DNA polymerase having ability to reduce innate selective discrimination against fluorescent dye-labeled dideoxynucleotides

• US 6,485,909 B1
• Filed: 02/24/2000
• Issued: 11/26/2002
• Est. Priority Date: 10/18/1995
• Status: Expired due to Term

• Current Assignee: Hong Kong S&T Limited (Guan Qiao Zhong Family)
• Original Assignee: Shanghai Mendel DNA Center Company Limited
• Inventors: Hong, GuoFan, Huang, Wei-hua
• Primary Examiner(s): Siew, Jeffrey
• Application Number:
  US09/512,021
• Time in Patent Office: 1,006 Days
• Field of Search: 435/6, 435/7.1, 435/91.1, 435/91.2, 536/22.1, 536/23.1, 536/24-- 2
• US Class Current: 435/6.12
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2535/101 Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators
• CPC Class Codes: C12Q 2563/107 fluorescence

Abstract

The invention relates to genetical modification of DNA polymerase to reduce its innate selective sequence-related discrimination against incorporation of fluorescent dye-labeled ddCTP and ddATP in the enzymatic reaction for preparation of samples for automated florescent dye-labeled terminator DNA sequencing. The modified DNA polymerases are more resistant to heat inactivation and are more effective in dideoxynucleotide incorporation than current DNA polymerases.

Claims

1. A method of sequencing a DNA strand comprising the steps of:

i) hybridizing a primer to a DNA template to be sequenced;

ii) extending the primer using a modified DNA polymerase in the presence of adequate amounts of nucleotide bases dATP, dGTP, dCTP and dTTP, or their analogs, and fluorescent dye-labeled dideoxynucleotide terminators,

wherein the modified DNA polymerase during DNA sequencing effectively incorporates fluorescent dye-labeled dideoxynucleotide terminators ddCTP, ddATP, ddTTP and ddGTP, and their analogs, and reduces selective discrimination against incorporation of fluorescent dye-labeled dideoxynucleotide terminators ddCTP and ddATP, which DNA polymerase in its unmodified state selectively discriminates against incorporation of fluorescent dye-labeled dideoxynucleotide terminators ddCTP and ddATP but does not discriminate against incorporation of fluorescent dye-labeled dideoxynucleotide terminators ddTTP and ddGTP,

under such conditions that the DNA strand is sequenced.

2. The method according to claim 1, wherein the DNA polymerase is a modified Bacillus stearothermophilus DNA polymerase.

3. The method according to claim 1, wherein the modified DNA polymerase has an amino acid sequence that shares not less than 95% homology of a DNA polymerase isolated from a strain of Bacillus stearothermophilus, Bacillus caldotenax or Bacillus caldolyticus.

4. The method according to claim 1, wherein the modified DNA polymerase has the amino acid sequence SEQ ID NO:

4.

5. The method according to claim 1, wherein the DNA polymerase is encoded by the nucleotide sequence SEQ ID NO:

3.

6. The method according to claim 1, wherein the DNA polymerase is a modified DNA polymerase obtained from a mesophilic bacterium.

7. The method according to claim 1, wherein the DNA polymerase is a thermostable DNA polymerase having proofreading 3′

-5′

exonuclease activity during DNA sequencing of a DNA strand from a template, such that the DNA polymerase functions to excise mismatched nucleotides from the 3′

terminus of the DNA strand at a faster rate than the rate at which the DNA polymerase functions to remove nucleotides matched correctly with nucleotides of the template.

8. The method according to claim 7, wherein the DNA polymerase is a modified Bacillus stearothermophilus, Bacillus caldotenax or Bacillus caldolyticus DNA polymerase.

9. The method according to claim 8, wherein the DNA polymerase has the amino acid sequence of SEQ ID NO:

4.

10. The method according to claim 8, wherein the DNA polymerase is encoded by a DNA segment having the nucleotide sequence of SEQ ID:

NO 3.

11. A method of dye-labeled primer sequencing of a DNA strand comprising the steps of:

i) hybridizing a fluorescent dye-labeled primer to a DNA template to be sequenced;

ii) extending the primer using a modified DNA polymerase in the presence of adequate amounts of nucleotide bases dATP, dGTP, dCTP and dTTP, or their analogs, and dideoxynucleotide terminators,

wherein the modified DNA polymerase during DNA sequencing effectively incorporates fluorescent dye-labeled dideoxynucleotide terminators ddCTP, ddATP, ddTTP and ddGTP, and their analogs, and reduces selective discrimination against incorporation of fluorescent dye-labeled dideoxynucleotide terminators ddCTP and ddATP, which DNA polymerase in its unmodified state selectively discriminates against incorporation of fluorescent dye-labeled dideoxynucleotide terminators ddCTP and ddATP but does not discriminate against incorporation of fluorescent dye-labeled dideoxynucleotide terminators ddTTP and ddGTP,

under such conditions that the DNA strand is sequenced.

12. The method according to claim 11, wherein the DNA polymerase is a modified Bacillus stearothermophilus DNA polymerase.

13. The method according to claim 11, wherein the modified DNA polymerase has an amino acid sequence that shares not less than 95% homology of a DNA polymerase isolated from a strain of Bacillus stearothermophilus, Bacillus caldotenax or Bacillus caldolyticus.

14. The method according to claim 11, wherein the modified DNA polymerase has the amino acid sequence SEQ ID NO:

4.

15. The method according to claim 11, wherein the DNA polymerase is encoded by the nucleotide sequence SEQ ID NO:

3.

16. The method according to claim 11, wherein the DNA polymerase is a modified DNA polymerase obtained from a mesophilic bacterium.

17. The method according to claim 11, wherein the DNA polymerase is a thermostable DNA polymerase having proofreading 3′

-5′

exonuclease activity during DNA sequencing of a DNA strand from a template, such that the DNA polymerase functions to excise mismatched nucleotides from the 3′

terminus of the DNA strand at a faster rate than the rate at which the DNA polymerase functions to remove nucleotides matched correctly with nucleotides of the template.

18. The method according to claim 17, wherein the DNA polymerase is a modified Bacillus stearothermophilus, Bacillus caldotenax or Bacillus caldolyticus DNA polymerase.

19. A method of ralioiostope-labeled sequencing a DNA strand comprising the steps of:

i) providing four separate tubes, each containing DNA template to be sequenced, a primer, dATP, dTTP, dCTP and dGTP, wherein one of dATP, dTTP, dCTP or dGTP is labeled with a radioisotope, and a modified DNA polymerase which during DNA sequencing effectively incorporates fluorescent dye-labeled dideoxynucleotide terminators ddCTP, ddATP, ddTTP and ddGTP, and their analogs, and reduces selective disc nation against incorporation of fluorescent dye-labeled dideoxynucleotide terminators ddCTP and ddATP, which DNA polymerase in its unmodified state selectively discriminates against incorporation of fluorescent dye-labeled dideoxynucleotide terminators ddCTP and ddATP but does not discriminate against incorporation of fluorescent dye-labeled dideoxynucleotide terminators ddTTP and ddGTP,

wherein each tube contains an appropriate amount of one of dATP, dTTP, dCTP or dGTP;

ii) allowing DNA sequencing of the DNA template to be carried out; and

iii) determining the sequence of the DNA template.

20. The method according to claim 19, wherein the DNA polymerase is a modified Bacillus stearothermophilus DNA polymerase.

21. The method according to claim 19, wherein the modified DNA polymerase has an amino acid sequence that shares not less than 95% homology of a DNA polymerase isolated from a strain of Bacillus stearothermophilus, Bacillus caldotenax or Bacillus caldolyticus.

22. The method according to claim 19, wherein the modified DNA polymerase has the amino acid sequence SEQ ID NO:

4.

23. The method according to claim 19, wherein the DNA polymerase is encoded by the nucleotide sequence SEQ ID NO:

3.

24. The method according to claim 19, wherein the DNA polymerase is a modified DNA polymerase obtained from a mesophilic bacterium.

25. The method according to claim 19, wherein the DNA polymerase is a thermostable DNA polymerase having proofreading 3′

-5′

exonuclease activity during DNA sequencing of a DNA strand from a template, such that the DNA polymerase functions to excise mismatched nucleotides from the 3′

terminus of the DNA strand at a faster rate than the rate at which the DNA polymerase functions to remove nucleotides matched correctly with nucleotides of the template.

26. The method according to claim 25, wherein the DNA polymerase is a modified Bacillus stearothermophilus, Bacillus caldotenax or Bacillus caldolyticus DNA polymerase.