Parallel polymorphism scoring by amplification and error correction

• US 20070009954A1
• Filed: 09/15/2006
• Published: 01/11/2007
• Est. Priority Date: 11/28/2001
• Status: Abandoned Application

• Current Assignee: Bio-Rad Laboratories Incorporated
• Original Assignee: Bio-Rad Laboratories Incorporated
• Inventors: Wang, Yan, Finney, Michael, Chen, Fan
• Application Number:
  US11/521,754
• Publication Number:
  US 20070009954A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07K 2319/80 containing a DNA binding domain, e.g. Lacl or Tet-repressor
• CPC Class Codes: C12Q 1/6823 Release of bound markers
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2521/319 Exonuclease
• CPC Class Codes: C12Q 2535/125 Allele specific primer extension
• CPC Class Codes: C12Q 2565/537 characterised by the capture oligonucleotide acting as a primer

Abstract

This invention provides a method of detecting polymorphisms, e.g., single nucleotide polymorphisms (SNPs), by amplification and error correction. The invention encompasses methods of performing amplification and error correction using an improved generation of nucleic acid polymerases, and methods of multiplexing the assay. The improvement to the polymerases is the joining of a sequence-non-specific nucleic-acid-binding domain to the enzyme in a manner that enhances the ability of the enzyme to bind and catalytically modify the nucleic acid.

Claims

1. A method of identifying a polymorphism using an error-correcting assay, the method comprising:

(a) contacting a target nucleic acid comprising a query sequence with a probe oligonucleotide under conditions in which the probe specifically hybridizes to the target nucleic acid, wherein the 3′

nucleotide of the probe is a labeled query nucleotide and is attached to a discrete surface location;

(b) providing an error-correcting polymerase;

(c) incubating the assay under conditions in which the probe is extended by the polymerase, wherein the labeled query nucleotide is cleaved from the probe when mismatched with the query sequence; and

(d) detecting the amount of probe in the discrete location that has been labeled.

2. The method of claim 1, wherein the polymerase is a family B polymerase.

3. The method of claim 2, wherein the family B polymerase is a Pyrococcus polymerase.

4. The method of claim 1, wherein the target nucleic acid is a PCR amplicon obtained using a PCR reaction performed with two unlabeled primers, wherein the query sequence is not present in either of the two primers.

5. The method of claim 4, wherein the target nucleic acid is obtained using a PCR reaction performed with two unlabeled primers, wherein the query sequence is not present in either of the two primers;

and further, wherein the PCR reaction is contacted with the probe oligonucleotide during amplification.

6. The method of claim 5, wherein the oligonucleotide probe is complementary to a region in the amplicon that does not comprise the two primers.

7. The method of claim 1, wherein the discrete surface location is on a microarray.

8. The method of claim 1, wherein the 3′

query nucleotide is labeled with a quencher.

9. The method of claim 1, further comprising a second probe oligonucleotide.

10. A method of identifying a polymorphism using an error-correcting assay, the method comprising:

(a) contacting a target nucleic acid comprising a query sequence with an oligonucleotide probe under conditions in which the probe specifically hybridizes to the target nucleic acid, wherein the 3′

nucleotide of the probe is a labeled query nucleotide;

(b) providing an error-correcting polymerase;

(c) incubating the assay under conditions in which the probe is extended by the polymerase thereby providing an extended product, wherein the labeled query nucleotide is cleaved from the probe when mismatched with the query sequence;

(d) providing a capture oligonucleotide attached to a discrete location and complementary to the extended product;

(e) hybridizing the extended product to the capture oligonucleotide and

(f) detecting the amount of label at the discrete location.

11. The method of claim 10, wherein the 3′

end of is labeled with a fluroescent label.

12. The method of claim 10, wherein the capture oligonucleotide is in a microarray.

13. The method of claim 10, wherein the 3′

end is labeled with a fluorescent quenching molecule.

14. The method of claim 13, further wherein the extended product is hybridized to a capture oligonucleotide that is labeled with a fluor.

15. The method of claim 10 wherein the polymerase is a family B polymerase.

16. The method of claim 10, wherein the family B polymerase is a Pyrococcus polymerase.

17. A method of identifying a polymorphism using an error-correcting assay, the method comprising:

(a) contacting a target nucleic acid comprising at least two query sequences with at least two oligonucleotide probes under conditions in which the probes specifically hybridize to the target nucleic acid at different sites, wherein the 3′

nucleotides of the probes are labeled query nucleotides, and further, wherein the labels are different;

(b) providing an error-correcting polymerase;

(c) incubating the assay under conditions in which the probes are extended by the polymerase thereby providing extended products, wherein the labeled query nucleotides are cleaved from the probes when mismatched with the query sequences;

(d) separating the extended products electrophoretically; and

(e) detecting the amount of label in the extended products.

18. The method of claim 17, wherein the polymerase is a family B polymerase.

19. The method of claim 18, wherein the family B polymerase is a Pyrococcus polymerase.