DNA sequencing

• US 4,994,372 A
• Filed: 12/06/1988
• Issued: 02/19/1991
• 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): Yarbrough, Amelia Burgess
• Application Number:
  US07/280,489
• Time in Patent Office: 805 Days
• Field of Search: 435/6, 435/91, 435/803, 436/501, 935/77, 935/78
• 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: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: Y10S 435/803 Physical recovery methods, e.g. chromatography, grinding

Abstract

This invention relates to processive DNA polymerases and methods for using them.

Claims

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

providing said DNA molecule annealed with a primer molecule able to hybridize to said DNA molecule;

incubating the annealed molecules in a vessel containing four different deoxynucleoside triphosphates, a processive DNA polymerase, wherein said polymerase is able to remain bound for at least 500 bases to said DNA molecule in an environmental condition used in the extension reaction of a DNA sequencing reaction, said polymerase 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 the 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 remains bound to said DNA molecule for at least 1,000 bases before dissociating.

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

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

5. The method of claim 4 wherein said modified polymerase has less than 1 unit of activity per mg of polymerase.

6. The method of claim 4 wherein said modified polymerase has less than 0.1 unit of activity per mg of polymerase.

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

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

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

10. 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.

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

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

13. The method of claim 12 wherein said pulse step comprises mixing said annealed molecules with all four deoxynucleoside triphosphates and a processive DNA polymerase, wherein at least one said deoxynucleoside triphosphate is labelled and present in limiting concentration.

14. The method of claim 13 wherein said pulse step incubation is carried out for 30 seconds to 20 minutes.

15. The method of claim 13 wherein said chase step comprises adding a different one of said chain terminating agents to each of four separate aliquots of the mixture after performing said pulse step.

16. The method of claim 15 wherein said chase step incubation is carried out for 1 to 60 minutes.

17. The method of claim 1 wherein said terminating agent is a dideoxynucleoside triphosphate.

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

19. The method of claim 1 wherein said primer is labelled.

20. The method of claim 19 wherein said incubating comprises a chase step.

21. The method of claim 19 wherein said primer is fluorescently labelled.

23. 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 sequencing reaction.

39. The method of claim 1 or 38 wherein said annealed molecules are incubated in at least four vessels containing four different deoxynucleoside triphosphates.

22. A kit for DNA sequencing, comprising:

a processive DNA polymerase, 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 the group consisting of

(a) dITP and

(b) a chain terminating agent.

24. The kit of claim 22 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.

25. The kit of claim 22 in which said reagent is a dideoxynucleoside triphosphate.

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

providing said DNA molecule annealed with a primer molecule able to hybridize to said DNA molecule;

incubating the annealed molecules in the presence of four different deoxynucleoside triphosphates, a processive DNA polymerase, wherein said polymerase is able to remain bound for at least 500 bases to said DNA molecule in an environmental condition used in the extension reaction of a DNA sequencing reaction, said polymerase having less than 50% of the exonuclease activity of the naturally associated level of exonuclease activity of said polymerase, and a DNA synthesis terminating agent which terminates DNA synthesis at a specific nucleotide base, and

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

27. The method of claim 26 wherein said polymerase has less than 1% of the exonuclease activity of the naturally associated level of exonuclease activity of said polymerase.

28. The method of claim 26 wherein said processive DNA polymerase is a T7-type DNA polymerase.

29. The method of claim 28 wherein said T7-type DNA polymerase has less than 1% of the exonuclease activity of the naturally associated level of exonuclease activity of said polymerase.

30. The method of claim 28 wherein said T7-type DNA polymerase is T7 DNA polymerase.

31. The method of claim 28 wherein said T7 DNA polymerase has less than 1% of the exonuclease activity of the naturally associated level of exonuclease activity of said polymerase.

32. A kit for DNA sequencing, comprising:

a processive DNA polymerase which remains bound to a DNA molecule for at least 500 bases before dissociating, said polymerase having less than 50% of the exonuclease activity of the naturally associated level of exonuclease activity of said 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 the group consisting of

(a) deoxyinosine triphosphate and

(b) a chain terminating agent.

33. The kit of claim 32 wherein said polymerase has less than 1% of the exonuclease activity of the naturally associated level of exonuclease activity of said polymerase.

34. The kit of claim 32 wherein said processive DNA polymerase is a T7-type DNA polymerase.

35. The kit of claim 34 wherein said T7-type DNA polymerase has less than 1% of the exonuclease activity of the naturally associated level of exonuclease activity of said polymerase.

36. The kit of claim 34 wherein said T7-type DNA polymerase is T7 DNA polymerase.

37. The kit of claim 36 wherein said T7 DNA polymerase has less than 1% of the exonuclease activity of the naturally-associated level of exonuclease activity of said polymerase.

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

providing said DNA molecule annealed with a primer molecule able to hybridize to said DNA molecule;

incubating the annealed molecules in the presence of four deoxynucleoside triphosphates, a processive DNA polymerase, wherein said polymerase is able to remain bound for at least 500 bases to said DNA molecule in an environmental condition used in the extension reaction of a DNA sequencing reaction, said polymerase having a level of exonuclease activity which is sufficiently low to permit the nucleotide base sequence of the DNA molecule to be determined, and a DNA synthesis terminating agent which terminates DNA synthesis at a specific nucleotide base, and

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

40. A kit for DNA sequencing, comprising:

a processive DNA polymerase which remains bound to a DNA molecule for at least 500 bases before dissociating, said polymerase having a level of exonuclease activity which is sufficiently low to permit the nucleotide base sequence of the DNA molecule to be determined, 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 the group consisting of

(a) deoxyinosine triphosphate

(b) a chain terminating agent.

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

providing said DNA molecule annealed with a primer molecule able to hybridize to said DNA molecule;

incubating the annealed primer molecule and the DNA molecule in the presence of at least four deoxynucleotide triphosphates, a T7-type DNA polymerase having less than 500 units of exonuclease activity per mg of said polymerase, and a DNA synthesis terminating agent which terminates DNA synthesis at a specific nucleotide base;

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

42. The method of claim 41 wherein said DNA polymerase is T7 DNA polymerase.

43. A kit for DNA sequencing, comprising:

a T7-type DNA polymerase having less than 500 units of exonuclease activity per mg of polymerase, and a reagent selected from the group consisting of dITP, and a chain terminating agent.

44. The kit of claim 43 wherein said DNA polymerase is T7 DNA polymerase.