T7 DNA polymerase

• US 4,795,699 A
• Filed: 01/14/1987
• Issued: 01/03/1989
• Est. Priority Date: 01/14/1987
• Status: Expired due to Term

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Tabor, Stanley, Richardson, Charles C.
• Primary Examiner(s): NOT, DEFINED
• Assistant Examiner(s): Jay, Jeremy M.
• Application Number:
  US07/003,227
• Time in Patent Office: 720 Days
• Field of Search: 435/5, 435/6, 435/91, 435/803, 435/172.3, 435/810, 436/501, 436/808, 935/78, 935/77
• US Class Current: 435/5
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• 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 2565/102 Multiple non-interacting labels
• CPC Class Codes: Y10S 435/803 Physical recovery methods, e.g. chromatography, grinding
• CPC Class Codes: Y10S 435/81 Packaged device or kit

Abstract

This invention relates to T7-type DNA polymerases and method for using them.

Claims

1. A method for determining the nucleotide base sequence of a DNA molecule, comprising:

annealing said DNA molecule with a primer molecule able to hybridize to said DNA molecule;

incubating separate portions of the annealed mixture in at least four vessels, each vessel containing four different deoxynucleoside triphosphates, a processive T7-type DNA polymerase, wherein said polymerase remains bound to said DNA molecule for at least 500 bases before dissociating in an environmental condition used in the extension reaction of a DNA sequencing reaction, said polymerse having less than 500 units of exonuclease activity per mg of said polymerase, and one of four DNA synthesis terminating agents which terminate DNA synthesis at a specific nucleotide base, wherein each said agent terminates DNA synthesis at a different nucleotide base, and

separating the DNA products of each incubating reaction according to their size, whereby at least a part of the nucleotide base sequence of said DNA molecule can be determined.

2. The method of claim 1, wherein said polymerase is unable to exhibit its processivity in a second environmental condition normally used in the pulse step of a DNA sequence reaction.

3. The method of claim 1 wherein said polymerase remains bound to said DNA molecule for at least 1,000 bases before dissociating.

4. The method of claim 1 wherein said polymerase is that polymerase in cells infected with a T7-type phage.

5. The method of claim 4 wherein said T7-type phage is T7, T3, Φ

I, Φ

II, H, W31, gh-1, Y, Al122 or Sp6.

6. The method of claim 1 wherein said polymerase is non-discriminating for dideoxy nucleotide analogs.

7. The method of claim 1 wherein said polymerase is a modified polymerase having less than 50 units of exonuclease activity per mg of polymerase.

8. The method of claim 7 wherein said modified polymerase has less than 1 unit of activity per mg of polymerase.

9. The method of claim 7 wherein said modified polymerase has less than 0.1 unit of activity per mg of polymerase.

10. The method of claim 1 wherein said polymerase has no detectable exonuclease activity.

11. The method of claim 1 wherein said polymerase is able to utilize primers of 10 base pairs or more.

12. The method of claim 1 wherein said polymerase is able to utilize primers of 4 base pairs or more.

13. The method of claim 1 wherein said primer comprises 4-20 base pairs and said polymerase is able to utilize primers of 4-20 base pairs.

14. The method of claim 4 wherein said polymerase is non-discriminating for nucleotide analogs, and is a modified polymerase having less than 500 units of exonuclease activity per mg of polymerase

said primer is single stranded RNA or DNA containing 4-10 bases and said polymerase is able to utilize primers of 4-10 bases,

and said incubating comprises a pulse and a chase step.

15. The method of claim 1 wherein said primer is single stranded DNA or RNA.

16. The method of claim 1 wherein said annealing comprises heating said DNA molecule and said primer to above 65°

C., and allowing the heated mixture to cool to 10°

C. to 30°

C.

17. The method of claim 1 wherein said incubating comprises a pulse and a chase step.

18. The method of claim 17 wherein said pulse step comprises mixing said annealed mixture with all four deoxynucleoside triphosphates and a processive DNA polymerase, wherein at least one said deoxynucleoside triphosphate is labelled and present in a limiting concentration.

19. The method of claim 18 wherein said pulse step incubation is carried out for 30 seconds to 20 minutes.

20. The method of claim 18 wherein said chase step comprises adding one said chain terminating agent to four separate aliquots of the mixture after performing said pulse step.

21. The method of claim 20 wherein said chase step incubation is carried out for 1 to 60 minutes.

22. The method of claim 1 wherein said terminating agent is a dideoxynucleotide.

23. The method of claim 1 wherein said terminating agent is a limiting level of one deoxynucleoside triphosphate.

24. The method of claim 1 wherein one said deoxynucleoside triphosphate is chosen from dITP or deazaguanosine.

25. The method of claim 1 wherein said primer is labelled prior to said annealing step.

26. The method of claim 25 wherein said incubating comprises a chase step.

27. The method of claim 25 wherein said primer is fluorescently labelled.

28. The method of claim 4 wherein said T7-type phage is T7.

29. The method of claim 12 wherein said mixture comprises T7 gene 2.5 or gene 4.

30. The method of claim 13 wherein said mixture comprises T7 gene 2.5 or gene 4.

31. A kit for DNA sequencing, comprising:

a processive T7-type DNA polymerase, wherein said polymerase remains bound to a DNA molecule for at least 500 bases before dissociating, said polymerase having less than 500 units of exonuclease activity per mg of polymerase, said polymerase being able to exhibit its processivity in an environmental condition normally used in the extension reaction of a DNA sequencing reaction, and

a reagent necessary for said sequencing, selected from

(a) dITP and

(b) a chain terminating agent.

32. The kit of claim 31 wherein said polymerase is unable to exhibit its processivity in a second environmental condition normally used in the pulse step of a DNA sequencing reaction.