PREVENTION AND ALLEVIATION OF STERIC HINDRANCE DURING SINGLE MOLECULE SYNTHESIS

• US 20100035269A1
• Filed: 08/04/2009
• Published: 02/11/2010
• Est. Priority Date: 08/05/2008
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Wyatt, Paul, Ma, Congcong
• Granted Patent: US 8,198,023 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]

Abstract

The present invention provides compositions and methods for reducing steric hindrance in the product of nucleic acid polymerase reaction. Methods and compositions of the invention encompass application of exonucleases, endonucleases, and uracil-DNA glycosylases to a nucleic acid polymerase reaction such that newly formed nucleic acid strands are modified (e.g., cleaved) while the polymerase reaction continues to proceed.

Claims

1. A method for reducing steric hindrance resulting from a product of a nucleic acid polymerase reaction, said method comprising conducting said nucleic acid polymerase reaction in the presence of an enzyme selected from the group consisting of an exonuclease, an endonuclease, and a uracil-DNA glycosylase, wherein said nucleic acid polymerase reaction includes a template nucleic acid, and wherein said enzyme modifies said product but does not modify said template nucleic acid.

2. The method of claim 1, wherein said enzyme is an exonuclease that is only able to initiate at a 5′

end of a single-stranded nucleic acid.

3. The method of claim 2, wherein said exonuclease is selected from the group consisting of RecJ, lambda exonuclease, and T7 exonuclease.

4. The method of claim 1, wherein said enzyme is a restriction endonuclease with a four-base-pair recognition site.

5. The method of claim 1, wherein the template nucleic acid comprises a modification such that said enzyme is unable to modify said template nucleic acid.

6. The method of claim 5, wherein said modification comprises a non-natural nucleotide at one or more termini of said template nucleic acid.

7. The method of claim 5, wherein said modification comprises methylation of said template nucleic acid.

8. The method of claim 5, wherein said modification comprises a protecting group on at least one nucleotide of said template nucleic acid.

9. The method of claim 5, wherein said modification comprises a chemically modified oligonucleotide extension ligated to an end of said template nucleic acid.

10. The method of claim 9, wherein said chemically modified oligonucleotide extension comprises a phosphorothioate bond or Spacer 18.

11. The method of claim 1, wherein said template nucleic acid is a circular nucleic acid molecule.

12. The method of claim 1, wherein said template nucleic acid is double-stranded template nucleic acid.

13. The method of claim 1, wherein said template nucleic acid is single-stranded template nucleic acid.

14. The method of claim 13, wherein said single-stranded template nucleic acid is protected by a phosphorothioate bond.

15. The method of claim 1, wherein said enzyme is a uracil-DNA glycosylase and the polymerase reaction further comprises uracil-containing nucleotides.

16. The method of claim 15, further comprising the step of hydrolyzing abasic sites created by removal of uracil residues from said product by said uracil-DNA glycosylase.

17. The method of claim 16, wherein said polymerase reaction is performed in the presence of at least one polyamine.

18. The method of claim 16, wherein said polymerase reaction is performed in the presence of at least one AP endonuclease.

19. The method of claim 15, wherein said polymerase reaction further comprises terminal-phosphate-labeled thymine-containing nucleotides.

20. The method of claim 1, wherein said template nucleic acid is immobilized within a zero mode waveguide.

21. The method of claim 1, wherein said nucleic acid polymerase reaction is conducted in a volume of less than one attoliter.

22. A method of determining a nucleotide sequence of a template nucleic acid comprising the steps of:

a. conducting a nucleic acid polymerase reaction in the presence of a set of detectable terminal-phosphate-labeled nucleotides and an enzyme selected from the group consisting of an exonuclease, an endonuclease, and a uracil-DNA glycosylase, wherein;

i. said reaction results in addition of members of said set of detectable terminal-phosphate-labeled nucleotides to a nascent nucleic acid strand, and

ii. said reaction results in production of labeled polyphosphates that are released from said members of said set of detectable terminal-phosphate-labeled nucleotides; and

b. sequentially identifying during said nucleic acid polymerase reaction said labeled polyphosphates or said members of said set of detectable terminal-phosphate-labeled nucleotides that yield said labeled polyphosphates in said reaction, thereby determining said nucleotide sequence of said template nucleic acid.

23. The method of claim 22, wherein said enzyme is an exonuclease or an endonuclease that cleaves said nascent nucleic acid strand but does not cleave said template nucleic acid.

28. The method of claim 22, wherein said template nucleic acid comprises a modification such that said enzyme is unable to modify said template nucleic acid.

38. The method of claim 22, wherein said enzyme is a uracil-DNA glycosylase and the set of detectable terminal-phosphate-labeled nucleotides comprises terminal-phosphate-labeled uracil-containing nucleotides.

39. The method of claim 38, further comprising the step of hydrolyzing abasic sites created by removal of uracil residues from said nascent nucleic acid strand by said uracil-DNA glycosylase.

24-27. -27. (canceled)

29-37. -37. (canceled)

40-44. -44. (canceled)

45. A composition comprising:

a) a nucleic acid polymerase, wherein said nucleic acid polymerase is immobilized on a substrate within a constricted volume reaction vessel;

b) an endonuclease, an exonuclease, or a uracil-DNA glycosylase; and

c) a template nucleic acid.

49. The composition of claim 45, comprising the uracil-DNA glycosylase, further comprising uracil-containing nucleotides.

46-48. -48. (canceled)

50-59. -59. (canceled)