Method and compositions for sequencing nucleic acid molecules

• US 20040126765A1
• Filed: 12/27/2002
• Published: 07/01/2004
• Est. Priority Date: 12/27/2002
• Status: Abandoned Application

• Current Assignee: Beckman Coulter Incorporated (Danaher Corporation)
• Original Assignee: Beckman Coulter Incorporated (Danaher Corporation)
• Inventors: Adams, Craig W.
• Application Number:
  US10/329,752
• Publication Number:
  US 20040126765A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/319 Exonuclease
• CPC Class Codes: C12Q 2525/125 incorporating agents resulting in resistance to degradation
• CPC Class Codes: C12Q 2535/101 Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators

Abstract

The invention relates to methods, compositions, kits and apparati for sequencing nucleic acid molecules. The invention particularly concerns the use of an exonuclease activity in concert with a polymerase activity to mediate such sequencing.

Claims

1. A method for determining the sequence of a region of one strand of a double-stranded nucleic acid target molecule, wherein said method comprises incubating said nucleic acid target molecule in the presence of an exonuclease activity, a polymerase activity and four differentially detectable, exonuclease activity-resistant, chain terminator nucleotide species.

2. The method of claim 1, wherein four differentially detectable, exonuclease activity-resistant, chain terminator nucleotide species are employed.

3. The method of claim 2, wherein at least one of said four differentially detectable, exonuclease activity-resistant, chain terminator nucleotide species is fluorescently labeled.

4. The method of claim 3, wherein said four differentially detectable, exonuclease activity-resistant, chain terminator nucleotide species are fluorescently labeled.

5. The method of claim 1, wherein said double-stranded nucleic acid target molecule possesses only one 3′

terminus that is a substrate for said exonuclease activity.

6. The method of claim 5, wherein said double-stranded nucleic acid target molecule possesses a 3′

terminus that extends beyond the 5′

terminus of the opposite strand.

7. The method of claim 5, wherein said double-stranded nucleic acid target molecule possesses a 3′

terminus that is sterically blocked from exonuclease activity degradation.

8. The method of claim 1, wherein both strands of said double-stranded nucleic acid target molecule possess a 3′

terminus that is a substrate for said exonuclease activity.

9. The method of claim 1, wherein one 5′

terminus of said double-stranded nucleic acid target molecule possesses a haptenic group.

10. The method of claim 9, wherein said haptenic group is biotin.

11. The method of claim 1, wherein both 5′

termini of said double-stranded nucleic acid target molecule possess a haptenic group.

12. The method of claim 11, wherein said haptenic group is biotin.

13. A method for determining the nucleotide sequence of a region of a double-stranded nucleic acid target molecule, wherein said method comprises the steps:

(A) incubating a preparation of said double-stranded target molecule in the presence of a 3′

to 5′

exonuclease activity, wherein said double-stranded nucleic acid target molecule possess at least one 3′

terminus that is a substrate for said exonuclease activity, wherein said incubation is conducted under conditions sufficient to permit said exonuclease activity to produce a nested population of double-stranded nucleic acid target molecule having at least one degraded 3′

termini;

(B) incubating said nested population of double-stranded nucleic acid target molecule in the presence of a polymerase activity and at least one detectably labeled, exonuclease activity-resistant, chain terminator nucleotide species, wherein said incubation is conducted under conditions sufficient to permit said polymerase activity to mediate the template-dependent incorporation of one of said nucleotide species onto the 3′

terminus of a nucleic acid target molecule whose 3′

terminus was degraded by said exonuclease activity; and

(C) determining the identity of the differentially detectable, exonuclease activity-resistant, chain terminator nucleotide species incorporated onto said 3′

terminus at said selected region.

14. The method of claim 13, wherein said steps A and B are conducted simultaneously, and wherein said conditions employed are sufficient to permit said exonuclease activity to degrade said substrate termini and sufficient to permit said polymerase activity to mediate said template-dependent incorporation of said nucleotide species.

15. The method of claim 13, wherein four differentially detectable, exonuclease activity-resistant, chain terminator nucleotide species are employed.

16. The method of claim 13, wherein at least one of said four differentially detectable, exonuclease activity-resistant, chain terminator nucleotide species is fluorescently labeled.

17. The method of claim 16, wherein said four differentially detectable, exonuclease activity-resistant, chain terminator nucleotide species are fluorescently labeled.

18. The method of claim 13, wherein said double-stranded nucleic acid target molecule possesses only one 3′

terminus that is a substrate for said exonuclease activity.

19. The method of claim 18, wherein said double-stranded nucleic acid target molecule possesses a 3′

terminus that extends beyond the 5′

terminus of the opposite strand.

20. The method of claim 18, wherein said double-stranded nucleic acid target molecule possesses a 3′

terminus that is sterically blocked from exonuclease activity degradation.

21. The method of claim 13, wherein both strands of said double-stranded nucleic acid target molecule possess a 3′

terminus that is a substrate for said exonuclease activity.

22. The method of claim 13, wherein one 5′

terminus of said double-stranded nucleic acid target molecule possesses a haptenic group.

23. The method of claim 22, wherein said haptenic group is biotin.

24. The method of claim 13, wherein both 5′

termini of said double-stranded nucleic acid target molecule possess a haptenic group.

25. The method of claim 24, wherein said haptenic group is biotin.

27. The in vitro composition of claim 13, wherein at least one of said four differentially detectable, exonuclease activity-resistant, chain terminator nucleotide species is fluorescently labeled.

28. The in vitro composition of claim 27, wherein said four differentially detectable, exonuclease activity-resistant, chain terminator nucleotide species are fluorescently labeled.

29. The in vitro composition of claim 13, wherein at least one 5′

terminus of said double-stranded nucleic acid target molecule possesses a haptenic group.

30. The in vitro composition of claim 29 wherein said haptenic group is biotin.

31. The in vitro composition of claim 13, wherein both 5′

termini of said double-stranded nucleic acid target molecule possesses a haptenic group.

32. The in vitro composition of claim 31, wherein said haptenic group is biotin.

26. An in vitro composition comprising a double-stranded nucleic acid target molecule, an exonuclease activity, a polymerase activity and four differentially detectable, exonuclease activity-resistant, chain terminator nucleotide species.

33. A kit specially adapted to facilitate the sequencing of a target nucleic acid molecule, said kit comprising a first container comprising a primer A, a second container comprising a primer B, and a third container containing an exonuclease activity, wherein said primers A and B mediate the amplification of a double-stranded nucleic acid molecule comprising said target nucleic acid molecule, and wherein at least one of said primer A or said primer B possesses a 5′

terminus having at least one modified nucleotide.

34. The kit of claim 33, wherein said modified nucleotide is a ribonucleotide, a dUridine nucleotide, a phosphothioate nucleotide, or a biotin-derivatized nucleotide.

35. The kit of claim 33, wherein said kit further comprises a fourth container containing four detectably labeled, exonuclease activity-resistant, chain terminator nucleotide species.

36. The kit of claim 35, wherein said four detectably labeled, exonuclease activity-resistant, chain terminator nucleotide species are fluorescently labeled.

37. A sequenator, comprising an apparatus for determining the identity of fluoresecently labeled exonuclease activity-resistant, chain terminator nucleotide species incorporated onto the 3′

termini of a nucleic acid target molecule whose 3′

terminus was degraded by said exonuclease; and

then extended by a template-dependent polymerase to incorporate said fluorescently labeled nucleotide species.