenesis of a cloned DNA
fragment comprising providing said cloned fragment and a T7-type DNA polymerase, contacting said cloned fragment with said polymerase under conditions suitable for synthesizing a DNA strand from said fragment, wherein said T7-type DNA polymerase has less than 500 units of exonuclease activity per mg of polymerase, and wherein said conditions cause formation of said DNA strand by incorporation of a plurality of contiguous bases able to base-pair with said fragment and incorporation of a nucleotide base unable to base-pair with said fragment.

10. A method for in vitro mutagenesis of a cloned DNA
fragment comprising providing a primer and a template, said primer and said template having a specific mismatched base, and extending said primer with a processive modified DNA polymerase.

11. A method for in vitro mutagenesis of a cloned DNA
fragment comprising providing said cloned fragment and synthesizing a DNA strand using a processive DNA polymerase, having less than 1 unit of exonuclease activity per mg of polymerase, under conditions which cause misincorporation of a nucleotide base.

12. A method of amplification of a DNA sequence comprising annealing a first and second primer to opposite strands of a double stranded DNA sequence and incubating the annealed mixture with a processive DNA polymerase having less than 500 units of exonuclease activity per mg of polymerase, wherein said first and second primers anneal to opposite strands of said DNA sequence.

13. The method of claim 12 wherein said first and second primers have their 3' ends directed toward each other after annealing.

14. The method of claim 12 wherein said method further comprises, after said incubation step, a cycle of denaturing the resulting DNA, annealing said first and second primers to said resulting DNA and incubating the annealed mixture with said polymerase.

15. The method of claim 14 wherein said cycle of denaturing, annealing and incubating is repeated from 10 to 40 times.

16. The method of claim 12 wherein said exonuclease activity is less than 1 unit per mg of polymerase.

17. A method of amplification of a DNA sequence comprising annealing a first and second primer to opposite strands of a double stranded DNA sequence and incubating the annealed mixture with a T7-type DNA polymerase having less than 500 units of exonuclease activity per mg of polymerase, wherein said first and second primers anneal to opposite strands of said DNA sequence with their 3' ends directed towards each other after annealing, and with the DNA sequence to be amplified located between the two annealed primers.

18. The method of claim 17 wherein said method further comprises, after said incubating step, denaturing the resulting DNA, annealing said first and second primers to said resulting DNA and incubating the annealed mixture with said polymerase.

19. The method of claim 18 wherein denaturing, then annealing and then incubating are repeated from 10 to 40 times.

20. The method of claim 17 wherein said exonuclease activity is less than 1 unit per mg of polymerase.

21. The method of claim 17, said T7-type DNA polymerase being selected from the group consisting of the DNA polymerase of phage T7, T3, ?, ?II, H, W31, gh-1, Y, A1122 and Sp6.

22. The method of claim 17 wherein said polymerase is non-discriminating for dideoxy nucleotide analogs.

23. The method of claim 17 wherein said polymerase is a modified polymerase having less than 50 units of exonuclease activity per mg of polymerase.

24. The method of claim 23 wherein said modified polymerase has less than 1 unit of activity per mg of polymerase.

25. The method of claim 23 wherein said modified polymerase has less than 0.1 unit of activity per mg of polymerase.

26. The method of claim 17 wherein said polymerase has no detectable exonuclease activity.

27. The method of claim 17 wherein said first and second primers include 10 bases or more.

28. The method of claim 17 wherein said first and second primers include 4 bases or more.

29. The method of claim 17 wherein said first and second primers comprise 4 to 20 bases.

30. The method of claim 17 wherein said polymerase is non-discriminating for nucleotide analogs and said first and second primers are single-stranded RNA or DNA comprising 10 bases.

31. The method of claim 17 wherein said first and second primers are single stranded DNA or RNA.

32. A method of amplification of a DNA sequence comprising annealing a first and second primer to opposite strands of a double stranded DNA sequence and incubating the annealed mixture with a processive T7-type DNA polymerase, having less than 50% of the exonuclease activity of the naturally occurring level of exonuclease activity of said polymerase.

33. The method of claim 32 wherein said polymerase has less than 1% of the exonuclease activity of the naturally occurring level of exonuclease activity of said polymerase.

34. The method of claim 32 wherein said T7-type DNA
polymerase is T7 DNA polymerase.

35. The method of claim 34 wherein said T7 DNA polymerase has less than 1% of the exonuclease activity of the naturally occurring level of exonuclease activity of said polymerase.

36. A method of amplification of a DNA sequence comprising annealing a first and second primer to opposite strands of a double stranded DNA sequence and incubating the annealed mixture with a processive T7-type DNA polymerase, said polymerase having a level of exonuclease activity which is sufficiently low to permit the nucleotide base sequence of the DNA molecule to be determined with said polymerase.

37. The method of claim 36 wherein said T7-type DNA
polymerase is T7 DNA polymerase.

38. The method of claim 36 wherein said processive DNA
polymerase has less than one percent of the exonuclease activity of the naturally occurring level of exonuclease activity of said polymerase.

39. The method of claim 38 wherein said polymerase has no detectable exonuclease activity.

40. The method of claim 38 or 39 wherein said polymerase is T7 DNA polymerase.