Method for multiplexed nucleic acid patch polymerase chain reaction

• US 8,586,310 B2
• Filed: 09/08/2009
• Issued: 11/19/2013
• Est. Priority Date: 09/05/2008
• Status: Active Grant

• Current Assignee: Washington University In St Louis
• Original Assignee: Washington University In St Louis
• Inventors: Mitra, Robi M., Varley, Katherine E.
• Primary Examiner(s): WOOLWINE, SAMUEL C
• Application Number:
  US12/555,627
• Publication Number:
  US 20100129874A1
• Time in Patent Office: 1,533 Days
• Field of Search: None
• US Class Current: 435/6.12
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]

Abstract

The invention encompasses a method for amplifying at least two different nucleic acid sequences. In particular, the method encompasses a multiplexed nucleic acid patch polymerase chain reaction.

Claims

1. A method of amplifying at least two different nucleic acid sequences, the method comprising:

a) creating known 5′ and

3′

ends of at least two nucleic acid sequences by;

i) annealing an upstream primer and a downstream primer to the at least two nucleic acid sequences to be amplified;

ii) amplifying the at least two nucleic acid sequences so as to create amplicons; and

iii) after amplifying, removing the upstream and downstream primer sequences from the amplicons of step ii);

b) annealing an upstream and a downstream nucleic acid patch to each of the amplicons of step iii), and annealing an upstream universal primer to the upstream patch, and a downstream universal primer to the downstream patch;

c) ligating the upstream universal primer and the downstream universal primer to the associated amplicon; and

d) amplifying the ligated nucleic acid sequences of step (c).

2. The method of claim 1, wherein each of the at least two nucleic acid sequences is encoded by genomic DNA.

3. The method of claim 1, wherein in step a(i) the upstream primer and the downstream primer comprise uracil instead of thymine.

4. The method of claim 1, wherein the upstream and downstream primer are removed from each amplicon of step ii) at least in part by the addition of uracil DNA glycosylase.

5. The method of claim 1, wherein the upstream and downstream primer are removed from each amplicon of step ii) at least in part by the addition of an endonuclease.

6. The method of claim 1, wherein the upstream and downstream primer are removed from each amplicon of step ii) at least in part by the addition of an exonuclease.

7. The method of claim 1, wherein the downstream universal primer comprises a protecting group.

8. The method of claim 1, further comprising degrading non-specific amplicons generated by step (a) after step (c) and before step (d).

9. The method of claim 8, wherein the non-specific amplicons are degraded by contacting the amplicons with an exonuclease.

10. The method of claim 1, further comprising sequencing the products of step (d).

11. The method of claim 10, wherein each upstream patch and each downstream patch comprises a tag specific for the nucleic acid sequence to which the upstream patch and the downstream patch are annealed.

12. The method of claim 11, wherein each universal primer further comprises a nucleic acid sequence to prime the sequencing.

13. The method of claim 1, wherein the at least two different nucleic acid sequences comprises at least 30 different nucleic acid sequences.

14. The method of claim 1, wherein the at least two different nucleic acid sequences are bisulfite treated DNA.

15. A method of amplifying one or more than one unique nucleic acid sequences, the method comprising:

a. annealing an upstream primer and a downstream primer to each unique nucleic acid sequence, wherein the upstream primer and the downstream primer comprise uracil instead of thymine;

b. amplifying each nucleic acid sequence so as to create amplicons of the nucleic acid sequence;

c. removing the upstream and downstream primer sequences from the amplicons of step (b) by contacting the amplicons with a uracil DNA glycosylase, an endonuclease, and an exonuclease;

d. annealing upstream and downstream nucleic acid patches to each unique amplicon of step (c), and annealing an upstream universal primer to the upstream patch of each unique amplicon, and a downstream universal primer to the downstream patch of each unique amplicon, wherein the downstream universal primer comprises a protecting group;

e. ligating the upstream universal primer and the downstream universal primer to each unique amplicon;

f. degrading non-specific amplicons of step (e); and

g. amplifying the amplicons of step (f).

16. The method of claim 15, further comprising sequencing the products of step (g).

17. The method of claim 15, wherein the non-specific amplicons are degraded by contacting the amplicons with an exonuclease.

18. A method of amplifying at least two different bisulfite treated nucleic acid sequences, the method comprising:

a) creating known 5′ and

3′

ends of at least two nucleic acid sequences, such that at least three nucleic acid bases are known at both the 5′ and

the 3′

ends,

b) annealing an upstream and a downstream nucleic acid patch to each nucleic acid sequence of step (a), and annealing an upstream universal primer to the upstream patch, and a downstream universal primer to the downstream patch;

c) ligating the upstream universal primer and the downstream universal primer to each nucleic acid sequence; and

d) amplifying the nucleic acid sequences of step (c).

19. The method of claim 18, wherein each of the at least two nucleic acid sequences are encoded by genomic DNA.

20. The method of claim 18, wherein the ends of at least two nucleic acid sequences are created in step (a) by

i) annealing an upstream primer and a downstream primer to the at least two nucleic acid sequences to be amplified;

ii) amplifying the at least two nucleic acid sequences so as to create amplicons; and

iii) after amplifying, removing the upstream and downstream primer sequences from the amplicons of step ii).

21. The method of claim 20, wherein the upstream primer and the downstream primer of step (i) comprise uracil instead of thymine.

22. The method of claim 20, wherein the upstream and downstream primer are removed from each amplicon of step ii) at least in part by the addition of uracil DNA glycosylase.

23. The method of claim 20, wherein the upstream and downstream primer are removed from each amplicon of step ii) at least in part by the addition of an endonuclease.

24. The method of claim 20, wherein the upstream and downstream primer are removed from each amplicon of step ii) at least in part by the addition of an exonuclease.

25. The method of claim 18, wherein the downstream universal primer comprises a protecting group.

26. The method of claim 18, further comprising sequencing the products of step (d).

27. The method of claim 26, wherein products of step (d) are sequenced using an upstream and a downstream universal primer, wherein each primer comprises a tag specific for the nucleic acid sequence to which the upstream patch and the downstream patch are annealed.

28. The method of claim 27, wherein each universal primer further comprises a nucleic acid sequence to prime the sequencing.

29. The method of claim 18, wherein the at least two different nucleic acid sequences comprises at least 30 different nucleic acid sequences.

30. The method of claim 18, wherein the ends of the at least two nucleic acid sequences are created by a method selected from the group consisting of multiplex PCR, enzyme restriction, exonuclease degradation, and triplex formation.

31. The method of claim 30, wherein the ends of the at least two nucleic acid sequences are created by type IIS restriction enzyme digestion.