Automated DNA sequencing technique

• US 5,821,058 A
• Filed: 12/21/1994
• Issued: 10/13/1998
• Est. Priority Date: 01/16/1984
• Status: Expired due to Term

• Current Assignee: California Institute of Technology
• Original Assignee: California Institute of Technology
• Inventors: Hood, Leroy E., Smith, Lloyd M., Connell, Charles R., Hunkapiller, Michael W., Hunkapiller, Tim J.
• Primary Examiner(s): Zitomer, Stephanie W.
• Application Number:
  US08/361,176
• Time in Patent Office: 1,392 Days
• Field of Search: 435/6, 435/71.1, 536/25.3, 536/25.31, 536/25.32, 536/24.3, 536/25.4, 935/77, 935/78
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• 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
• CPC Class Codes: G01N 27/44721 by optical means
• CPC Class Codes: G01N 27/44726 using specific dyes, markers or binding molecules

Abstract

A process for the electrophoretic analysis of DNA fragments produced in DNA sequencing operations wherein chromophores or fluorophores are used to tag the DNA fragments produced by the sequencing chemistry and permit the detection and characterization of the fragments as they are resolved by electrophoresis through a gel. Preferably four different fragment sets are tagged with the fluorophores fluorescein, Texas Red, tetramethyl rhodamine, and 7-nitrobenzofurazan. A system for the electrophoretic analysis of DNA fragments produced in DNA sequencing operations comprising: a source of chromophore or fluorescent tagged DNA fragments; a zone for contacting an electrophoresis gel; means for introducing said tagged DNA fragments to said zone; and photometric means for monitoring said tagged DNA fragments as they move through said gel.

Claims

1. A method of separating and detecting tagged polynucleotides which comprises:

providing a plurality of polynucleotides, each tagged with a chromophore or fluorophore;

resolving to separate one of the plurality of tagged polynucleotides from other tagged polynucleotides differing in length by a single nucleotide using an electrophoretic procedure capable of resolving tagged polynucleotides differing by a single nucleotide; and

detecting the resolved tagged polynucleotides by means of the chromophore or fluorophore.

2. The method according to claim 1, wherein the polynucleotide is DNA.

3. The method according to claim 1, wherein the tagged polynucleotides are obtained from a chemical degradation reaction.

4. The method according to claim 1, wherein the step of detecting the tagged polynucleotides is performed during the electrophoresis.

5. The method of claim 1, wherein the detecting of the resolved tagged polynucleotides comprises exposure of the tagged polynucleotides to a high-intensity monochromatic light source.

6. The method of claim 1, wherein the detecting of the resolved tagged polynucleotides comprises exposure of the tagged polynucleotides to a laser.

7. A method of detecting a tagged polynucleotide, which comprises:

providing a plurality of polynucleotides, wherein each of said plurality of polynucleotide comprises an identical primer oligonucleotide tagged with a chromophore or fluorophore;

resolving the plurality of tagged polynucleotides using an electrophoretic procedure capable of resolving tagged polynucleotides differing in length by a single nucleotide; and

detecting each of the plurality of resolved tagged polynucleotides by means of the chromophore or fluorophore.

8. The method according to claim 7, wherein the tagged polynucleotides are obtained from one of a set of primer extension reactions in which each of the tagged primer oligonucleotides used in one of said sets is distinguishable by its spectral characteristics from the tagged primer oligonucleotides used in the other said sets.

9. The method according to claim 7, wherein prior to electrophoresis, each primer oligonucleotide is coupled to the chromophore or fluorophore by an amine linkage.

10. The method according to claim 7, wherein the primer oligonucleotide is tagged with the chromophore or fluorophore to allow chain extension by a polymerase.

11. The method according to claim 7, wherein the primer oligonucleotide is tagged such that the primer oligonucleotide has a free 3'"'"' hydroxyl group.

12. The method of claim 7, wherein the detecting of the resolved tagged polynucleotides comprises exposure of the tagged polynucleotides to a high-intensity monochromatic light source.

13. The method of claim 7, wherein the detecting of the resolved tagged polynucleotide comprises exposure of the tagged polynucleotide to a laser.

14. A method of determining the sequence of a polynucleotide by analyzing polynucleotide fragments generated by a polynucleotide sequencing technique, each of said polynucleotide fragments being tagged with a chromophore or fluorophore, comprising:

introducing the tagged polynucleotide fragments into an electrophoretic medium;

separating the tagged polynucleotide fragments in said electrophoretic medium using an electrophoretic procedure capable of resolving said polynucleotide fragments differing in length by a single nucleotide;

detecting the separated tagged polynucleotide fragments by means of the chromophore or fluorophore; and

determining the polynucleotide sequence from the polynucleotide fragments detected.

15. The method according to claim 14, wherein the polynucleotide is DNA.

16. The method according to claim 14, wherein each tagged polynucleotide fragment comprises an identical primer oligonucleotide sequence, and the primer oligonucleotide sequence is tagged with a chromophore or fluorophore to generate a tagged primer oligonucleotide.

17. The method according to claim 16, wherein the tagged fragment is obtained from one of a set of primer extension reactions in which at least one of the tagged primer oligonucleotides used in the set is distinguishable by its spectral characteristics from another of the tagged primer oligonucleotides used in the set.

18. The method according to claim 17, wherein the fragments are provided with a protected amino group which is deblocked and coupled to a chromophore or fluorophore subsequent to the sequencing reaction.

19. The method according to claim 14, wherein the method is a chemical degradation method and the polynucleotide fragments are tagged with the chromophores or fluorophores.

20. The method according to claim 14, wherein the tagged fragments are obtained from a primer extension reaction in which the primer is tagged with a chromophore or fluorophore such that the tagged fragments resulting from at least one of the sequencing reactions A, C, G and T are distinguishable from other fragments by the spectral characteristics of the tag.

21. The method according to claim 14, wherein the tagged fragments from at least two of the sequencing reactions A, C, G and T are distinguishable from one another and from other fragments by the spectral characteristics of the tags.

22. The method according to claim 14, wherein the tagged fragments from all of the sequencing reactions A, C, G and T are distinguishable from one another by the spectral characteristics of the tags.

23. The method according to claim 14, wherein prior to electrophoresis, at least one of the polynucleotide fragments is coupled to the chromophore or fluorophore by an amine linkage.

24. The method according to claim 14, wherein the step of detecting the polynucleotide fragments is performed during the electrophoresis.

25. The method according to claim 14, wherein the polynucleotide sequencing technique comprises a Maxam/Gilbert chemical degradation reaction or a Sanger chain-termination reaction.

26. The method of claim 14, wherein the detecting of the separated tagged polynucleotide fragments comprises exposure of the tagged polynucleotide to a high-intensity monochromatic light source.

27. The method of claim 14, wherein the detecting of the separated tagged polynucleotide comprises exposure of the tagged polynucleotide to a laser.

28. A method for determining the sequence of a polynucleotide which comprises:

providing polynucleotide fragments tagged with chromophores or fluorophores, wherein the chromophores or fluorophores are distinguishable from others by their spectral characteristics;

resolving the polynucleotide fragments by electrophoresis; and

detecting the resolved fragments by means of the chromophores or fluorophores, and thereby determining the sequence based on the polynucleotide fragments detected.

29. The method according to claim 28 wherein the polynucleotide is DNA.

30. The method according to claim 28, wherein each tagged polynucleotide fragment comprises an identical primer oligonucleotide sequences, and the primer oligonucleotide sequence is tagged with a chromophore or fluorophore.

31. The method according to claim 28, wherein the method is a chemical degradation method and the polynucleotide fragments are labeled with the chromophores or fluorophores.

32. The method according to claim 29, wherein the tagged fragments are obtained from a primer extension reaction in which the primer is tagged with a chromophore or fluorophore such that the tagged fragments resulting from at least one of the sequencing reactions A, C, G and T are distinguishable from other fragments by the spectral characteristics of the tag.

33. The method according to claim 28, wherein the tagged fragments from at least two of the sequencing reactions A, C, G and T are distinguishable from one another and from other fragments by the spectral characteristics of the tags.

34. The method according to claim 28, wherein the tagged fragments from all of the sequencing reactions A, C, G and T are distinguishable from one another by the spectral characteristics of the tags.

35. The method according to claim 34, wherein the fluorophore is fluorescein, tetramethyl rhodamine, or substituted rhodamine.

36. The method according to claim 28, wherein prior to electrophoresis, at least one of the polynucleotide fragments are coupled to the chromophore or fluorophore by an amine linkage.

37. The method according to claim 28, wherein one or more polynucleotide fragments are provided with a protected amino group prior to electrophoresis, which is deblocked and coupled to a chromophore or fluorophore subsequent to the sequencing reaction.

38. The method according to claim 37, wherein the fragments generated by each of the different sequencing reactions are coupled with a different chromophore or fluorophore.

39. The method of claim 28, wherein the detecting of the resolved fragments comprises exposure of the resolved fragments to a high-intensity monochromatic light source.

40. The method of claim 28, wherein the detecting of the resolved fragments comprises exposure of the resolved fragments to a laser.

41. A method for determining the sequence of a polynucleotide which comprises:

providing polynucleotide fragments generated by a polynucleotide sequencing technique, which are tagged with chromophores or fluorophores, wherein the fragments from one or more of the four sequencing reactions A, C, G or T are distinguishable from fragments of the other reactions by their spectral characteristics;

resolving the fragments by electrophoresis;

detecting the fragments as they are being resolved by means of the spectral characteristics of the chromophores or fluorophores, and thereby determining the polynucleotide sequence based on the polynucleotide fragments detected.

42. The method according to claim 41 wherein the polynucleotide is DNA.

43. The method according to claim 41, wherein each tagged polynucleotide fragment comprises an identical primer oligonucleotide sequence, and the primer oligonucleotide sequence is tagged with a chromophore or fluorophore.

44. The method according to claim 41, wherein the method is a chemical degradation method and the polynucleotide fragments are labeled with the chromophores or fluorophores.

45. The method according to claim 41, wherein the tagged fragments are obtained from a primer extension reaction in which the primer is tagged with a chromophore or fluorophore such that the tagged fragments resulting from at least one of the sequencing reactions A, C, G and T are distinguishable from other fragments by the spectral characteristics of the tag.

46. The method according to claim 41, wherein the tagged fragments from at least two of the sequencing reactions A, C, G and T are distinguishable from one another and from other fragments by the spectral characteristics of the tags.

47. The method according to claim 41, wherein the tagged fragments from all of the sequencing reactions A, C, G and T are distinguishable from one another by the spectral characteristics of the tags.

48. The method according to claim 47, wherein the fluorophore is fluorescein, tetramethyl rhodamine, or substituted rhodamine.

49. The method according to claim 41, wherein the fragments from one or more of four sequencing reactions A, C, G or T are coupled to the chromophore or fluorophore by an amine linkage prior to electrophoresis.

50. The method according to claim 49, wherein the fragments generated by each of the different sequencing reactions are coupled with a different chromophore or fluorophore.

51. The method according to claim 41, wherein the fragments from one or more of four sequencing reactions A, C, G or T are provided with a protected amino group, which is deblocked and coupled to a chromophore or fluorophore subsequent to the sequencing reaction.

52. The method of claim 41, wherein the detecting of the fragments as they are being resolved comprises exposure of the fragments to a high-intensity monochromatic light source.

53. The method of claim 41, wherein the detecting of the fragments as they are being resolved comprises exposure of the fragments to a laser.

54. A method of determining the sequence of a single-stranded polynucleotide comprising:

obtaining a set of fragments of the complement of said single-stranded polynucleotide, wherein each fragment is tagged with a chromophore or fluorophore such that fragment differing by a single nucleotide are capable of being resolved;

separating the tagged fragments according to size, using an electrophoretic procedure capable of resolving tagged fragments differing by a single nucleotide, to obtain separated tagged fragments;

detecting each separated tagged fragment by means of its chromophore or fluorophore; and

analyzing the sizes of the separated tagged fragments, thereby determining the sequence of the single-stranded polynucleotide.

55. The method of claim 54, wherein the detecting of the separated tagged fragments comprises exposure of the separated tagged fragments to a high-intensity monochromatic light source.

56. The method of claim 54, wherein the detecting of the separated tagged fragments comprises exposure of the separated tagged fragments to a laser.