CA2174944A1 - Cloned dna polymerases from thermotoga neapolitana and mutants thereof - Google Patents

Abstract

The invention relates to a substantially pure thermostable DNA polymerase from Thermotoga neapolitana (Tne) and mutants thereof. The Tne DNA polymerase has a molecular weight of about 100 kilodaltons and is more thermostable than Taq DNA polymerase. The mutant Tne DNA polymerase has at least one mutation selected from the group consisting of (1) a first mutation that substantially reduces or eliminates 3' 5' exonuclease activity of said DNA polymerase; (2) a second mutation that substantially reduces or eliminates 5' 3' exonuclease activity of said DNA polymerase; (3) a third mutation in the O helix of said DNA polymerase resulting in said DNA polymerase becoming non-discriminating against dideoxynucleotides. The present invention also relates to the cloning and expression of the wild type or mutant Tne DNA polymerase in E. coli, to DNA molecules containing the cloned gene, and to host cells which express said genes. The Tne DNA polymerase of the invention may be used in well-known DNA
sequencing and amplification reactions.

Description

Wo 96110640 2 1 7 ~ 9 4 4 PCTIUS95112358 .
CLONI~D DNA pO~.YM~UA~ FROM 1nl~lc~O'rOGA
NEAPOLITANA AND IIUTASTS Tlll~Rl~O~
Backgroun~l of tlte Inven~ion F~eld o~r~ e ~nPenr~on The present invenfiorl relates to a :~ub~lL~lly pure ~h~rsn~hl~ DNA
polymerase. ~r ~ , the DNA ~1~ ~ of t~e present mverltion is a ~ennotoga ..~. yu~ Y DNA pcl~ having a molecular weight of about 100 l~lodaltons. The present invention also }elates to the dorlirlg and expression of the 171ermologa r,of~ r DNA pol~ a~ in ~ coli, to DNA molecules containing the doned gerle, and to hosts whidl express said genes. The DNA
pOlyTnerase of the present invention may he used in DNA sequencing and ..).. reactions.
This irlvention also relates to mutants of the lhe 7no~oga ~ ; T
~Trle) DNA ~1~ , having ~ reduced 3'-5' ~ , activity, mutants of 17~ermofoga neapoli~ana containing a Phe6' -Ty~ (as numbered in l~ig 5) mutation resulting in the ability of the mutant DNA p~ly~.~ to incorporate dld~;u~...~clo~Ldes into a DNA molecule about as efficiently as deo~y~ c; and mutants having ~ reduced 5'-3' ~ k~
activjty. The Tne mutants of this invention can llave one or more of these propesties These Tne DNA polymerase mutants may also be used in DNA
c~ n~;n~ and ~r~ifi~tion reactions.

~8 wo 96110640 PCTlUSgS1123

-2-2'~7 4944 P ~,, J ~,~f ,.
DNA pul~ ,.~ synthesize the formation of DNA molecules which are , ' y to a DNA template. Upon ~.J~Ii ii~i;oa of a primer to the single-stranded DNA template, p~lr....,.~,i. synthesize DNA in the 5' to 3' direction, ~u~,c~ adding nucleotides to the 3 '-hydroxyl group of the growing strand.
Thus, in the presence of d~l il YJ ,. !~ i . ' . ' (dNTPs) and a primer, a new DNA molecule, . . ' y to the single stranded DNA template, can be synthesized.
A number of DNA pol~ have been isolated from mesophilic u~ ~ suchasl~ coli. AnumberofthesemesophilicDNApoly.. ~
have aiso been cloned. Lin et aL cloned and expressed T4 DNA polymerase m ~; coli (Proc. Natl. Aca~ Sci. USA 84:7000-7004 (1987)). Tabor et al. (U.S.
Patent No. 4,795,699) describes a cloned T7 DNA p.,l~ , while Miniciey ef aL (J. Biol. Chem. 259:10386-10392 (1984)) and Chatterjee (U.S. Patent No.
5,047,342) described 1~ coli DNA polymerase I and the cloning of T5 DNA
pol-ymerase, respectively.
Aithough DNA ~ ,~ from ;' . ' ' are icnown, relatively little ill . ~,~Li~iull has been done to isolate and even clone these enzymes. Chien et aL, J. BacterioL 127:1550-1557 11976) describe a ~Ju~ir~ Oli scheme for obtaining a polymerase from Thermus aquatiCUs (Taf~). The resulting protein had a molecuiar weight of about 63,000 daitons by gel filtration analysis and 68,000 daitons by sucrose glaciient f~ ;. ,.. Kaiedin ef aL, Biokhymiya 45:644-51 (1980) disclosed a purifcation procedure for isolating DNA polymerase from T. aquaticus YTI strain. The puri~ied enzyme was reported to be a 62,000 dalton protein. Gelfand et al. (~J.S. Patent No. 4,889,818) cloned a gene encodmg a Ih -"~ DNA polymerase from Thermus a(lua~iCus. The molecuiar weight of this protein was found to be about 86,000 to 90,000 daitons.Simpson et aL purified and partiaily I -.,~- lr,;, J a lh.. ,.,..-~-l,l. DNA
puly from a Thermotoga species (l~iochem. CelL BioL 86:1292-1296 (1990)). The purified DNA polymerase isolated by Simpson ef aL exhibited a . = . = ~

WO 96/10640 2 1 7 4 ~t 4 ~ o molecular weight of 85,000 daitons as determined by SDS-p~,ly~ ' gel cl~~ ;a and ~ CA~IU~;V~ The enzyme eAhibited haif-iives of 3 minutes at 95C and 60 minutes at 50C in the absence of substrate and its pH optimum was in the range of pH 7.5 to 8Ø Triton X-100 appeared to enhance the ;' ' ".y of this enzyme. The strain used to obtain the Ih~ v ,lhW~, DNA polymerase described by Simpson et al. was Thermotoga si~ esstrainFiss3-B l (Hussaretal., FEMSMi~"o~.olog,~Letters37:121-127 (1986)). Other DNA pc,4,.,.,,~,~, haYe been isolated from ~ .lllv~ lfil;~. bacteria including Baeillus ' ' , ' ' (Stenesh et aL, Biochim. Biophys. Aeta 272:156-166 (1972); and Kaboev et aL, J. Baetenol. 145:21-26 (1981)) and severai ~ ' species (Rossi et aL, System. ~ppL MicrobioL ?:337-341 (1986); Kiimczaic etaL, P .~ .y 25:48504855 (1986); and Elie etaL, Eur.
J. Biochem. 178:619-626 (1989)). The most eAtensively purified ~
DNA iivl~ had a reported haif-life of 15 minutes at 87C (Elie et aL
(1989), supra). Innis et aL, In PCR Protocol: A Guide To Methods and Al I 'i," , Academic Press, Inc., San Diego (1990) noted that there are several eAtreme Ih~ ' ' eubacteria and ~ ' ' that are capable of growth at very high . ~lu1~3 (Bergquist et al., Bioteeh Genet. Eng Rev.
5:199-244 (1987); and Kelly et aL, Biotechnol Prog. 4:47-62 (1988)) and suggested that these organisms may contain very ;' ' ' DNA p~l~.r.. ,.~cs.
In many of the i~nown p~ l.,,",e~, the 5'-3'; ' activity is presen~intheN-terminairegionofthepoiymerase. (Oiiis,etal.,Nature313:762-76v (1985); Freemont et al., Proteins 1:66-73 (1986); Joyce, Cur. Opin. Struet.
BioL 1:123-129 (1991).) There are some conserved amino acids that are thought to be responsible for the 5'-3 ' ~ activity. (Gutman & Olinton, NueL
Aeids iRes. 21:44064407 (1993).) These amino acids include TyrZ2, Glyl03, Gly~8', and Gly!-2 in E. eoli poly nerase L Any mutation of these amino acids would reduce 5'-to-3' . ' activity. It is icnown that the 5' l -.. 1. -- -- domain j5 r~ ,1, The best icnown exarnple is the Kienow fragment of E. eoli pU4~ ~ I. The Kienow fragment is a natural proteolytic firagment devoid of WO 96110640 PCT/US951123~8 O
.~7 d~44 5'-~. ' activjty (Joyce et. aL, J. BioL Chem. 257:1958-64 (1990).) rUl~,...,.a.,~ lacking this activity are useful for DNA s'T''nri 'g Most DNA pol~ ., also contain a 3'-5' ' activity. This activity provides a l~uurl~ ' ~ ability to the DNA p~ ,.aD~;. A
T5 DNA pul~ aD~ that lacks 3'-5' ' activity is disclosed in U.S.
Patent No. 5,270,179. ru7~ ~"., Iacking this activity are useful for DNA
5frl~f~nring The polymerase active site, including the dNTP binding domain is usually present at the carboxyl terminal region ol~ the polymerase (Ollis et al., Natl~re 313:762-766 (1985); Freemont et al., Proteins 1:66-73 (1986)). It has been shown that Phe~62 of ~ coli polymerase I is one of the amino acids that directlyinteracts with the nucleotides (Joyce & Steitz, Ann Rev. Biochem. 63:777-822 (1994); Astatke, J. BioL Chem. 270:1945-54 (1995)). Converting this amino acid to a Tyr results in a mutant DNA pOI~nl~ DC that does not ~' against d;~w~ . ~ f'~ and is highly processive. See copending U.S. Application No.
08/525,087, of Deb K. Chatterjee, filed September 8, 1995, entitled "Mutant DNA
Pul~ laDeD and the Use Thereof." which is expressly ~u~L~i herein by reference.
Thus, there exists a need in the art to develop l ~... ,..., _I ~l~ processive DNA p~l~ There also exists a need in the art to obtain wild type or mutant DNA POI~ DI~D that are devoid of; ' activities and are non-' ~ against .I;I:L,~,~. ~ l~ ,,l; l.
.~ ~y of t~e Invent~on The present mvention satisfes these needs in the art by providing additional DNA p~l~ useful in molecular biology. ~, - r ~ this invention mcludes a Ih. . 1~ DNA polymerase having a molecular weight of about 1001 ' - ' ' More specifically, the DNA polymerase of the invention is isolated from Thermotoga ~ (Tne). The 7'hermotoga species preferred for isolating the DNA polymerase of the present invention was isolated WO 96/10640 PCrtUS95112358 5 21 7~9~
from an African continental sohfataric spring (Wi~ ,.,, et aL, Arch. MicrobioL
151. 506-512, (19~9)).
The Tne DNA polymerase of the present invention is extremely , showing more than 50% of activity after being heated for 60 S minutes at 90C with or without detergent. Thus, the DNA p~ l c a~, of the present invention is more ~ than Taq DNA p~ly~ ae The present invention is also directed to cloning a gene encoding a Thermotoga I , ,ul~. ~ DNA polymerase enyme. DNA molecules containing the Tne DNA ~ol),lll~C gene, according to the present invention, can be ~ amd expressed in a host cell to produce a Tne DNA pGI~
having a molecular weight of 1001~ ' - ' ' ~ Any number of hosts may be used to express the Thermotoga DNA polymerase gene of the present invention;
including prokaryotic and eukaryotic cells. Preferably, I~uk~uyulic cells are used toexpresstheDNApolymeraseoftheinvention. Thepreferredl,.ul~lyuLi~,hosts according to the present invention is ~ coli.
The Tne DNA polymerase of the invention may be used in well known DNA sequencing (dideoxy DNA ~5ell~ nr~-~r,, cycle DNA sequencing of plasmid DNAs, etc.) and DNA ~ reactions.
The present invention is also directed to mutant 1~ DNA
p JI~ ~. More specifically, the mutant DNA ~ ,.,,,,.,., of the invention are derived from 7~ ~ ~ ~ ' and are ' "y reduced or devoid of

3'-5' P~ activity, 5'-3' . ~ activity, or is ' ,, against d;JW~ l - The present invention also relates to mutants having more than one ofthese properties, and DNA molecules containing the mutant Tne DNA polymerase en~me genes. These mutants may also be used in well kno vn DNA sequencing and DNA .~ reactions.

wo 96/10640 PCTlUSgS1123~8 ~1 49 44 Brief Description of flte Figures Figure I i the heat stability of Tne DNA polymerase at 90C
oYer time. Crude extract firom Thermotoga ~....1,.,1... ~ cells was used in the assay.
S Figure 2 shows the DNA polymerase activity in crude extracts from an E coli host containing the cloned Tne DNA polymerase gene.
Flgure 3 compares the ability of various DNA p~ to ~,~" al~
radioactive dATP and [aS]dATP. Tne DNA polymerase is more effective at nl/u~ [aS]dATP than was Taq DNA polymerase.
Figure 4 shows the restriction map of the ~ DNA fragment which contains the Tne DNA porymerase gene in pSport I and pUCI9. This figure also shows the region containing the O-helix 1..-"~ sequences.
Figure 5 shows the nucleotide and deduced amino acid sequences, in all 3 reading frames, for the carboxyl terminal portion, including the O-helix region, 15 of the Thermotoga , ~ polymerase gene.
Figure 6A ' ~ depicts the c.~- :.... 1;..A of plasmids pUC-Tne (3'-5') and pUC-Tne FY.
Figure 6B ' "~v depicts the ~,o.. ,.l u~,l;.,.l of plasmids pTrc Tne35 and pTrcTne FY.
Flgure 7 ~ ~t,~ depicts the l,OIIaLI U~ l- of plasmid pTrcTne35 FY
and pTrcTne535FY.
Flgure 8 L ' '- "~ depicts the .,. .- - ~., ,1 1;.~1- of plasmid pTTQTneS FY.

WO96110640 2 1 7~ P~ ;L~sS~

Detailed Descrip~ion of thePreferred F~ 5~;mor~t~
Def nitions In the description that follows, a number of terms used in ICI ' ' DNA t ' ' O.r are extensively utilized. In order to provide a clearer and consistent I ' ~ ofthe ~p~ and claims, including the scope to be given such terms, the fol]owing defmitions are provided.
Cloning vector. ~ plasmid, cosmid or phage DNA or other DNA
molecule which is able to replicate _ 'y in a host cell, and which is J by one or a smaU nun~ber of ~ . l. ^ . recognition sites at which such DNA sequences may be cut in a d ' '- fashion without loss of an essential biological function of the vector, and into which DNA may be spliced in order to bring about its replication and cloning. The cloning vector may furthercontain a marker suitable for use in the ~ r ' of cells l, '` ' with the cloning vector. Markers, for example, are IcL.a..y~.lillc resistance or ampicillin resistance.
vector. A vector similar to a cloning vector but which is capable of enhancing the expression of a Oene which has been cloned into it, after f~ into a host. The cloned gene is usually placed under the control of (i.e., operabb livked to) certain control sequences such as promoter sequences.
~ host. Any ~luh~yuL;~. or eukaryotic or llf~luulo~lhD-l which contains the desired cloned genes in an expression vector, cloning vector or any DNA molecule. The term "le ....l..., -- ' host" is also meant to include those host cells which have been genetically engineered to contain the desired gene on the host ' u"l~ ,o~..c or genome.
Elost. Any~,.uk~yul;voreukaryotic uulL thatistherecipient of a replicable expression vector, cloning vector or any DNA molecule. The DNA
molecule may contain, but is not limited to, a structural gene, a promoter and/or am origin of replication.

~1 4q44 Promoter. A DNA sequence generally described as the 5 ' region of a gene, located proximal to the start codon. At the promoter region, ~1....~_. ;~.liu..
of an adjacent gene(s) is initiated.
Gene. A DNA sequence that contains r '- necessary for expression of a polypeptide or protein. It includes the promoter and the structural gene as weD AS other sequences involved in expression of the protein.
Structural gene. A DNA sequence that is transcribed into messenger RNA that is then translated into a sequence of amino acids ,~ ,L~ L;c of a specific P~JIJ~ iJ~.
Iû Operably linked. As used herein means that the promoter is positioned to control the iA~itiatioD of expression of the l)Gl.~ ,L;dC encoded by the structural gene.
F. . ~ - Expression is the process by which a gene produces a polypeptide. It includes i . of the gene into messenger RNA (mRNA) and the translation of such mRNA into poly~ ,Li.:lc(s).
Pure. As used herein " ' "~ pure" means that the desired purified protein is essentially free from f.",~ cellular which are associated with the desired protein in nature.
~ ,, cellular r ' may include, but are not limited to, 1,~ ,,~,1. ,.. ,~, .. ".,,lrA r~, . :~.I.,.. ~rlr~ ~ or undesirable DNA p~l~.. -~
enzymes.
Prinner. As used herein "primer" refers to a single-stranded ;"", lrlr ~ Ar that is extended by covalent bonding of nucleotide monomers during , . r ' or p~l~ of a DNA molecule.
Template. rlhe term "template" as used herein refers to a double-stranded or single-stranded DNA molecule which is to be amplified, synthesized or sequenced. In the case of a double-stranded DNA molecule, d~,..A iu~ aliull of its strands to form a first and a second strand is performed before these molecules may be amplified, synthesized or sequenced. A primer,, , ' y to a portion of a DNA template is hybridized under appropriate conditions and the DNA pGI.~ ,r,~i of the invention may then synthesize a DNA molecule WO 96/10640 2 1 7 ~ 9 ~ ~ PCTrUS95,~2358 g

4 ~ y to said template or a portion thereo The newly synthesized DNA
molecule, according to the invention, may be equal or shorter in length than theoriginai DNA template. Mismatch , during the synthesis or extension of the newly synthesized DNA molecule may result in one or a number of S rnicn~ A base pairs. Thus, the synthesized DNA molecule need not be exactly y to the DNA template.
T r ~ ~ The term " ~UI.~ " as used herein means becoming a part of a DNA molecule or primer.
As used herein "~ ;...." refers to any in vifro method for increasing the number of copies of a nucleotide sequence with the useof a DNA pOly~ ac. Nucleic acid ~ . ~ r ' results in the illcul lJuld~iull of nucleotides mto a DNA molecule or primer thereby forming a new DNA molecule ~ - "~ to a DNA template. The formed DNA molecule and its tempiate can be used as tempiates to synthesrze additionai DNA molecules. As used herein,one , ' ~ reaction may consist of many rounds of DNA replication. DNA
;.... reactions include, for example, polymerase chain reactions (PCR).
One PCR reaction may consist of 30 to 100 "cycles" of .1 and synthesis of a DNA molecuie.
O~ " refers to a synthetic or natural molecule complising a covaientiy iini~ed sequence of nucleotides which are joined by a l.l~ - bond between the 3' position ofthe pentose of one nucleotide and the 5 ' position of the pentose of the adjacent nucleotide.
Nucleotide. As used ~erein "~lu~,lwl~J~" refers to a base-sugar-phosphate Nucieotides are units of a nucleic acid sequence (DNA
and RNA). The term nucleotide includes d~Ayl;bl~ such as dATP, dCTP, dUTP, dGTP, dTTP, or derivatives thereof Such derivatives include, for eAample, dm, [aS]dATP amd 7-deaza-dGTP The term nucleotide as used herein aiso refers to didevA~ - (ddNTPs) amd their derivatives lliustratedexamples of i;~i~,uAy~ "J~ include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. According to the present invention, a "llu~ ;d~,l' may be uniabeled or detectably labeled by o 7 ~9 ~ 4 -lo-well knovvn techniques. Detectable labels include, for eAvample, radioactive isotopes, fluorescent labels, ' ' labels, ' ' Iabels and enzyme labels.
T'' '- As used herein "Ih.. ~ " refers to a DNA
pol~ which is resistant to i"a.,liv~.LiOI. by heat. DNA pol~,l.. ,.~es synthesize the formation of a DNA molecule ~ y to a single-str~mded DNA template by eAvtending a primer in the S'-to-3 ' direction. This activity for mesophilic DNA pu~ may be inactivated by heat treatment. For example, T5 DNA pol~ activity is totally inactivated by exposing the enzyme to a i . ~ of 90C for 30 seconds. As used herein, a Ih. .".. ,~lAl,lr. DNA
polymerase activity is more resistant to heat ill~ iva~iUII than a mesophilic DNA
pUI,~ ,l a~e However, a Ih .,... ,~1 Al ,Ir DNA polymerase does not mean to refer to an enzyme which is totaUy resistant to heat illA~,~iv~,Lio.~ and thus heat treatment may reduce the DNA polymerase activity to some extent. A Ih- .1, ,. .~I ,.I ,li DNA
polymerase typicai~y wii~ also have a higher optimum t~ . aLul ~; than mesophilic DNA p~l~
II~- ; - - The terr,As h,S.Uli~Lio.." and ~yL ' ~" refers to the pairing oft~vo ~ y single-stranded nucleic acid molecules ~RNA and/or DNA) to give a double-stranded molecule. As used herein, two nucleic acid molecules may be hybridized, although the base pairing is not completely y. Accordingly, ' ' bases do not prevent llyblkli~iun of two nucleic acid molecules provided that ~ lul conditions, well known in the art, are used.
3'-to-5' F - Activity. "3'-to-5' 1- ,..~ activity" is an enzymatic activity well knowrl to the art. This activity is often associated with DNA ~,c.l~ , and is thought to be in~olved in a DNA replication "editing"
or correction S'-t~3~ F Act*ib. -5 '-to-3 ' I ,.. ,1 - - activity' is also an erlzymatic acti-vity well known in the art. This activity is often associated with DNA pol~.. ~,.~.. , such as E. cûli PolI and PolIII.

WO 96/10640 PcTtusssllt358 2 ~ 74 q4 ~
A "DNA pc,l~ , reduced in 3 '-to-S ' ~
activity" is defined herein as either (I) a mutated DNA polymerase that has about orlessthanlO%,orpreferablyaboutorlessthanl%,ofthe3'-to-S'. -, ~
activity of the GUII~ r 1' g unmutated, wild-type enzymel or (2) a DNA
polymerase having a 3'-to-S'; I specific activity which is less than about I unitlmg protein, or preferably about or less than 0.1 unitslmg protein. A unit of activity of 3 '-to-5 '; I is defned as the amount of activity that solubilizes 10 nmoles of substrate ends irl 60 min at 37C, assayed as described in the "BRL1989 Catalogue & Reference Guide", page S, with ~haI fragments of lambda DNA 3'-end labeled with [3HldTTP by terminal dc~,~.. u~,lc~ l transferase (TdT). Protein is measured by the method of Bradford, ~naL Biochem. 72:248 (1976). As a mGanS of . natural, wild-type T5-DNAP or T5-DNAP
encoded by pTTQI9-T5-2 has a specific activity of about 10 units/mg protein while the DNA polymerase encoded by pTTQ I 9-T5-2(Exo ) (IJ. S . 5,270,179) has IS a specific activity of about 0.0001 units/mg protein, or 0.001% of the specific activity of the unmodifiGd enzyme, a 105-fold reduction.
A "DNA ~ reduced in 5 '-to-3 ' i l activity" is defined herein as either (I) a mutated DNA p~l~ that has about or less than lO/O, or preferably about or less than l%, of the S '-to-3 ' ~ 4 activity of the .,~ ' g ummutated, wild-type enzyme, or (2) a DNA
polymerase having 5'-to-3'; -' specific activity which is less than about I unit mg proteinl or preferably about or less than 0.1 units/mg protein.
Both of these activities, 3'-to-5'; ' activity and 5'-to-3' rlr- activity, can be observed on sequencing gels. Active 5'-to-3' 1 ' activity will produce nonspecific ladders in a sequencing gel by removing nuclGotides from growing primers. 3'-to-S' .... ,.1...~ activity can bemeasured by following the ~. ,~, ,..1 ~;"" of ""l~ 1 d primers in a sequencing gel. Thus, the relative amounts of these activities, e.g. by comparing wild-typeand mutant ~vl, , can be deternnined from these ~ of the sequencing gel.

wo 96/10640 PCr/USg5112358 4 -12- ~
A Cloning and r of Therrnotoga 1- ' - DNA
r Ij, The Therr"otoga DNA polymerase of the invention can be isolated from any strain of Therrnotoga which produces a DNA polymerase having the molecular weight of about 100 kilodaltons. The preferred strain to isolate the gene encoding Therntotoga DNA polymerase of the present invention is T~rerrnotoga r~qn ' ~ The most preferred 'rfrerrnotoga neapolifana for isolating the DNA polymerase of the invention was isolated from an African continental solfataric spring (W;lld~ et aL, Arch MicrobioL 151:506-512 (1989) and may be obtained from Deutsche Sammalung von M;V~V~U~L und 7.1"' ' GmbH (DSM; German Collection of Mivlv~ul~ and Cell Culture) ~vl.v.v~dv. Weg Ib D-3300 Bl.lul.~vh.._;~, Federal Republic of Germany, as Deposit No. 5068.
To clone a gene encoding a 7'herrnotoga n~ or DNA pvl~ ~e of the invention, isolated DNA which contains the polymerase gene, obtained from Ihermotoga .-~ cells, is used to construct a I, ' DNA library in a vector. Any vector, well known in the art, can be used to clone the wild type or mutant Thermotoga .~ ) DNA pol,vmerase of the present invention.
However, the vector used must be compatible with the host in which the ., ' DNAlibrarywillbe '' Prokaryotic vectors for w~ l uvLi..L the plasmid library include plasmids such as those capable of replication ~n E. coli such as, for example, pBR322, CoEI, pSC101, pUC-vectors (pUC18, pUCI9, etc.: In: Molecular Cloning"4 LaboratoryManual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1982); and Sambrook et al., In: Molecular Cloning A Laboratory al (2d ed.) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989)). Bacillus plasmids include pC194, pC221, pC217, etc. Such plasmids are disclosed by Glyczan, T. In: T71e Molecular Biology Bacilli, Academic Press, York (1982), 307-329. Suitable Sl, ~ .; , plasmids include pLT101 (Kendall et al., J. Bacteriol 169:4177-4183 (1987)). r2 'l plasmids are reviewed by John et al., (Rad. Insec. DisO. 8:693-704 (1986)), and -WO96/10640 2~4944 ~ US9S112358 Igaki, (Jpn. J. BacterioL 33:729-742 (1978)). Broad-host range plasmids or cosmids, such as pCP13 (Da~zins and C1~41~4lb~.ly, J. Bac~erio~. 159:9-18, 1984) can also be used far the present invention. The preferred vectors for cloning the genes of the present invention are ~,. ukalyu~ic vectors. Preferably, pClP13 and pUC vectors are uæd to clone the genes of the present invention.
The preferred host for cloning the wild type or mutant DNA pUIy~ ,.~e genes of the invention is a prolc~uyotic host. The most preferred 1~ ul~yu~.., host is E. coli. However, the wild type or mutant DNA ~ol.~ genes of the present invention may be cloned in other y.u~4 yulic hosts including, but not lû limited to, Escherichia, Bacillus, Sl, r , P~- , Salmonella, Serrafia, and Proteus. Bacterial hosts of particular intere$ include E. coli DH10B, which may be obtained from Life T~r~ , Inc. (LTI) ((~ ' ' ~5, MD).
Eukaryotic hosts for cloning and expression of the wild type or mutant DNA pGI~ of the preænt invention include yeast, fungi, and cells. Expression of the desired DNA polymerase in such eukaryotic cells may require the use of eukaryotic regulatory regions which include eukaryotic promoters. Cloning arld expressing the wild type or mutsnt DNA pu~
gene of the invention in eukaryotic cells may be ~ . u~ I by well known techniques using well known eukaryotic vector systems.
Once a DNA library has been ~u.l,l-u~,~cd in a palticular vector, an appropriate host ;5 r ~ by well known techniques. T r I colonies are plated at a density of ayu~ 200-300 colonies per petri dish. Colorlies are then screened for the expression of a heat stable DNA ~ul.~ , by L r - i r I E. coli coloniesto ucellulosc ' Afterthe transferred cells are grown on . " ' (r~ / 12 hours), the cells are Iyæd by stsndard techniques, and the membranes are then treated at 95C for 5minutestoinactivatethe Iir~ E. coli enzyme. Other ~ ,u~41u~t~ may be used to inactivate the host p~lJII..,.,.,,~,~ depending on the host used and the : . ,.IL.l~ stability of the DNA polymerase to be cloned. Stable DNA
p~l,,,...~"4~i activity is then detected by assaying for the presence of DNA

WO 96/10640 PCTtUS95tl23~8 q ~ 4 -14~
polymerase activity using weii known techniques. Sagner et aL, Gene 9 ':119-123 (1991), which is hereby ;lI~UllJUlalC i by reference in its entirety. The gene encoding a DNA pvly of the present invention can be cloned using the procedure described by Sagner et aL, suprcl.
The .~ ' host containing the wild type gene encoding DNA
pc)l~ , E. coli DHIOB (pUC-Tne), was deposited on September 30, 1994, with the Patent Culture Collection, Northern Regionai Research Center, USDA, 1815 North University Street, Peoria, IL 61604 USA as Deposit No. NRRL
B-21338.
If the Tne DNA polymerase has 3 '-to-5 ' . . . " ,. ,. 1. --- activity, this activity may be reduced, ' "~, reduced, or eliminated by mutating the Tne DNA
polymerase gene. Such rnutations include point mutations, frame shift mutations,deletions and insertions. Preferably, the region of the gene encoding the 3 '-to-5 ' ." " 1 .1 activity is deleted using techniques well known in the art (Sambrooket aL, (1989) in: Molecular Cloning, A raborator,v Manual ~2nd Ed.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).
The 3 '-to-5 ', ' activity can be deleted by creating site specific mutants within the 3'-5' . ' domain. See inJra. In a specific ; b. " of the invention Asp3n of Tne DNA polymerase was changed to Aia3n to ' '~ reduce 3~-to-5~ activity.
The 5'-3' .. 1.-~- activity of the Tne DNA polymerase can be reduced or eliminated by mutating the ~ne DNA polymerase gene. Such mutations inciude point mutations, fr~ne shifr mutations, deletions, and insertions.
Preferably, the region of the gene encoding the 5'-3', ... ...lf A ~' activity is deleted using techniques weii known in the art. In c~bG " ' ofthis invention, certain conserved amino acids that are associated with the 5'-3' .... ..1~ .
activity can be mutated. Exarnples of these conserved amino acids include Gly3'.In other . ~ " , the entire 5 '-3 '; ' domain of e.g, Tne or Tma polymerase can be deleted by proteolytic cleavage or by genetic c ~ 2 For example, a unique SphI restriction site carl be used to obtain a clone devoid of W096/10640 2 1 7 4 9 4 4 PcrlUS95112358 ~ -15-PotiAP~ encoding the 219 arnino terminal amino acids of Tne DNA
polyrnerase. Examples of such a clone are pTTQTne53 5FY and pTTOTne5FY.
Tne DNA polymerase mutants can also be made to render the polymerase non-~" ~ against ~ r-.rlPotiA~ such as did~,u~y. ~
By way of example, one Tne DNA polymerase mutant having this property substitutes a Tyr for Phe at amino acid 67 as numbered in Figure 5. Other changes within the O helix of various pc,l~...."~ such as other point mutations,deletions, and insertions can also be made.
B. F~' ' r, Of rhermotoga ~ D~A
l o rt /j - ~e To optimize expression of the wild type or mutant Therr~otoga DNA
yul~ ~ ofthe present inventio4 mducible or constitutive promoters are well known and may be used to express high levels of a polymerase structural gene in a 1~ ' host. Similarly, high copy number vectors, well known in the art, may be used to achieve high levels of expression. Vectors having an inducible high copy number may also be useful to enhance expression of 17termo~oga DNA
polymerase in a It ' ' ' host.
To express the desired structural gene in a I~IULlyU~iC cell ~such as, ~ coli, B. sub~ilis, ~ ' , etc.), it is necessary to operably link the desired structural gene to a functional ~ yulic promoter. However, the natural 17~err,totoga r y~ promoter rnay function in ~uk~l-yuL;c hosts allowing expression of the ~ gene. Thus, the natural Therr~otoga promoter or other promoters may be used to express the DNA polymerase gene. Such other promoters may be used to enharlce expression and may either be cù...,.ill.Liv~ or regulat~ble ~I.e., inducible or J~,.~,.~,.. ,;~lc) promoters. Examples of Cull~LiLuLiv~
promoters include the in~ promoter of l . ~ A, and the bla promoter of the~-lac~amasegeneofpBR322. Examplesofinducible~.lukd.yuLi~,promoters include the major right and lef promoters of ~ ,. A (PL and PR)~ bp, recA, lacZ, lacL gal, trc, and tac promoters of ~: coli. The B. subtilis promoters include a-arnylase (IJlmanen etal., 1 Bacteriol 162:176-182 (1985)) and Bacillus _ _ _ . . . ... . _ WO 96/10640 PCr/US9~/12358 ~74944 -16~
UP~ promoters (Gryczan, T., In: The Molecular Bio~ogy Of ~acilli, Academic Press, New York (1982)). Sl" ~ .. promoters are described by Ward ef aL, MoL Gen Genef. 203:468478 (1986)). P~U~YUI;G promoters are also reviewed by Glick, J. Ind. Microbiol. 1:277-282 (1987); ~t~ tit~rt~l, Y., Biochimie 68:505-516 (1986); and Gottesman, Ann. Rev. Genet. 1~:415-442 (1984). EApression in a prokalyotic cell also requires the presence of a ribosomal binding site upstream of the gene-encoding sequence. Such ribosomal binding sites are disclosed, for example, by Gold ef aL, Ann Rev. Microbiol. 35:365404 (1981). --~
To enhance the expression of Tne DNA polymerase in a eukaryotic cell, well known eukaryotic promoters and hos~s may be used. Preferably, however, enhanced eApression of Tne DNA polymerase is . ' ' ' in a ~,. uk~uyuli~, host. The preferred ~: l uh~uyuli~. host for U . _~A~ ' r this enzyme is ~ coli.
C Isolat~'on and 1~,;^ ' of Thermotoga ~ DNA
P. ~ ~e The enzyme(s) of the present invention (Thermo~oga I , ' . DNA
polymerase, Tne, and mutants thereof) is preferably produced by r . . of the ,t, ' host containing and GApressing the cloned DNA polymerase gene.
However, the wild type and mutant Tne DNA pGI.~ of the present invention may be isolated from any Thermofoga strain which produces the polymerase of the present invention. Fragments of the Tne polymerase are also included in the present invention. Such fragments include proteolytic fragments and fragments having polymerase activity.
Any nutrient that can be assimilated by Thermofoga non.~ ;t~ttt or a host containing the cloned Tne DNA polymerase gene may be added to the culture medium. Optimal culture conditions should be selected case by case according to the strain used and the, , of the culture medium. Antibiotics may also be addGd to the growth media to insure of vector DNA containing the desired gene to be expressed. Culture conditions for Thermo~oga ~en~ ' have, for example, been described by Huber ef aL, Arch MicrobioL 144:324-333 wo 96/10640 2 1 7 ~ 9 4 ~ Pcrlu595112358 (1986). Media 1~ are also described in DSM or ATCC Catalogs and Sarnbrook e~ al., In: Molecl~lar Cloning"4 Labora~oryMan~al (2nd ed.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).
Thermo~oga ~ , ' - and .~ ' host cells producing the DNA
polyrnerase ofthis invention can be separated from liquid culture, for example, by ~ g In general, the collected microbial cells are dispersed in a suitable buffer, and then broken down by ultrasonic treatment or by other well known procedures to allow extraction of the enzymes by the buffer solution. After removal of cell debris by 1' '- ' _ the DNA
p~ can be purified by standard protein purification techniques such as extraction, ~,.~,;~,;L~,Lio." ~,1"..-- ~ ,~,, .1.l.~, affinity ull~ .hy, cl~L~ or the like. Assays to detect the presence of the DNA polymerase during purification are well known in the art and can be used during .,u....
b;u~ ;. . methods to determine the presence of these enzymes.
D. Usesof Thermotoga r . ' ~DNA 1~ S
The wild type and mutant Ihermotoga r~ DNA pGI~
(Tne) ofthe present invention may be used in well known DNA ~PqllPnri~ DNA
labehng, and DNA , ' ' reactions. Tne DNA polymerase mutants devoid of or 9 ' '~ reduced in 3'- 5' ~ A`- activity, devoid of or ' ' '~y reduced in 5 '- 3 ' ~ activity, or containing a Phe6~-Tyr6~
mutation are especially useful for DNA seqll~pnri~g~ DNA labelmg, and DNA
~" r ~ .. reactions. Moreover, Tne pul~ .la~, mutants contairling two or more of these properties are also especially useful for DNA ~Pq~Pnring, DNA
labeling, ûn DNA . -r ~' reactions. As is well known, sequencing reactions (dideoxy DNA sequenGing and cycle DNA sequencing of plasmid DNA) require the use of DNA ~vl~ Dideoxy-mediated sequencing involves the use of a chain-termination technique which uses a specitic polymer for extension by DNApGI,~ aO~ a base-specific chain terminator and the use of pul~ ' ' - gels to separate the newly synt-hesized ~ ' : ' DNA molecules by size so that ~7 4944 -18- O~D
at least a part of the nucleotide sequence of the original DNA molecule can be d~ ' Specifically, a DNA molecule is sequenced by using four separate DNA sequence reactions, each of which contains different base-specific For example, the first reaction will contain a G-specific terminator, the second reaction will contain a T-specific terminator, the third reaction will contain an A-specific terminator, and a fourth reaction may contain a C-specificterminator. Preferred terminator nucleo~ides include did~A,~Ii~ ' '~
i . ' . ' (ddNTPs) such as ddATP, ddTTP, ddGTP, and ddCTP. Analogs of d;d~ . i . ' . ' may also be used and are well known in the art.
When sequencing a DNA molecule, ddNTPs lack a hydroxyl residue at the 3 ' position of the dc~,~.il,~, .~ base and thus, although they can be ~,. J
by DNA ~ ,.~ into the growing DNA chain, the absence of the 3 '-hydroxy residue prevents formation of a i ' . ' ' bond resulting in i of extension of the DNA molecule. Thus, when a small amount of one ddNTP is included in a sequencing reaction mixture, there is . . between extension ofthe chain and base-specific termination resulting in a population of synthesized DNA molecules which are shorter in length than the DNA template to be sequenced. By using four different ddNTPs in four separate enzymatic reactions, populations ofthe synthesized DNA molecules can be separated by size so that at least a part of the nucleotide sequence of the original DNA molecule can be d: ' DNA sequencing by dideoxy-nucleotides is well known and is described by Sambrook ef aL, In: Molecular Cloning, A Laborafory Mam al, ColdSpringHarborLaboratoryPress,ColdSpringHarbor,N.Y.(1989). Aswill be readily recognized, the Tne DNA polymerase of the present invention may be used in such sequencing reactions.
As is well known, detectably labeled nucleotides are typically included in ~, ~ reactions. Any number of labeled nucleotides can be used in sequencing (or labeling) reactions, includmg, but not limited to, radioactive isotopes, fluorescent labels, ' ' Iabels, ' ' Iabels, and enzyme labels. It has been discovered that the wild type and mutant Tne DNA

W096110C40 2 ~ 7 4 9, 4 ~ 5~
'9- .
P('l.t .,~C of the present invention may be useful for ;.~.,ù.~,u.~.Lil.~ aS
nucleotides ([aS]dATP, [aS]dTTP, [aS]dCTP and [aS]dGTP) during sequencing (or labeling) reactions. For example, [a35S]dATP, a commoniy used detectably labeled nucleotide in sequencing reactions, is ;..c~.l,u.~ i tLlree times more efficientiy with the Tne DNA p~,l~ .. ,.~ ,., of the present invention, than with Taq DNA polymerase. Thus, the enzyme ofthe present invention is pal ' I~, suited for sequencing or labeling DNA molecules with [a3sS]dNTPs.
rùl.~ chain reaction (PCR), a well known DNA ~
technique, is a process by which DNA polymerase and d~ y~ Ir i , ' , ' are used to ampiify a target DNA template. In such PCR reactions, two primers, one ~ . ' y to the 3 ' termini (or near the 3 '-termini) of the first strand of the DNA molecule to be amplified, and a second primer , ' y to the 3 ' termini (or near the 3 '-termini) of the second strand of the DNA molecule to be amplified, are hybridized to their respective DNA
molecules. After ~ iUll~ DNA polymerase, in the presence of d~y~ ;, ' , ' , ailows the synthesis of a third DNA molecule '~"" .1,1~ " " - y to the first strand and a fourth DNA molecule ~ , ' y to the second strand of the DNA molecule to be ampiified. This synthesis results intwo double stranded DNA molecuies. Such double stranded DNA molecuies rnay then be used as DNA templates for synthesis of additionai DNA molecules by providing a DNA p~l~ , primers, and d~,u~LyliL ' '- i, ' , ' As is weil known, the additional synthesis is carried out by "cycling" the original reaction (with excess primers and dw~ ,1,.,...,. l....,:~l~ i , ' , ' ) allowingmuitiple denaturing and synthesis steps. Typicaily, denaturing of double stranded DNA molecuies to form single stranded DNA templates is: , ' ' ' by high ~,.,. The wild type and mutant Thermologa DNA pGI~ of the present invention are heat stable DNA pûlymerases, and thus will survive such thermal cycling during DNA ~ , ' ' reactions. Thus, the wiid type amd mutant Tne DNA pvl~ ~,.. of the invention are ideaily suited for PCR
reactions, pcll~ uLly where high t~ d~UI~i are used to denature the DNA
molecules during , ,-r .

C~ 4 4 -20~
E. ~its The wl1d type and mutant 77termotoga 1:, ' (Tne) DNA
POIJ of the invention are suited ~or the preparation of a kit. Kits e the wild type or mutant Tne DNA pul~ 's) may be used for detectably labeling DNA molecules, DNA seqll~nrin~ or amplifying DNA
molecules by well known tecbniques, depending on the content of the kit. Such kits may comprise a carlying means being UUIII~J- i '' ' to receive in close one or more container means such as vials, test tubes and the like.
Each of such container means comprises uu , ' or a mixture of .
needed to perform DNA ~ ~ ~ DNA labeling, or DNA ~
A kit for sequencing DNA may comprise a number of container means.
A frst container means may, for example, comprise a ' '1~, purified sample of Tne DNA polymerase having the molecular weight of about 100 kilodaltons or a mutant thereof. A second container means may comprise one or a number of types of nucleotides needed to synthesize a DNA molecule ~.. ,l.l~ .. ~l_~y to DNA template. A third container means may comprise one or a number different types of d;dcv~.. ,l~idc i . ' . ' In addition to the above container means, ad&tional container means may be included in the kit which comprise one or a number of DNA primers.
A kit used for amplifying DNA will comprise, for example, a first container means comprising a ' '1~ pure mutant or wild type Tne DNA polymerase and one or a number of additional container means which comprise a single type of nucleotide or mr~tures of mlrl.ootir~ Various primers may or may not be included in a kit for amplifying DNA.
When desired, the kit of the present invention may also include .,ontainer means which comprise detectably labeled nucleotides which may be used during the synthesis or sequencing of a DNA molecule. One of a number of labels may be used to detect such n-l.-lPoti~c Illustrative labels include, but are not limited to, radioactive isotopes, fluorescent labels, ' ' Iabels, ' ' Iabels and enzyme labels.

wo96110640 ,~llO~ sa~
~ 21 21 74'344 HaYing now generally described the invention, the saune will be more readily understood through reference to the following Elxamples which are proYided by way of illustration, and are not intended to be limiting of the present inYention, unless specified.
S Example 1: Bacter~al StrainsAnd Growtl~ C'o. ~;R~-, Thermofoga ~ r DSM No. 5068 was grown under anaerobic conditions as described in the DSM catalog (addition of res~urin, Na2S, and sulfurgranuleswhilespargingthemediawithnitrogen)at85Cinanoilbathfrom 12 to 24 hours. The cells were harvested by filtering the broth through Whatman #1 filter paper. The supernatant was collected in an ice bath and then centrifuged in a refiigerated centrifuge at 8,000 rpms for twenty minutes. The cell paste was stored at -70C prior to total genomic DNA isolation.
~. coli strains were grown in 2X LB broth base (Lermox L broth base:
GIBCOIBRL) medium. T r ~ cells were incubated in SOC (2% tryptone, 0.5% yeast extract, yeast 10 mM NaCI, 2.5 M KCI, 20mM glucose, IOrnM MgCI2, and lOmM MgS04 per liter) before plating. When ~,..,~.ir~le antibiotic --rrl~ were 20 mg/l l~ ~. " and 100 mg/l ampicillin. E coli strain DHIOB (Lorow ef aL, Focus 12:19-20 (1990)) was used as host strain.
Competent DHIOB may be obtained from Life T ' ' ~, Inc. (LTI) (~ 1JUI~;7 MD).
Example 2: DNA Isolation ~ermofoga ~ k~ IUUI~ VUI~I DNA was isolated firom l.lg of cells by suspending the cells in 2.5 ml TNE r50mM Tris-HCI, pH 8.0, SOmM
NaCI, IOrnM EDTA) and treated with 1% SDS for 10 minutes at 37C. DNA
was extracted with phenol by gently rocking ~he Iysed cells overnight at 4~C. The next day, the Iysed cells were extracted with .,I.lo., r ' yl alcohol. The resulting .,1.. , ' DNA was further purified by c~lILI î,, in a CsCI

~ ~ 7 ~q 4 4 -22- ~ID
density gradient. CLIulllosulllal DNA isolated from the density gradient was extracted three times with ;sv~,.u,u~...ul and dialyzed overrlight against a buffer containing 10 mM Tris-HCI (pH 8.0) and I mM EDTA.
F~ 3: Construction ~7f Geno~nic Libraries The ~hlU.IIOSOIII~II DNA isolated in Example 2 was used to construct a genomic library in the plasmid pCP 13 . Brieily, 10 tubes each containing I llg f ~henno~oga~ ,I..ulll~ulllalDNAwasdigestedwithO.OI to lOunits of Sau3AI for I hour at 37C. A portion of the digested DNA was tested in an agarose (1 2%) gel to determine the extent of digestion. Samples with less than 50% digestion were pooled, ethanol pl~ alt;d and dissolved in TE. 6.5 ,ug of partially digested ,LI UllluSullldl DNA was ~igated mto 1.5 ~g of pCP13 cosmid which had been digested with BaniHI restriction .. 1,.".. 1. . and L~d with calf intestinal alkaline l' .' Ligation of the partially digested Therrnotoga DNA and BamHI cleaved pCP13 was carried out with T4 DNA ligase at 22C for 16 hours. After ligation, about l~g of ligated DNA was packaged using A-packaging extract (obtained from Life T~ ç,: , Inc., t~ ' ' ," MD). DHIOB cells (Life Tech. Inc.) were then infected with 100 ~1 ofthe packaged material. The infected cells were plated on l~LIa~,y. ' containing plates. Serial dilutions were made so that a~ 'y 200 to 300 ~la~l ' resistant colonies were obtaine~ per plate.
Exan~71e4: ScreeningforClonesE~ T~t ".~lv~., ~ - ~,7, '-~r~ DNA Poly~nerase T ' " - ofthe Thennotoga ~ DNA polymerase gene of the invention was cloned using the method of Sanger et aL, Gene 97:119-123 (1991) which reference is herein IIIWIIJI ' ~ i111 its entirety. Briefly, the E cofi tetracycline resistant colonies from Example 3 were transferred to IU~I u~ uh~e membranes amd aUowed to grow for 12 hours. The cells were then Iysed with the WO 96110640 2 17 4 9 4 4 r ~

fumes of ' ~ P (1:1) for 20 minutes and dried for 10 minutes at room l~ L~. The membranes were then treated at 9~C for S minutes to inactivate the . ~.. ,~ E coli enzymes. Surviving DNA polymerase activity was detected by ~ul,.~;;ng the membranes in 15 mi of polymerase reaction mix (50 mM Tris-HCI (pH 8.8), I mM MgCk, 3 mM ~ .,. ' l, 10 ~M
dCTP, dGTP, dTTP, and 15 ~lci of 3,000 Ci/mmol [a32P]dATP) for 30 minutes at 65C.
Using - lo~, l;.J~,-l,l.y, three colonies were identifed that expressed a l~ermotoga I , ' DNA P~IJ The cells were grown in iiquid culture and the protein extract was made by sonication. The presence of the cloned Ih . .~ polymerase was confrmed by treatment at 90C followed by ,a~ul~ of DNA p~ ,la~l, activity by il~ ioll of radioactive d~. i~ i . ' . ' into acid insoluble DNA. One of the clones, expressing Tne DNA polymerase, contained a plasmid designated pCP13-32 was used for further study.
Example 5: Subcloning of Tne DNA poly--- ,~e Since the pCP13-32 clone expressing the Tne polymerase gene contains about 25 kb of 7~ J~ DNA, we attempted to subclone a smailer fragment oftheTnepolymerasegene. ThemolecularweightoftheTnepolyl"~"a~epurified from E coli/pCP13-32 was about 100 kd. Therefore, a 2.5-3.0 kb DNA fragment will be suf'dcient to code for full-length polymerase. A second round of 5~u3A
partiai digestion similar to Example 3 was done usirlg pCP13-32 DNA. In this case, a 3 .5 kb region was cut out from the agarose gel, purif ed by Gene Clean tBIO 101, La Joiia, CA) and iigated into plasmid pSport I (Life T-. I.,..~l..~,. c, Inc.) which had been linearized with BamHI and d~llc,~L,l.~J~' ' with caif intestinai I ' . ' After iigation, DHIOB was i '' and colonies were tested for DNA pvl~ a ,~ activity as described in Example 4. Severai clones were identifed that expressed Tne DNA polymerase. One of the clones (pSport-Tne) containing about 3 kb insert was fulther ~,ll~a~ ;liL~i. A restriction ~ ~ 7 ~ ~ 4 4 -24- O~ID
map of the DNA fragment is shown in Fig. 4. Further, a 2.7 Kb l~indIII-Sstl fragment was subcloned into pUC19 to generate pUCI9-Tne. Æ coli/pUCI9-Tne aiso produced Tne DNA pG~
The Tne ~ul~ aa~ clone was sequenced by methods i~nown in the art.
The nucleotide seciuence obtained of the 5' end prior to the start ATG is shown in SEQ ID NO:I. The nucleotide sequence obtained which encodes the Tne polymerase is shown m SEQ ID NO:2. When SEQ ID NO:2 is translated it does not produce the entire amino acid sequence of the Tne polymerase due to frame shift errors in the nucleotide sequence set forth in SEQ ID NO:2. However, an amino acid sequence of the Tne polymerase was obtained by transiating ail three reading frames of SEQ ID NO:27 comparing these sequences with i~nown pol~ amino acid sequences, amd splicing the Tne polymerase sequence together to form the amino acid sequence set forth in SEQ ID NO:3.
Example 6: Purif cation oSTI2ermotoga .~ I;f~r1~ DNA
P~l~ ,~efromE. coli Twelve grams of ~ coli ceUs expressing cloned Tne DNA p~ ,.aa~
(DHlOB/pSport-Tne) were Iysed by sonication (four thirty-second bursts with a medium tip at the set~ing of nine with a Heat Systems Uitrasonics Inc., model 375 sonicator) in 20 mi of ice cold extraction buffer (50 mM Tris HCIl pH 7.4, 8%
glycerol, 5 mM l ~-r ~- " 1, 10 mM NaCI, I mM EDTA, 0.5 mM PMSF).
The sonic$ed extract was heated at 80C for 15 min. and then cooled in ice for

5 min. 50 mM KCi and PEI (0.4%) was added to remove nucleic wids. The extract was centrifuged for ~ ;.... Arnmonium sulfate was added at 60%, the pellet was coliected by ~ n ;( ll l and ~.,~ cd in 10 mi of column buffer (25 mM Tris-HCI, pH 7.4, 8% glycerol, 0.5% EDTA, 5mM
2-merrqrtoet~ -l, 10 mM KCI). A Blue-Sepharose (Pharmacia) column, or preferably a Toso heparin (Tosohaqs) column, was washed with 7 column volumes of column buffer and eluted with a 15 column volume gradient of buffer A from lOmM to 2 M KCI. Frwtions containing polymerase activity were pooled. The . . , , _ _ _ _ _ _ _ . . _ . _ . . _ . . _ WO96/10640 2 1 7 1~ 9 ~ ~ p~~ aa~

fractions were dialyzed against 20 volumes of column buffer. The pooled firactions were applied to a Toso650Q column (Tosohaas). The column was washed to baseline ODz, o and elution effected with a linear 10 column volume gradient of 25 mM Tri$ pH 7.4, 8% glycerol, 0.5 mM EDTA, 10 mM KCI, 5 mM
,~ l to the same buffer plus 650 mM KCI. Active fractions were pooled.
Example 7: Characterization of Purif ed Tne DNA Polytnerase 1. 1~ ofthcMolecular Weightof Thermotoga DNA ~L 'j ~-The molecular weight of 100 kilodaltons was determined by elc~,l.o, ' ~,~;a in a 12.5% SDS gel by the method of Laemmli, U.K., Na~ure (Lond ) 227:680-685 (1970). Proteins were detected by staining with Coomassie brilliant blue. A 10 kd protein ladder (Life T,~ , Inc.) was used as standard.
2. Mrthodfor MeasuAng r , _ . of 1a~551-dATP
Relatlve to /I~-~ArP
L.~ la~;ull of [~S]dATP was evaluated in a final volume of 500 ~l of reaction mix, which was ~ ' ' at 72C for five minutes, containing either a [3H]TTP nucleotide cocktail (100 ,uM[ each TTP, dATP, dCTP, dGTP with [3H~TTP at 90.3 cpm/pmol), a nucleotide cocktail containing ~aS]dATP as the only source of dATP (100 ,uM each [aS]dATP, dCTP, dGTP, TTP with [~35S]dATP at 235 cpm/pmol), or a mixed cocktail (50 ,uM [aS]dATP, 50 ,uM
dATP, 100 ,uM TTP, 100 IlM dCTP, 100 ,uM dGTP with [35aS] dATP at 118 cpm/pmol and [3HlTTP at 45.2 cpm/pmol). The reaction was initiated by the addition of 0.3 units of T. ~ , ' DNA ~ ae or T. aquaticus DNA
polymerase. At the times indicated a 25 111 aliquot was removed and quenched by addition of ice cold EDTA to a final, of 83 mM. 20 ~1 aliquots of _ _ _ _ wo 961106~0 r~
~1 49~-4 -26- 0 the quenched reaction samples were spotted onto GF/C fiiters. Rates of v~lioll were compared and expressed as a ratio of T. `~ ! / to T. aquaticus. The ~ol~lLio.. of ta3'S]dATP by T. . ~ DNA
was three-fold higher than that of T. aquaticus DNA polymerase.
s Example8: Reverse Tr ~ Ac~ivi~y (A)~:(dT)12 ~8 is the synthetic tempiate primer uscd most frequently to assay for reverse ~ activity of DNA pGI~ oco. It is not specific fo}
rewroviral-like reverse l . ~ , howe~er, being copied by many ~nui d-y~
andeukaryoticDNApGI~ wO(ModalcandMarcus,J. BioL Chem. 252:11-19 (1977);Gerardetal.,Biochem. 13:1632-1641 ~1974);SpadariandWeissbach7J.
BioL Chem. 249:5809-5815 (1974)). (A)n:(dT)Iz.l, is copied particularly well by cellular, replicative DNA pGI~ o in the presence of Mn~7 and much less effiwentiy in the presence of Mg~ (Modaic and Marcus7 J. BioL Chem. 252:11-19 (1977);Gerardefal.7Biochem. 13:1632-1641 (1974);SpadariandWeissbach,J.
BioL Chem. 249:5809-5815 (1974)). In contrast, most cellular7 replicative DNA
pc Iy~w~o do not copy the synthetic template primer (C)~:(dG)Iz l, efficiently in presence of either Mn~ or Mg~7 but retroviral reverse i , do.
Therefore7 in testing for the reverse ~ hCf' activity of a DNA polymerase with synthetic template primers, the stringency of the test increases in the foDowing manner from least to most st~ingent: (A)~:(dT)12.,3 (Mn~) < (A)n:(dT),z.
(Mg~) (C)n:(dG)Iz ~ (Mn~) < (C)~ (dG)~z ~ (Mg~.
The reverse l . hl I - .. ;1.l h~ activity of Thermotoga r~n~ 7 (Tne) DNA
pOI,~ wao~ was compared with Thermus .,1~, , ' ' (Tth) DNA polymerase utiiizing both (A)~:(dT)20 and (C)n:(dG)Iz l,. Reaction mixtures (50 111) with (A)n (dT)20 contained 50 mM Tris-HCI (pH 8.4)7 100 IIM (A)n7 100 ,uM (dT)Zo~
and either 40 mM KC17 6 mM MgCI27 10 rnM dithi. " ~ ' 17 and 500 ~lM
[3H]dTTP (85 cpm/pmole)7 or 100 nlM KC17 I mM MnCI27 and 200 !IM
[3H]dTTP (92 cpm/pmole). Reaction mixtures (50 111) with (C)n:(dG)Iz.l, contained 50 mM Tris-HCI (pH 8.4)7 60 ,uM (C)~, 24 yM (dG)I2.l,, and either 50 Ss /12358 WO g611~640 PCTIU S
2 ~ 4 rbM KCI, 10 mM MgC12, 10 mM J;Il:OLh.~;.OI~ and 100 ,uM [3HldGTP (132 ;, 'ylllol~), or 100 rnM KCI, 0.5 mM MnCI2, and 200 ,uM [3HldGTP (107 cpm/pmole). Reaction rnixtures also contained either 2.5 units of the Tth DNA
polymerase (perkin-Elmer) or 2.5 units of the Tne DNA p~lJ~ . Trlrvh~ nc S were at 45 C for 10 min followed by 75 C for 20 min.
The table shows the results of ~' ~ the relative levels of of Tne amd Tth DNA polymerase with (A)~:(dT)2o and (c)n:(dG)l2-l~
in the presence of Mg' t and Mn~. Tne DNA polyrnerase appears to be a better reverse ll , than Tth DNA p~ymerase under reaction conditions more specific for reverse ~ i ~, i.e., rn the presence of (A)n:(dT)2c with Mg~ and (C)n:(dG),2." with Mn~ or Mg~.
DNA Pobmerase Activity of Tth nrld Tne DNA Polymerase with (A).:(dT)20 nnd (C~:(dG),. ,.
DNA Polymerase Activity (pMoles ~, ' y [3HldNTP Incorpor,2ted) ~ne (A),:(dT)20 (C)~:(dG) M~ Mn~ M~ Mn~
Tne 16~.8 188.7 0.6 4.2 Tth 44.8 541.8 0 0.9 9: Construction of Tllermotoga N~lrolifnr~ 3 '-to-S ' F - '~ ~ Mutant The amino acid sequence of portions of the Tne DNA ~..,1~ was compared with other known DNA p~lyll~ ,3 such as E. col~ DNA pol~
1, Taq DNA ~,It...~..~e, T5 DNA polymerase, and T7 DNA p~ .~ to localize the regions of 3'-to-5' l - ,.,~ activity, and the dNTP binding domains within the DNA polymerase. We have determined that one of the 3'-to-5' domains based on the comparison of the amino acid sequences of ~ ;1 4 -28- PCTNS9S/12358 various DNA ~ .o (Blanco, L., et al. Gene 112: 139-144 (1992);
Braithwaite and Ito, NucleicAcidsRes 21: 787-802 (1993)) is as follows:
*
Tne 317 PSFALDLETSS 327 (SEQ.lDNO.4) S PolI 350 PVFAFDTETDS 360 (SEQ. IDNO. s;r ~ ~.. andIto, s~lpra) T~ 133 GPVAFDSETSA 143 (SEQ.ID.NO 6;BraithwaiteandIto, supra) T7 1 MIVSDIEANA 10 (SEQ. ID.NO. 7; r~. .. andIto, supra).
As a first step to make the Tne DNA polymerase devoid of 3'-5' eY~ 2e activity, a 2kb Sph fragment from pSport-Tne was cloned into M13mpl9 (LTI, C~L~ OIJUI~ MD). The l~.u~L;~ ; clone was selected in E.
coli DH5c~F'IQ (LTI, (' ' ' ~, MD). One of the clones with the proper insert was used to isolate uracilated smgle-stranded DNA by infecting E. coli CJ236 (Biorad, California) with the phage particle obtained from E. coli DHSaF'IQ. An n~ ;ul;~lr~ GA CGT TTC AAG CGC TAG GGC AAA
AGA (SEQ ID ~O. 8) was used to perform site directed ., ...~ This site-directed .,.~ converted Asp323 (indicated as * above) to Ala3''. An Eco47III restriction site was created as part of this ~ to facilitate screeningofthemutantfollowing~ l c, \r-~ The"l,ll.c,..., :~wasperformed using a protocol as described in the Biorad manual (1987) except T7 DNA
polymerase was used instead of T4 DNA polymerase (~JSB, Cleveland, OH). The mutant clones were screened for the Eco47m restriction site that was created m themutagenic.,l;g.. ".l~l~vl:~l~ OneofthemutantshavingthecreatedEco47III
restriction site was used for further study.
To incorporate the 3 '-to-~ ', . . " " ,. .l. .. 2 rnutation in an expression vector, the mutant phage was digested with spm and Hindm. A 2 kb fragment containing the mutation was isolated. This fragment was cloned in pUC-Tne to replace the wild type fragment. See Figure 6A. The desired clone, pUC-Tne RECrIFIED SHEET (RULE gl) Wo 96/10640 P~ s~s ~1 7494~

(3'-5'), was isolated. The presence ofthe mutant sequence was conflrmed by the presence of the unique ~047m site. The plasrnid was then digested with SstI and E~xl[lI. The entire mutant polymerase gene (2.6 kb) was purified and cloned intoSstI and H~rumI digested pTrc99 expression vector (Pharmacia, Sweden). The S clones were sele~ted in DHIOB (LTI, C ' ' ~5, MD). The resulting plasmid was designated pTrcTne35. See Figure 6B. This clone produced active heat stable DNA p~l~
1;~ , '? I O: P~ '~;r - to Tyrosine Mutant As discussed supra~ the polymerase active site including the dNTP binding domain is usually present at the carboxyl terminal region of the p~l~ The p~ y and partial sequence of the Tne ~,c.l~..~.~, gene suggests that the amino acids that ~ contact and interact ~vith the dNTPs are present within the 694 bases starting at the internal BamHI site. see Figure 4. This conclusion is based on homology with a prototype pol~ e E. coli pG4~ ~c 1. See Polisky et al., .l BioL C~lem. 265:14579-14591 (1990). The sequence of the carboxyl tenninal portion ofthe polymerase gene is shown in Figure 5. Based upon this sequence, it is possible to compare the amino acid sequence within theO-helix for various pG~, Tne 63 KMVNFSUYG 72 (SEQ IDNO. 9) PolI 758 KAINFGLIYG 767 (SEQ lDNO. 10) T5 566 KAITFGlLYG 575 (SEQlDNO. Il) T7 522 KTFIYGFLYG 531 (SEQIDNO. 12) Taq 663 KTINFGVLYG 672 (SEQIDNO. 13) It was shown that by replacing the l ' ,' ' residue of Taq DNA
pu~ , ( ndicated as ~ above) the pul.~ becomes against non-natural nucleotides such as ~li.l~ ' ' See application Serial No. 08/525,087 enti~ded '~utant DNA Pul~ and Use Thereof' of Deb K.
30Chatterjee, filed September 8, 1995, specificaDy ~J.a~ .i herein by reference.
ÆCrlFJE~ StlEET (RllLE 91) ,,, . , , . ,, , . ,,,, , _ _ _ . , _ wo 96/10640 PcT/USgS/I2358 7-rq44 30 The mutation was based on the assumption that T7 DNA pU4~ contains a tyrosine residue in place of the ~ e, and T7 DNA pC~4..1~ is non-~.' _ against d;d~ The ~ r ~- _ residue, Phe762 of E. coli PolI is an amino acid that directly interacts with ~llrl~nti~l~c (Joyce and S Steitz, Ann Rev. Biochem. 63:777-822 (1994); Astake, ~.J., J. BioL Chem.
270:1945-1954 (1995)). We prepared a sirl~ilar mutant of Tne DNA pu4 ...~
In order to change Phe67 of the Tne polymerase to a Tyr67 as numbered in Figure 5, we performed a -;" c~,t~,~ _ using the 'i,, GTA TAT TAT AGA GTA GTT AAC CAT CTT TCC A. (SEQ lD NO. 14) Aspartofthis,li" ' ' directed _ , a HpaI restrictionsitewas created in order to screen mutants easily. The same uracilated single-stranded DNA and _ procedure described in Example 9 were used for this Following _ the mutants were screened for the ~IpaI cite.
Mutants with the desired HpaI cite were used for further study.
The Phe67 to Tyr67 mutation was , 1 into pUC-Tne by replacing the wild type SphI -Hindm fragment with the mutant fragment obtained from the mutant phage DNA. The presence of the desired clone, pUC-TneFY, was confirmed by the presence of the unique Hpal site, see Figure 6A. The entire mutant pol~ gene was subcloned into pTrc99 as an Ss~I-Hindm as described above in DHIOB. The resulting plasmid was designated pTrcTneFY.
(Figure oB) The clone produced active heat stable polymerase.
, '? 11: 3 '-to-S ' F~- ~. - and Phe'7~Tyf ' Dol ble Mr~tan~s In order to introduce the 3'-5'; ' mutation and the Phe67~Tyr6r mutation in the same expression vector, pTrc99, it was necessary to first reconstitute both mutations in the pUC-Tne clone. See Figure 7. Both the pUC-Tne (3 '-5 ') and the pUC-TneFY were digested with BamE~. The digested pUC-Tne (3'-5') was ~ to avoid ICI,UCUI~ in the following ligations. The resulting fragments were puriiied on a 1% agarose gel. The largest BamHI fragment (4.4 kb) was purified from pUC-Tne (3 '-5') digested DNA and IlEt:llF:lED SHEEt (RULE 91) WO 96/I0640 PCTIUS95~12358 -31- 2 1 74q44 the smailest BamE~ fragment (0.8 kb) containing the Phe6'~Tyr67 mutation was purified and ligated to generate pUC-Tne35FY. The proper orientation and the presence of both mutations in the sqme plasmid was confirmed by Eco47III, Hpal, and Sphl-Hindm restriction digests. See Figure 7.
The entire poiymerase contqining both mutations was subcloned as a Sstl-Hindlll fragment in pTrc99 to generate pTrcTne35FY in DHIOB. The clone produced active heat stable p~ ~aae.
E~cample 12: 3 '-to- 5 'F~ n~, 5'- to-3' F~7r~ n~q, and Phe6'~Tyr6' Triple Mutan~s In most of the known PO~ the 5'-to-3'; ' activity is present at the amino terminal region of the pol~ (oiiis~ D~L~ et aL, Nafure 313, 762-766, 1985; Freemont, P.S., ef al., Pro~eins 1, 66-73, 1986; Joyce, C.M., Curr. Opin. Struct. Biol. 1, 123-129, 1991). There are some conserved amino acids that are implicated to be responsible for 5'-to-3' . -~l .. Ir,. -~ activity (Gutman and Minton, Nucl. ~cidsRes. 21, 44064407, 1993). See supra It is known that 5'-to-3' -~ domain is ~ rPn~ The best known example is the Kienow fragment of E. coli Pol 1. The Kienow fragment is a naturai proteolytic fragment devoid of 5'-to-3' . .A~ activity (Joyce, C.M., et aL, 1 Biol. Chem. 257, 1958-1964, 1990). In order to generate an equivaient mutant for Tne DNA polymerase devoid of 5'-to-3'; ' activity we exploited the presence of a unique Sphl site present 680 bases from the Ssfl site.
pUC-Tne35FY was digested with f~in~lIL filled-in with Klenow fragment to generate a blunt-end, amd digested with Sphl. The 1.9 kb fragment was cloned mto an expression vector pTTQI9 (St. rk MJ.R, Gene 51, 255-267, 1987) at the Sphl-Smal sites and was mtroduced into DHIOB. This cloning strategy generated an in-frame ~GI,~ i clone with an initiation codon for methionine from the vector. The resulting clone is devoid of 219 amino terminal amino acids of Tne DNA POIJ...~. This clone is designated as pTTQTne535FY. The clone produced active heat stable polymerase. No l--.. ~lf~ activity could be _ 4 4 -32- ~
detected in the mutant polymerase as evidenced by lack of primer (labeled with ) fi. r,.A.~ in the sequencing rcaction. This particular mutant p~ is highly suitable for DNA e~
EYa~Ple 13 S~-t0-3~ F.Y.^I~ n~^ Deletion and Phe6'-Tyr67 ,~1~hctitti~ Mutant In order to generate the 5'-3' . ' .~ deletion mutant of the Tne DNA ~VI~ Phe6~Tyr6~ mutant, the 1.8 kb SphI-SpeI fragment of pTTQTne35FY was replaced with the identical fragment of pUC-Tne FY. See Fig. 8. A resulting clone, pTTQTne5~Y, produced active heat stable DNA
pGI?.. ~.A~c. As measured by the rate of ~ct5.dl~.Lio.l of a labeled primer, this mutant has a modulated, low but detectable, 3' - 5': ' activity compared to wild type Tne DNA polymerase. M13 sequencing primer, obtainable from LTI, ~~ ' sl u-~;, MD, was labeled at the 5' end with [p32] ATP and T4 kinase, also obtainable from LTI, ~ ' ' ~, MD, as described by the l~ urA~Lul tl . The reaction mixtures contained 20 units of either wild-type or mutant Tne DNA l.ol~..l.,._ ,~" 0.25 pmol of labeled primer, 20 mM tricine, pH
8.7, 85 mM potassium acetate, 1.2 mM magnesium acetate, and 8% glycerol.
Incubation was carried out at 70C. At various time points, 10 ml aliquots were removed to 5 ml cyde sequencing stop solution and were resolved in a 6 %
pu'~ )' ' sequencinggelfollowedby All.'i.)li~ 1y, Whilethewild-type polymerase de~raded the primer in 5 to 15 minutes, it took the mutant pulyl~ e more than 60 minutes for the same amount of d~.ddd~ of the primer.
Preliminary results suggest that this mutant polymerase is able to amplify more than 12 kb of genomic DNA when used in, ; with ~aq DNA
p~ . Thus, the mu polymerase is suitable for large fragment PCR.

Wo 96/10640 33 21 ~a944 E:xample 4: Putif cation of tl~e MutantPol~ ~
The ~ ;.. of the mutant PU J..._.~.S~j was done essentiaily as descrl~ed in U.S. Patent Appiication Seriai No. 08/370,190, filed January 9, 1995, entitled "Cloned DNA rul~ for Tl~err,20~0ga ~ ," and as in Example 6, sl~pra, with minor . "'' Specificaily, 5 to 10 grams of celis ei-cpressing cloned mutant Tne DNA po,l~ a..~ were Iysed by sonication with a ~eat Systems Uitrasonic, Inc. Model 375 machine in a sonication buffer comprising 50 mi~ Tris-HCI, pH 7.4; 8% glycerol; 5 m~i 2~ 10 rnM NaCL I mM EDTA, and 0.5 mM PMSF. The sonication sample was heated at 75C for 15 minutes. Following heat treatment, 200 mM NaCI and 0.4% PEI
was added to remove nucleic acids, The extract was centrifuged for ~
Ammonium sulfate was added to 48%, the pellet was ItD~lD~J~,..;iC;i in a column buffer consisting of 25 rnM Tris-HCI, pH 7.4; 8% glycerol; 0.5% EDTA; 5 mM
2-~ ,a~ ; 10 mM KCI and loaded on a Heparin agarose column. The column was washed with 10 column volumes using the loading buffer and eluted with a 10 column volume buffer gradient from 10 mM to I M KCI. Fractions containing PUI-~ D; activity were pooled and diaiyzed in column buffer as âbove with the pH adjusted to 7.8. The diaiyzed pool of fractions were loaded onto a mono Q column. The coiumn was washed and eluted as described above for the Heparin column. The active fractions are pooled and a unit assay was performed.
The urlit assay reaction mixture contained 25 mM TAliS pH 9.3, 2 mM
MgCI2, 50 mM KCI, I mM DTT, 0.2 mM dNTPs, 500 llg/mi DNAse I treated saimon sperm DNA, 21 mCi/mi [aP32] dCTP and various amounts of p.,l~ laD~i in a finai volume of 50 mi. After 10 minutes incubation at 70C, 10 mi of 0.5 M
~)TA was added to the tube. TCA ~l t~ lc counts were measured in GF/C
filters using 40 mi of the reaction mixture.
.

~74q44 O
Exarnple 15: DNA Sequencing with tlte Mutant /~
Cycle sequencing reactions using p32 end-labeled primers were prepared using wild-type Tne DNA polymerase and each of the three mutants, TneFY, Tne35FY, and Tne535FY. All four of the polymerases produced sequencing S ladders. The TneFY mutant gave only a 9 base sequencing ladder when the Taqcycle sequencing reaction conditions were used. Diluting the didc~,~.,u~ ,vLiJ~,s by a factor of 100 extended the ladder to about 200 bases. The F-Y mutation in the Tne FT polymerase therefore allowed J;J~o~...~ vLi~.., to be ~uldl~d at a much higher frequency than for wild-type p~ . The Tne35FY mutant ~ asimilarabilitytoincorporateJid~VA.~. ~ I vl;l . Inthiscase,the sequence extended to bevond 400 bases and the excess p32 end-labeled M13/pUC
for~vard 23-Base sequencing primer band remained at the 23-base position in the ladder. The persistence of the 23-base primer band confirmed that the 3 ' - 5' , activity had been ~;~lf~Lw.~ly reduced. The Tne535FY mutant performed similarly to the Tne35FY mutant except that the signal intensity increased by at least fivefold. The b~ uu~l was very low and relative band intensities were extremely even, showing no patterns of sequence-dependent intensity variation.

WO 96/10640 PCIIUS95~11358 ( 1 ) GENERAL INFORMATION:
(i) APPLICaNT:LIFE ~r~r~rlNnr,n~.~ T r~`.C, INC.
(ii) TITLE OF INVENTION: Cloned DNA Polymerases from Thermotoga Neapolitana and Mutants Thereof (iii) NUMBER OF SEQIJENCES: 3 (iV) ~ ; ADDRESS:
(A) ADDRESSEE: STERNE, KESSLER, GOLDSTEIN ~ FOX
(B) STREET: 1100 New York Avenue, N.W.
(C) CITY: nashington (D) STATE: DC
( E ) COUNTRY: USA
(F) ZIP: 20005 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy di~k (B) COMPUTER: IBM PC ~ompatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTnARE: PatentIn Relea~e #1.0, Version #1.30 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: TO BE ASSIGNED
(B) FILING DATE: 02-OCT-1995 (C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NOMBER: US Ob/316,423 (E) FILING DATE: 30-SEP-1994 (vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/370,190 (B) FILING D~TE: O9-;rAN-l99S
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Esmond, Robert W.
(B) REGISTRATION NUMBER: 32,893 (C) REFERENCE/DOCRET NCMBER: 09~2.280PCO2 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 202-371-2600 (B) TELEFAX: 202-371-2540 (2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTE~ISTICS:

WO 96/10640 r~
~7 4q 44 . -36- --(A) LENGTH: 23 ba~e pair~
(S) TYPE: nucleic acid ~C) STR~NnRr~N~c~c both (D) TOPOLOGY: both (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
GAGCTCACGG GGGATGCAGG AaA 23 (2) INFORMATION FOR SEQ ID NO:2:
(1) SEQUENCE rTll~R~rTERT~cTIcs:
(A) LENGTE~ 1310 base pair~
(S) TYPE: nucleic acid (C) STR~mET~M~cq both (D) TOPOLOGY: linear (xi) SEQ~ENCE DESCRIPTION: SEQ ID NO:2:
ATGGCGAGAC TATTTCTCTT TGATGGCACA ~iU ~~ r~r.r.r.r~Tz~ TTACGCCCTC 60 GACAGATCCC TTTCCACATC CACAGGAATT rr~rQ~ArQ CCGTCTATGG CGTTGCCAGG 120 A~ A AATTCATTAA GGAACACATT ~TLrrrr.~ AGGACTACGC ~ 180 TTCGACAAGA AGGCAGCr~AC GTTCAGACAC A~ACTGCTCG T~nrr.~r~ r.r.rr.r~r.r. 240 rr~2 ~r.~rr,C U~ AGTTCAGCAG CTACCTTACA TCAAGCGGCT GATAGAAGCT 300 ~ lll~ Aa-GTGcTGGA GCTGGAPGGG Tz~rQ~l~r~rAn ACGATATCAT CGCCACGCTT 360 Gr~r.r~r.n GCTGCACGTT TTTTGATGAG ATTTTCATAA TAACCGGTGA CZ~AGGATATG 420 CTTC~ACTTG T~ rn~n~ GATAaAGGTc TGGAGAATCG TCAAGGGGAT ATCGGATCTT 480 GAGCTTTACG ATTCGAaAAA GGTGAiiAGAA AGATACGGTG Tr,n~rr~r~ TCAQATACCG ~i40 GATCTTCTAG CACTGACGGG ~nArr.~r~T~ GACAACATTC CCGGTGTAAC nQn~T~r.r.T 600 r.~ r.~rrn CTGTACAGCT TCTCGGCAAG TATAGA~ATC TTGAATACAT TCTGGAGCAT 660 GCCCGTGAAC TCCCCCAGAG AGTGAGA~AG r,~, ~ ~ r.~n~r~nQr.~ AGTTGCCATC 720 CTCAGTA~AA AACTTGCAAC TCTGGTGACG AACGCACCTG TTGA~GTGGA CTGGGAAGAG 7 8 0 WO 96110640 ~ l 7 4 S ~ 4 PCT ~S9SI123S8 ATGAAATACA GAr~r~ATArrA rAAr.~rAAA~ CTACTTCCGA TATTGAAAGA ACTGGAGTTT 840 GCTTCCATCA TGAAGGAACT TCAACTGTAC rAAr.~Arr~r. AArrrarrnr. ATACGAaATC 900 r~rrrTGr~Arr TTGAAACGTC CTCCTTGGAC CCGTTCAACT GTGAGATAGT CGGCATCTCC 1020 JlL~ AArrrAA~Ar AGCTTATTAC ATTCCACTTC ATCACAGAAA rr.rrr~r~T 1080 CTTGATGAAA CACTGGTGCT GTCGAAGTTG AaAGAGATCC TCGAAGACCC GTCTTCGAAG 1140 A~ ~ AGAACCTGAA rTArrArTAr AAGGTTCTTA Tr.r.TAA~r.r.r TATATCGCCA 1200 GTTTATCCGC ATTTTGACAC GATGATAGCT GCATATTTGC TGGAGCCAAA rr~Ar~AAAAAA 1260 TTCAATCTCG AAGATCTGTC :TTTGAAATTT CTCGGATACA AAATGACGTC 1310 (2) INFORMATION FOF~ SEQ ID NO:3:
(i) sEQlrENcE rT~ rT~TCTICS:
(A) LENGTH: 436 amino acids (B) TYPE: amlno acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESC~IPTION: SEQ ID NO:3:
et Ala Arg Leu Phe Leu Phe A~p Gly Thr Ala Leu Ala Tyr Arg Ala yr Tyr Ala Leu Asp Arg ser Leu Ser Thr Ser Thr Gly Ile Pro Thr sn Ala Val Tyr Gly Val Ala Arg Met Leu Val Lys Phe Ile Lys Glu His Ile Ile Pro Glu Lys Asp Tyr Ala Ala Val Ala Phe A~p Lys Lys Ala Ala Thr Phe Arg His Ly~3 Leu Leu Val Ser ALP Lys Ala Gln Arg ro Lys Thr Pro Ala Leu Leu Val Gln Gln Leu Pro Tyr Ile Ly~ Arg Leu Ile Glu Ala Leu Gly Phe Ly~ Val Leu Glu Leu Glu Gly Tyr Glu WO 96110640 r~ ,3112.~
~1 7 4~4 -3~- O
Ala Asp Asp IIe }le Ala Thr Leu Ala Ala Lys Gly Cy8 Thr Phe Phe Asp Glu Ile Phe }le Ile Thr Gly Asp Lys Asp Met Leu Gln Leu Val Asn Glu Lys Ile Lys Val Trp Arg Ile Val LYB Gly Ile Ser Asp Leu lu Leu Tyr Asp Ser Lys LYB Val Lys Glu Arg Tyr Gly Val Glu Pro 165 170 , 175 is Gln Ile Pro Asp Leu Leu Ala Leu Thr Gly A0p Asp Ile Asp Asn 150 185 = 190 Ile Pro Gly Val Thr Gly Ile Gly Glu Lys Thr Ala Val Gln Leu Leu Gly Lys Tyr Arg Asn Leu Glu Tyr Ile Leu Glu His Ala Arg Glu Leu Pro Gln Arg Val Arg Lys Ala Leu Leu Arg Asp Arg Glu Val Ala Ile eu Ser Lys Lys Leu Ala Thr Leu Val Thr Agn Ala Pro Val Glu Val sp Trp Glu Glu ~qet Lys Tyr Arg Gly Tyr Asp Lys Arg Lys Leu Leu Pro Ile Leu LYG Glu Leu Glu Phe Ala Ser ne Met Lys Glu Leu Gln Leu Tyr Glu GlU Ala Glu Pro Thr Gly Tyr Glu Ile Val Lys Asp His Ly~ Thr Phe Glu Asp Leu Ile Glu Lys Leu Lvs Glu Val Pro Ser Phe 305 310 315 = 320 la Leu Asp Leu Glu Thr Ser Ser Leu Asp Pro Phe Asn Cys Glu Ile al Gly Ile Ser Val 8er Phe Lys Pro Lys Thr Ala Tyr Tyr Ile Pro Leu His His Arg Asn Ala His Asn Leu Asp Glu Thr Leu Val Leu Ser Lys Leu Lys Glu Ile Leu Glu Asp Pro Ser Ser LYG Ile Val Gly Gln Asn Leu Lys Tyr Asp Tyr Lys Val Leu Met Val Lys Gly Ile Ser Pro WO 96/10640 PCTIUS9~ 8 2 1 749~4 Val Tyr Pro ~is Phe A~p Thr Met Ile Ala Ala Tyr Leu Leu Glu Pro 405 410 41~
Asn Glu Lys Lys Phe Asn Leu Glu }~p Leu Ser Leu Lys Phe Leu Gly Tyr Lys Me t Thr 43~

Claims (70)

What Is Claimed Is:

1. A substantially pure Thermotoga neapolitana (Tne) DNA
polymerase having a molecular weight of about 100 kilodaltons, or fragments thereof.

2. The DNA polymerase of claim 1, which is isolated from Thermologa neapolitana,

3. The DNA polymerase of claim 2, which is isolated from Thermotoga neapolitana DSM 5068.

4. An isolated DNA molecule comprising a gene encoding a Tne DNA polymerase having a molecular weight of about 100 kilodaltons

5. An isolated DNA molecule of claim 4, wherein the gene is modified to reduce 3'-5' exo activity.

6. The isolated DNA molecule of claim 4, wherein the promoter of said gene is an inducible promoter.

7. The isolated DNA molecule of claim 6, wherein said inducible promoter is selected from the group consisting of .alpha. ?-P? promoter, a tac promoter, a trp promoter, and a trc promoter.

8 A recombinant host comprising a gene encoding Tne DNA
polymerase having a molecular weight of 100 kilodaltons.

9. The recombinant host of claim 8, wherein said gene is obtained from Thermotoga neapolitana.

10. The recombinant host of claim 9, wherein said gene is obtained from Thermotoga neopolitana DSM 5068.

11 The recombinant host of claim 8, wherein said host is prokaryotic.

12. The recombinant host of claim 11, wherein said host is E.coli.

13. A method of producing a Tne DNA polymerase having a molecular weight of about 100 kilodaltons, said method comprising:
(a) culturing a cellular host comprising a gene encoding said DNA polymerase;
(b) expressing said gene; and (c) isolating said DNA polymerase from said host.

14. The method of claim 13, wherein said host is a eukaryotic host.

15. The method of claim 13, wherein said host is a prokaryotic host.

16. The method of claim 15, wherein said prokaryotic host is E. coli.

17. A method of synthesizing a double-stranded DNA molecule comprising:
(a) hybridizing a primer to a first DNA molecule; and (b) incubating said DNA molecule of step (a) in the presence of one or more deoxyribonucleoside triphosphates and Tne DNA polymerase having a molecular weight of about 100 kilodaltons, under conditions sufficient to synthesize a second DNA molecule complementary, to all or a portion of said first DNA molecule.

18. The method of claim 17, wherein said DNA polymerase is isolated from Thermotoga neapolitana.

19. The method of claim 18, wherein said DNA polymerase is isolated from Thermotoga neapolitana. DSM 5068.

20. The method of claim 17, wherein said DNA polymerase is isolated from a recombinant host expressing a gene encoding said DNA polymerase.

21. The method of claim 20, wherein said host is a eukaryotic host.

22. The method of claim 20, wherein said host is a prokaryotic host.

23. The method of claim 22, wherein said prokaryotic host is E. coli.

24. The method of claim 17, wherein said deoxyribonucleoside triphosphates are selected from the group consisting of dATP, dCTP, dGTP, dTTP, dITP, 7-deaza-dGTP, dUTP, ddATP, ddCTP, ddGTP, ddlTP, ddTTP, [.alpha.S]dATP, [.alpha.S]dTTP,[.alpha.S]dGTP, and [.alpha.S]dCTP.

25. The method of claim 24, wherein one or more of said deoxyribonucleoside triphosphates are detectably labeled.

26. The method of claim 25, wherein said detectable label is selected from the group consisting of a radioactive isotope, a fluorescent label, a chemiluminescent label, a bioluminescent label, and an enzyme label.

27. A method of sequencing a DNA molecule, comprising:
(a) hybridizing a primer to a first DNA molecule;
(b) contacting said DNA molecule of step (a) with deoxyribonucleoside triphosphates, DNA polymerase having a molecular weight of about 100 kilodaltons, and a terminator nucleotide;

(c) incubating the mixture of step (b) under conditions sufficient to synthesize a random population of DNA molecules complementary to said first DNA molecule, wherein said synthesized DNA molecules are shorter in length than said first DNAmolecule and wherein said synthesized DNA molecules comprise a terminator nucleotide at their 5' termini; and (d) separating said synthesized DNA molecules by size so that at least a part of the nucleotide sequence of said first DNA
molecule can be determined.

28. The method of claim 27, wherein said terminator nucleotide is ddTTP.

29. The method of claim 27, wherein said terminator nucleotide is ddATP.

30. The method of claim 27, wherein said terminator nucleotide is ddGTP.

31. The method of claim 27, wherein said terminator nucleotide is ddCTP.

32. The method of claim 27, wherein said DNA polymerase is isolated from Thermotoga neapolitana.

33 . The method of claim 32, wherein said DNA polymerase is isolated from Thermotoga neapolitana DSM 5068.

34. The method of claim 27, wherein said DNA polymerase is isolated from a recombinant host expressing a gene encoding said DNA polymerase.

35. The method of claim 27, wherein one or more of said deoxyribonucleoside triphosphates is detectably labeled.

36. The method of claim 35, wherein said labeled deoxyribonucleoside triphosphate is [.alpha.35S]dATP.

37. A method for amplifying a double stranded DNA molecule, (a) providing a first and second primer, wherein said first primer is complementary to a sequence at or near the 3'-termini of the first strand of said DNA molecule and said second primer is complementary to a sequence at or near the 3'-termini of the second strand of said DNA molecule;
(b) hybridizing said first primer to said first strand and said second primer to said second strand in the presence of Tne DNA polymerase having a molecular weight of about 100 kilodaltons, under conditions such that athird DNA molecule complementary to said first strand and a fourth DNA
molecule complementary to said second strand are synthesized;
(c) denaturing said first and third strand, and said second and fourth strands with heat; and (d) repeating steps (a) to (c) one or more times.

38. The method of claim 37, wherein said DNA polymerase is isolated from Thermotoga neapolitana.

39. The method of claim 38, wherein said DNA polymerase is isolated from Thermotoga neapolitana DSM 5068.

40. The method of claim 37, wherein said DNA polymerase is isolated from a recombinant host expressing a gene encoding said DNA Polymerase.

41. A kit for sequencing a DNA molecule, comprising:

(a) a first container means comprising a Tne DNA polymerase having a molecular weight of about 100 kilodaltons;
(b) a second container means comprising one or more dideoxyribonucleoside triphosphates; and (c) a third container means comprising one or more deoxyribonucleoside triphosphates.

42. The kit of claim 41, wherein said DNA polymerase is isolated from Thermotoga neapolitana.

43 . The kit of claim 42, wherein said DNA polymerase is isolated from Thermotoga neapolitana DSM 5068.

44. The kit of claim 41, wherein said DNA polymerase is isolated from a recombinant host expressing a gene encoding said DNA polymerase.

45. A kit for amplifying a DNA molecule, comprising:
(a) a first container means comprising a Tne DNA polymerase having a molecular weight of about 100 kilodaltons; and (b) a second container means comprising one or more deoxyribonucleoside triphosphates.

46. The kit of claim 45, wherein said DNA polymerase is isolated from Thermotoga neapolitana.

47. The kit of claim 46, wherein said DNA polymerase is isolated from Thermotoga neapolitana DSM 5068.

48. The kit of claim 45, wherein said DNA polymerase is isolated from a recombinant host expressing a gene encoding said DNA polymerase.

49. A mutant Thermotoga neapolitana DNA polymerase having at least one mutation selected from the group consisting of (1) a first mutation that substantially reduces or eliminates 3'-5' exonuclease activity of said DNA
polymerase; (2) a second mutation that substantially reduces or eliminates 5'-3'exonulease activity of said DNA polymerase; and (3) a third mutation in the O
helix of said DNA polymerase resulting in said DNA polymerase becoming non-discriminating against dideoxynucleotides, or fragments thereof

50. The mutant Thermotoga neapolitana DNA polymerase as claimed in claim 49, wherein said third mutation is a Phe67 ? Tyr67 substitution.

51. The mutant Thermotoga neapolitana DNA polymerase as claimed in claim 49, wherein said first mutant is a Asp323-Ala322 substitution.

52. The mutant Thermotoga neapolitana DNA polymerase as claimed in claim 49, wherein said mutant polymerase comprises both a Phe67 - Tyr67 substitution and a Asp322 ? Ala322- substitution.

53 The mutant Thermotoga neapolitana DNA polymerase as claimed in claim 49, wherein said mutant polymerase is devoid of the N- terminal 5'- 3' exonuclease domain.

54. The mutant Thermotoga neapolitana DNA polymerase as claimed in claim 53, wherein said mutant polymerase is devoid of the 219 N-terminal amino acids of Thermotoga neapolitana DNA polymerase.

55. An isolated DNA molecule comprising a DNA sequence encoding a mutant Thermotoga neapolitana DNA polymerase having at least one mutation selected from the group consisting of (1) a first mutation that substantially reduces or eliminates 3'-5' exonuclease activity of said DNA polymerase; (2) a second mutation that substantially reduces or eliminates 5'-3' exonuclease activity of said DNA polymerase; and (3) a third mutation in the O helix of said DNA polymerase resulting in said DNA polymerase becoming non-discriminating against dideoxynucleotides, or fragments thereof.

56. The isolated DNA molecule as claimed in claim 55, wherein said DNA molecule is selected from the group consisting of pTrcTne35, pTrcTneFY, pTrcTne35FY, and pTTQTne535FY.

57. The isolated DNA molecule as claimed in claim 55, wherein said DNA molecule further comprises expression control elements.

58. The isolated DNA molecule as claimed in claim 57, wherein said expression control elements comprise an inducible promoter selected from the group consisting of ?PL promoter, a tac promoter, a trp promoter, and a trc promoter.

59. A recombinant host comprising a DNA sequence encoding a mutant Thermotoga neapolitana DNA polymerase having at least one mutation selected from the group consisting of(I) a first mutation that substantially reduces or eliminates 3'?5' exonuclease activity of said DNA polymerase; (2) a second mutation that substantially reduces or eliminates 5'-3' exonuclease activity of said DNA polymerase; and (3) a third mutation in the O helix of said DNA polymerase resulting in said DNA polymerase becoming non-discriminating against dideoxynucleotides, or fragments thereof.

60. A method of producing a Tne DNA polymerase, said method comprising:
(a) culturing a cellular host comprising a gene encoding a mutant Thermotoga neapolitana DNA polymerase having at least one mutation selected from the group consisting of (1) a first mutation that substantially reduces or eliminates 3'5'exonuclease activity of said DNA polymerase; (2) a second mutation that substantially reduces or eliminates 5'-3' exonuclease activity of said DNA polymerase; and (3) a third mutation in the O helix of said DNA polymerase resulting in said DNA polymerase becoming non-discriminating against dideoxynucleotides, or fragments of said mutant Thermotoga neapolitana DNA
polymerase;
(b) expressing said gene; and (c) isolating said mutant Thermotoga neapolitana DNA
polymerase from said host.

61. The method of producing a Thermotoga neapolitana DNA
polymerase as claimed in claim 60, wherein said host is E. coli.

62. A method of synthesizing a double-stranded DNA molecule, (a) hybridizing a primer to a first DNA molecule; and (b) incubating said DNA molecule of step (a) in the presence of one or more deoxyribonucleoside and a mutant Thermotoga neapolitana DNA polymerase under conditions sufficient to synthesize a second DNA molecule complementary to all or a portion of said first DNA molecules;
wherein:
said mutant Thermotoga neapolitana DNA polymerase has at least one mutation selected from the group consisting of (1) a first mutation that substantially reduces or eliminates 3'-5' exonuclease activity of said DNA
polymerase; (2) a second mutation that substantially reduces or eliminates 5'-3'exonuclease activity of said DNA polymerase; and (3) a third mutation in the O
helix of said DNA polymerase resulting in said DNA polymerase becoming non-discriminating against dideoxynucleotides, or fragments thereof.

63. The method of a synthesizing a double-stranded DNA molecule as claimed in claim 62, wherein said deoxyribonucleoside triphosphates are selectedfrom the group consisting of: dATP, dCTP, dGTP, dTTP, dITP, 7-deaza-dGTP, dUTP, ddATP, ddCTP, ddGTP, ddITP, ddTTP, [.alpha.S]dATP, [.alpha.S]dTTP, [.alpha.S]dGTP, and [.alpha.S]dCTP.

64. The method of synthesizing a double-stranded DNA molecule as claimed in claim 63, wherein one or more of said deoxyribonucleoside are detectably labelled.

65. The method of synthesizing a double-stranded DNA molecule as claimed in claim 64, wherein said label is selected from the group consisting of a radioactive isotape, a fluorescent label a chemiluminessant label, a bioluminescent label, and an enyzme label.

66. A method of sequencing a DNA molecule, comprising:
(a) hybridizing a primer to a first DNA molecule;
(b) contacting said DNA molecule of step (a) with deoxyribonucleoside triphosphates, a mutant Thermotoga neapolitana DNA
polymerase, and a terminator nucleotide;
(c) incubating the mixture of step (b) under conditions sufficient to synthesize a random population of DNA molecules complementary to said first DNA molecule;
wherein said synthesized DNA molecules are shorter in length than said first DNA molecule and wherein said synthesized DNA molecules comprise a terminator nucleotide at their 5' termini; and (d) separating said synthesized DNA molecules by size so that at least a part of the nucleotide sequence of said first DNA molecule can be determined.
wherein said mutant Thermotoga neapolitana DNA polymerase has at least one mutation selected from the group consisting of (1) a first mutation that substantially reduces or eliminates 3'-5' exonuclease activity of said DNA
polymerase; (2) a second mutation that substantially reduces or eliminates 5'-3'exonuclease activity of said DNA polymerase; and (3) a third mutation in the O

helix of said DNA polymerase resulting in said DNA polymerase becoming non-discriminating against dideoxynucleotides, or fragments thereof.

67. The method sequencing a DNA molecule as claimed in claim 66, wherein said terminator nucleotide is selected from the group consisting of ddTTP, ddATP, ddGTP, and ddCTP.

68. A method for amplifying a double stranded DNA molecule, comprising:
(a) providing a first and second primer, wherein said first primer is complementary to a sequence at or near the 3'-termini of the first strand of said DNA molecule and said second primer is complementary to a sequence at or near the 3'-termini of the second strand of said DNA molecule;
(b) hybridizing said first primer to said first strand and said second primer to said second strand in the presence of a Thermotoga neapolitana DNA
polymerase, under conditions such that a third DNA molecule complementary to said first strand and a fourth DNA molecule complementary to said second strand are synthesized;
(c) denaturing said first and second strand, and said second and fourth strands with heat; and (d) repeating steps (a) to (c) one or more times, wherein:
said mutant Thermotoga neapolitana DNA polymerase has at least one mutation selected from the group consisting of (1) a first mutation that substantially reduces or eliminates 3'-5' exonuclease activity of said DNA
polymerase; (2) a second mutation that substantially reduces or eliminates 5'-3'exonuclease activity of said DNA polymerase; and (3) a third mutation in the O
helix of said DNA polymerase resulting in said DNA polymerase becoming non-discriminating against dideoxynucleotides.

69. A kit for sequencing a DNA molecule, comprising:

(a) a first container means comprising a mutant Thermotoga neapolitana DNA polymerase;
(b) a second container means comprising one or more dideoxyribonucleoside triphosphates; and (c) a third container means comprising one or more wherein:
said mutant Thermotoga neapolitana DNA polymerase has at least one mutation selected from the group consisting of (1) a first mutation that reduces or eliminates 3'-5' exonuclease activity of said DNA
polymerase; (2) a second mutation that substantially reduces or eliminates 5'-3'activity of said DNA polymerase; and (3) a third mutation in the O
helix of said DNA polymerase resulting in said DNA polymerase becoming non-discriminating against dideoxynucleotides.

70. A kit for amplifying a DNA molecule, comprising:
(a) a first container means comprising a mutant Thermotoga neapolitana DNA polymerase; and (b) a second container means comprising one or more deoxyribonucleoside triphosphates, wherein:
said mutant Thermotoga neapolitana DNA polymerase has at least one mutation selected from the group consisting of (1) a first mutation that substantially reduces or eliminates 3'-5' exonuclease activity of said DNA
polymerase; (2) a second mutation that substantially reduces or eliminates 5'-3'exonuclease activity of said DNA polymerase; and (3) a third mutation in the O
helix of said DNA polymerase resulting in said DNA polymerase becoming non-discriminating against dideoxynucleotides.