DNA sequencing

• US 5,674,716 A
• Filed: 04/13/1995
• Issued: 10/07/1997
• Est. Priority Date: 07/12/1988
• 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): Jones, W. Gary
• Assistant Examiner(s): REES, DIANE
• Application Number:
  US08/422,147
• Time in Patent Office: 908 Days
• Field of Search: 435/6, 435/5, 435/91.1, 435/174, 435/91.2, 435/91.5, 435/21, 435/194, 435/195, 204/182.8, 204/299, 536/24.3, 536/24.31, 536/24.33, 422/116, 422/68.1, 436/94
• US Class Current: 435/91.1
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/125 Methyl transferase, i.e. methylase
• CPC Class Codes: C12Q 2527/137 Concentration of a component of medium
• 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 2537/157 A reaction step characterised by the number of molecules incorporated or released
• CPC Class Codes: Y10S 435/803 Physical recovery methods, e.g. chromatography, grinding
• CPC Class Codes: Y10S 435/81 Packaged device or kit
• CPC Class Codes: Y10S 436/808 Automated or kit
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

A method for sequencing a strand of DNA, including the steps of: providing the strand of DNA; annealing the strand with a primer able to hybridize to the strand to give an annealed mixture; incubating the mixture with a deoxyribonucleoside triphosphate, a DNA polymerase, and a chain terminating agent under conditions in which the polymerase causes the primer to be elongated to form a series of DNA products differing in length of the elongated primer, each DNA product having a chain terminating agent at its elongated end; the number of each DNA product being approximately the same for substantially all DNA products differing in length from 1 to 20 bases.

Claims

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

providing said strand of DNA,

annealing said strand with a primer able to hybridize to said strand, to give an annealed mixture, and

incubating said annealed mixture with a deoxyribonucleoside triphosphate, a DNA polymerase, and a first chain terminating agent, under conditions in which said polymerase causes said primer to be elongated to form a first series of first DNA products differing in the length of the elongated primer, each said first DNA product having a said chain terminating agent at its elongated end, the number of each said first DNA products being approximately the same for substantially all DNA products differing in length from 1 to 20 bases.

2. The method of claim 1 further including the steps of:

separating said first DNA products by gel permeation according to molecular weight to form a first series of bands, each said first series band representing a said first DNA product of a given molecular weight, wherein the intensity of each nearby first series band is approximately the same for substantially all said first series bands, and

determining the position of each said first series band.

3. The method of claim 1 further comprising the steps of:

providing a second chain terminating agent in said annealed mixture at a concentration different from said first chain terminating agent, wherein said DNA polymerase causes production of a second series of second DNA products each said second DNA product having said second chain terminating agent at its elongated end, the number of each said second DNA product being approximately the same for substantially all DNA products differing in length from 1 to 20 bases, wherein the number of substantially all said second DNA products differing in length from 1 to 20 bases is distinctly different from that of all said first DNA products.

4. The method of claim 3 further including the steps of:

separating said first and second DNA products by gel permeation according to molecular weight to form a first and second series of bands respectively, each said first or second series band representing a said first or second DNA product, respectively, of a given molecular weight, wherein the intensity of each nearby first series band is approximately the same for substantially all said first series bands, and wherein the intensity of substantially all nearby second series bands is approximately the same, and wherein the intensity of substantially all bands of the first series is distinctly different from the intensity of each nearby band of the second series, and

determining the position and intensity of each said first and second series band.

5. The method of claim 1 further comprising the steps of:

providing two other said chain terminating agents wherein said polymerase causes production of a second and third series of second and third DNA products, the number of each said second DNA products and said third DNA products being approximately the same for substantially all said second DNA products and said third DNA products, repsectively, differing in length from 1 to 20 bases, wherein the numbers of substantially all said first, all said second and all said third DNA products are distinctly different from all those of a different series.

6. The method of claim 5, further including the steps of:

separating said first, second and third DNA products by gel permeation according to molecular weight to form a first, second and third series of bands, respectively, each said first, second or third series band representing a said first, second or third DNA product, respectively, of a given molecular weight, wherein the intensity of each nearby first series band is approximately the same for substantially all said first series bands, wherein the intensity of substantially all nearby second series bands is approximately the same, and the intensity of substantially all nearby third series bands is approximately the same, and wherein the intensity of substantially all nearby bands of different series is distinctly different, and

determining the position and intensity of each said band.

7. The method of claim 1 further comprising the steps of:

providing in said annealed mixture four different deoxyribonucleoside triphosphates and four different chain terminating agents, wherein said DNA polymerase causes production of a second, third and fourth series of second, third and fourth DNA products, the number of each said second, third and fourth DNA products being approximately the same for substantially all DNA products of the same series differing in length from 1 to 20 bases, wherein the number of substantially all said DNA products differing in length from 1 to 20 bases is distinctly different from all those of a different series.

8. The method of claim 7, further including the steps of:

separating said first, second, third and fourth DNA products by gel permeation according to molecular weight to form a first, second, third and fourth series of bands respectively, each said first, second, third and fourth series band representing a said first, second, third or fourth DNA product of a given molecular weight, wherein each said second, third and fourth series produce second, third and fourth bands, wherein the intensity of substantially all nearby first series bands, or substantially all nearby second series bands, or substantially all nearby third series bands, or substantially all nearby fourth series bands is approximately the same, and wherein the intensity of substantially all nearby bands in a different series is distinctly different, and

determining the position and intensity of each said band.

9. The method of claim 1, 2, 3, 4, 5, 6, 7 or 8 wherein said annealed mixture is provided with a manganese or iron ion, wherein said ion causes said polymerase to be non-discriminatory for a said chain terminating agent.

10. The method of claim 7 wherein said DNA products are separated according to molecular weight in less than four lanes of a gel.

11. The method of claim 10 wherein the intensity of each band is measured by a gel reading apparatus.

12. The method of claim 1 wherein said DNA polymerase is chosen from a T7 -type DNA polymerase, the large fragment of E. coli DNA polymerase I, and Taq polymerase.

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

14. The method of claim 9 wherein said annealed mixture is provided with a chelator.

15. The method of claim 14, said chelator being citrate or isocitrate.

16. A method for sequencing a strand of DNA, comprising the steps of:

providing a DNA polymerase, and

incubating said polymerase and said strand of DNA in a solution comprising a manganese or iron ion and a chain terminating agent.

18. The method of claim 16 or 17 wherein said solution comprises a chelator.

19. The method of claim 18, said chelator being citrate or isocitrate.

21. The solution of claim 16 wherein said ion is at a concentration from 0.005-100 millimolar.

17. A method for producing a DNA polymerase useful for DNA sequencing, comprising the step of mixing said DNA polymerase in a solution comprising a manganese or iron ion.

20. A solution comprising a T7-type DNA polymerase, or Taq polymerase, and a manganese or iron ion.

22. The solution of claim 20 further comprising a chelator.

23. The solution of claim 20, said chelator being citrate or isocitrate.

24. A kit for sequencing DNA comprising:

a DNA polymerase,

a chain terminating agent, and

a manganese or iron ion.

25. The kit of claim 24 wherein said polymerase is a T7-type DNA polymerase, Klenow, or Taq polymerase.

26. The kit of claims 24 or 25, wherein said chain terminating agent is a dideoxynucleoside triphosphate.

27. The kit of claim 25 further comprising a deoxyribonucleoside triphosphate.

28. The kit of claim 24 further comprising a chelator.

29. The kit of claim 28, said chelator being citrate or isocitrate.

30. A method for sequencing DNA comprising:

providing a DNA polymerase which is substantially non-discriminating for a chain terminating agent.

31. A method for automated sequencing of DNA, comprising the steps of:

providing a polymerase which is substantially non-discriminating for a chain terminating agent and causes production of a series of DNA products differing in molecular weight and terminating with the same chain terminating agent, and separating said DNA products by gel permeation according to molecular weight to form a series of bands, wherein said DNA products produce substantially all nearby bands of approximately the same intensity.

32. The method of claim 31 wherein said intensity differs by at most two-fold.

33. An automated DNA sequencing apparatus comprising:

a reactor for providing at least two series of DNA products formed from a single primer and a DNA strand, each said DNA product of a said series differing in molecular weight and having a chain terminating agent as one end,

separating means for separating said DNA products to form a series of bands, the intensity of substantially all nearby bands in a series being approximately the same, and the intensity of substantially all nearby bands in any one series being different from those of other series,

band reading means for determining the position and intensity of each said band after said separating, and

computing means for determining the DNA sequence of said DNA strand directly from said position and intensity of said bands.

34. The apparatus of claim 33 wherein said reactor comprises a manganese or iron ion.

35. The apparatus of claim 33 wherein said reactor comprises T7-type polymerase and a manganese or iron ion.

36. The apparatus of claim 33, 34 or 35, said reactor further comprising a chelator.

37. The apparatus of claim 36, said chelator being citrate or isocitrate.

38. A solution for use in a DNA sequencing reaction comprising a DNA polymerase together with an unbound pyrophosphatase.

39. The solution of claim 38 comprising a chain terminating agent, or dITP.

40. The solution of claim 38 or 39 further comprising a chelator.

41. The solution of claim 40, said chelator being citrate or isocitrate.

42. A kit for use in DNA sequencing reaction comprising a DNA polymerase and a pyrophosphatase together in a solution.

43. The kit of claim 42 comprising a chain terminating agent, or dITP.

44. The kit of claim 42 comprising a chelator.

45. The kit of claim 44, said chelator being citrate or isocitrate.

46. Method for sequencing DNA comprising the steps of:

providing a DNA molecule to be sequenced,

contacting said DNA molecule with a DNA polymerase and a pyrophosphatase in the presence of deoxyribonucleoside triphosphates and one or more chain terminating agents to form a mixture of chain terminated DNA fragments, and

separating said DNA fragments according to size so that at least a portion of said sequence can be determined.

47. T7 DNA polymerase Δ

Lys 118-Arg 145.

48. DNA encoding the polymerase of claim 47.