METHOD FOR MULTIPLEXED NUCLEIC ACID PATCH POLYMERASE CHAIN REACTION

• US 20100129874A1
• Filed: 09/08/2009
• Published: 05/27/2010
• Est. Priority Date: 09/05/2008
• Status: Active Grant

• Current Assignee: Washington University In St Louis
• Original Assignee: Washington University In St Louis
• Inventors: Varley, Katherine E., Mitra, Robi M.
• Granted Patent: US 8,586,310 B2
• Time in Patent Office: Days
• US Class Current: 435/91.200
• 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. defining the ends of at least two nucleic acid sequences,

b. annealing upstream and downstream nucleic acid patches 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).

2. The method of claim 1, wherein each nucleic acid sequence is encoded by genomic DNA.

3. The method of claim 1, wherein the ends of at least two nucleic acid sequences are defined by

a. annealing an upstream primer and a downstream primer to each unique nucleic acid sequence to be amplified;

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

c. removing the upstream and downstream primer sequences from the amplicons of step (b).

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

5. The method of claim 3, wherein the upstream and downstream primer sequences are removed from each amplicon of step (b) in part by the addition of uracil DNA glycosylase.

6. The method of claim 3, wherein the upstream and downstream primer sequences are removed from each amplicon of step (b) in part by the addition of an endonuclease.

7. The method of claim 3, wherein the upstream and downstream primer sequences are removed from each unique amplicon of step (b) in part by the addition of an exonuclease.

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

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

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

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

12. The method of claim 11, wherein products of step (d) are sequenced using an upstream and a downstream universal primer, wherein each primer comprises a tag specific for the unique nucleic acid sample.

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

14. The method of claim 1, wherein at least 30 unique nucleic acid sequences are amplified.

15. The method of claim 1, wherein the nucleic acid sequence is bisulfite treated DNA.

16. The method of claim 1, wherein the ends of at least two nucleic acid sequences are defined by a method selected from the group consisting of multiplex PCR, enzyme restriction, exonuclease degradation, and triplex formation.

17. The method of claim 16, wherein the ends of at least two nucleic acid sequences are defined by type IIS restriction enzyme digestion.

18. 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).

19. The method of claim 18, further comprising sequencing the products of step (g).

20. The method of claim 18, wherein the non-specific amplicons are degraded by contacting the amplicons with an exonuclease.