Title: Compositions and methods utilizing DNA polymerases
Abstract: The invention features a novel isolated Family B DNA polymerase, a Thermococcus polymerase JDF-3, and mutant recombinant forms thereof. Mutant polymerases of the invention are deficient in 3′ to 5′ exonuclease activity and/or exhibit reduced discrimination against non-conventional nucleotides relative to the wild-type form of the polymerase.
Patent Number: 6,946,273 Issued on 09/20/2005 to Sorge, et al.
| Inventors:
|
Sorge; Joseph A. (Wilson, WY);
Hogrefe; Holly Hurlbut (San Diego, CA);
Hansen; Connie Jo (San Diego, CA)
|
| Assignee:
|
Stratagene California (La Jolla, CA)
|
| Appl. No.:
|
698341 |
| Filed:
|
October 27, 2000 |
| Current U.S. Class: |
435/194; 424/94.5; 435/183; 530/350; 536/23.2 |
| Intern'l Class: |
C12N 009/12; C07K 001/00 |
| Field of Search: |
435/194,183
530/350
536/232
|
References Cited [Referenced By]
U.S. Patent Documents
| 5674679 | Oct., 1997 | Fuller.
| |
| 5882904 | Mar., 1999 | Riedl et al.
| |
| Foreign Patent Documents |
| 0655506 | May., 1995 | EP.
| |
| 0727496 | Aug., 1996 | EP.
| |
| WO 97/3915/0 | Oct., 1997 | WO.
| |
| WO 99/0653/8 | Feb., 1999 | WO.
| |
Other References
Dong et al., Mutational Studies of Human DNA Polymerase alpha, Journal of Biological
Chemistry, vol. 268, No. 15, pp. 24163-24174, 1993.
Stryer, Biochemistry, Third Edition, 1988, W.H. Freeman and Co., New York, p. 72.
International Search Report of International Application No. PCT/US02/20562.
Gardner and Jack, Nucleic Acids Research, 1999, vol., 27, No. 12 2545-2553.
|
Primary Examiner: Hutson; Richard
Attorney, Agent or Firm: Williams; Kathleen M., Palmer & Dodge, LLP
Parent Case Text
This application claims the benefit of U.S. Provisional Application No. 60/162,600,
filed Oct. 29, 1999.
Claims
1. A purified thermostable DNA polymerase having an amino acid sequence presented
in SEQ ID NO: 2 from residue 1 to 776.
2. An isolated recombinant polypeptide comprising the amino acid sequence of
SEQ ID NO: 2.
3. An isolated recombinant JDF-3 DNA polymerase comprising a sequence of SEQ
ID NO: 2 and further comprising a mutation at D141 and/or E143 within said sequence
of SEQ ID NO: 2.
4. The isolated recombinant JDF-3 DNA polymerase of claim 3, wherein said mutation
at D141 is an aspartic acid (D) to threonine (T) or alanine (A) mutation, and said
mutation at E143 is a glutamic acid (E) to alanine (A) mutation.
5. The isolated recombinant JDF-3 DNA polymerase of 4, further comprising a mutation
at L408 and/or P410.
6. The isolated recombinant JDF-3 DNA polymerase of claim 5, wherein said mutation
at L408 is a leucine (L) to histidine (H) or phenylalanine (F) mutation and said
mutation at P410 is a proline (P) to leucine (L) mutation.
7. The isolated recombinant JDF-3 DNA polymerase of 4, further comprising a mutation
at one or more additional amino acids selected from the group consisting of: A485,
S345, T604, Y497, I630, E645, E578, R465, V401, N424, P569. E617. V640, S651, L396,
E459, L456, E658, V437, L478, Y496, Y409 and A490 within the sequence of SEQ ID
NO: 2.
8. The isolated recombinant JDF-3 DNA polymerase of claim 7, wherein said mutation
at S345 is serine (S) to proline (P), said mutation at A485 is alanine (A) to threonine
(T), cysteine (C), serine (S), leucine (L), isoleucine (I), phenylalanine (F) or
valine (V), said mutation at Y497 is tyrosine (Y) to cysteine (C), said mutation
at I630 is isoleucine (I) to valine (V), said mutation at E645 is glutamic acid
(E) to lysine (L), said mutation at E578 is glutamic acid (E) to lysine (L), said
mutation at R465 is arginine (R) to methionine (M), said mutation at L396 is leucine
(L) to glutamine (Q) or to proline (P), said mutation at S651 is serine (S) to
asparagine (B), said mutation at L456 is leucine (L) to histidine (H), said mutation
at Y496 is tyrosine (Y) to asparagine (B) or leucine (L), said mutation at Y409
is tyrosine (Y) to valine (V), said mutation at A490 is alanine (A) to tyrosine (Y).
9. The isolated recombinant JDF-3 DNA polymerase of 4, wherein said JDF-3 DNA
polymerase has reduced discrimination against a non-conventional nucleotide selected
from the group consisting of: dideoxynucleotides, ribonucleotides and conjugated nucleotides.
10. The isolated recombinant JDF-3 DNA polymerase of claim 9, wherein said conjugated
nucleotide is selected from the group consisting of radiolabeled nucleotides, fluorescently
labeled nucleotides, biotin labeled nucleotides, chemiluminescently labeled nucleotides
and quantum dot labeled nucleotides.
11. An isolated JDF-3 DNA polymerase comprising a sequence of SEQ ID NO: 2 and
further comprising the following mutations: D141T or D141A, E143A, L408H or L408F,
P410L, and A485T within said SEQ ID NO: 2.
12. An isolated JDF-3 DNA polymerase comprising a sequence of SEQ ID NO: 2 and
further comprising the following mutations: D141T or D141A and E143A within said
SEQ ID NO: 2.
13. An isolated JDF-3 DNA polymerase comprising a sequence of SEQ ID NO: 2 and
further comprising the following mutations: D141T or D141A and E143A, and further
comprising one or more mutations selected from the group consisting of: L408H or
L408F, P410L, and S345P within said SEQ ID NO: 2.
14. An isolated JDF-3 DNA polymerase comprising a sequence of SEQ ID NO: 2 and
further comprising mutations at: D141, E143, P410, and A485 within said SEQ ID
NO: 2.
15. An isolated JDF-3 DNA polymerase comprising a sequence of SEQ ID NO: 2 and
further comprising the following mutations of: D141T or D141A, E143A, P410L, and
A485T within said SEQ ID NO: 2.
16. A kit comprising an isolated recombinant polypeptide of claim 2, and packaging
material thereof.
17. A kit comprising an isolated recombinant DNA polymerase of 4, and packaging
material thereof.
18. A kit comprising an isolated recombinant DNA polymerase of claim 5, and packaging
material thereof.
19. A kit comprising an isolated recombinant DNA polymerase of claim 6, and packaging
material thereof.
20. A kit comprising an isolated recombinant DNA polymerase of claim 7, and packaging
material thereof.
21. A kit comprising an isolated recombinant DNA polymerase of claim 8, and packaging
material thereof.
22. A method of making a purified thermostable DNA polymerase of claim 1, comprising
a) transfecting a host cell with the nucleic acid sequence presented in SEQ ID
NO: 1, and b) culturing said host cell under conditions which permit production
of said DNA polymerase.
23. The method of claim 22, wherein said host cell is
E. Coli or
Thermococcus.
24. A method of making a purified thermostable DNA polymerase of 4, comprising
a) transfecting a host cell with a nucleic acid sequence encoding said polymerase
and b) culturing said host cell under conditions which permit production of said
DNA polymerase.
25. The method of claim 24, wherein said host cell is
E. Coli or
Thermococcus.
Description
FIELD OF THE INVENTION
The present invention relates to compositions and methods utilizing DNA polymerase
enzymes with reduced discrimination for non-conventional nucleotides. The enzymes
of the invention are useful in many applications calling for the detectable labeling
of nucleic acids and are particularly useful in DNA sequencing applications.
BACKGROUND OF THE INVENTION
Detectable labeling of nucleic acids is required for many applications
in molecular biology, including applications for research as well as clinical diagnostic
techniques. A commonly used method of labeling nucleic acids uses one or more unconventional
nucleotides and a polymerase enzyme that catalyzes the template-dependent incorporation
of the unconventional nucleotide(s) into the newly synthesized complementary strand.
The ability of a DNA polymerase to incorporate the correct deoxynucleotide is
the basis for high fidelity DNA replication in vivo. Amino acids within the active
site of polymerases form a specific binding pocket that favors the placement of
the correct complementary nucleotide opposite the template nucleotide. If a mismatched
nucleotide, ribonucleotide, or nucleotide analog fills that position, the precise
alignment of the amino acids contacting the incoming nucleotide may be distorted
into a position unfavorable for DNA polymerization. Because of this, the unconventional
nucleotides or nucleotide analogs used to label DNA tend to be incorporated into
the elongated strand less efficiently than do the standard deoxynucleotide triphosphates
(dNTPs; the so-called "standard" dNTPs include deoxyadenosine triphosphate (dATP),
deoxycytosine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), and thymidine
triphosphate (TTP)).
The reduced efficiency with which unconventional nucleotides are incorporated
by the polymerase increases the amount of the unconventional nucleotide necessary
for DNA labeling. The reduced efficiency of incorporation of a particular nucleotide
can also adversely affect the performance of techniques or assays, such as DNA
sequencing, that depend upon unbiased incorporation of unconventional nucleotides
for homogeneous signal strength.
The identity and exact arrangement of the amino acids of a DNA polymerase that
contact an incoming nucleotide triphosphate determine the nature of the nucleotides,
both conventional and unconventional, that may be incorporated by that polymerase
enzyme. Changes in the exact placement of the amino acids that contact the incoming
nucleotide triphosphate at any stage of binding or chain elongation can dramatically
alter the polymerase's capacity for utilization of unusual or unconventional nucleotides.
Sometimes changes in distant amino acids can influence the incorporation of nucleotide
analogs due to indirect global or structural effects. Polymerases with increased
capacity to incorporate nucleotide analogs are useful for labeling DNA or RNA strands
with nucleotides modified with signal moieties such as dyes, reactive groups or
unstable isotopes.
In addition to labeled nucleotides, an extremely important class of modified
nucleotides
is the dideoxynucleotides. The so-called "Sanger" or "dideoxy" DNA sequencing method
(Sanger et al., 1977, Proc. Natl. Acad. Sci. USA 74: 5463, which is incorporated
herein by reference) relies upon the template-directed incorporation of nucleotides
onto an annealed primer by a DNA polymerase from a mixture containing deoxy- and
dideoxynucleotides. The incorporation of a dideoxynucleotide results in chain termination,
the inability of the enzyme to catalyze further extension of that strand. Electrophoretic
separation of reaction products results in a "ladder" of extension products wherein
each extension product ends in a particular dideoxynucleotide complementary to
the nucleotide opposite it in the template. The distance of the dideoxynucleotide
analog from the primer is indicated by the length of the extension product. When
four reactions, each containing one of the four dideoxynucleotide analogs ddA,
ddC, ddG, or ddT (ddNTPs) are separated on the same gel, the sequence of the template
may be read directly from the ladder patterns. Extension products may be detected
in several ways, including for example, the inclusion of isotopically- or fluorescently-labeled
primers, deoxynucleotide triphosphates or dideoxynucleotide triphosphates in the reaction.
Fluorescent labeling has the advantages of faster data collection, since
detection may be performed while the gel is running, and longer reads of sequence
data from a single reaction and gel. Further, fluorescent sequence detection has
allowed sequencing to be performed in a single reaction tube containing four differentially-labeled
fluorescent dye terminators (the so-called dye-terminator method, Lee et al., 1992,
Nucleic Acids Res. 20: 2471, incorporated herein by reference).
A desirable quality of a polymerase useful for DNA sequencing is improved incorporation
of dideoxynucleotides. Improved incorporation of dideoxynucleotides can make processes
such as DNA sequencing more cost effective by reducing the requirement for expensive
radioactive or fluorescent dye-labeled dideoxynucleotides. Moreover, unbiased dideoxynucleotide
incorporation provides improved signal uniformity, leading to increased accuracy
of base determination. The even signal output further allows subtle sequence differences
caused by factors like allelic variation to be detected. Allelic variation, which
produces two different half strength signals at the position of relevance, can
easily be concealed by the varied signal strengths caused by polymerases with non-uniform
ddNTP utilization.
Incorporation of ribonucleotides by the native form of DNA polymerase
is a rare event. Mutants that incorporate higher levels of ribonucleotides can
be used for applications such as sequencing by partial ribosubstitution. In this
system, a mixture of ribonucleotides and deoxynucleotides corresponding to the
same base are incorporated by the mutant polymerase (Barnes, 1978 J. Mol. Biol.
119:83-99). When the ribosequencing reactions are exposed to alkaline conditions
and heat, fragmentation of the extended strand occurs. If the reactions for all
four bases are separated on a denaturing acrylamide gel, they produce a sequencing
ladder. there is a need in the art for polymerase mutants with higher utilization
of ribonucleotides for this alternative method of sequencing.
Alternatively, the incorporation of ribonucleotides followed by alkaline
hydrolysis could be utilized in a system that requires random cleavage of DNA molecules
such as DNA shuffling ((Stemmer, 1994, Nature, 370: 389-391) which has also been
called molecular breeding, sexual PCR and directed evolution).
Another desirable quality in a DNA labeling enzyme is thermal stability.
DNA polymerases exhibiting thermal stability have revolutionized many aspects of
molecular biology and clinical diagnostics since the development of the polymerase
chain reaction (PCR), which uses cycles of thermal denaturation, primer annealing,
and enzymatic primer extension to amplify DNA templates. The prototype thermostable
DNA polymerase is Taq polymerase, originally isolated from the thermophilic eubacterium
Thermus aquaticus. So-called "cycle sequencing" reactions using thermostable
DNA polymerases have the advantage of requiring smaller amounts of starting template
relative to conventional (i.e., non-cycle) sequencing reactions.
There are three major families of DNA polymerases, termed families A, B and
C. The classification of a polymerase into one of these three families is based
on structural similarity of a given polymerase to
E. coli DNA polymerase
I (Family A), II (Family B) or III (family C). As examples, Family A DNA polymerases
include, but are not limited to Klenow DNA polymerase,
Thermus aquaticus DNA
polymerase I (Taq polymerase) and bacteriophage T7 DNA polymerase; Family B DNA
polymerases, formerly known as α-family polymerases (Braithwaite and Ito,
1991, Nuc. Acids Res. 19:4045), include, but are not limited to human α,
δ and ε DNA polymerases, T4, RB69 and Φ29 bacteriophage DNA polymerases,
and
Pyrococcus furiosus DNA polymerase (Pfu polymerase); and family C DNA
polymerases include, but are not limited to
Bacillus subtilis DNA polymerase
III, and
E. coli DNA polymerase III α and ε subunits (listed
as products of the dnaE and dnaQ genes, respectively, by Brathwaite and Ito, 1993,
Nucleic Acids Res. 21: 787). An alignment of DNA polymerase protein sequences of
each family across a broad spectrum of archaeal, bacterial, viral and eukaryotic
organisms is presented in Braithwaite and Ito (1993, supra), which is incorporated
herein by reference.
The term used to describe the tendency of DNA polymerases to not to carry the
incorporation of unnatural nucleotides into the nascent DNA polymer is "discrimination".
In Family A DNA polymerases, the effective discrimination against incorporation
of dideoxynucleotide analogs is largely associated with a single amino acid residue.
The majority of enzymes from the Family A DNA polymerases have a phenylalanine
(phe or F) residue at the position equivalent to F762 in
E. coli Klenow
fragment of DNA polymerase and demonstrate a strong discrimination against dideoxynucleotides.
A few polymerases (e.g. T7 DNA polymerase) have a tyrosine (tyr or Y) residue at
the corresponding position and exhibit relatively weak discrimination against dideoxynucleotides.
Family A polymerases with tyrosine at this position readily incorporate dideoxynucleotides
at levels equal to or only slightly different from the levels at which they incorporate
deoxynucleotides. Conversion of the tyrosine or phenylalanine residues in the site
responsible for discrimination reverses the dideoxynucleotide discrimination profile
of the Family A enzymes (Tabor and Richardson, 1995, Proc. Natl. Acad. Sci. USA 92:6449).
Among the thermostable DNA polymerases, a mutant form of the Family A DNA polymerase
from
Thermus aquaticus, known as AmpliTaq FS® (Perkin Elmer), contains
a F667Y mutation at the position equivalent to F762 of Klenow DNA polymerase and
exhibits increased dideoxynucleotide uptake (i.e., reduced discrimination against
ddNTPs) relative to the wild-type enzyme. The reduced discrimination for dideoxynucleotide
uptake makes it more useful for fluorescent and labeled dideoxynucleotide sequencing
than the wild-type enzyme.
The F667Y mutant of Taq DNA polymerase is not suited, however, for use with fluorescein-labeled
dideoxynucleotides, necessitating the use of rhodamine dye terminators. Rhodamine
dye terminators that are currently utilized with Taq sequencing reactions, however,
stabilize DNA secondary structure, causing compression of signal. Efforts to eliminate
compression problems have resulted in systems that use high amounts of the nucleotide
analog deoxyinosine triphosphate (dITP) in place of deoxyguanosine triphosphate.
While incorporation of (dITP) reduces the compression of the signal, the presence
of dITP in the reaction produces additional complications including lowered reaction
temperatures and increased reaction times. Additionally, the use of rhodamine dyes
in sequencing requires undesirable post-reaction purification (Brandis, 1999 Nuc.
Acid Res. 27:1912).
Family B DNA polymerases exhibit substantially different structure compared
to Family A DNA polymerases, with the exception of the position of acidic residues
involved in catalysis in the so-called palm domain (Wang et al., 1997, Cell 89:1087;
Hopfner et al., 1999, Proc. Natl. Acad. Sci. USA 96:3600). The unique structure
of Family B DNA polymerases may permit a completely different spectrum of interactions
with nucleotide analogs, perhaps allowing utilization of analogs which are unsuitable
for use with Family A DNA polymerases due to structural constraints. Thermostable
Family B DNA polymerases have been identified in hyperthermophilic archaea. These
organisms grow at temperatures higher than 91° C. and their enzymes demonstrate
greater themostability (Mathur et al., 1992, Stratagies 5:11) than the thermophilic
eubacterial Family A DNA polymerases. Family B polymerases from hyperthermophilic
archaea may be well suited starting substrates for modification(s) to reduce discrimination
against non-conventional nucleotides.
Although the crystal structures of three Family B DNA polymerases have been
solved (Wang et al., 1997, supra; Hopfner, K.-P. et al., 1999, Proc. Natl. Acad.
Sci. 96: 3600; Zhao, 1999, Structure Fold Des., 7:1189), the structures of DNA-polymerase
or dNTP-polymerase co-complexes have not yet been reported. At present, identification
of amino acid residues contributing to nucleotide analog discrimination can only
be inferred from extrapolation to Family A-dNTP structures or from mutagenesis
studies carried out with related Family B DNA polymerases (e.g., human polα,
phage T4, phage Φ29,
T. litoralis DNA polymerase).
Sequence comparison of the Family B DNA polymerases indicate six conserved
regions numbered I-VI (Braithwaite and Ito, 1993, supra). The crystal structure
of bacteriophage RB69 DNA polymerase (Family B) proposed by Wang et al. (Wang et
al., 1997, supra) shows that Y416 in region II ( which corresponds to Y409 in the
Family B DNA polymerase of
Thermococcus species JDF-3) has the same position
as Y115 in HIV reverse transcriptase (RT) and E710 in the Klenow fragment (Family
A polymerases). Modeling of the dNTP and primer template complex in RB69 was carried
out using the atomic coordinates of the reverse transcriptase-DNA cocrystal. This
model predicts the RB69 Y416 packs under the deoxyribose portion of the dNTP. Tyrosine
at this position has been implicated in ribose selectivity, contributing to polymerase
discrimination between ribonucleotides and deoxribonucleotides in mammalian reverse
transcriptases (Y115) (Gao et al., 1997, Proc. Natl. Acad. Sci. USA 94:407; Joyce,
1994, Proc. Natl. Acad. Sci. USA 94:1619) and in Family A DNA polymerases where
modification of the corresponding invariable glutamate residue (E710) reduces discrimination
against ribonucleotides (Gelfand et al., 1998, Pat. No. EPO823479; Astatke et al.,
1998, Proc. Natl. Acad. Sci. USA 96:3402).
Mutagenesis studies done in Family B DNA polymerases also implicate the
region containing the analogous Y in region II in dNTP incorporation and ribose
selectivity. Mutations at the corresponding Y865 in human DNA polymerase α
affect polymerase fidelity and sensitivity to dNTP nucleotide inhibitors such as
AZT-TP, which has a bulky 3′-azido group in place of the 3′-OH group,
BuPdGTP, which contains a butylphenyl group attached to the amino group at the
C-2 position in the guanine base of dGTP (resulting in a bulkier and more hydrophobic
purine base nucleotide) and aphidicolin, a competitive inhibitor of pyrimidine
deoxynucleotide triphosphate. Interestingly, the mutants showed no difference in
their uptake of ddCTP (Dong et al., 1993, J. Biol. Chem. 268: 24163). Additionally,
mutants of bacteriophage T4 DNA polymerase, which have converted L412 to methionine
(M) or isoleucine (I) just one amino acid before the analogous Y (Y411), show extreme
and mild sensitivity, respectively, to the inorganic pyrophosphate analog phosphonoacetic
acid (PAA). Alterations in PAA sensitivity have been shown to predict polymerase
interactions with nucleotide analogs. L412 in T4 DNA polymerase corresponds to
L410 in
Thermococcus species JDF-3 DNA polymerase. The L412M T4 DNA polymerase
mutant was inhibited with 50-fold less ddGTP than wild-type polymerase while the
K
ms for dGTP was similar. As stated by the authors in that study, "[d]espite
the sensitivity of the L412M DNA polymerase to ddGTP, there was no difference found
in the incorporation of ddNTPs by wild-type and L412M DNA polymerase." (Reha-Krantz
et al., 1993, J. Virol. 67:60). In bacteriophage Φ29, mutations in region
II (LYP where Y is analogous to
Thermococcus species JDF3 DNA polymerase
Y409) produce mixed results when challenged with PAA; P255S was hypersensitive
to PAA while L253V was shown to be less sensitive than the wild-type enzyme (Blasco
et al., 1993, J. Biol. Chem. 268: 24106). These data support the role of the LYP
region (region II) in polymerase-nucleotide interactions, but improved incorporation
of ddNTPs was not achieved in these references.
In another study, extensive mutation of region II in the archaeal Family B DNA
polymerase from
Thermococcus litoralis DNA polymerase (VENT™ polymerase,
New England Biolabs) was performed. In that study, 26 different site-directed mutants
were made for the sole intent of examining nucleotide analog discrimination (Gardner
and Jack, 1999, Nucleic Acids Res. 27: 2545). Site-directed mutagenesis of VENT™
DNA polymerase demonstrated that three mutations at Y412 (which corresponds to
JDF-3 DNA polymerase Y409) could alter nucleotide binding (Gardner and Jack, 1999,
supra). Y412V was most significant with a 2 fold increase in dideoxynucleotide
incorporation and a 200 fold increase in the incorporation of ribonucleotide ATP.
The mutation Y412F showed no change in analog incorporation.
Region III of the Family B polymerases (also referred to as motif B) has also
been demonstrated to play a role in nucleotide recognition. This region, which
corresponds to AA 487 to 495 of JDF-3 Family B DNA polymerase, has a consensus
sequence KX
3NSXYG (SEQ ID NO: 5) (Jung et al., 1990, supra; Blasco et
al., 1992, supra; Dong et al., 1993, J. Biol. Chem. 268:21163; Zhu et al., 1994,
Biochem. Biophys. Acta 1219:260; Dong and Wang, 1995, J. Biol. Chem. 270:21563),
and is functionally, but not structurally (Wang et al., 1997, supra), analogous
to KX
3(F/Y)GX
2YG (SEQ ID NO: 6) in helix O of the Family
A DNA polymerases. In Family A DNA polymerases, such as the Klenow fragment and
Taq DNA polymerases, the O helix contains amino acids that play a major role in
dNTP binding (Astatke et al., 1998, J. Mol. Biol. 278:147; Astatke et al., 1995,
J. Biol. Chem. 270:1945; Polesky et al., 1992, J. Biol. Chem 267:8417; Polesky
et al., 1990, J. Biol. Chem. 265:14579; Pandey et al., 1994, J. Biol. Chem. 269:13259;
Kaushik et al., 1996, Biochem. 35:7256). Specifically, helix O contains the F (F763
in the Klenow fragment; F667 in Taq) which confers ddNTP discrimination in Family
A DNA polymerases (KX
3(
F/Y)GX
2YG SEQ
ID NO: 6) (Tabor and Richardson, 1995, supra).
Directed mutagenesis studies in region III of VENT™ DNA polymerase
also targeted an alanine analogous to A485 of the
Thermococcus species JDF-3
DNA polymerase (Jung et al., 1990, supra). These mutants (A→C, A→S,
A→L, A→I, A→F and A→V) exhibited a range of specific
activities from 0.12 to 1.2 times the polymerase activity of the progenitor enzyme
(Gardner and Jack, 1999, Nucl. Acids Res. 27:2545). The dideoxynucleotide incorporation
ranged from 4 to 15 times the unmutated enzyme. Interestingly, the mutant with
the highest dideoxynucleotide incorporation (15×) had a specific activity
of only 0.12× of the original enzyme.
Site-directed mutagenesis studies on the Family B DNA polymerase from
Thermococcus barossii modified each residue independently in the sequence
ILANSF, which corresponds to AA residues 488-493 of the JDF-3 DNA polymerase, to
tyrosine (Reidl et al., U.S. Pat. No. 5,882,904). That study indicated that an
L489Y mutant exhibits approximately 3 times greater incorporation of dideoxynucleotides
relative to an enzyme bearing the wild-type leucine residue at this site.
One area of active research involves the use of nucleic acid arrays, often referred
to as nucleic acid or DNA "chips", in the simultaneous analyses of multiple different
nucleic acid sequences. Many of these applications, such as those described in
U.S. Pat. No. 5,882,904 (Reidl et al., issued Mar. 16, 1999) will benefit from
DNA polymerases exhibiting reduced discrimination against non-conventional nucleotides,
particularly fluorescently-labeled non-conventional nucleotides. Applications being
addressed in the chip format include DNA sequencing and mutation detection, among
others. For example, the "mini-sequencing" methods (e.g., Pastinen et al., 1997,
Genome Res. 7: 606; Syvanen, 1999, Human Mutation 13: 1-10) and the arrayed primer
extension (APEX) mutation detection method (Shumaker et al., 1996, Hum. Mutat.
7: 346) and methods like them can benefit from DNA polymerases with reduced discrimination
against fluorescently-labeled or other non-conventional nucleotides. There is a
need in the art for a non-discriminating DNA polymerase for use in chip or gel
based mini-sequencing systems. Such a system would advantageously permit detection
of multiplexed single nucleotide polymorphisms (SNPs) and allow for quantitative
genotyping. Identification of sequence variation permits the diagnosis and treatment
of genetic disorders, predisposition to multifactorial diseases, and sensitivity
to new or existing pharmaceutical products.
There is a need in the art for DNA polymerases with reduced discrimination
against unconventional nucleotides. There is particularly a need in the art for
thermostable DNA polymerases exhibiting reduced discrimination against dideoxynucleotides,
and further, for DNA polymerases exhibiting reduced discrimination against fluorescently
labeled dideoxynucleotides.
SUMMARY OF THE INVENTION
The present invention relates to compositions and methods utilizing DNA polymerase
enzymes exhibiting reduced discrimination against non-conventional nucleotides.
Enzymes with this quality are useful in many applications calling for the detectable
labeling of nucleic acids and are particularly useful in DNA sequencing applications.
The invention further relates to a Family B DNA polymerase having one or more
mutations at a site or sites corresponding to L408, P410, S345, and/or A485 of
SEQ ID NO: 2, or a fragment thereof which retains the ability to direct the template-dependent
polymerization of nucleic acid. The invention also encompasses mutants and modified
versions (e.g., reversibly inactivated versions of a Family B polymerase prepared,
for example, by chemical modification or antibody complexing) of a Family B polymerase
mutated at sites corresponding to L408, P410 and or A485 of SEQ ID NO: 2.
In one embodiment, the DNA polymerase has a dual mutation comprising comprising
a serine to proline mutation at a site corresponding to S345 of SEQ ID NO: 2; and
a proline to leucine mutation at a site corresponding to P410 of SEQ ID NO: 2.
The invention encompasses purified thermostable DNA polymerase having an amino
acid sequence presented in SEQ ID NO: 2 from residue 1 to 776.
In one embodiment, the thermostable DNA polymerase is isolated from
Thermococcus
species JDF-3.
In another embodiment, the thermostable polymerase is isolated from a recombinant
organism transformed with a vector that codes for the expression of
Thermococcus
species JDF-3 DNA polymerase.
The invention further encompasses a recombinant vector comprising the nucleotide
sequence presented in SEQ ID NO: 1.
The invention further encompasses an isolated recombinant polypeptide comprising
the amino acid sequence of SEQ ID NO: 2 or a functional fragment thereof.
The invention further encompasses an isolated recombinant DNA polymerase from
Thermococcus species JDF-3 that is 3′ to 5′ exonuclease deficient.
In one embodiment, the isolated recombinant DNA polymerase of has an aspartic
acid to threonine or alanine mutation at the amino acid corresponding to D141 of
SEQ ID NO: 2 or a glutamic acid to alanine mutation at the amino acid corresponding
to E143 of SEQ ID NO: 2.
In another embodiment, the isolated recombinant DNA polymerase has an aspartic
acid to threonine or alanine mutation at the amino acid corresponding to D141 of
SEQ ID NO: 2 and a glutamic acid to alanine mutation at the amino acid corresponding
to E143 of SEQ ID NO: 2.
The invention further encompasses an isolated recombinant DNA polymerase having
reduced discrimination against non-conventional nucleotides.
In one embodiment, the DNA polymerase is a Family B DNA polymerase.
In another embodiment, the DNA polymerase further comprises a mutation selected
from the group consisting of: a leucine to histidine mutation at a site corresponding
to L408 of SEQ ID NO: 2; a leucine to phenylalanine mutation at a site corresponding
to L408 of SEQ ID NO: 2; a proline to leucine mutation at a site corresponding
to P410 of SEQ ID NO: 2; and an alanine to threonine mutation at a site corresponding
to A485 of SEQ ID NO: 2.
The invention further encompasses an isolated recombinant DNA polymerase having
the alanine to threonine mutation at the site corresponding to A485 of SEQ ID NO:
2 further comprising a mutation selected from the group consisting of: a leucine
to histidine mutation at a site corresponding to L408 of SEQ ID NO: 2; a leucine
to phenylalanine mutation at a site corresponding to L408 of SEQ ID NO: 2; and
a proline to leucine mutation at a site corresponding to P410 of SEQ ID NO: 2.
The invention further encompasses an isolated recombinant DNA polymerase having
the a proline to leucine mutation at a site corresponding to P410 of SEQ ID NO:
2, further comprising of serine to proline mutation at a site corresponding to
S345 of SEQ ID NO: 2
In another embodiment, the DNA polymerase has reduced discrimination against a
non-conventional nucleotide selected from the group consisting of: dideoxynucleotides,
ribonucleotides and conjugated nucleotides.
In another embodiment, conjugated nucleotide is selected from the group consisting
of radiolabeled nucleotides, fluorescently labeled nucleotides, biotin labeled
nucleotides, chemiluminescently labeled nucleotides and quantum dot labeled nucleotides.
The invention further encompasses an isolated recombinant Family B DNA polymerase
comprising an alanine to threonine mutation at the site corresponding to A485 of
SEQ ID NO: 2 or a mutation at a site corresponding to L408 or P410 of SEQ ID NO:
2, wherein the DNA polymerase has reduced discrimination against non-conventional
nucleotides relative to the wild-type form of that polymerase.
In one embodiment, the Family B DNA polymerase is 3′ to 5′ exonuclease deficient.
In another embodiment, the Family B DNA polymerase has a mutation at an amino
acid corresponding to D141 or E143 of SEQ ID NO: 2.
In another embodiment, the Family B DNA polymerase has an aspartic acid to threonine
or alanine mutation at a site corresponding to D141 of SEQ ID NO: 2.
In another embodiment, the Family B DNA polymerase has a glutamic acid to alanine
mutation at a site corresponding to E143 of SEQ ID NO: 2.
In another embodiment, the Family B DNA polymerase has a glutamic acid to alanine
mutation at a site corresponding to E143 of SEQ ID NO: 2 and has an aspartic acid
to threonine or alanine mutation at the amino acid corresponding to D141 of SEQ
ID NO: 2.
In another embodiment, the Family B DNA polymerase is thermostable.
In another embodiment, the Family B DNA polymerase is archaeal.
In another embodiment, the Family B DNA polymerase comprises a leucine to histidine
mutation at a site corresponding to L408 of SEQ ID NO: 2.
In another embodiment, the Family B DNA polymerase comprises a leucine to phenylalanine
mutation at a site corresponding to L408 of SEQ ID NO: 2.
In another embodiment, the Family B DNA polymerase comprises a proline to leucine
mutation at a site corresponding to P410 of SEQ ID NO: 2.
In another embodiment, the Family B DNA polymerase comprises an alanine to threonine
mutation at a site corresponding to A485 of SEQ ID NO: 2.
In another embodiment, the Family B DNA polymerase comprising an alanine to threonine
mutation at a site corresponding to A485 of SEQ ID NO: 2 comprises a leucine to
histidine mutation at a site corresponding to L408 of SEQ ID NO: 2.
In another embodiment, the Family B DNA polymerase comprising an alanine to threonine
mutation at a site corresponding to A485 of SEQ ID NO: 2 comprises a leucine to
phenylalanine mutation at a site corresponding to L408 of SEQ ID NO: 2.
In another embodiment, the Family B DNA polymerase comprising an alanine to threonine
mutation at a site corresponding to A485 of SEQ ID NO: 2 comprises a proline to
leucine mutation at a site corresponding to P410 of SEQ ID NO: 2.
In another embodiment, the Family B DNA polymerase comprising a proline to leucine
mutation at a site corresponding to P410 of SEQ ID NO: 2, further having a serine
to proline mutation at a site corresponding to S345 of SEQ ID NO: 2.
In another embodiment, the Family B DNA polymerase comprises a serine to proline
mutation at a site corresponding to S345 of SEQ ID NO: 2, and may further comprise
a mutation at a site corresponding to T604 of SEQ ID NO: 2.
In another embodiment, the Family B DNA polymerase comprises a tyrosine to cysteine
mutation at a site corresponding to Y497 of SEQ ID NO: 2, and may further comprise
an isoleucine to valine mutation at a site corresponding to I630 of SEQ ID NO: 2.
In another embodiment, the Family B DNA polymerase comprises a glutamic acid
to
lysine mutation at a site corresponding to E645 of SEQ ID NO: 2.
In another embodiment, the Family B DNA polymerase comprises a glutamic acid
to
lysine mutation at a site corresponding to E578 of SEQ ID NO: 2, and may further
comprise an arginine to methionine mutation at a site corresponding to R465 of
SEQ ID NO: 2.
In another embodiment, the Family B DNA polymerase comprises a leucine to glutamine
mutation at a site corresponding to L396 of SEQ ID NO: 2, and may further comprise
a mutation at a site corresponding to V401, N424, P569, E617, or V640 of SEQ ID
NO: 2.
In another embodiment, the Family B DNA polymerase comprises a serine to asparagine
mutation at a site corresponding to S651 of SEQ ID NO: 2.
In another embodiment, the Family B DNA polymerase comprises a leucine to proline
mutation at a site corresponding to L396 of SEQ ID NO: 2, and may further comprise
a mutation at a site corresponding to E459 of SEQ ID NO: 2.
In another embodiment, the Family B DNA polymerase comprises a leucine to proline
mutation at a site corresponding to L456 of SEQ ID NO: 2, and may further comprise
a mutation at a site corresponding to E658 of SEQ ID NO: 2.
In another embodiment, the Family B DNA polymerase comprises a leucine to histidine
mutation at a site corresponding to L408 of SEQ ID NO: 2, and may further comprise
a mutation at a site corresponding to V437, or L478 of SEQ ID NO: 2. The L408H
mutation was isolated both in the dideoxynucleotide and the dye-dideoxynucleotide
screens described herein.
In another embodiment, the Family B DNA polymerase comprises an tyrosine to asparagine
mutation at a site corresponding to Y496 of SEQ ID NO: 2.
In another embodiment, the Family B DNA polymerase has reduced discrimination
against a non-conventional nucleotide selected from the group consisting of: dideoxynucleotides,
ribonucleotides and conjugated nucleotides.
In another embodiment, the conjugated nucleotide is selected from the group consisting
of radiolabeled nucleotides, fluorescently labeled nucleotides, biotin labeled
nucleotides, chemiluminescently labeled nucleotides and quantum dot labeled nucleotides.
In another embodiment, an isolated recombinant DNA polymerase having reduced
discrimination
against non-conventional nucleotides or an isolated recombinant Family B DNA polymerase
comprising an alanine to threonine mutation at the site corresponding to A485 of
SEQ ID NO: 2 or a mutation at a site corresponding to L408 or P410 of SEQ ID NO:
2, wherein the DNA polymerase has reduced discrimination against non-conventional
nucleotides relative to the wild-type form of that polymerase further comprises
a mutation at an amino acid residue in the polymerase that corresponds to a mutation
selected from the group consisting of: a Y to V mutation at amino acid 409 of SEQ
ID NO: 2; an A to C, S, L, I, F, or V mutation at amino acid 485 of SEQ ID NO:
2; a Y to S mutation at amino acid 494 of SEQ ID NO: 2; a Y to L mutation at amino
acid 496 of SEQ ID NO: 2; and an A to Y mutation at amino acid 490 of SEQ ID NO: 2.
In another embodiment, an isolated recombinant DNA polymerase having reduced
discrimination
against non-conventional nucleotides or an isolated recombinant Family B DNA polymerase
comprising an alanine to threonine mutation at the site corresponding to A485 of
SEQ ID NO: 2 or a mutation at a site corresponding to L408 or P410 of SEQ ID NO:
2, wherein the DNA polymerase has reduced discrimination against non-conventional
nucleotides relative to the wild-type form of that polymerase further comprises
a mutation at an amino acid of the polymerase corresponding to one of amino acids
483 to 496, inclusive, of SEQ ID NO: 2.
In one embodiment, the mutation is at an amino acid of the polymerase corresponding
to one of amino acids 485, 490, 494, or 496 of SEQ ID NO: 2.
The invention further encompasses an isolated recombinant Family B DNA polymerase
comprising an alanine to threonine mutation at an amino acid corresponding to A485T
of SEQ ID NO: 2 and at least one substitution in the polymerase of an amino acid
corresponding to L408, Y409, or P410, respectively, of SEQ ID NO: 2.
The invention further encompasses an isolated recombinant Family B DNA polymerase
comprising an amino acid other than A at an amino acid of the polymerase corresponding
to A485 of SEQ ID NO: 2, and at least one substitution in the polymerase of an
amino acid corresponding to L408, Y409, or P410, respectively, of SEQ ID NO: 2.
The invention further encompasses a recombinant vector comprising a nucleic acid
sequence encoding the Family B DNA polymerase.
The invention further encompasses a method of labeling a complementary strand
of DNA, the method comprising the step of contacting a template DNA molecule with
a recombinant Family B DNA polymerase from
Thermococcus species JDF-3, wherein
the DNA polymerase has reduced discrimination against non-conventional nucleotides,
and a non-conventional nucleotide, under conditions and for a time sufficient to
permit the DNA polymerase to synthesize a complementary DNA strand and to incorporate
the non-conventional nucleotide into the synthesized complementary DNA strand.
The invention further encompasses a method of labeling a complementary strand
of DNA, the method comprising the step of contacting a template DNA molecule with
a recombinant Family B DNA polymerase comprising an alanine to threonine mutation
at a site corresponding to A485 of SEQ ID NO: 2 or a mutation at a site corresponding
to L408 or P410 of SEQ ID NO: 2, wherein the DNA polymerase has reduced discrimination
against non-conventional nucleotides, and a non-conventional nucleotide, under
conditions and for a time sufficient to permit the DNA polymerase to synthesize
a complementary DNA strand and to incorporate the non-conventional nucleotide into
the synthesized complementary DNA strand.
In one embodiment, the recombinant Family B DNA polymerase is 3′ to 5′
exonuclease deficient.
In another embodiment, the recombinant Family B polymerase comprises a leucine
to histidine mutation at a site corresponding to amino acid L408 of SEQ ID NO: 2.
In another embodiment, the recombinant Family B polymerase comprises a leucine
to phenylalanine mutation at a site corresponding to amino acid L408 of SEQ ID
NO: 2.
In another embodiment, the recombinant Family B polymerase comprises a proline
to leucine mutation at a site corresponding to amino acid P410 of SEQ ID NO: 2.
In another embodiment, the recombinant Family B polymerase comprises an alanine
to threonine mutation at a site corresponding to amino acid A485 of SEQ ID NO: 2.
In another embodiment, the recombinant Family B polymerase comprising an alanine
to threonine mutation at a site corresponding to amino acid A485 of SEQ ID NO:
2 comprises a leucine to histidine mutation at an amino acid corresponding to L408
of SEQ ID NO: 2.
In another embodiment, the recombinant Family B polymerase comprising an alanine
to threonine mutation at a site corresponding to amino acid A485 of SEQ ID NO:
2 comprises a leucine to phenylalanine mutation at an amino acid corresponding
to L408 of SEQ ID NO: 2.
In another embodiment, the recombinant Family B polymerase comprising an alanine
to threonine mutation at a site corresponding to amino acid A485 of SEQ ID NO:
2 comprises a proline to leucine mutation at an amino acid corresponding to P410
of SEQ ID NO: 2.
In another embodiment, the recombinant Family B polymerase has reduced discrimination
against a non-conventional nucleotide selected from the group consisting of: dideoxynucleotides,
ribonucleotides, and conjugated nucleotides.
In another embodiment, the conjugated nucleotide is selected from the group consisting
of radiolabeled nucleotides, fluorescently labeled nucleotides, biotin labeled
nucleotides, chemiluminescently labeled nucleotides and quantum dot labeled nucleotides.
The invention further encompasses a method of sequencing DNA comprising the steps
of contacting a DNA strand to be sequenced with a sequencing primer, a recombinant
Family B DNA polymerase from
Thermococcus species JDF-3, wherein the DNA
polymerase has reduced discrimination against non-conventional nucleotides, and
a chain-terminating nucleotide analog, under conditions that permit the DNA polymerase
to synthesize a complementary DNA strand, and to incorporate nucleotides into the
synthesized complementary DNA strand, wherein incorporation of a chain-terminating
nucleotide analog results in the termination of chain elongation, such that the
nucleotide sequence of the template DNA strand is determined.
The invention further encompasses a method of sequencing DNA comprising the steps
of contacting a DNA strand to be sequenced with a sequencing primer, a recombinant
Family B DNA polymerase comprising an alanine to threonine mutation at a site corresponding
to A485 of SEQ ID NO: 2 or a mutation at a site corresponding to L408, S345 or
P410 of SEQ ID NO: 2, where the DNA polymerase has reduced discrimination against
non-conventional nucleotides, and a chain-terminating nucleotide analog, under
conditions that permit the DNA polymerase to synthesize a complementary DNA strand,
and to incorporate nucleotides into the synthesized complementary DNA strand, wherein
incorporation of a chain-terminating nucleotide analog results in the termination
of chain elongation, such that the nucleotide sequence of the template DNA strand
is determined.
In one embodiment, the recombinant DNA polymerase is deficient in 3′ to
5′ exonuclease activity.
In another embodiment, the recombinant Family B polymerase has a leucine to histidine
mutation at a site corresponding to amino acid L408 of SEQ ID NO: 2.
In another embodiment, the recombinant Family B polymerase has a leucine to phenylalanine
mutation at a site corresponding to amino acid L408 of SEQ ID NO: 2.
In another embodiment, the recombinant Family B polymerase has a proline to leucine
mutation at a site corresponding to amino acid P410 of SEQ ID NO: 2.
In another embodiment, the Family B DNA polymerase comprising a proline to leucine
mutation at a site corresponding to P410 of SEQ ID NO: 2, further having a serine
to proline mutation at a site corresponding to S345 of SEQ ID NO: 2.
In another embodiment, the recombinant Family B polymerase has an alanine to
threonine
mutation at a site corresponding to amino acid A485 of SEQ ID NO: 2.
In another embodiment, the recombinant Family B polymerase having an alanine
to
threonine mutation at a site corresponding to amino acid A485 of SEQ ID NO: 2 has
a leucine to histidine mutation at a site corresponding to L408 of SEQ ID NO: 2.
In another embodiment, the recombinant Family B polymerase having an alanine
to
threonine mutation at a site corresponding to amino acid A485 of SEQ ID NO: 2 has
a leucine to phenylalanine mutation at a site corresponding to L408 of SEQ ID NO: 2.
In another embodiment, the recombinant Family B polymerase having an alanine
to
threonine mutation at a site corresponding to amino acid A485 of SEQ ID NO: 2 has
a proline to leucine mutation at a site corresponding to P410 of SEQ ID NO: 2.
In another embodiment, the chain-terminating nucleotide analog is a dideoxynucleotide.
In another embodiment, the dideoxynucleotide is detectably labeled.
In another embodiment, the dideoxynucleotide is fluorescently labeled.
In another embodiment, the dideoxynucleotide is labeled with a moiety selected
from the group consisting of fluorescein and rhodamine.
The invention also encompasses a kit for performing the methods disclosed herein.
The invention also encompasses methods of making a recombinant DNA polymerase
as disclosed here, comprising culturing a host cell containing a nucleic acid sequence
encoding said polymerase under conditions which permit production of said DNA polymerase.
The invention encompasses a mixture of a mutant DNA polymerase described herein
and another DNA polymerase such as Taq DNA polymerase (preferably the mutant form,
F667Y). Such a mixture is useful in that it may increase signal uniformity generated
from polymerization of a labeled nucleotide into a synthetic nucleotide.
As used herein, "discrimination" refers to the tendency of DNA polymerase to
not
incorporate non-conventional nucleotides into a nascent DNA polymer. DNA polymerase
has the ability to sense nucleotide structure, including but not limited to nucleotide
base complementarity, and structural features of the sugar and heterocyclic base,
thereby allowing DNA polymerase to preferentially utilize conventional deoxynucleotides
rather than non-conventional nucleotides for incorporation into a nascent polymer.
DNA polymerase strongly prefers to incorporate the conventional deoxynucleotides
dATP, dCTP, dGTP and TTP into DNA polymers; the polymerase is unlikely to progress
with an unconventional nucleotide in its binding pocket.
As used herein, "reduced discrimination" refers to a reduction of at least 50%
in the tendency of a DNA polymerase to exclude a non-conventional nucleotide from
(that is, to not incorporate non-conventional nucleotides into) a nascent DNA polymer,
relative to a parental or wild type DNA polymerase which does not exhibit reduced
discrimination. The preference of DNA polymerase to incorporate the conventional
deoxynucleotides dATP, dCTP, dGTP and TTP rather than non-conventional nucleotides
into DNA polymers is thereby reduced compared to the natural level of preference,
such that non-conventional nucleotides are more readily incorporated into DNA polymers
by DNA polymerase. According to the invention, a polymerase exhibiting reduced
discrimination will exhibit reduced discrimination against at least one non-conventional
nucleotides, but may not exhibit reduced discrimination against all non-conventional nucleotides.
According to the invention, discrimination is quantitated by measuring
the concentration of a non-conventional nucleotide required to inhibit the incorporation
of the corresponding conventional nucleotide by 50%. This concentration is referred
to herein as the "I
50%" for a non-conventional nucleotide. Discrimination
against a given non-conventional nucleotide is "reduced" if the I
50%
for that non-conventional nucleotide is reduced by at least two fold (50%) relative
to an identical assay containing, in place of the mutant DNA polymerase, a parental
DNA polymerase.
Alternatively, reduced discrimination may be quantitated by determining
the amount of a non-conventional nucleotide (for example, a dideoxynucleotide,
ribonucleotide, or cordycepin) required in a reaction with a mutant polymerase
having reduced discrimination to generate a sequencing ladder identical to a sequencing
ladder produced using the wild-type or parental enzyme. The sequencing ladder can
be examined, for example, in the range of 1 to 400 bases from the primer terminus,
and the ladders will be identical in the number of extension products generated
as well as the lengths of extension products generated in the sequencing reaction.
For this type of assay, a constant amount of dNTPs and varying amounts of non-conventional
nucleotides are used to generate a sequencing ladder with both the wild-type (or
parental) enzyme and the mutant polymerase (for ribonucleotides, a sequencing ladder
is generated by alkali cleavage of the polymerization products). See Gardner &
Jack, 1999, supra. A mutant exhibits reduced discrimination if it requires at least
two-fold (50%) less, five-fold (80%) less, ten-fold (100%) less, etc. of the amount
of the non-conventional nucleotide used by the wild-type or parental polymerase
to produce a sequencing ladder identical (with respect to the number and length
of extension products generated) to that generated by the wild-type or parental enzyme.
As used herein, the term "parental" or "progenitor" refers to a polymerase used
as the starting material in generating a mutant polymerase having reduced discrimination.
The term "parental" is meant to encompass not only a so-called "wild-type" enzyme
as it occurs in nature, but also intermediate forms, for example, an exonuclease
deficient enzyme that is used as the starting material for generating an enzyme
with reduced discrimination against non-conventional nucleotides.
As used herein, "non-conventional nucleotide" refers to a) a nucleotide structure
that is not one of the four conventional deoxynucleotides dATP, dCTP, dGTP, and
TTP recognized by and incorporated by a DNA polymerase, b) a synthetic nucleotide
that is not one of the four conventional deoxynucleotides in (a), c) a modified
conventional nucleotide, or d) a ribonucleotide (since they are not normally recognized
or incorporated by DNA polymerases) and modified forms of a ribonucleotide. Non-conventional
nucleotides include but are not limited to those listed in Table III, which are
commercially available, for example, from New England Nuclear. Any one of the above
non-conventional nucleotides may be a "conjugated nucleotide", which as used herein
refers to nucleotides bearing a detectable label, including but not limited to
a fluorescent label, isotope, chemiluminescent label, quantum dot label, antigen,
or affinity moiety.
As used herein, the term "cell", "cell line" and "cell culture" can be used interchangeably
and all such designations include progeny. Thus, the words "transformants" or "transformed
cells" includes the primary subject cell and cultures derived therefrom without
regard for the number of transfers. It is also understood that all progeny may
not be precisely identical in DNA content, due to deliberate or inadvertent mutations.
Mutant progeny that have the same functionality as screened for in the originally
transformed cell are included.
As used herein, the term "organism transformed with a vector" refers to an organism
carrying a recombinant gene construct.
As used herein, "thermostable" refers to a property of a DNA polymerase, such
that the enzyme active at elevated temperatures and is resistant to DNA duplex-denaturing
temperatures in the range of about 93° C. to about 97° C. "Active" means
the enzyme retains the ability to effect primer extension reactions when subjected
to elevated or denaturing temperatures for the time necessary to effect denaturation
of double-stranded nucleic acids. Elevated temperatures as used herein refer to
the range of about 70° C. to about 75° C., whereas non-elevated temperatures
as used herein refer to the range of about 35° C. to about 50° C.
As used herein, "archaeal" refers to an organism or to a DNA polymerase from
an
organism of the kingdom Archaea
As used herein, "sequencing primer" refers to an oligonucleotide, whether natural
or synthetic, which serves as a point of initiation of nucleic acid synthesis by
a polymerase following annealing to a DNA strand to be sequenced. A primer is typically
a single-stranded oligodeoxyribonucleotide. The appropriate length of a primer
depends on the intended use of the primer, but for DNA sequencing applications
typically ranges from about 15 to about 40 nucleotides in length.
As used herein, "Family B DNA polymerase" refers to any DNA polymerase that is
classified as a member of the Family B DNA polymerases, where the Family B classification
is based on structural similarity to
E. coli DNA polymerase II. The Family
B DNA polymerases, formerly known as α-family polymerases, include, but are
not limited to those listed as such in Table I.
As used herein, "Family A DNA polymerase" refers to any DNA polymerase that is
classified as a member of the Family A DNA polymerases, where the Family A classification
is based on structural similarity to
E. coli DNA polymerase I. Family A
DNA polymerases include, but are not limited to those listed as such in Table I.
As used herein, "3′ to 5′ exonuclease deficient" or "3′
to
5′ exo
-" refers to an enzyme that substantially lacks the ability
to remove incorporated nucleotides from the 3′ end of a DNA polymer. DNA
polymerase exonuclease activities, such as the 3′ to 5′ exonuclease
activity exemplified by members of the Family B polymerases, can be lost through
mutation, yielding an exonuclease-deficient polymerase. As used herein, a DNA polymerase
that is deficient in 3′ to 5′ exonuclease activity substantially
lacks 3′ to 5′ exonuclease activity. "Substantially lacks" encompasses
a complete lack of activity, or a "substantial" lack of activity. "Substantial"
lack of activity means that the 3′ exonuclease activity of the mutant polymerase
relative to the parental polymerase is 0.03%, and also may be 0.05%, 0.1%, 1%,
5%, 10%, or 20%, but is not higher than 50% of the 3′ exonuclease activity
of the parental or wild type polymerase.
As used herein, "mutation" refers to a change introduced into a starting parental
DNA sequence that changes the amino acid sequence encoded by the DNA. The consequences
of a mutation include but are not limited to the creation of a new character, property,
function, or trait not found in the protein encoded by the parental DNA.
As used herein, "wild-type" refers to the typical state of an organism, strain,
gene, protein or characteristic as it occurs in nature. The wild-type is therefore
the natural state that is distinguished from a mutant, which was derived from the
wild type by introduction of change(s) to the wild-type.
As used herein, "corresponding" refers to sequence similarity in a comparison
of two or more nucleic acids or polypeptides, where functionally equivalent domains
or sub-sequences are identified; such functionally equivalent domains or sub-sequences
or amino acids within such a domain or sub-sequence are said to "correspond". That
is, two or more sequences are compared through a comparative alignment analysis
in which an entire sequence is examined for regions of sequence that are similar
or identical, and thus regions likely
