U.S. patent application number 10/052417 was filed with the patent office on 2002-10-03 for thermostable dna polymerases incorporating nucleoside triphosphates labeled with fluorescein family dyes.
This patent application is currently assigned to F. Hoffmann-La Roche AG.. Invention is credited to Gelfand, David Harrow, Kalman, Lisa Vivian, Myers, Thomas W., Reichert, Fred Lawrence, Sigua, Christopher Lim.
Application Number | 20020142333 10/052417 |
Document ID | / |
Family ID | 22017355 |
Filed Date | 2002-10-03 |
United States Patent
Application |
20020142333 |
Kind Code |
A1 |
Gelfand, David Harrow ; et
al. |
October 3, 2002 |
Thermostable DNA polymerases incorporating nucleoside triphosphates
labeled with fluorescein family dyes
Abstract
Modified thermostable DNA polymerases having enhanced efficiency
for incorporating unconventional nucleotides such as those labeled
with fluorescein family dyes are advantageous in many in vitro DNA
synthesis applications. Such enzymes are particularly useful for
use in chain termination nucleic acid sequencing protocols, as are
native forms of such enzymes. Genes encoding the modified enzymes
and methods for their production and use offer cost and efficiency
advantages for DNA sequencing.
Inventors: |
Gelfand, David Harrow;
(Oakland, CA) ; Kalman, Lisa Vivian; (San
Francisco, CA) ; Reichert, Fred Lawrence; (Oakland,
CA) ; Sigua, Christopher Lim; (Antioch, CA) ;
Myers, Thomas W.; (Alameda, CA) |
Correspondence
Address: |
PENNIE & EDMONDS LLP
1155 Avenue of the Americas
New York
NY
10036
US
|
Assignee: |
F. Hoffmann-La Roche AG.
|
Family ID: |
22017355 |
Appl. No.: |
10/052417 |
Filed: |
January 17, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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10052417 |
Jan 17, 2002 |
|
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|
09146631 |
Sep 3, 1998 |
|
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60058525 |
Sep 11, 1997 |
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Current U.S.
Class: |
435/6.12 ;
435/199; 435/91.2 |
Current CPC
Class: |
C12N 9/1252
20130101 |
Class at
Publication: |
435/6 ; 435/199;
435/91.2 |
International
Class: |
C12Q 001/68; C12P
019/34; C12N 009/22 |
Claims
We claim:
1. A recombinant thermostable DNA polymerase which is characterized
in that a) in its native form said polymerase comprises the amino
acid sequence LeuSerXaaXaaLeuXaaXaaProXaaXaaGlu (SEQ ID NO: 1),
whereby "Xaa" at positions 3, 4, 6, 9, and 10 of said sequence are
any amino acid residue, and "Xaa" at position 7 of said sequence is
Val or Ile; b) said "Xaa" at position 4 is mutated in comparison to
said native sequence, except that "Xaa" at position 4 is not
mutated to Glu; and c) said thermostable DNA polymerase has a level
of discrimination against incorporation of nucleotides labeled with
fluorescein family dyes which is reduced in comparison to the
native form of said polymerase.
2. The recombinant thermostable DNA polymerase of claim 1 wherein
said nucleotide is a dideoxynucleotide and said level of
discrimination is at least 3-fold lower than that of said native
form of said polymerase.
3. The recombinant thermostable DNA polymerase of claim 2 wherein
said level of discrimination is measured by determining the
concentration of a dideoxynucleotide labeled with a fluorescein dye
that is required for 50% inhibition of DNA synthesis.
4. The thermostable DNA polymerase of claim 2 wherein said
polymerase is from a thermophilic species selected from the group
consisting of Thermosipho africanus, Bacillus caldotenax, and
Bacillus stearothermophilus.
5. The thermostable DNA polymerase of claim 2 wherein said
polymerase is from a Thermus species.
6. The recombinant thermostable DNA polymerase of claim 5 which is
characterized in that a) in its native form said polymerase
comprises the amino acid sequence LeuSerXaaXaaLeuXaaIleProTyrGluGlu
(SEQ ID NO: 2), whereby "Xaa" at position 3 is Gln or Gly, "Xaa" at
position 4 is any amino acid, and "Xaa" at position 6 is Ser or Ala
b) said "Xaa" at position 4 is mutated in comparison to said native
sequence, except that "Xaa" at position 4 is not mutated to Glu;
and c) said thermostable DNA polymerase has a level of
discrimination against incorporation of nucleotides labeled with
fluorescein family dyes which is reduced in comparison to the
native form of said polymerase.
7. The recombinant thermostable DNA polymerase of claim 6 which is
characterized in that a) in its native form said polymerase
comprises the amino acid sequence LeuSerGlnXaaLeuAlaIleProTyrGluGlu
(SEQ ID NO:3), whereby "Xaa" at position 4 is any amino acid b)
said "Xaa" at position 4 is mutated in comparison to said native
sequence, except that "Xaa" at position 4 is not mutated to Glu;
and c) said thermostable DNA polymerase has a level of
discrimination against incorporation of nucleotides labeled with
fluorescein family dyes which is reduced in comparison to the
native form of said polymerase.
8. The recombinant thermostable DNA polymerase of claim 7 which is
characterized in that said "Xaa" at position 4 is mutated to
Lys.
9. The thermostable DNA polymerase of claim 2 wherein said
polymerase is from a Thermotoga species.
10. The recombinant thermostable DNA polymerase of claim 9 which is
characterized in that a) in its native form said polymerase
comprises the amino acid sequence LeuSerValXaaLeuGlyXaaProValLysGlu
(SEQ ID NO: 4), whereby "Xaa" at position 4 is any amino acid and
"Xaa" at position 7 is Val or Ile. b) said "Xaa" at position 4 is
mutated in comparison to said native sequence, except that "Xaa" at
position 4 is not mutated to Glu; and c said thermostable DNA
polymerase has a level of discrimination against incorporation of
nucleotides labeled with fluorescein family dyes which is reduced
in comparison to the native form of said polymerase.
11. A nucleic acid sequence encoding a recombinant thermostable DNA
polymerase which is characterized in that a) in its native form
said polymerase comprises the amino acid sequence
LeuSerXaaXaaLeuXaaXaaProXaaX- aaGlu (SEQ ID NO: 1), whereby "Xaa"
at positions 3, 4, 6, 9, and 10 of said sequence are any amino acid
residue, and "Xaa" at position 7 of said sequence is Val or Ile; b)
said "Xaa" at position 4 is mutated in comparison to said native
sequence, except that "Xaa" at position 4 is not mutated to Glu;
and c) said thermostable DNA polymerase has a level of
discrimination against incorporation of nucleotides labeled with
fluorescein family dyes which is reduced in comparison to the
native form of said polymerase.
12. The nucleic acid sequence of claim 11 wherein said nucleotide
is a dideoxynucleotide and said level of discrimination is at least
3-fold lower than that of said native form of said polymerase.
13. The nucleic acid sequence of claim 12 wherein said level of
discrimination is measured by determining the concentration of a
dideoxynucleotide labeled with a fluorescein dye that is required
for 50% inhibition of DNA synthesis.
14. The nucleic acid sequence of claim 12 wherein said polymerase
is from a thermophilic species selected from the group consisting
of Thermosipho africanus, Bacillus caldotenax, and Bacillus
stearothermophilus.
15. The nucleic acid sequence of claim 12 wherein said polymerase
is from a Thermus species.
16. The nucleic acid sequence of claim 15 which is characterized in
that a) in its native form said polymerase comprises the amino acid
sequence LeuSerXaaXaaLeuXaaIleProTyrGluGlu (SEQ ID NO: 2), whereby
"Xaa" at position 3 is Gln or Gly, "Xaa" at position 4 is any amino
acid, and "Xaa" at position 6 is Ser or Ala b) said "Xaa" at
position 4 is mutated in comparison to said native sequence, except
that "Xaa" at position 4 is not mutated to Glu; and c) said
thermostable DNA polymerase has a level of discrimination against
incorporation of nucleotides labeled with fluorescein family dyes
which is reduced in comparison to the native form of said
polymerase.
17. The nucleic acid sequence of claim 15 which is characterized in
that a) in its native form said polymerase comprises the amino acid
sequence LeuSerGlnXaaLeuAlaIleProTyrGluGlu (SEQ ID NO:3), whereby
"Xaa" at position 4 is any amino acid b) said "Xaa" at position 4
is mutated in comparison to said native sequence, except that "Xaa"
at position 4 is not mutated to Glu; and c) said thermostable DNA
polymerase has a level of discrimination against incorporation of
nucleotides labeled with fluorescein family dyes which is reduced
in comparison to the native form of said polymerase.
18. The nucleic acid sequence of claim 17 which is characterized in
that said "Xaa" at position 4 is mutated to Lys.
19. The nucleic acid sequence of claim 12 wherein said polymerase
is from a Thermotoga species.
20. The nucleic acid sequence of claim 19 which is characterized in
that a) in its native form said polymerase comprises the amino acid
sequence LeuSerValXaaLeuGlyXaaProValLysGlu (SEQ ID NO: 4), whereby
"Xaa" at position 4 is any amino acid and "Xaa" at position 7 is
Val or Ile. b) said "Xaa" at position 4 is mutated in comparison to
said native sequence, except that "Xaa" at position 4 is not
mutated to Glu; and c) said thermostable DNA polymerase has a level
of discrimination againstincorporation of nucleotides labeled with
fluorescein family dyes which is reduced in comparison to the
native form of said polymerase.
21. A method of DNA sequencing which comprises a) providing a
thermostable DNA polymerase characterized in that i) said
polymerase comprises the amino acid sequence
LeuSerXaaXaaLeuXaaXaaProXaaXaaGlu (SEQ ID NO: 1), whereby "Xaa" at
positions 3, 6, 9, and 10 of this sequence are any amino acid
residue, and "Xaa" at position 4 can be any amino acid except Glu,
and "Xaa" at position 7 of this sequence is Val or Ile ii) said
polymerase has a reduced level of discrimination against
incorporation of nucleotides labeled with fluorescein family dyes,
and b) providing a dye-terminator labeled with a negatively charged
fluorescent dye, and c) performing a dye-terminator sequencing
reaction.
22. The method of claim 21 wherein said nucleotide is a
dideoxynucleotide and said level of discrimination is measured by
determining the ratio of the concentration of a dideoxynucleotide
labeled with a fluorescein dye required for 50% inhibition of DNA
synthesis versus the concentration of an unlabeled
dideoxynucleotide required for 50% inhibition.
23. The method of claim 22 wherein said ratio is 4 or less.
24. The method of claim 22 wherein said polymerase is from a
thermophilic species selected from the group consisting of
Thermosipho africanus, Bacillus caldotenax, and Bacillus
stearothermophilus.
25. The method of claim 22 wherein said thermostable DNA polymerase
is from a Thermus species.
26. The method of claim 25 wherein said amino acid sequence
comprises: LeuSerGlnXaaLeuAlaIleProTyrGluGlu (SEQ ID NO:3), whereby
"Xaa" at position 4 is any amino acid except Glu.
27. The method of claim 26 wherein said "Xaa" at position 4 is
Lys.
28. The method of claim 22 wherein said polymerase is from a
Thermotoga species.
29. The method of claim 28 wherein said amino acid sequence
comprises: LeuSerValXaaLeuGlyXaaProValLysGlu (SEQ ID NO: 4),
whereby "Xaa" at position 4 is any amino acid except Glu and "Xaa"
at position 7 is Val or Ile.
30. The method of claim 29 wherein said "Xaa" at position 4 is
Arg.
31. A method of producing labeled DNA which comprises: a) providing
a thermostable DNA polymerase characterized in that i) said
polymerase comprises the amino acid sequence
LeuSerValXaaLeuGlyXaaProValLysGlu (SEQ ID NO: 4), whereby "Xaa" at
position 4 can be any amino acid except Glu, and "Xaa" at position
7 of this sequence is Val or Ile. ii) said polymerase has a reduced
level of discrimination against incorporation of nucleotides
labeled with fluorescein family dyes; b) providing a nucleotide
labeled with a fluorescein family dye, and c) performing a DNA
synthesis reaction.
32. A method of producing labeled primer extension products which
comprises: a) providing a thermostable DNA polymerase characterized
in that i) said polymerase comprises the amino acid sequence
LeuSerValXaaLeuGlyXaaProValLysGlu (SEQ ID NO: 4), whereby "Xaa" at
position 4 can be any amino acid except Glu, and "Xaa" at position
7 of this sequence is Val or Ile. ii) said polymerase has a reduced
level of discrimination against incorporation of nucleotides
labeled with fluorescein family dyes; iii) said polymerase also
comprises the second amino acid sequence SQIXLR(V/I) (SEQ ID NO:
18) where "X" is any amino acid except E, iv) said polymerase has
reduced discrimination against incorporation of ribonucleotides
labeled with fluorescein family dyes; b) providing a ribonucleotide
labeled with a fluorescein family dye, and c) performing a primer
extension reaction.
33. A kit for DNA sequencing which comprises a) a thermostable DNA
polymerase characterized in that i) said polymerase comprises the
amino acid sequence LeuSerXaaXaaLeuXaaXaaProXaaXaaGlu (SEQ ID NO:
1), whereby "Xaa" at positions 3, 6, 9, and 10 of this sequence are
any amino acid residue, and "Xaa" at position 4 can be any amino
acid except Glu, and "Xaa" at position 7 of this sequence is Val or
Ile ii) said polymerase has reduced discrimination against
incorporation of nucleotides labeled with fluorescein family dyes,
and b) a terminator labeled with negatively-charged fluorescent
dye.
34. The kit of claim 33 wherein said reduced level of
discrimination is measured by determining the ratio of the
concentration of ddNTP labeled with a fluorescein family dye
required for 50% inhibition of DNA synthesis compared to that for
an unlabeled ddNTP and said ratio is 4 or less.
35. The kit of claim 34 wherein said amino acid sequence comprises:
LeuSerGlnXaaLeuAlaIleProTyrGluGlu (SEQ ID NO:3), whereby "Xaa" at
position 4 is any amino acid except Glu.
36. Kit of claim 35 wherein said "Xaa" at position 4 is Lys.
37. The kit of claim 34 wherein said amino acid sequence comprises:
LeuSerValXaaLeuGlyXaaProValLysGlu (SEQ ID NO: 4), whereby "Xaa" at
position 4 is any amino acid except Glu and "Xaa" at position 7 is
Val or Ile.
38. Kit of claim 37 wherein said "Xaa" at position 4 is Arg.
39. A kit for DNA sequencing which comprises a) a mutant
thermostable DNA polymerase characterized in that i) in its native
form said polymerase comprises the amino acid sequence
LeuSerXaaXaaLeuXaaXaaProXaaXaaGlu (SEQ ID NO: 1), whereby "Xaa" at
positions 3, 4, 6, 9, and 10 of this sequence are any amino acid
residue, and "Xaa" at position 7 of this sequence is Val or Ile ii)
said amino acid sequence is mutated, except that "Xaa" at position
4 is not mutated to Glu; and iii) said thermostable DNA polymerase
has a level of discrimination against incorporation of nucleotides
labeled with fluorescein family dyes which is reduced in comparison
to the native form of said polymerase.
40. The kit of claim 39 wherein said level of discrimination is at
least 5-fold lower than that of said native form of said
polymerase.
41. The kit of claim 40 wherein said recombinant thermostable DNA
polymerase is characterized in that in its native form said
polymerase comprises the amino acid sequence
LeuSerGlnXaaLeuAlaIleProTyrGluGlu (SEQ ID NO:3), whereby "Xaa" at
position 4 is any amino acid.
42. Kit of claim 41 wherein said "Xaa" is mutated to Lys.
43. The kit of claim 40 wherein said recombinant thermostable DNA
polymerase is characterized in that in its native form said
polymerase comprises the amino acid sequence
LeuSerValXaaLeuGlyXaaProValLysGlu (SEQ ID NO: 4), whereby "Xaa" at
position 4 is any amino acid and "Xaa" at position 7 is Val or
Ile.
44. Kit of claim 43 wherein said "Xaa" is Arg.
45. A kit for producing labeled DNA which comprises a) a
thermostable DNA polymerase characterized in that i) said
polymerase comprises the amino acid sequence
LeuSerXaaXaaLeuXaaXaaProXaaXaaGlu (SEQ ID NO: 7), whereby "Xaa" at
positions 3, 6, 9, and 10 of this sequence are any amino acid
residue, and "Xaa" at position 4 can be any amino acid except Glu,
and "Xaa" at position 7 of this sequence is Val or Ile ii) said
polymerase has reduced discrimination against incorporation of
nucleotides labeled with fluorescein family dyes, and b) a
nucleotide labeled with a negatively-charged fluorescent dye.
46. The kit of claim 45 wherein said amino acid sequence comprises:
LeuSerGlnXaaLeuAlaIleProTyrGluGlu (SEQ ID NO:14), whereby "Xaa" at
position 4 is any amino acid except Glu.
47. Kit of claim 45 wherein said "Xaa" at position 4 is Lys.
48. The kit of claim 45 wherein said amino acid sequence comprises:
LeuSerValXaaLeuGlyXaaProValLysGlu (SEQ ID NO: 15), whereby "Xaa" at
position 4 is any amino acid except Glu and "Xaa" at position 7 is
Val or Ile.
49. Kit of claim 48 wherein said "Xaa" at position 4 is Arg.
50. A kit for producing labeled primer extension products which
comprises a) a thermostable DNA polymerase which is characterized
in that i) in its native form, the polymerase comprises the first
amino acid sequence LeuSerXaaXaaLeuXaaXaaProXaaXaaGlu (SEQ ID NO:
1), whereby "Xaa" at positions 3, 6, 9, and 10 of this sequence are
any amino acid residue, and "Xaa" at position 4 can be any amino
acid except Glu, and "Xaa" at position 7 of this sequence is Val or
Ile; ii) the polymerase has reduced discrimination against
incorporation of nucleotides labeled with fluorescein family dyes;
iii) the polymerase also comprises the second amino acid sequence
SQIXLR(V/I) where "X" is any amino acid except; iv) the polymerase
has reduced discrimination against incorporation of ribonucleotides
labeled with fluorescein family dyes; and b) a ribonucleotide
labeled with a fluorescein family dye.
51. The kit of claim 50 wherein said amino acid sequence comprises:
LeuSerGlnXaaLeuAlaIleProTyrGluGlu (SEQ ID NO:3), whereby "Xaa" at
position 4 is any amino acid except Glu.
52. Kit of claim 51 wherein said "Xaa" at position 4 is Lys.
53. The kit of claim 50 wherein said amino acid sequence comprises:
LeuSerValXaaLeuGlyXaaProValLysGlu (SEQ ID NO: 4), whereby "Xaa" at
position 4 is any amino acid except Glu and "Xaa" at position 7 is
Val or Ile.
54. Kit of claim 53 wherein said "Xaa" at position 4 is Arg.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to thermostable DNA
polymerases which have enhanced efficiency for incorporating
nucleoside triphosphates labeled with fluorescein family dyes. The
present invention provides means for isolating and producing such
altered polymerases. The enzymes of the invention are useful for
many applications in molecular biology and are particularly
advantageous for nucleic acid sequencing.
BACKGROUND OF THE INVENTION
[0002] Incorporation of nucleoside triphosphates (dNTPs) labeled
with fluorescent dyes is important for many in vitro DNA synthesis
applications. For example, dye-terminator DNA sequencing reactions
require the incorporation of fluorescent dideoxynucleotide
analogues for termination and labeling. In addition, in vitro
synthesis of labeled products may involve incorporation of
fluorescent nucleotides or nucleotide analogues. For example,
fluorescently labeled DNA has been used in hybridization assays
using microarrays of immobilized probes (Cronin et al., 1996, Human
Mutation 7:244).
[0003] To assure fidelity of DNA replication, DNA polymerases have
a very strong bias for incorporation of their normal substrates,
referred to herein as conventional deoxynucleoside triphosphates
(dNTPs), and against incorporation of unconventional dNTPs
including dNTPs and dNTP analogues labeled with fluorescent dyes.
In the cell, this property attenuates the incorporation of abnormal
bases such as dUTP in a growing DNA strand. In vitro, this
characteristic is particularly evident where both conventional and
unconventional fluorescently-labeled nucleoside triphosphates are
present, such as in DNA sequencing reactions using a version of the
dideoxy chain termination method that utilizes dye-terminators (Lee
et al., 1992, Nuc. Acids. Res. 20:2471 which is incorporated herein
by reference).
[0004] Commercially available DNA cycle sequencing kits for
dye-terminator methods use chain terminator ddNTPs labeled with
fluorescent dyes of the rhodamine family.
[0005] However, rhodamine dyes are zwitterionic in charge and
nucleoside triphosphates labeled with these dyes migrate
anomalously in the electrophoretic gels used to separate the
sequencing products for detection. This property of rhodamine
family dyes necessitates making modifications in the standard
sequencing protocol which include the use of dITP and an additional
processing step before electrophoresis.
[0006] In contrast, negatively charged fluorescent dyes such as
fluorescein family dyes allow 1) better separation between the
labeled nucleoside triphosphates and labeled primer extension
products, and 2) better electrophoretic migration of the labeled
sequencing products than neutral or positively charged fluorescent
dyes. Thus, the use of fluorescein family dyes avoids the need for
additional processing steps required with the use of rhodamine
family dyes. However, available dyes of the fluorescein family are
not ideal for use in current commercially available DNA cycle
sequencing formats because ddNTPs labeled with these dyes are not
efficiently incorporated into sequencing products using these
formats. Consequently, there is a need for commercially available
thermostable DNA polymerases that can efficiently incorporate both
conventional and fluorescein-labeled nucleotides. The present
invention serves to meet that need. Further, an unexpected property
of the mutant enzymes of this invention is the increased rate of
primer extension relative to the corresponding wild-type enzyme.
Another unexpected property is the increased uniformity of
incorporation of the various terminator nucleotides in automated
DNA sequence analysis.
SUMMARY OF THE INVENTION
[0007] The present invention provides template-dependent
thermostable DNA polymerase enzymes having reduced discrimination
against incorporation of nucleotides labeled with fluorescein
family dyes compared to previously characterized enzymes. These
enzymes incorporate nucleotides, including deoxynucleotides (dNTPs)
and base analogues such as dideoxynucleotides (ddNTPs), that are
labeled with fluorescein family dyes more efficiently than
conventional thermostable enzymes. Genes encoding these enzymes are
also provided by the present invention, as are recombinant
expression vectors for providing large amounts of purified
enzymes.
[0008] By the present invention, a region of criticality within
thermostable DNA polymerases is identified which affects the
polymerase's ability to incorporate nucleotides labeled with
fluorescein family dyes, while retaining the ability to incorporate
faithfully natural nucleotides. This region of criticality, or
Critical Motif, can be introduced into genes for thermostable DNA
polymerases by recombinant DNA methods such as site-specific
mutagenesis to provide the advantages of the invention.
[0009] Thus, in one aspect, the invention provides recombinant
thermostable DNA polymerase enzymes which are characterized in that
the enzymes have been mutated to produce the Critical Motif and
have reduced discrimination against incorporation of nucleotides
labeled with fluorescein family dyes, in comparison to the
corresponding wild-type enzyme.
[0010] In this aspect, the invention provides recombinant
thermostable DNA polymerase enzymes which are characterized in that
a) in its native form said polymerase comprises the amino acid
sequence (given in one-letter code) LSXXLX(V/I)PXXE (SEQ ID NO: 1),
where X is any amino acid; b) the X at position 4 in said sequence
is mutated in comparison to said native sequence, except that X is
not mutated to E; and c) said thermostable DNA polymerase has
reduced discrimination against incorporation of nucleotides labeled
with fluorescein family dyes in comparison to the native form of
said enzyme. In the three-letter code, this amino acid sequence is
represented as LeuSerXaaXaaLeuXaaXaaProXaaXaaGlu (SEQ ID NO: 1),
whereby "Xaa" at positions 3, 4, 6, 9 and 10 of this sequence are
any amino acid residue, and "Xaa" at position 7 of this sequence is
Val or Ile.
[0011] In another embodiment, the recombinant thermostable DNA
polymerases are characterized in that a) the native form of the
polymerase comprises the amino acid sequence LS(Q/G)XL(S/A)IPYEE
(SEQ ID NO: 2), where X is any amino acid; b) the X at position 4
in said sequence is mutated in comparison to said native sequence,
except that X is not mutated to E; and c) said thermostable DNA
polymerase has reduced discrimination against incorporation of
nucleotides labeled with fluorescein family dyes in comparison to
the native form of said enzyme. In the three-letter code, this
amino acid sequence is represented as
[0012] LeuSerXaaXaaLeuXaaIleProTyrGluGlu (SEQ ID NO: 2), whereby
"Xaa" at position 3 is Gln or Gly, "Xaa" at position 4 is any amino
acid, and "Xaa" at position 6 is Ser or Ala. In a preferred
embodiment, the amino acid sequence is LSQXLAIPYEE (SEQ ID NO:3),
where X is any amino acid. In the three-letter code, this amino
acid sequence is represented as
[0013] LeuSerGlnXaaLeuAlaIleProTyrGluGlu (SEQ ID NO:3), whereby
"Xaa" at position 4 is any amino acid. In a more preferred
embodiment, the "Xaa" at position 4 is Lys.
[0014] In yet another embodiment, the recombinant thermostable DNA
polymerases are characterized in that a) the native form of the
polymerase comprises the amino acid sequence LSVXLG(V/I)PVKE (SEQ
ID NO: 4); b) the X at position 4 in said sequence is mutated in
comparison to said native sequence, except that X is not mutated to
E; and c) said thermostable DNA polymerase has reduced
discrimination against incorporation of nucleotides labeled with
fluorescein family dyes in comparison to the native form of said
enzyme. In the three-letter code, this amino acid sequence is
represented as LeuSerValXaaLeuGlyXaaProValLys- Glu (SEQ ID NO: 4),
whereby "Xaa" at position 4 is any amino acid and "Xaa" at position
7 is Val or Ile. In a preferred embodiment, the amino acid sequence
is LSVXLGVPVKE (SEQ ID NO: 5) where X at position 4 is any amino
acid. In the three-letter code, this amino acid sequence is
represented as LeuSerValXaaLeuGlyValProValLysGlu (SEQ ID NO: 5),
whereby "Xaa" at position 4 is any amino acid. In a more preferred
embodiment, the "Xaa" at position 4 is Arg. In another preferred
embodiment, the amino acid sequence is LSVXLGIPVKE (SEQ ID NO: 6)
where X at position 4 is any amino acid. In the three-letter code,
this amino acid sequence is represented as
LeuSerValXaaLeuGlyIleProValLysGlu (SEQ ID NO: 6), whereby "Xaa" at
position 4 is any amino acid. In a more preferred embodiment, the
"Xaa" at position 4 is Arg.
[0015] In another aspect of this invention, the particular region
of criticality of this invention can be combined with motifs in
other regions of the polymerase gene that are known to provide
thermostable DNA polymerases with reduced discrimination against
incorporation of unconventional nucleotides such as rNTPs and
ddNTPs. As exemplified herein, a recombinant Thermus aquaticus
(Taq) DNA polymerase enzyme containing two mutations was
constructed. The first mutation was an E to K mutation in the X
residue at position 4 of the critical motif of this invention. The
second mutation was a mutation allowing more efficient
incorporation of ddNTPs known as the F667Y mutation. This mutation
is a phenylalanine to tyrosine mutation at position 667 of Taq DNA
polymerase (described in U.S. Pat. No. 5,614,365 and U.S. Ser. No.
8/448,223 and herein incorporated by reference). When used in a
sequencing reation with fluorescein dye family-labeled ddNTPs, the
E681K F667Y double mutant enzyme was found to produce a readable
sequencing ladder. Thus, in one embodiment, a motif conferring
reduced discrimination toward dideoxynucleotides is combined with
the critical motif of this invention to provide an enzyme having an
increased efficiency of incorporation of both labeled and unlabeled
ddNTPs.
[0016] In addition, the E681K F667Y mutant enzyme was unexpectedly
found to exhibit a significantly increased extension rate relative
to an enzyme with the F667Y mutation alone. Thus, in another
embodiment of the invention, introduction of the critical motif
into a thermostable DNA polymerase enzyme, alone or in combination
with other motifs, produces enzymes having an increased extension
rate. The double mutant enzyme was also unexpectedly found to
produce more uniform peak heights in dye-terminator dideoxy
sequencing using rhodamine-labeled terminators. Thus, in yet
another embodiment, introduction of the critical motif into a
thermostable DNA polymerase enzyme produces enzymes displaying more
uniform peak heights in DNA sequencing methods using rhodamine dye
family labeled terminators.
[0017] In another embodiment, a mutation allowing more efficient
incorporation of rNTPs, such as the glutamic acid to glycine
mutation at position 615 of Taq DNA polymerase, or E615G mutation
(described in U.S. Ser. No. 60/023,376, filed Sep. 6, 1996, and
herein incorporated by reference), is combined with the critical
motif of this invention to provide an enzyme having an increased
efficiency of incorporation of ribonucleotides labeled with
fluorescein family dyes.
[0018] In another aspect of this invention, genes encoding the
polymerases of this invention are also provided. Specifically,
genes encoding recombinant thermostable polymerases comprising the
critical motif of this invention are provided. Also included in
this aspect are genes encoding combinations of two or more
mutations that include mutations producing the critical motif of
this invention.
[0019] In yet another aspect, the invention also provides improved
methods of DNA sequencing that allow the use of lower
concentrations of fluorescein dye family-labeled ddNTPs, thereby
reducing the cost of performing the reactions. The improved methods
of the invention also allow the use of lower ratios of fluorescein
dye family-labeled ddNTPs to dNTPs. Use of these methods results in
numerous advantages, including more efficient polymerization, lower
concentrations of template nucleic acid being required, and a
decreased likelihood of introducing inhibitors into the reaction
mix. These advantages also facilitate the sequencing of long
templates. The invention also provides improved methods of
sequencing wherein sequencing reactions can be loaded directly onto
sequencing gels for subsequent electrophoresis without intermediate
purification.
[0020] Thus, in one embodiment of the invention, the invention
provides improved methods for determining the sequence of a target
nucleic acid using a recombinant enzyme which has a) a mutation at
position 4 which produces the critical motif of this invention and
b) has reduced discrimination against incorporation of nucleotides
labeled with fluorescein family dyes in comparison with the
corresponding wild-type enzyme. Also within the scope of this
invention are improved sequencing methods using thermostable DNA
polymerase enzymes derived from thermophilic species, where the
enzymes contain naturally occurring sequence variations that
produce the critical motif of this invention. These native enzymes
can also provide reduced discrimination against incorporation of
unconventional nucleotides. In this embodiment, the invention
provides improved methods of sequencing using a native thermostable
DNA polymerase a) having the critical motif of this invention
wherein the amino acid in position 4 is not Glu and b) having
reduced discrimination against incorporation of nucleotides labeled
with fluorescein family dyes.
[0021] Also within the scope of this invention are improved methods
of producing DNA labeled with fluorescein family dyes. The enzymes
of the invention efficiently incorporate fluorescein-labeled dNTPs
in a polymerase chain reaction method, producing amplified products
that are labeled at various sites with fluorescein family dyes.
Thus, in one embodiment, an improved method of labeling DNA
comprises a) providing a reaction mixture comprising dNTPs labeled
with fluorescein family dyes and an enzyme of the invention and b)
performing a nucleic acid amplification reaction.
[0022] The enzymes of the invention, and genes encoding these
enzymes, provide additional aspects of the invention which are kits
for DNA sequencing that comprise a recombinant enzyme of the
invention and may additionally include a negatively charged
fluorescent terminator compound. Other kits for DNA sequencing
comprise a) a negatively charged fluorescent terminator compound
and b) a native enzyme of the invention.
[0023] The invention also provides kits for producing labeled DNA
which comprise a recombinant enzyme of the invention. Other kits
for producing labeled DNA comprise a) a negatively charged
fluorescent nucleoside triphosphate compound and b) a native enzyme
of the invention.
BRIEF DESCRIPTION OF THE DRAWING
[0024] FIG. 1 is a schematic representation of the Taq DNA
polymerase gene. Restriction sites are indicated that relate to
Example I and the description of methods for preparing additional
mutants and expression vectors provided herein.
DETAILED DESCRIPTION OF THE INVENTION
[0025] To facilitate understanding of the invention, a number of
terms are defined below.
[0026] The term "gene" refers to a DNA sequence that comprises
control and coding sequences necessary for the production of a
recoverable bioactive polypeptide or precursor. The polypeptide can
be encoded by a full-length gene sequence or by any portion of the
coding sequence so long as the enzymatic activity is retained.
[0027] The term "native" refers to a gene or gene product which is
isolated from a naturally occurring source. This term also refers
to a recombinant form of the native protein produced by molecular
biological techniques which has an amino acid sequence identical to
that of the native form.
[0028] The term "mutant" refers to a gene that has been altered in
its nucleic acid sequence or a gene product which has been altered
in its amino acid sequence, resulting in a gene product which may
have altered functional properties when compared to the native or
wild-type gene or gene product. Such alterations include point
mutations, deletions and insertions.
[0029] The term "host cell(s)" refers to both single-cellular
prokaryote and eukaryote organisms such as bacteria, yeast, and
actinomycetes and single cells from higher order plants or animals
when being grown in cell culture.
[0030] The term "expression system" refers to DNA sequences
containing a desired coding sequence and control sequences in
operable linkage, so that host cells transformed with these
sequences are capable of producing the encoded proteins. To effect
transformation, the expression system may be included on a vector;
however, the relevant DNA may also be integrated into the host
chromosome.
[0031] The term "oligonucleotide" as used herein is defined as a
molecule comprised of two or more deoxyribonucleotides or
ribonucleotides, preferably more than three, and usually more than
ten. The exact size of an oligonucleotide will depend on many
factors, including the ultimate function or use of the
oligonucleotide.
[0032] Oligonucleotides can be prepared by any suitable method,
including, for example, cloning and restriction of appropriate
sequences and direct chemical synthesis by a method such as the
phosphotriester method of Narang et al., 1979, Meth. Enzymol.
68:90-99; the phosphodiester method of Brown et al., 1979, Meth.
Enzymol. 68:109-151; the diethylphosphoramidite method of Beaucage
et al., 1981, Tetrahedron Lett. 22:1859-1862; the triester method
of Matteucci et al., 1981, J. Am. Chem. Soc. 103:3185-3191 or
automated synthesis methods; and the solid support method of U.S.
Pat. No. 4,458,066, which publications are each incorporated herein
by reference.
[0033] The term "primer" as used herein refers to an
oligonucleotide, whether natural or synthetic, which is capable of
acting as a point of initiation of synthesis when placed under
conditions in which primer extension is initiated. A primer is
preferably a single-stranded oligodeoxyribonucleotide. The
appropriate length of a primer depends on the intended use of the
primer but typically ranges from 15 to 35 nucleotides. Short primer
molecules generally require cooler temperatures to form
sufficiently stable hybrid complexes with the template. A primer
need not reflect the exact sequence of the template but must be
sufficiently complementary to hybridize with a template for primer
elongation to occur.
[0034] A primer can be labeled, if desired, by incorporating a
label detectable by spectroscopic, photochemical, biochemical,
immunochemical, or chemical means. For example, useful labels
include .sup.32P, fluorescent dyes, electron-dense reagents,
enzymes (as commonly used in ELISA assays), biotin, or haptens and
proteins for which antisera or monoclonal antibodies are
available.
[0035] The term "thermostable polymerase," refers to an enzyme
which is stable to heat, is heat resistant and retains sufficient
activity to effect subsequent primer extension reactions and does
not become irreversibly denatured (inactivated) when subjected to
the elevated temperatures for the time necessary to effect
denaturation of double-stranded nucleic acids. The heating
conditions necessary for nucleic acid denaturation are well known
in the art and are exemplified in U.S. Pat. Nos. 4,683,202 and
4,683,195, which are incorporated herein by reference. As used
herein, a thermostable polymerase is suitable for use in a
temperature cycling reaction such as the polymerase chain reaction
("PCR"). Irreversible denaturation for purposes herein refers to
permanent and complete loss of enzymatic activity. For a
thermostable polymerase, enzymatic activity refers to the catalysis
of the combination of the nucleotides in the proper manner to form
primer extension products that are complementary to a template
nucleic acid strand.
[0036] The term "conventional" or "natural" when referring to
nucleic acid bases, nucleoside triphosphates, or nucleotides refers
to those which occur naturally in the polynucleotide being
described (i.e., for DNA these are dATP, dGTP, dCTP and dTTP).
Additionally, dITP, and 7-deaza-dGTP are frequently utilized in
place of dGTP and 7-deaza-dATP can be utilized in place of dATP in
in vitro DNA synthesis reactions, such as sequencing. Collectively
these may be referred to as dNTPs.
[0037] The term "unconventional" or "modified" when referring to a
nucleic acid base, nucleoside, or nucleotide includes modification,
derivations, or analogues of conventional bases, nucleosides, or
nucleotides that naturally occur in a particular polynucleotide.
The deoxyribonucleotide form of uracil is an unconventional or
modified base in DNA (dUMP), whereas, the ribonucleotide form of
uracil is a conventional base in RNA (UMP). As used herein,
unconventional nucleotides include but are not limited to compounds
used as terminators for nucleic acid sequencing. Terminator
compounds include but are not limited to those compounds which have
a 2',3' dideoxy structure and are referred to as dideoxynucleoside
triphosphates. The dideoxynucleoside triphosphates ddATP, ddTTP,
ddCTP and ddGTP are referred to collectively as ddNTPs. Other
unconventional nucleotides include phosphorothioate dNTPs
([.alpha.-S]dNTPs), 5'-.alpha.-borano-dNTPs,
.alpha.-methyl-phosphonate dNTPs, and ribonucleoside triphosphates
(rNTPs). Unconventional bases may be labeled with radioactive
isotopes such as .sup.32P, .sup.33P, or .sup.35S; fluorescent
labels; chemiluminescent labels; bioluminescent labels; hapten
labels such as biotin; or enzyme labels such as streptavidin or
avidin. Fluorescent labels may include dyes that are negatively
charged, such as dyes of the fluorescein family, or dyes that are
neutral in charge, such as dyes of the rhodamine family, or dyes
that are positively charged, such as dyes of the cyanine family.
Dyes of the fluorescein family include e.g., FAM, HEX, TET, JOE,
NAN and ZOE. Dyes of the rhodamine family include Texas Red, ROX,
R110, R6G, and TAMRA. FAM, HEX, TET, JOE, NAN, ZOE, ROX, R110, R6G,
and TAMRA are marketed by Perkin-Elmer (Foster City, Calif.), and
Texas Red is marketed by Molecular Probes. Dyes of the cyanine
family include Cy2, Cy3, Cy5, and Cy7 and are marketed by Amersham
(Amersham Place, Little Chalfont, Buckinghamshire, England).
[0038] The term "DNA synthesis reaction" refers to methods of
producing copies of DNA including but not limited to PCR, strand
displacement amplification, transcription mediated amplification,
primer extension and reverse transcription.
[0039] In order to further facilitate understanding of the
invention, specific thermostable DNA polymerase enzymes and
fluorescent dyes are referred to throughout the specification to
exemplify the invention, and these references are not intended to
limit the scope of the invention.
[0040] The present invention provides novel and improved
compositions which are thermostable DNA polymerases. The enzymes of
the invention include recombinant polymerases which more
efficiently incorporate nucleoside triphosphates labeled with
fluorescein family dyes in comparison to the corresponding
wild-type enzymes. The thermostable DNA polymerases of the
invention are more suitable and desirable for use in processes such
as DNA sequencing and in vitro synthesis of labeled products than
prior art polymerases. Improved DNA sequencing methods of the
invention include the use of these recombinant polymerases as well
as the use of native enzymes which more efficiently incorporate
nucleoside triphosphates labeled with fluorescein family dyes than
previously characterized enzymes. DNA sequences encoding these
enzymes, and vectors for expressing the proteins are also
provided.
[0041] The thermostable DNA polymerases of the invention possess a
region of criticality within the amino acid sequence of the
polymerase activity domain of the enzyme. The critical region
within the amino acid sequence of a thermostable DNA polymerase
provided by the present invention is shown below using the
conventional single-letter amino acid code (Lehninger,
Biochemistry, New York, N.Y., Worth Publishers Inc., 1970, page 67,
which is incorporated herein by reference).
[0042] SEQ ID NO: 7 LSXXLX(V/I)PXXE where the "X" at position 4
indicates any amino acid except E. In the three-letter code for
amino acids, this sequence is represented as
[0043] LeuSerXaaXaaLeuXaaXaaProXaaXaaGlu (SEQ ID NO: 7) whereby
"Xaa" at positions 3, 6, 9, and 10 is any amino acid, "Xaa" at
position 4 of this sequence is any amino acid but not a glutamic
acid residue (Glu) and "Xaa" at position 7 is Val or Ile. This
region of criticality provides thermostable DNA polymerase enzymes
characterized by the ability to efficiently incorporate nucleotides
labeled with fluorescein family dyes.
[0044] For example, in a derivative of the Thermus aquaticus (Taq)
DNA polymerase gene which already contains a glycine to aspartic
acid mutation at position 46 (G46D) and an F667Y mutation, a
mutation of G to A in the first position of the codon for glutamic
acid at residue 681 sequence of the full length Taq DNA polymerase
sequence (corresponding to position 4 of the critical motif)
results in an enzyme having the critical motif. This enzyme
displays 1) an approximately a 2- to 10-fold increase in the
efficiency of incorporation of nucleotides labeled with fluorescein
family dyes with no impairment of the enzyme's ability to mediate
PCR in the presence of conventional nucleotides and 2) a 3 to
4.3-fold increase in the extension rate. In Taq DNA polymerase this
particular mutation results in an amino acid change of E (glutamic
acid) to K (lysine).
[0045] Although this particular amino acid change produced the
critical motif and significantly alters the ability of the enzyme
to incorporate unconventional nucleotides, it is expected that the
specific change of E to K is not as critical to the invention as is
the now identified position within the region of criticality. Thus,
in a preferred embodiment, the invention provides recombinant
thermostable DNA polymerase enzymes which are characterized in that
a) in its native form said polymerase comprises the amino acid
sequence LSXXLX(V/I)PXXE (SEQ ID NO: 1), where X is any amino acid;
b) the X at position 4 in said sequence is mutated in comparison to
said native sequence, except that the X at position 4 is not
mutated to E; and c) said thermostable DNA polymerase has reduced
discrimination against incorporation of nucleotides labeled with
fluorescein family dyes in comparison to the native form of said
enzyme. In a more preferred embodiment, the X at position 4 is
replaced by an amino acid having a positive charge, such as K, R or
H, or by a polar amino acid such as Q or N. In a most preferred
embodiment, the X at position 4 is replaced by K.
[0046] In another preferred embodiment of the invention, the of the
invention is characterized in that the enzyme (a) has reduced
discrimination against fluorescein dye family labeled nucleotides
and (b) comprises the amino acid sequence LS(Q/G)XL(S/A)IPYEE where
X is any amino acid (SEQ ID NO: 2). In three-letter code, this
amino acid sequence is represented as
[0047] LeuSerXaaXaaLeuXaaIleProTyrGluGlu, whereby "Xaa" at position
3 is Gln or Gly, "Xaa" at position 4 is any amino acid, and "Xaa"
at position 6 is Ser or Ala.
[0048] In a more preferred embodiment of the invention, the enzyme
having reduced discrimination against fluorescein dye family
labeled nucleotides comprises the amino acid sequence LSQXLAIPYEE
where X is any amino acid (SEQ ID NO: 3). In the three-letter code,
this amino acid sequence is represented as
LeuSerGlnXaaLeuAlaIleProTyrGluGlu, whereby "Xaa" at position 4 is
any amino acid. In a most preferred embodiment of the invention,
the X is a K residue.
[0049] In another preferred embodiment of the invention, the the
enzyme having reduced discrimination against fluorescein dye family
labeled nucleotides comprises the amino acid sequence
LSVXLG(V/I)PVKE where X is any amino acid (SEQ ID NO: 4). In the
three-letter code, this amino acid sequence is represented as
LeuSerValXaaLeuGlyXaaProValLysGlu, whereby "Xaa" at position 4 is
any amino acid and "Xaa" at position 7 is Val or Ile.
[0050] In a more preferred embodiment of the invention, the enzyme
having reduced discrimination against fluorescein dye family
labeled nucleotides comprises the amino acid sequence LSVXLGVPVKE
where X is any amino acid (SEQ ID NO: 5). In the three-letter code,
this amino acid sequence is represented as
LeuSerValXaaLeuGlyValProValLysGlu, whereby "Xaa" at position 4 is
any amino acid. In a most preferred embodiment, the X is an R
residue.
[0051] In another more preferred embodiment, the enzyme having
reduced discrimination against fluorescein dye family labeled
nucleotides comprises the amino acid sequence LSVXLGIPVKE where X
is any amino acid (SEQ ID NO: 6). In the three-letter code, this
amino acid sequence is represented as
LeuSerValXaaLeuGlyIleProValLysGlu, whereby "Xaa" at position 4 is
any amino acid. In a most preferred embodiment, the X is an R
residue.
[0052] The characterization of the E681K mutation described herein
identified a region in the DNA polymerase gene that affects the
ability of the polymerase to interact with negatively charged
fluorescent nucleotides. This site, distal to helix O, is at the
end of the O.sub.a helix and the beginning of the O.sub.b helix of
the polymerase (Kim, et al., 1995, Nature, 376:612). Based on
molecular modeling principles well-known in the art, changes in the
structure of the O.sub.a-O.sub.b helix other than E to K at
position 681 are also expected to produce changes in the ability of
the polymerase to discriminate against nucleotides labeled with
fluorescein family dyes. Thus, mutations at positions in the
critical motif other than those in the X residue at position 4 are
also within the scope of this invention. In this embodiment, the
invention provides a recombinant thermostable DNA polymerase enzyme
which is characterized in that (a) in its native form, the
polymerase comprises the amino acid sequence LSXXLX(V/I)PXXE (SEQ
ID NO: 1) where X is any amino acid, (b) the recombinant polymerase
comprises at least one mutation within the amino acid sequence,
except that X at position 4 is not mutated to E, and c) the enzyme
has reduced discrimination against incorporation of nucleotides
labeled with fluorescein family dyes, in comparison to the
corresponding native enzyme.
[0053] Similarly, thermostable DNA polymerases that comprise
critical motifs that are similar, but not identical to the critical
motif that is amino acid sequence LSXXLX(V/I)PXXE (SEQ ID NO: 7)
where X at position 4 is any amino acid except E, are within the
scope of this invention. Specifically, in one embodiment, the
critical motif is the amino acid sequence LXXXXXXXXXE (SEQ ID NO:
8) where X at position 4 is any amino acid except E. In the three
letter code, this amino acid sequence is represented as
[0054] LeuXaaXaaXaaXaaXaaXaaXaaXaaXaaGlu (SEQ ID NO: 8), whereby
"Xaa" at positions 2, 3, 5, 6, 7, 8, 9 and 10 are any amino acid
and "Xaa" at position 4 is any amino acid except Glu.
[0055] In another embodiment, the critical motif is amino acid
sequence L(S/A)XX(L/I)XXXXXE (SEQ ID NO: 9) where X at position 4
is any amino acid except E. In the three-letter code, this amino
acid sequence is represented as LeuXaaXaaXaaXaaXaaXaaXaaXaaXaaGlu
(SEQ ID NO: 9), whereby "Xaa" at positions 3, 6, 7, 8, 9, and 10
are any amino acid, "Xaa" at position 2 is Ser or Ala, "Xaa" at
position 4 is any amino acid except Glu, and "Xaa" at position 5 is
Leu or Ile.
[0056] In yet another embodiment, the critical motif is amino acid
sequence LSXXLXXXXXE (SEQ ID NO: 10) where X at position 4 is any
amino acid except E. In the three-letter code, this amino acid
sequence is represented as LeuSerXaaXaaLeuXaaXaaXaaXaaXaaGlu (SEQ
ID NO: 10), whereby "Xaa" at positions 3, 6, 7, 8, 9, and 10 are
any amino acid and "Xaa" at position 4 is any amino acid except
Glu.
[0057] The ability of the enzymes of this invention to efficiently
incorporate nucleotides labeled with fluorescein family dyes is
measured by ddNTP incorporation assays. One such assay is a primer
extension competition assay conducted under conditions of limiting
template. In this assay, a primer DG48
(5'-GGGAAGGGCGATCGGTGCGGGCCTCTTCGC), (SEQ ID NO: 11), bound to
M13mp 18 template (Innis et al., 1988, Proc. Natl. Acad. Sci. USA
85:9436) is extended in the presence of [.alpha.-.sup.33P]dCTP and
excess enzyme with various levels of a fluorescently labeled ddNTP,
Zowie-ddCTP. Because the incorporation of a ddCTP residue
terminates the extension reaction, the more readily a DNA
polymerase incorporates a ddCTP into an extended primer, the less
[.alpha.-.sup.33P]dCTP can be incorporated. Thus, as the efficiency
of fluorescently labeled ddCTP incorporation increases, the extent
of inhibition of DNA synthesis is increased. The reactions were
also performed with various levels of an unlabeled ddCTP. The
concentrations of ddCTP and Zowie-ddCTP needed for 50% inhibition
were calculated and compared to give a relative measure of the
ability of the enzyme to incorporate the fluorescently-labeled
nucleotide. The details of the ddNTP incorporation assay are
provided in Example II.
[0058] Thus, in one embodiment of the invention, the characteristic
of reduced discrimination against incorporation of nucleotides
labeled with fluorescein family dyes is measured by the fluorescent
ddNTP incorporation assay described in Example II. In a preferred
embodiment, the concentration of a ddNTP labeled with a fluorescein
dye, Zowie-ddCTP, required for 50% inhibition of DNA synthesis is
reduced at least 3-fold for a mutant enzyme of the invention,
relative to the wild-type enzyme. In a more preferred embodiment,
the concentration is reduced at least 5-fold. In a most preferred
embodiment, the concentration is reduced at least 10-fold. In
another embodiment, the characteristic of reduced discrimination is
assayed by measuring fluorescent dNTP incorporation.
[0059] In another aspect of the invention, the thermostable DNA
polymerase gene sequence and enzyme are derived from various
thermophilic species. In one embodiment, the polymerase gene
sequence and enzyme are from a species of the genus Thermus. In
other embodiments of the invention, the gene sequence and enzyme
are from thermophilic species other than Thermus. The full nucleic
acid and amino acid sequence for numerous thermostable DNA
polymerases are available. The sequences each of Taq, Thermus
thermopilus (Tth), Thermus species Z05, Thermus species sps17,
Thermotoga maritima (Tma), and Thermosipho africanus (Taf)
polymerase have been published in PCT International Patent
Application No. PCT/U.S.91/07035 which published as PCT Patent
Publication No. WO 92/06200 on Apr. 16, 1992, and is incorporated
herein by reference. The sequences for the DNA polymerase from
Thermus flavus, Bacillus caldotenax, and Bacillus
stearothermophilus have been published in Akhmetzjanov and
Vakhitov, 1992, Nucleic Acids Research 20 (21):5839, Uemori et al.,
1993, J. Biochem. 113:401-410, and as accession number BSU23149.ng
from the NG: New GenBank database, respectively, which are each
incorporated herein by reference. The sequence of the thermostable
DNA polymerase from Thermus caldophilus is found in EMBL/GenBank
Accession No. U62584. The sequence of the thermostable DNA
polymerase from Thermus filiformis can be recovered from ATCC
Deposit No. 42380 using the methods provided in U.S. Pat. No.
4,889,818, as well as the sequence information provided in Table 1.
The sequence of the Thermotoga neapolitana DNA polymerase is from
GeneSeq Patent Data Base Accession No. R98144 and PCT WO
97/09451
1TABLE I Critical Amino Acid Organism Critical Motif Position
.dwnarw. Consensus L S/a - - L/i - - - - - E Thermus aquaticus L S
Q E L A I P Y E E 681 Thermus flavus L S G E L A I P Y E E 679
Thermus thermophilus L S Q E L A I P Y E E 683 Thermus specie Z05 L
S Q E L A I P Y E E 683 Thermus specie sps17 L S Q E L S I P Y E E
679 Thermus caldophilus L S Q E L A I P Y E E 683 Thermus
filiformis L S Q E L S I P Y E E 679 Thermotoga maritima L S V R L
G V P V K E 744 Thermotoga neapolitana L S V R L G I P V K E 744
Thermosipho africanus L S K R I G L S V S E 743 Bacillus
caldotenax.sup.1 L A Q N L N I S R K E 725, 724 Bacillus
stearothermophilus.sup.2 L A Q N L N I T R K E 724, 727, 802
[0060] 1. Protein sequence from Accession No. D12982, Uemori T.,
Ishino Y., Fujita K., Asada K., Kato I. "Cloning of the DNA
polymerase gene of Bacillus caldotenax and characterization of the
gene product" J. Biochem. 113:401 (1993). The critical residue in
that sequence is 725. An almost identical protein sequence is
provided as a putative "Bacillus stearothermophilus" DNA Polymerase
in Accession No. R45155 and WPI 93-408323/51. The critical residue
in that sequence is 724.
[0061] 2. There are several sequence submissions for Bacillus
stearothermophilus DNA polymerase in the GeneBank, or SwissProt/PIR
databases. Although these sequences are highly related, but
somewhat different from one another, each contains the identical
L(S/A)XX(L/I)XXXXXE (SEQ ID NO: 9) motif, where X is any amino acid
except E. In the three-letter code, this amino acid sequence is
represented as LeuXaaXaaXaaXaaXaaXaaXaaXaaXaaGlu (SEQ ID NO: 9),
whereby "Xaa" at positions 3, 6, 7, 8, 9, and 10 are any amino
acid, "Xaa" at position 2 is Ser or Ala, "Xaa" at position 4 is any
amino acid except Glu, and "Xaa" at position 5 is Leu or Ile. In
the table above, protein sequences comprising the Critical Residue
in the Critical Motif at position 724 are provided by Japanese
patent publication J 05 304 964A, EP No. 699,760, and Accession No.
U33536. Another highly related, but somewhat different, protein
sequence was published in Gene 163:65-68 (1995), contains the
Critical Residue in the Critical Motif at position 727. Another
highly related, but somewhat different, protein sequence, Accession
No. U23149, for Bst DNA polymerase contains the Critical Residue in
the Critical Motif at position 802.
[0062] Because the DNA polymerases of each thermophilic species are
unique, the amino acid position of the region of criticality is
distinct for each enzyme. Amino acid and nucleic acid sequence
alignment programs are readily available and, given the particular
region identified herein, serve to assist in the identification of
the exact sequence region of the invention. Such sequence alignment
programs are available from the Genetics Computer Group, 575
Science Drive, Madison, Wis. Given the particular motif identified
herein, these programs, including "GAP," "BESTFIT," and "PILEUP,"
serve to assist in the localization of the critical motif. The
position of the regions of criticality are shown in Table I for
thermostable DNA polymerases from exemplary thermophilic
species.
[0063] Regardless of the exact position of the critical motif
LSXXLX(V/I)PXXE (SEQ ID NO: 7), where X at position 4 is any amino
acid except E, within the polymerase domain of a thermostable DNA
polymerase, the presence of the motif serves to provide
thermostable DNA polymerases having the ability to efficiently
incorporate nucleotides labeled with fluorescein family dyes.
Therefore, mutation of the conserved glutamic acid of the
thermostable DNA polymerases of Thermus flavus (Glu 679), Thermus
thermophilus (Glu 683), Thermus species Z05 (Glu 683), Thermus
species sps17 (Glu 679) Thermus caldophilus (Glu 683), Thermus
filiformis (Glu 679) to produce the critical motif will provide an
enhancing effect on the ability of the polymerase to efficiently
incorporate nucleotides labeled with fluorescein family dyes.
[0064] In addition, in view of the highly conserved nature of the
now identified critical motif, novel thermostable DNA polymerases
may be identified based upon their homology to, for example, Taq
DNA polymerase or the sequences of other DNA polymerases in Table I
(see for example U.S. Pat. Nos. 5,618,711 and 5,624,833 which are
herein incorporated by reference). Such polymerases, so long as
their peptide sequence is at least 45% and most preferably greater
than 80% homologous to the Taq polymerase amino acid sequence, as
determined by the methods described herein, are within the scope of
the present invention. Consequently, the invention relates to a
class of enzymes which also includes, for example, the thermostable
DNA polymerase, and corresponding gene and expression vectors from
Thermus oshimai (Williams R A, et al, 1996, Int J Syst Bacteriol 46
(2): 403-408); Thermus silvanus and Thermus chliarophilus (Tenreiro
S, et al., 1995, Int. J. Syst. Bacteriol 45 (4): 633-639); Thermus
scotoductus (Tenreiro S et al., 1995, Res. Microbiol 146 (4):
315-324); Thermus ruber ATCC 35948, (L. G. Loginova, 1984, Int. J.
Syst. Bacteriol 34: 498-499); and Thermus brockianus (Munster, M.
J., 1986, J. Gen. Microbiol 132: 1677), which publications are each
incorporated herein by reference.
[0065] Those of skill in the art will recognize that the above
thermostable DNA polymerases with enhanced efficiency for
incorporating fluorescein-labeled nucleotides are most easily
constructed by recombinant DNA techniques such as site-directed
mutagenesis. See for example Sambrook et al., Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor, 1989, second edition,
chapter 15.51, "Oligonucleotide-mediated mutagenesis," which is
incorporated herein by reference. This technique is now standard in
the art, and can be used to create all possible classes of base
pair changes at any determined site in a gene. The method is
performed using a synthetic oligonucleotide primer complementary to
a single-stranded phage or plasmid DNA to be mutagenized except for
a limited mismatching, which represents the desired mutation.
Briefly, the synthetic oligonucleotide is used as a primer to
direct synthesis of a strand complementary to the phage or plasmid,
and the resulting double-stranded DNA is transformed into a phage-
or plasmid-supporting host bacterium. The resulting bacteria can be
assayed by, for example, DNA sequence analysis or probe
hybridization to identify those plaques or colonies carrying the
desired mutated gene sequence.
[0066] Subsequent to the invention of PCR, primer-directed
mutagenesis (described in U.S. Pat. No. 4,683,195, which is herein
incorporated by reference) and "overlap PCR" (Higuchi, 1989, in PCR
Technology, ed. Erlich, Stockton Press, New York, N.Y., pp.61-70)
have become routine means of introducing any mutation at any
position of a gene.
[0067] The mutated DNA can be recovered from the plasmid, phasmid,
phage or amplification reaction by conventional means and ligated
into an expression vector for subsequent culture and purification
of the resulting enzyme. Numerous cloning and expression vectors
are suitable for practicing the invention, including mammalian and
bacterial systems, as described in, for example, Sambrook et al.,
1989 supra. For convenience, the present invention is exemplified
utilizing the lambda derived P.sub.L promoter (Shimatake et al.,
1981, Nature 292:128). Use of this promotor is specifically
described in U.S. Pat. Nos. 4,711,845 and 5,079,352, which are
incorporated herein by reference.
[0068] Plasmid pCS1 has been deposited with the ATCC, on Aug. 28,
1997, and given accession No. 98521. This plasmid contains a gene
encoding a thermostable DNA polymerase which gene is mutated at the
codon at position 681 such that glutamic acid is replaced with
lysine in the resulting polypeptide and provides a means for
providing thermostable DNA polymerases having an enhanced
efficiency for incorporating nucleotides labeled with fluorescein
family dyes. Example I illustrates the use of flanking restriction
sites suitable for subcloning the E681K mutation to create other
thermostable DNA polymerase enzymes. Alternatively, because the
complete gene sequence for numerous thermostable DNA polymerases
are known, other means for introducing the E681K mutation, such as
restriction digestion and fragment replacement, are readily
available to those of skill in the art, having the availability of
ATCC deposits and the sequence information provided herein.
[0069] When one desires to produce one of the mutant or native
enzymes of the present invention, or a derivative or homologue of
those enzymes, the production of the enzyme typically involves the
transformation of a host cell with the expression vector, and
culture of the transformed host cell under conditions such that
expression will occur. Means for transforming and culturing
transformed host cells are well known in the art and are described
in detail in, for example, Sambrook et al., 1989, supra.
[0070] The thermostable DNA polymerases of the present invention
are generally purified from E. coli strain DG116 (deposited as ATCC
53606 on Apr. 7, 1987) which has been transformed with an
expression vector operably linked to a gene encoding a wild-type or
modified thermostable DNA polymerase. Methods for purifying the
thermostable DNA polymerase are described in, for example, Example
I and Lawyer et al., 1993, PCR Methods and Applications 2:275-87,
which is incorporated herein by reference.
[0071] The thermostable enzymes of the invention may be used for
any purpose in which such enzyme activity is necessary or desired.
Examples of uses include DNA sequencing, DNA labeling, and labeling
of primer extension products. DNA sequencing by the Sanger
dideoxynucleotide method (Sanger et al, 1977, Proc. Natl. Acad.
Sci. 74: 5463) is particularly improved by the present invention.
Advances in the basic Sanger et al. method have provided novel
vectors (Yanisch-Perron et al, 1985 Gene 33:103-119) and base
analogues (Mills et al., 1979, Proc. Natl. Acad. Sci. 76:2232-2235,
and Barr et al., 1986, Biotechniques 4:428-432). In general, DNA
sequencing requires template-dependent primer extension in the
presence of chain-terminating base analogs, resulting in a
distribution of partial fragments which are subsequently separated
by size. The basic dideoxy sequencing procedure involves (i)
annealing an oligonucleotide primer, optionally labeled, to a
template; (ii) extending the primer with DNA polymerase in four
separate reactions, each containing a mixture of unlabeled dNTPs
and a limiting amount of one chain terminating agent such as a
ddNTP, optionally labeled; and (iii) resolving the four sets of
reaction products on a high-resolution denaturing
polyacrylamide/urea gel. The reaction products can be detected in
the gel by autoradiography or by fluorescence detection, depending
on the label used, and the image can be examined to infer the
nucleotide sequence. These methods utilize DNA polymerase such as
the Klenow fragment of E. coli Pol I or a modified T7 DNA
polymerase.
[0072] The availability of thermoresistant polymerases, such as Taq
DNA polymerase, resulted in improved methods for sequencing with
thermostable DNA polymerase (see Innis et al., 1988, supra) and
modifications thereof referred to as "cycle sequencing" (Murray,
1989, Nuc Acids Res. 17:8889). As an alternative to basic dideoxy
sequencing, cycle sequencing is a linear, asymmetric amplification
of target sequences complementary to the template sequence in the
presence of chain terminators. A single cycle produces a family of
extension products of all possible lengths. Following denaturation
of the extension reaction product from the DNA template, multiple
cycles of primer annealing and primer extension occur in the
presence of terminators such as ddNTPs. Cycle sequencing requires
less template DNA than conventional chain-termination sequencing.
Thermostable DNA polymerases have several advantages in cycle
sequencing; they tolerate the stringent annealing temperatures
which are required for specific hybridization of primer to nucleic
acid targets as well as tolerating the multiple cycles of high
temperature denaturation which occur in each cycle, i.e.,
90-95.degree. C. For this reason, AmpliTaq.RTM. DNA Polymerase and
its derivatives and descendants have been included in Taq cycle
sequencing kits commercialized by companies such as Perkin-Elmer,
Norwalk, Conn.
[0073] Two variations of chain termination sequencing methods
exist--dye-primer sequencing and dye-terminator sequencing. In
dye-primer sequencing, the ddNTP terminators are unlabeled, and a
labeled primer is utilized to detect extension products (Smith et
al., 1986, Nature 32:674-679). In dye-terminator DNA sequencing, a
DNA polymerase is used to incorporate dNTPs and fluorescently
labeled ddNTPs onto the end of a DNA primer (Lee et al., supra.).
This process offers the advantage of not having to synthesize dye
labeled primers. Furthermore, dye-terminator reactions are more
convenient in that all four reactions can be performed in the same
tube.
[0074] Both dye-primer and dye-terminator methods may be automated
using an automated sequencing instrument produced by Applied
Biosystems, Foster City, Calif. (U.S. Pat. No. 5,171,534, which is
herein incorporated by reference). When using the instrument, the
completed sequencing reaction mixture is fractionated on a
denaturing polyacrylamide gel mounted in the instrument. A laser at
the bottom of the instrument detects the fluorescent products as
they are electrophoresed according to size through the gel.
[0075] Two types of fluorescent dyes are commonly used to label the
terminators used for dye-terminator sequencing--negatively charged
and zwitterionic fluorescent dyes. Negatively charged fluorescent
dyes include those of the fluorescein and BODIPY families. BODIPY
dyes (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) are described
International patent application WO 97/00967, which is incorporated
herein by reference. Zwitterionic fluorescent dyes include those of
the rhodamine family. Commercially available cycle sequencing kits
use terminators labeled with rhodamine derivatives. However, the
rhodamine-labeled terminators are rather costly and the product
must be separated from unincorporated dye-ddNTPs before loading on
the gel since they co-migrate with the sequencing products.
Rhodamine dye family terminators seem to stabilize hairpin
structures in GC-rich regions, which causes the products to migrate
anomalously. This requires the use of dITP which relaxes the
secondary structure but also affects the efficiency of
incorporation of terminator.
[0076] In contrast, fluorescein-labeled terminators eliminate the
separation step prior to gel loading since they have a greater net
negative charge and migrate faster than the sequencing products. In
addition, fluorescein-labeled sequencing products have better
electrophoretic migration than sequencing products labelled with
rhodamine. Although wild-type Taq DNA polymerase does not
efficiently incorporate terminators labeled with fluorescein family
dyes, this can now be accomplished efficiently by use of the
modified enzymes provided herein.
[0077] Thus, the scope of this invention includes novel methods for
dideoxy sequencing using enzymes having the critical motif, as well
as kits for performing the method. In one embodiment, the
sequencing method of the invention comprises:
[0078] a) providing a recombinant thermostable DNA polymerase
enzyme which is characterized in that
[0079] i) in its native form said polymerase comprises the amino
acid sequence LSXXLX(V/I)PXXE (SEQ ID NO: 1), where X is any amino
acid,
[0080] ii) the X at position 4 in said sequence is mutated in
comparison to said native sequence, except that X is not mutated to
E; and
[0081] iii) said thermostable DNA polymerase has reduced
discrimination against incorporation of nucleotides labeled with
fluorescein family dyes in comparison to the native form of said
enzyme; and
[0082] b) performing a dye-terminator sequencing reaction.
[0083] In a preferred embodiment of the above method, the native
form enzyme has the amino acid sequence LS(Q/G)XL(S/A)IPYEE (SEQ ID
NO: 2), where X is any amino acid. In the three-letter code, this
amino acid sequence is represented as
LeuSerXaaXaaLeuXaaIleProTyrGluGlu (SEQ ID NO: 2), whereby "Xaa" at
position 3 is Gln or Gly, "Xaa" at position 4 is any amino acid,
and "Xaa" at position 6 is Ser or Ala. In a more preferred
embodiment, the native form amino acid sequence is LSQXLAIPYEE (SEQ
ID NO:3), where X is any amino acid. In the three-letter code, this
amino acid sequence is represented as
[0084] LeuSerGlnXaaLeuAlaIleProTyrGluGlu (SEQ ID NO:3), whereby
"Xaa" at position 4 is any amino acid. In a most preferred
embodiment, the "Xaa" at position 4 is Lys.
[0085] As described above, DNA sequencing with thermostable DNA
polymerases requires a mixture of unconventional base analogues
that act as chain-terminators and conventional nucleotides at a
specified ratio of concentrations that insures that a population of
extension products would be generated representing all possible
fragment lengths over a distance of several hundred bases. Some
thermostable DNA polymerases previously used for sequencing, such
as wild-type Taq polymerase, are characterized in that they
preferentially incorporate conventional nucleotides in the presence
of a mixture of conventional and unconventional nucleotides.
However, some recently described thermostable DNA polymerases allow
the ratio of unconventional base analogues to conventional bases to
be reduced from a hundred to several hundred fold, or up to over a
thousand fold.
[0086] One such polymerase is the F667Y mutant of Taq DNA
polymerase. Another such mutant is a Taq DNA polymerase having an
F667Y mutation and a mutation at position 46 which changes a
glycine residue to an aspartic acid residue (G46D) mutation. This
mutant polymerase, known as AmpliTaq, FS, is manufactured by
Hoffmann-La Roche and marketed by Perkin-Elmer. F730Y Tma30 DNA
Polymerase is another such polymerase. This mutant polymerase is a
combination of 1) nucleotides 1-570 of Taq DNA polymerase modified
to encode a G46D mutation and 2) nucleotides 571-2679 of Tma DNA
polymerase modified to encode an aspartic acid to alanine mutation
at position 323, a glutamic acid to alanine mutation at position
325, and a phenylalanine to tyrosine mutation at position 730 (U.S.
application Ser. No. 60/05265, which is hereby incorporated by
reference). Another polymerase that incorporates unconventional
base analogues is a F730Y mutant DNA polymerase from Thermotoga
neapolitana (International patent applications WO 96/10640, WO
96/41014, and WO 97/09451, which are hereby incorporated by
reference). Using these enzymes, for a given dNTP concentration,
the rhodamine-ddNTP concentration can be decreased by about 50- to
several hundred-fold compared to thermostable DNA polymerases
previously available.
[0087] The E681K mutation of the invention was combined using
recombinant DNA methods with an F667Y mutation to produce the
double mutant Taq DNA polymerase enzyme used in the sequencing
reactions described in Example IV. The double mutant was used in a
dye-terminator sequencing reaction with fluorescein-labeled dye
terminators. The results, described in Example IV, show that the
enzyme is capable of incorporating fluorescein-labeled dye
terminators in a sequencing reaction and produces sequencing
ladders that can be accurately read in an automated sequencing
instrument. Unexpectedly, the combination of the E681K and the
F667Y mutations was also found to produce a thermostable DNA
polymerase enzyme with a 3- to 4-fold increased extension rate
relative to an enzyme with the F667Y mutation alone, as measured by
the assay described in Example III.
[0088] Thus, in another aspect of this invention, the critical
motif identified in this invention can be combined with motifs
conferring reduced discrimination against ddNTPs to produce
polymerases having an increased efficiency of incorporation of both
labeled and unlabeled ddNTPs. These polymerases are useful in DNA
sequencing methods. In one embodiment of the present invention, a
thermostable DNA polymerase having the critical motif defined
herein also comprises the critical motif that includes the F667Y
mutation, described in U.S. Ser. No. 08/448,223. In this
embodiment, the thermostable DNA polymerase is characterized in
that
[0089] i) in its native form said polymerase comprises a first
amino acid sequence LSXXLX(V/I)PXXE (SEQ ID NO: 1), where X is any
amino acid,
[0090] ii) the X at position 4 in said first amino acid sequence is
mutated in comparison to said native sequence, except that X is not
mutated to E; and
[0091] iii) said thermostable DNA polymerase has reduced
discrimination against incorporation of nucleotides labeled with
fluorescein family dyes in comparison to the native form of said
enzyme; and
[0092] iv) said polymerase comprises a second amino acid sequence
MRRXXKXXNYXXXYG (SEQ ID NO: 12) where X is any amino acid;
[0093] v) said thermostable DNA polymerase also has reduced
discrimination against incorporation of unconventional nucleotides
in comparison to the native form of said enzyme. In the
three-letter code, the second amino acid sequence is represented
by
[0094] MetArgArgXaaXaaLysXaaXaaAsnTyrXaaXaaXaaTyrGly (SEQ ID NO:
12), where "Xaa" at positions 4, 5, 7, 8, 11, 12, and 13 is any
amino acid. In a preferred embodiment, the "Xaa" at position 4 in
the first amino acid sequence is mutated to Lys. In a more
preferred embodiment, the enzyme is Taq DNA polymerase and it
comprises the E681K and F667Y mutations. Also within the scope of
this invention are methods of sequencing using the above
polymerases.
[0095] Also within the scope of this invention is the improved
sequencing method of the invention performed using thermostable DNA
polymerase enzymes having a critical motif which is not derived by
mutation, but which critical motif exists as a natural variant. In
this aspect, the DNA polymerase of a thermophilic bacterial species
has a critical motif in which the residue at position 4 is not Glu.
For example, in the thermostable DNA polymerase from Thermotoga
neapolitana, the X at position 4 in the motif LSXXLX(V/I)PXXE (SEQ
ID NO: 7), where X is any amino acid except E, is an arginine
residue. Thus, the invention provides for improved methods of DNA
sequencing using a native thermostable DNA polymerase which
comprises the amino acid sequence LSXXLX(V/I)PXXE (SEQ ID NO: 7)
where X can be any amino acid except E. In the three-letter code,
this amino acid sequence is represented as
[0096] LeuSerXaaXaaLeuXaaXaaProXaaXaaGlu (SEQ ID NO: 7), where
"Xaa" at positions 3, 6, 9, and 10 are any amino acid and "Xaa" at
position 4 is any amino acid except Glu and "Xaa" at position 7 is
Val or Ile. In this embodiment, the sequencing method of the
invention comprises:
[0097] a) providing a thermostable DNA polymerase which is
characterized in that
[0098] i) said polymerase comprises the amino acid sequence
[0099] LSXXLX(V/I)PXXE (SEQ ID NO: 7), where X at position 4 is any
amino acid except E,
[0100] ii) said thermostable DNA polymerase has reduced
discrimination against incorporation of nucleotides labeled with
fluorescein family dyes; and
[0101] b) providing a dye-terminator labeled with a fluorescein
family dye, and
[0102] c) performing a dye-terminator sequencing reaction.
[0103] In a more preferred embodiment, the sequencing method of the
invention comprises:
[0104] a) providing a thermostable DNA polymerase which is
characterized in that
[0105] i) said polymerase comprises a first amino acid sequence
[0106] LSXXLX(V/I)PXXE (SEQ ID NO: 7), where X at position 4 is any
amino acid except E,
[0107] ii) said thermostable DNA polymerase has reduced
discrimination against incorporation of nucleotides labeled with
fluorescein family dyes;
[0108] iii) said polymerase comprises a second amino acid sequence
MRRXXKXXNYXXXYG (SEQ ID NO: 12) where X is any amino acid.
[0109] iv) said thermostable DNA polymerase has reduced
discrimination against incorporation of unconventional nucleotides;
and
[0110] b) providing a dye-terminator labeled with a fluorescein
family dye, and
[0111] c) performing a dye-terminator sequencing reaction.
[0112] In another preferred embodiment, the enzyme comprises the
amino acid sequence LS(Q/G)XL(S/A)IPYEE (SEQ ID NO: 13), where X at
position 4 is any amino acid except E. In the three-letter code,
this amino acid sequence is represented as
[0113] LeuSerXaaXaaLeuXaaIleProTyrGluGlu (SEQ ID NO: 13) whereby
"Xaa" at position 3 is Gln or Gly, "Xaa" at position 4 is any amino
acid except Glu, and "Xaa" at position 6 is Ser or Ala. In a more
preferred embodiment, the amino acid sequence is LSQXLAIPYEE (SEQ
ID NO:14), where X is any amino acid except E. In the three-letter
code, this amino acid sequence is represented as
LeuSerGlnXaaLeuAlaIleProTyrGluGlu (SEQ ID NO: 14), whereby "Xaa" at
position 4 is any amino acid except Glu.
[0114] In yet another preferred embodiment, the enzyme has the
amino acid sequence LSVXLG(V/I)PVKE (SEQ ID NO: 15), where X is any
amino acid except E. In the three-letter code, this amino acid
sequence is represented as LeuSerValXaaLeuGlyXaaProValLysGlu (SEQ
ID NO: 15), whereby "Xaa" at position 4 is any amino acid except
Glu and "Xaa" at position 7 is Val or Ile. In a more preferred
embodiment, the amino acid sequence is LSVXLGVPVKE (SEQ ID NO: 16),
where X is any amino acid except E. In the three-letter code, this
amino acid sequence is represented as
LeuSerValXaaLeuGlyValProValLysGlu (SEQ ID NO: 16), whereby "Xaa" at
position 4 is any amino acid except Glu. In a most preferred
embodiment, the "Xaa" at position 4 is Arg. In another more
preferred embodiment, the amino acid sequence is LSVXLGIPVKE (SEQ
ID NO: 17), where X is any amino acid except E. In the three-letter
code, this amino acid sequence is represented as
LeuSerValXaaLeuGlyIleProValLysGlu (SEQ ID NO: 17), whereby "Xaa" at
position 4 is any amino acid except Glu. In a most preferred
embodiment, the "Xaa" at position 4 is Arg.
[0115] In another embodiment of the invention, the sequencing
methods are performed using a native enzyme which has a reduced
level of discrimination against nucleotides labeled with
fluorescein family dyes which level is measured using a ddNTP
incorporation assay such as that described in Example II. The
concentration of ddCTP required for 50% inhibition of DNA synthesis
is determined, as is the concentration of Zowie-ddCTP needed for
50% inhibition. The ratio of the concentration for Zowie-ddCTP to
the concentration for ddCTP is calculated. In a preferred
embodiment, the ratio is 10 or less. In a more preferred
embodiment, the ratio is 4 or less. In a most preferred embodiment,
the ratio is 1.2 or less.
[0116] Although the examples provided herein use dideoxynucleotides
labeled with fluorescein family dyes, the use of other
unconventional nucleotides and fluorescent dyes is also within the
scope of this invention. Other unconventional nucleotides include
fluorescently-labled dNTPs, which can be used to label the products
of DNA synthesis, and fluorescently-labled rNTPs, which can be used
to label the primer extension products. Other dyes include other
negatively charged fluorescent dyes, such as BODIPY, which are
structurally and chemically similar to fluorescein. Other dyes also
include cyanine dyes. Cyanine-labeled dNTPs were added to a
standard PCR reaction which included a Taq DNA polymerase with the
E681K mutation (and a G46D mutation). The cyanine-labeled dNTPs
were unexpectedly found to be incorporated into amplification
products at a level that was higher than for the wild-type enzyme.
Thus, in this aspect, a method of labeling DNA of the invention
uses a native or mutant polymerase of the invention in combination
with a nucleotide labeled with either a negatively charged
fluorescent dye or a cyanine dye. In one embodiment, the DNA
labeling method of the invention comprises:
[0117] a) providing a thermostable DNA polymerase characterized in
that
[0118] i) said polymerase comprises the amino acid sequence
[0119] LSXXLX(V/I)PXXE (SEQ ID NO: 7) where X at position 4 can be
any amino acid except E
[0120] ii) said polymerase has reduced discrimination against
incorporation of unconventional nucleotides, and
[0121] b) providing a nucleotide labeled with a negatively charged
fluorescent dye, and
[0122] c) performing a DNA synthesis reaction.
[0123] In another embodiment, the DNA labeling method of the
invention comprises:
[0124] a) providing a thermostable DNA polymerase characterized in
that
[0125] i) said polymerase comprises the amino acid sequence
[0126] LSXXLX(V/I)PXXE (SEQ ID NO: 7) where X at position 4 can be
any amino acid except E
[0127] ii) said polymerase has reduced discrimination against
incorporation of unconventional nucleotides, and
[0128] b) providing a nucleotide labeled with a cyanine dye,
and
[0129] c) performing a DNA synthesis reaction.
[0130] In another aspect of the invention, a thermostable DNA
polymerase is provided which combines a mutation allowing more
efficient incorporation of rNTPs, such as the glutamic acid to
glycine mutation at position 615 of Taq DNA polymerase, and the
critical motif of this invention. The resulting enzyme is expected
to have an increased efficiency of incorporation of ribonucleotides
labeled with fluorescein family dyes. Thus, in one embodiment, the
invention provides a recombinant thermostable DNA polymerase which
(1) in its native form comprises the amino acid sequence
LSXXLX(V/I)PXXE (SEQ ID NO: 1) where X is any amino acid and (2)
the X at position 4 is mutated such that X at position 4 is not
mutated to E and (3) also comprises the region of criticality which
is amino acid sequence SQIXLR(V/I) (SEQ ID NO: 18) where "X" is any
amino acid except E, and (4) is capable of efficient incorporation
of ribonucleotides labeled with fluorescein family dyes. In the
three-letter code, the latter sequence is represented as
SerGlnIleXaaLeuArgXaa, where "Xaa" at position 4 is any amino acid
except Glu and "Xaa" at position 7 is Val or Ile
[0131] Mutant polymerase domains such as that for Taq containing
the E615G and E681K mutations are useful in improved methods of
producing primer extension products labeled with fluorescein family
dyes. For example, in a primer extension reaction such as PCR,
rNTPs labeled with fluorescein family dyes are substituted at least
partially for one of the 4 standard dNTPs and a double mutant
polymerase such as E681K E615G Taq DNA polymerase is included. The
mutant polymerase synthesizes primer extension products that have
fluorescein-labeled ribonucleotide residues at various positions
along their lengths. Upon heat or alkali treatment, the primer
extension products are fragmented at each ribonucleotide residue,
producing a population of end-labeled fragments. This population of
uniformly labeled fragments represents a distribution of the
fluorescent label across the length of the primer extension
product. Labeled fragments of these characteristics are useful in
nucleic acid detection formats based on silicon chips, such as that
of (Cronin et al., supra.). Thus, in one embodiment, the invention
provides a method of labeling primer extension products which
comprises (1) providing a thermostable DNA polymerase which (a) in
its native form comprises the amino acid sequence LSXXLX(V/I)PXXE
(SEQ ID NO: 1) where X is any amino acid, (b) the X at position 4
is mutated such that X at position 4 is not mutated to E, (c) also
comprises the region of criticality which is amino acid sequence
SQIXLR(V/I) (SEQ ID NO: 18) where "X" is any amino acid except E
and (d) is capable of efficient incorporation of ribonucleotides
labeled with fluorescein and/or cyanine family dyes, and (2)
performing a primer extension reaction.
[0132] In yet another aspect, enzymes having the critical motif of
this invention display an increased rate of extension relative to
the wild-type enzyme as shown in Example IV for a E681K F667Y
mutant. In one embodiment, the enzyme is characterized in that (1)
in its native form, it comprises the amino acid sequence
LSXXLX(V/I)PXXE (SEQ ID NO: 1) where X is any amino acid, (2) the
amino acid sequence is mutated at position 4 such that X at
position 4 is not mutated to E, and (3) it has an increased
extension rate relative to the wild-type enzyme. In a preferred
embodiment, the enzyme is characterized in that (1) in its native
form, it comprises the amino acid sequence LSXXLX(V/I)PXXE (SEQ ID
NO: 1) where X is any amino acid, (2) the amino acid sequence is
mutated at position 4 such that X at position 4 is not mutated to E
and (3) it also comprises the amino acid sequence MRRXXKXXNYXXXYG
(SEQ ID NO: 12) where X is any amino acid and (4) has an increased
extension rate. In a more preferred embodiment, the enzyme is
characterized in that (1) in its native form it contains the amino
acid sequence LSXXLX(V/I)PXXE (SEQ ID NO: 1) where X is any amino
acid and (2) the amino acid sequence is mutated at position 4 such
that X at position 4 is mutated to K. In a most preferred
embodiment, the enzyme is Taq DNA polymerase and contains the E681K
mutation and the F667Y mutation. Also included within this aspect
are methods of sequencing and labeling of DNA using the polymerases
with increased extension rate as well as kits for doing the
same.
[0133] In a preferred method for DNA sequencing according to the
invention, thermostable pyrophosphatase is included in the reaction
mixture. Pyrophosphatase has been shown to enhance sequencing data
using mesophilic as well as the mutant thermostable DNA polymerases
described in U.S. Ser. No. 08/528,384.
[0134] In an exemplified embodiment, the thermostable DNA
polymerase of the invention also contains a mutation in the
5'-nuclease domain that serves to greatly attenuate this nuclease
activity. Modified forms of Taq polymerase have been described in
PCT Patent Publication No. WO 92/06200, published Apr. 16, 1992 and
in U.S. Pat. No. 5,466,591. In one embodiment of that invention,
the codon for the glycine residue at amino acid position 46 has
been replaced with a codon for aspartic acid (G46D mutation). The
resulting enzyme has enhanced utility in cycle sequencing reactions
due to the decreased 5'-nuclease activity. The polymerase domain
amino acid sequence and polymerase activity are both unchanged in
the G46D mutant in comparison to the wild-type enzyme.
[0135] In a commercial embodiment of the invention, kits for
practicing methods that are improved by use of the present
invention are considered to be an additional aspect of the
invention. One such kit for DNA sequencing comprises
[0136] a) a thermostable DNA polymerase characterized in that
[0137] i) said polymerase comprises the amino acid sequence
[0138] LSXXLX(V/I)PXXE (SEQ ID NO: 7) where X at position 4 can be
any amino acid except E
[0139] ii) said polymerase has reduced discrimination against
incorporation of nucleotides labeled with fluorescein family dyes,
and
[0140] b) a dye-terminator labeled with a negatively charged
fluorescent dye and may additionally include other reagents for DNA
sequencing such as dNTPs, thermostable pyrophosphatase and
appropriate buffers. In another embodiment, the enzyme in the kit
has the amino acid sequence LSVXLG(V/I)PVKE (SEQ ID NO: 15),
whereby X is any amino acid except E. In the three-letter code,
this amino acid sequence is represented as
LeuSerValXaaLeuGlyXaaProValLysGlu (SEQ ID NO: 15), whereby "Xaa" at
position 4 is any amino acid except Glu and "Xaa" at position 7 is
Val or Ile. In a preferred embodiment, the amino acid sequence is
LSVXLGVPVKE (SEQ ID NO: 16), where X is any amino acid except E. In
the three-letter code, this amino acid sequence is represented as
LeuSerValXaaLeuGlyValPro- ValLysGlu (SEQ ID NO: 16), whereby "Xaa"
at position 4 is any amino acid except E. In a more preferred
embodiment, the "Xaa" at position 4 is Arg. In another preferred
embodiment, the amino acid sequence is LSVXLGIPVKE (SEQ ID NO: 17)
where X is any amino acid except E. In the three-letter code, this
amino acid sequence is represented as
[0141] LeuSerValXaaLeuGlyIleProValLysGlu (SEQ ID NO: 17), whereby
"Xaa" at position 4 is any amino acid except Glu. In a more
preferred embodiment, the "Xaa" at position 4 is Arg.
[0142] Other kits for DNA sequencing comprise a mutant thermostable
DNA polymerase characterized in that
[0143] a) in its native form said polymerase comprises the amino
acid sequence LSXXLX(V/I)PXXE (SEQ ID NO: 1) where X is any amino
acid,
[0144] b) said amino acid sequence is mutated at position 4, except
that X at position 4 is not mutated to E; and
[0145] c) said thermostable DNA polymerase has reduced
discrimination against incorporation of nucleotides labeled with
fluorescein family dyes in comparison to the native form of said
enzyme and may additionally include reagents for DNA sequencing
such as chain terminating compounds, dNTPs, thermostable
pyrophosphatase and appropriate buffers. Where the terminators are
labeled, preferable labels are fluorescent dyes, more preferable
labels are negatively charged fluorescent dyes or cyanine dyes, and
the most preferable labels are fluorescein family dyes. In a
preferred embodiment, the enzyme in the kit has the amino acid
sequence LS(Q/G)XL(S/A)IPYEE (SEQ ID NO: 2), where X is any amino
acid. In the three-letter code, this amino acid sequence is
represented as LeuSerXaaXaaLeuXaaIleProTyrGluGlu (SEQ ID NO: 2),
whereby "Xaa" at position 3 is Gln or Gly, "Xaa" at position 4 is
any amino acid, and "Xaa" at position 6 is Ser or Ala. In a more
preferred embodiment, the amino acid sequence is LSQXLAIPYEE (SEQ
ID NO:3), where X is any amino acid. In the three-letter code, this
amino acid sequence is represented as
LeuSerGlnXaaLeuAlaIleProTyrGluGlu (SEQ ID NO:3), whereby "Xaa" at
position 4 is any amino acid. In a most preferred embodiment, the
"Xaa" at position 4 is Lys.
[0146] Kits for labeling DNA comprise a thermostable DNA polymerase
which is characterized in that (a) in its native form, the
polymerase comprises the amino acid sequence LSXXLX(V/I)PXXE (SEQ
ID NO: 1) where X is any amino acid, (b) the X at position 4 in
said sequence is mutated in comparison to said native form, except
that X at position 4 is not mutated to E, and c) the enzyme has
reduced discrimination against incorporation of nucleotides labeled
with fluorescein family dyes, in comparison to the corresponding
wild-type enzyme and may additionally include dNTPs and appropriate
buffers. In a preferred embodiment, the X at position 4 is mutated
to K. Other kits for producing labeled DNA comprise a) a nucleotide
or nucleotide analog labeled with a negatively charged fluorescent
compound and b) a native thermostable DNA polymerases having the
following critical motif:
[0147] LSXXLX(V/I)PXXE (SEQ ID NO: 7)
[0148] where X at position 4 can be any amino acid except E and
said polymerase has reduced discrimination against incorporation of
fluorescein-labeled nucleotides, and may additionally include dNTPs
and appropriate buffers. In a preferred embodiment, the X at
position 4 is K. In another preferred embodiment, the X at position
4 is R.
[0149] Kits for labeling primer extension products comprise a
thermostable DNA polymerase which is characterized in that (a) in
its native form, the polymerase comprises the amino acid sequence
LSXXLX(V/I)PXXE (SEQ ID NO: 1) where X is any amino acid, (b) the X
at position 4 in said sequence is mutated in comparison to said
native sequence, except that X at position 4 is not mutated to E,
c) the polymerase also comprises the second amino acid sequence
SQIXLR(V/I) (SEQ ID NO:18) where "X" is any amino acid except E, d)
the enzyme has reduced discrimination against incorporation of
ribonucleotides labeled with fluorescein family dyes, in comparison
to the corresponding wild-type enzyme, and may additionally include
a ribonucleotide or ribonucleotide analog labeled with a negatively
charged fluorescent compound or cyanine compound, dNTPs, and
appropriate buffers. In a preferred embodiment, the polymerase
contains a E681K mutation and a E615G mutation. Other kits for
producing labeled primer extension products comprise a) a
ribonucleotide or ribonucleotide analog labeled with a negatively
charged fluorescent compound or cyanine compound and b) a native
thermostable DNA polymerase characterized in that it (i) comprises
the critical motif which is the amino acid sequence:
[0150] LSXXLX(V/I)PXXE (SEQ ID NO: 7)
[0151] where X at position 4 can be any amino acid except E, (ii)
comprises the second amino acid sequence SQIXLR(V/I) where "X" is
any amino acid except E, and (iii) has reduced discrimination
against incorporation of fluorescein-labeled ribonucleotides, and
may additionally include dNTPs and appropriate buffers. In a
preferred embodiment, in the first amino acid sequence, the X at
position 4 is a K and in the second amino acid sequence, the X is a
G. In another preferred embodiment, in the first amino acid
sequence, the X at position 4 is a R and in the second amino acid
sequence, the X is a G.
[0152] The following examples are offered by way of illustration
only and are by no means intended to limit the scope of the claimed
invention.
EXAMPLE I
Expression of a Modified Tag Polymerase Gene Having Reduced
Discrimination Against Nucleotides Labeled with Fluorescein Family
Dyes
[0153] The C-terminal amino acid portion of Taq DNA polymerase
encodes the polymerase active site domain (Lawyer et al., 1993, PCR
Methods and Applications 2:275-287, Freemont et al., 1986,
Proteins: Structure, Function and Genetics 1:66-73, which are
incorporated herein by reference). A DNA fragment containing this
region was isolated from the full-length Taq gene and mutagenized
by PCR amplification in the presence of manganese (Leung et al,
1989, Technique 1(1):11-15). For this example, all restriction
enzymes were purchased from New England Biolabs, Beverly Mass. The
mutagenized fragments were digested with PstI and BglII and cloned
into a Taq expression plasmid, pLK102, which had been digested with
PstI and BglII. Plasmid pLK102 is a derivative of pLK101 in which
the 900 bp PstI-BglII fragment is replaced by a short PstI-BglII
linker. Plasmid pLK101 is a modified form of pSYC1578 (Lawyer et
al., 1993, supra and U.S. Pat. No. 5,079,352 ), in which the small
HincII/EcoRV fragment located 3' to the polymerase coding region
was deleted.
[0154] The resulting expression plasmids were transformed into E.
coli strain N1624 (available from the E. coli Genetic Stock Center
at Yale University, strain No. CGSC#5066) and the resulting
transformants were screened for the ability to more efficiently
incorporate [.alpha.-.sup.32P]Tet-dCTP in comparison to the
wild-type enzyme. Using this procedure Mutant CS1, was identified
as having the ability to more efficiently incorporate
[.alpha.-.sup.32P]Tet-dCTP. The mutagenized Taq expression plasmid
of mutant CS1 was digested with HindIII/NheI and the resulting
restriction fragment was subcloned into the wild-type gene of
pLK101, replacing the unmutagenized HindIII/NheI fragment, to
determine which portion of the mutagenized Taq polymerase gene was
responsible for the altered phenotype. Subclones containing the
HindIII/NheI restriction fragment conferred the altered phenotype
on the wild-type enzyme, indicating that the mutation was within
this fragment. Subsequent subclone analysis determined that the
mutation was located in the 265 bp BamHI-NheI fragment.
[0155] DNA sequence analysis of the 265 NheI-BamHI fragment was
performed on pCS1 using the TaqFS DyeDeoxy.TM. Terminator Cycle
Sequencing Kit from Applied Biosystems, Foster City, Calif., and
the Applied Biosystems Model Prism 377 DNA Sequencing System. The
sequence analysis identified a missense mutation in the Taq
polymerase gene at amino acid position 681, that caused a Glutamic
acid (E) residue to be replaced by a Lysine (K) residue. Numbering
is initiated at the codon encoding the first methionine residue of
the mature protein, as in U.S. Pat. No. 5,079,352, which is herein
incorporated by reference. This mutation, E681K, specifically was
caused by a GAG to AAG change in the codon sequence. Plasmid pCS1
was deposited with the ATCC on Aug. 28, 1997, and given accession
No. 98521.
[0156] Plasmid pCS1 may contain additional mutations in the coding
sequence for Taq polymerase; however, by further subcloning
experiments, the E681K mutation was determined to be solely
responsible for the increased efficiency in incorporation of
nucleoside triphosphates labeled with fluorescein dyes. This point
mutation is located in the 265 base pair BamHI-NheI DNA fragment
shown in FIG. 1. Within the 265 bp DNA fragment, the E681K mutation
is the only change from the wild-type Taq polymerase gene
sequence.
[0157] For further analysis and quantitation of the efficiency of
incorporation of nucleotide analogues, the 265 bp BamHI-NheI
fragment of plasmid pCS1 was cloned into a Taq expression vector
that contained the wild-type sequence within the polymerase domain,
pRDA3-2. Plasmid pRDA3-2 referred to as clone 3-2, is fully
described in PCT Patent Publication No. WO 92/06200, which is
incorporated herein by reference. A second clone encoding both the
E681K mutation as well as a F667Y mutation was created by
primer-directed mutagenesis and subsequent cloning of a PCR product
containing both mutations into the BamHI-NheI sites of plasmid
pRDA3-2.
[0158] Expression vector pRDA3-2 contains the full-length Taq DNA
polymerase gene operably linked to the phage lambda P.sub.L
promoter. In vector pRDA3-2, the 5'-nuclease domain of the Taq DNA
polymerase gene contains a point mutation at the codon encoding
glycine at position 46 that reduces 5'-nuclease activity (G46D
mutation). However, the gene sequence within the polymerase domain
of the expression vector pRDA3-2 is identical to the wild-type Taq
DNA polymerase gene sequence. Plasmids, pRDA3-2, pCS1 and the E681K
F667Y PCR product were digested with BamHI and NheI and the 265 bp
DNA fragment from plasmid pCS1 or the PCR product was ligated into
vector pRDA3-2 by conventional means. The resulting plasmids,
pLK112 and pLK113, respectively, were transformed into E. coli
strain DG116 (ATCC No. 53606). These plasmids encode thermostable
DNA polymerases herein referred to as G46D E681K Taq and G46D E681K
F667Y Taq, respectively. The expressed thermostable DNA polymerase
protein G46D E681K F667Y Taq was purified according to the method
described by Lawyer et al., 1993, supra.
[0159] The G46D E681K Taq enzyme was purified using a similar, but
smaller scale preparation method as follows: All steps were
preformed at 4.degree. C. unless indicated otherwise. Cells from a
475 ml culture were resuspended in 30 ml of buffer (50 mM Tris-HCl,
pH 7.5, 10 mM EDTA, pH 8.0, 0.5 mM Pefabloc.COPYRGT.SC, 0.5
.mu.g/ml leupeptin, 0.1 mM N.alpha.-p-tosyl-L-Lysine Chloromethyl
Ketone, 1 mM DTT). Cells were sonicated at 50% duty cycle, setting
5 for 1 minute, and cooled on ice for 1 minute. This step was
repeated twice more. Then 1.5 ml of 4.0 M ammonium sulfate was
added and the mixture heated in a 75.degree. C. water bath for 15
minutes, followed by cooling on ice. Polyethyleneimine was added to
0.6% and the mixture was incubated on ice for 10 minutes. The
mixture was centrifuged at 16,000 xg for 30 minutes. The
supernatant was loaded on a 1.8 ml volume phenyl-sepharose column
(Bio-rad Polyprep chromatography column) equilibrated with a
solution of 50 mM Tris-HCl, pH 7.5, 10 mM EDTA, pH 8.0, 1 mM DTT,
0.2 M (NH.sub.4).sub.2SO.sub.4. The column was washed with 6 ml.
each of three solutions: 1) 25 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM
DTT, 0.2 M (NH.sub.4).sub.2SO.sub.4, 2) 25 mM Tris-HCl, pH 7.5, 1
mM EDTA, 1 mM DTT, and 3) 25 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM
DTT, 20% ethylene glycol. The polymerase was eluted with 6 ml of 25
mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM DTT, 20% ethylene glycol, 2.5
M urea. After adjusting the polymerase preparation to 100 mM KCl
with 3M KCl, the mixture was loaded on a heparin-sepharose column
(1.8 ml volume, Bio-rad Poly-prep column) equilibrated in 25 mM
Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM DTT, 100 mM KCl. After a wash
with the same buffer, the sample was eluted in a buffer of 25 mM
Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM DTT, 400 mM KCl.
[0160] Following purification, the activity of the modified enzymes
was determined by the activity assay described in Lawyer et al.,
1989, J. Biol. Chem. 264:6427-6437, which is incorporated herein by
reference. The activity of the purified enzymes was calculated as
follows: one unit of enzyme corresponds to 10 nmoles of product
synthesized in 30 min. DNA polymerase activity is linearly
proportional to enzyme concentration up to 80-100 pmoles dCMP
incorporated (diluted enzyme at 0.024-0.03 units/.mu.l). The
purified enzymes were utilized in the incorporation and sequencing
reactions described in Examples II-IV.
EXAMPLE II
Assay to Compare Efficiency of Incorporation of ddNTPs
[0161] The relative abilities of G46D F667Y Taq, G46D F667Y E681K
Taq and F730Y Tma30 DNA polymerases to incorporate a fluorescein
dye family-labeled ddCTP were compared by use of a limiting
template, primer extension competition assay. F730Y Tma30 DNA
polymerase is described in Example I of U.S. Ser. No. 60/05265,
filed Jul. 6, 1997, and is herein incorporated by reference. In
this competition assay, because the incorporation of a ddCTP
terminates the extension reaction, the more readily the polymerase
incorporates a ddCTP into an extended primer, the less
[.alpha.-.sup.33P]dCTP can be incorporated. Thus, as the efficiency
of ddCTP incorporation increases, the extent of inhibition of DNA
synthesis is increased. The efficiency of incorporation of ddCTP is
then compared to the efficiency of incorporation of fluorescently
labeled ddCTP to give a relative measurement of the efficiency of
incorporation of fluorescently-labeled ddNTPs for a given
enzyme.
[0162] The assay was performed as previously described (Lawyer et
al., 1989, J. Biol. Chem. 264:6427) including the following
modifications. The assay mixture was composed so the final
concentration was 50 mM Bicine pH 8.3, 25.degree. C., 2.5 mM
MgCl.sub.2, 1 mM .beta.-mercaptoethanol, 20 .mu.M each of dATP,
dGTP and dTTP (Perkin-Elmer), 20 .mu.M dCTP (Perkin-Elmer) and
[.alpha.-.sup.33P]dCTP (New England Nuclear, Boston, Mass.). M13 mp
18 (Perkin-Elmer) was annealed to primer DG48, (SEQ ID NO: 10) and
the equivalent of 0.085 pmoles of the annealed template was added
to the assay mixture for each reaction. Thirty-five .mu.l of the
assay mixture with template DNA was added to each of 38 0.5 ml
eppendorf tubes. Dilutions of Zowie-ddCTP in 25 mM CAPSO buffer, pH
9.6 were prepared such that when 10 .mu.l of each was added to the
reaction tube, the final concentration of Zowie-ddCTP would be 3,
1, 0.5, 0.25, 0.125, or 0.0625 .mu.M. For G46D F667Y Taq DNA
polymerase, two tubes each of the 3, 1, 0.5, 0.25, 0.125 .mu.M
Zowie-ddCTP were prepared. For G46D F667Y E681K Taq and F730Y Tma30
DNA polymerases, two tubes each of the 1, 0.5, 0.25, 0.125, and
0.0625 .mu.M Zowie-ddCTP were prepared. The eight remaining
reaction tubes received 10 .mu.l of 25 mM CAPSO buffer, pH 9.6.
Thus, each of the thirty-eight tubes contained 35 .mu.l of assay
mix and 10 .mu.l of either 25 mM CAPSO buffer, pH 9.6 or one of the
Zowie-ddCTP dilutions.
[0163] For each enzyme to be tested, polymerization was initiated
in one tube of each Zowie-ddCTP dilution and two tubes containing
the CAPSO buffer alone using 5 .mu.l of the enzyme. The following
concentrations of the enzymes were used, each predetermined to be
an excess amount of enzyme for the amount of substrate in the
assay: 2.5 units of F667Y G46D Taq DNA polymerase prepared as in
Example I; 1.25 units of G46D, F667Y, E681K Taq DNA polymerase,
prepared as in Example I; or 2 units of F730Y Tma30 DNA polymerase.
As a control for the level of background, the remaining negative
control was initiated with enzyme dilution buffer rather than
polymerase. All reaction tubes were immediately vortexed briefly
and incubated for 10 minutes at 75.degree. C. The reactions were
stopped by addition of 10 .mu.l 60 mM EDTA and stored at 0.degree.
C.
[0164] In an analogous experiment, ddCTP was diluted in 25 mM CAPSO
buffer, pH 9.6 such that when 10 .mu.l of each dilution was added
to the reaction tubes, the final concentration would be 0.5, 0.25,
0.125, 0.0625, or 0.0312 .mu.M. Ten .mu.l of each dilution was
pipetted into each of three 0.5 ml Eppendorf tubes containing 35
.mu.l of the assay mixture as described above. Four tubes
containing 35 .mu.l of the assay mix plus 10 .mu.l of 25 mM CAPSO
buffer, pH 9.6 were also prepared. Thus, each of the 19 tubes
contained 35 .mu.l of assay mix and 10 .mu.l each of either 25 mM
CAPSO, pH 9.6 or one of the ddCTP dilutions.
[0165] Polymerization was initiated in one tube of each ddCTP
dilution and one tube of CAPSO buffer with 2.5 units of G46D F667Y
Taq DNA polymerase, 1.25 units of G46D F667Y E681K Taq DNA
polymerase or 2 units of F730Y Tma30 DNA polymerase. The remaining
tube containing CAPSO was initiated with enzyme dilution buffer
rather than the polymerase-containing buffer as a negative control.
All reactions were immediately vortexed and incubated 10 minutes at
75.degree. C. The reactions were stopped by addition of 10
microliters of 60 mM EDTA and stored at 0.degree. C.
[0166] For each reaction, a 50 .mu.l aliquot of the 60 .mu.l
reaction was diluted with 1 ml 2 mM EDTA, 50 .mu.g/ml sheared
salmon sperm DNA as a carrier. The DNA was precipitated with TCA
using standard procedures and collected on GF/C filter discs
(Whatman). The amount of incorporated [.alpha.-.sup.33P]dCMP was
determined for each sample and normalized to the CAPSO samples
without ddNTP (0% inhibition). The concentration of ddCTP or
Zowie-ddCTP needed for 50% inhibition was calculated for each
sample and is shown in Table 2. Comparison of the amount of ddCTP
needed to inhibit synthesis 50% with the amount of Zowie-ddCTP
required to inhibit synthesis by 50% for a particular enzyme
reflects the relative ability of each enzyme to incorporate
fluorescently-labeled analog. These data show that G46D F667Y Taq
DNA polymerase incorporates Zowie-ddCTP least efficiently of the
three enzymes tested (ratio of concentrations for 50% inhibition by
Zowie-ddCTP vs. ddCTP=25). F730Y Tma30 DNA polymerase incorporates
this labeled analog more efficiently than G46D F667Y Taq DNA
polymerase (ratio of concentrations for 50% inhibition by
Zowie-ddCTP vs. ddCTP=4), while G46D F667Y E681K Taq DNA polymerase
incorporates labeled and unlabeled ddCTP with nearly equal
efficiency (ratio of concentrations for 50% inhibition by
Zowie-ddCTP vs. ddCTP=1.2).
2TABLE 2 Concentration (.mu.M) of Zowie-ddCTP or ddCTP needed for
50% inhibition Zowie- DNA polymerase Zowie-ddCTP ddCTP ddCTP/ddCTP
G46D F667Y Taq 1.4 0.056 25 G46D F667Y E681K Taq 0.14 0.116 1.2
F730Y Tma3O 0.236 0.057 4
EXAMPLE III
Extension Rate Assay
[0167] The extension rate of G46D F667Y Taq and G46D F667Y E681K
Taq were determined using an extension rate assay. In this
experiment, the enzymes were used to extend a primer annealed to an
M13 template in the presence of [.alpha.-.sup.33P]dCTP. The
extension reactions were denatured and the products analyzed by
denaturing agarose gel electrophoresis.
[0168] The assay was performed as previously described (Lawyer et
al., 1989, J. Biol. Chem. 264:6427) including the following
modifications. The assay mixture was composed so the final
concentration was 50 mM Bicine pH 8.3, 25.degree. C., 2.5 mM
MgCl.sub.2, 1 mM .beta.-mercaptoethanol, 200 .mu.M each of dATP,
dGTP and dTTP (Perkin-Elmer), 100 .mu.M dCTP (Perkin-Elmer)
containing [.alpha.-.sup.33P]dCTP (New England Nuclear, Boston,
Mass.). M13 mp 18 (Perkin-Elmer) was annealed to primer DG48, (SEQ
ID NO: 11), and the equivalent of 0.085 pmoles of the annealed
template was added to the assay mixture for each reaction.
Forty-five .mu.l of the assay mixture with template DNA was added
to each of fourteen 0.5 ml eppendorf tubes. Each tube was
preincubated at 75.degree. C. for at least 30 seconds before the
start of the polymerase reaction.
[0169] Polymerization was initiated in six of the fourteen assay
tubes with 5 .mu.l of G46D F667Y Taq DNA polymerase (2.5 units) or
G46D F667Y E681K Taq DNA polymerase (1.25 units). Both enzymes were
prepared as in Example I and the concentration used represents a
predetermined excess amount of enzyme for the amount of substrate
in the assay. As a control for the level of background, the
remaining negative control was initiated with enzyme dilution
buffer rather than polymerase. All reaction tubes were immediately
vortexed briefly and incubated at 75.degree. C. Two of the six
tubes containing G46D F667Y Taq DNA polymerase were incubated 3
minutes, two for 6 minutes and two for 10 minutes. Similarly, two
of the tubes started with G46D F667Y E681K Taq DNA polymerase were
incubated for 30 seconds, two for 1 minute and two for 2 minutes.
The control tubes were incubated for 3 minutes. The reactions were
stopped by addition of 10 .mu.l 60 mM EDTA and stored at 0.degree.
C.
[0170] For each reaction, a 25 .mu.l aliquot of the 60 .mu.l
reaction was diluted with 1 ml 2 mM EDTA, 50 .mu.ml sheared salmon
sperm DNA as a carrier. The DNA was precipitated with TCA using
standard procedures and collected on GF/C filter discs (Whatman).
The amount of incorporated [.alpha.-.sup.33P]dCMP was determined
for each sample.
[0171] The remaining 35 .mu.l of each duplicated were combined and
the 70 .mu.l sample was ethanol precipitated, dried and resuspended
in 50 mM NaOH, 1 mM EDTA. Aliquots were removed from these samples
such that an equal number of .alpha.-.sup.33P] counts were taken
from each. These aliquots were loaded on an 0.9% alkaline agarose
gel, electrophoresed, dried and autoradiographed as previously
described (Maniatis et al., 1982, In Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring
Harbor, N.Y.) Bacteriophage lambda DNA cut with restriction enzyme
HindIII (BRL) and 5' end-labeled with [.sup.32P] was used as a
molecular weight standard.
[0172] The length in base pairs of the extension product in each
sample was determined by comparison of the migration distance of
each sample with the distance migrated by the lambda DNA size
standard. The number of base pairs in each product was divided by
the number of seconds each extension reaction incubated to give the
extension rate as shown below.
3 DNA Polymerase Time Base Pairs/Sec G46D F667Y Taq 3 min. 125 G46D
F667Y Taq 6 min. 12.2 G46D F667Y Taq 10 min. 11.8 G46D F667Y E681K
Taq 30 sec. 36.7 G46D E667Y E681K Taq 1 min. 41.7 G46D F667Y E681K
Taq 2 min 52.9
[0173] These results indicate that the presence of the E681K
mutation increases the extension rate of a G46D F667Y enzyme by 3-
to 4.3- fold.
EXAMPLE IV
Cycle Sequencing with G46D F667Y E681K Tag DNA Polymerase and
Fluorescein Labeled ddNTPs
[0174] This example demonstrates the application of the modified
polymerase of the invention to fluorescein dye labeled dideoxy
terminator cycle sequencing, utilizing 1 .mu.M or less ddNTP and a
ratio of ddNTP:dNTP of at least 1:100. The fluorescein dye labeled
dideoxy terminators are reagents from the Applied Biosystems PRISM
Sequenase.RTM. Terminator Sequencing Kits (Perkin-Elmer, Norwalk,
Conn.) and were optimized for use with Sequenase DNA polymerase and
alpha-thio dNTPs. Cycle sequencing reactions were performed in a 20
.mu.l volume containing 50 mM Tris-HCl (pH 8.8), 2.0 mM MgCl.sub.2,
100 .mu.M each dATP, dCTP, and dTTP (Perkin-Elmer, Norwalk, Conn.),
500 .mu.M dITP (Pharmacia Biotech, Piscataway, N.J.), 0.2 .mu.g
M13mp 18 single-strand DNA template (Perkin-Elmer), 0.15 .mu.M LacZ
Forward Primer (Perkin-Elmer), 5 units of G46D F667Y E681K Taq DNA
polymerase, 20 units of rTth Thermostable Pyrophosphatase (U.S.
Ser. No. 08/528,384), 0.05 .mu.M Sequenase A Dye Terminator, 0.80
.mu.M Sequenase C Dye Terminator, 0.08 .mu.M Sequenase G Dye
Terminator, and 1.0 .mu.M Sequenase T Dye Terminator. All four
Sequenase Dye Terminators were purchased from Perkin-Elmer.
Reactions were placed in a preheated (75.degree. C.) Perkin-Elmer
GeneAmp.RTM. PCR System 9600 thermal cycler and subjected to 25
cycles of 96.degree. C. for 10 seconds, 50.degree. C. for 5
seconds, and 60.degree. C. for 4 minutes. Dye labeled fragments
were purified with Centri-Sep.TM. columns (Princeton Separations,
Adelphia, N.J.) following the manufacturer's instructions and dried
in a vacuum centrifuge. Pellets were resuspended in 6 .mu.l of
deionized formamide: 50 mg/mL Blue dextran (in 25 mM EDTA, pH 8.0)
5:1 (v/v), heated at 90.degree. C. for 3 minutes, and directly
loaded onto a pre-electrophoresed 4% polyacrylamide/6 M urea gel
and electrophoresed and analyzed on a Perkin-Elmer ABI PRISM` 377
DNA Sequencer according to the manufacturer instructions (ABI PRISM
377 DNA Sequencer User's Manual). Automated base-calling by the
Perkin-Elmer ABI PRISM 377 DNA Sequencer analysis software resulted
in greater than 98.5% accuracy for 450 bases (6 errors for bases
+10 to +460 from primer).
* * * * *