Multiplex targeted amplification using flap nuclease

• US 9,528,148 B2
• Filed: 03/23/2015
• Issued: 12/27/2016
• Est. Priority Date: 01/17/2007
• Status: Active Grant

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Zheng, Jianbiao, Weng, Li, Faham, Malek
• Primary Examiner(s): THOMAS, DAVID C
• Application Number:
  US14/665,506
• Publication Number:
  US 20150259726A1
• Time in Patent Office: 645 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2531/125 Rolling circle

Abstract

Methods for multiplex amplification of a plurality of targets of distinct sequence from a complex mixture are disclosed. In one aspect targets are circularized using a single circularization probe that is complementary to two regions in the target that flank a region to be amplified. The targets may hybridize to the circularization probe so that 5′ or 3′ flaps are generated and methods for removing flaps and circularizing the resulting product are disclosed. In another aspect targets are hybridized to dU probes so that 5′ and 3′ flaps are generated. The flaps are cleaved using 5′ or 3′ flap endonucleases or 3′ to 5′ exonucleases. The target sequences are then ligated to common primers, the dU probes digested and the ligated targets amplified.

Claims

1. A method for amplifying a plurality of target sequences comprising:

fragmenting a nucleic acid to obtain a plurality of target fragments including the plurality of target sequences;

mixing the plurality of target fragments with (1) a plurality of probes, wherein each probe comprises a central target region, a 5′

first common sequence and a 3′

second common sequence, wherein the central target region is complementary to a target region in a target fragment, wherein the target fragment hybridizes to the central target region of the probe and one or more of a single stranded 5′

flap structure or a single stranded 3′

flap structure is formed; and

(2) a first oligonucleotide that is complementary to the first common sequence and hybridizes thereto and a second oligonucleotide that is complementary to the second common sequence and that hybridizes thereto, to form a mixture;

adding one or more of a 5′

flap nuclease or a 3′

flap nuclease to the mixture, wherein one or more of the 5′

flap structure or the 3′

flap structure is cleaved;

adding a ligase to the mixture to ligate the first oligonucleotide to a 5′

end of the target fragment and the second oligonucleotide to a 3′

end of the target fragment;

removing the probes; and

amplifying the target sequences using primers for the first and second common sequences.

2. The method of claim 1 wherein the probes include one or more deoxyuracils and the probes are degraded by uracil-DNA glycosylase or an AP endonuclease.

3. The method of claim 1 wherein the nucleic acid is fragmented by one or more restriction enzymes.

4. The method of claim 1 wherein the central target region is perfectly complementary to the target region in the target fragment.

5. The method of claim 1 wherein the target fragment hybridized to the central target region forms the single stranded 5′

flap structure and a 5′

flap nuclease is added to cleave the 5′

flap structure.

6. The method of claim 1 wherein the target fragment hybridized to the central target region forms the single stranded 3′

flap structure and a 3′

flap nuclease is added to cleave the 3′

flap structure.

7. The method of claim 1 wherein the target fragment hybridized to the central target region forms the single stranded 5′

flap structure and the single stranded 3′

flap structure and the 5′

flap nuclease and the 3′

flap nuclease are added the cleave the 5′

flap structure and the 3′

flap structure.

8. The method of claim 1 wherein the nucleic acid is fragmented by mechanical shearing, chemical fragmentation or enzymatic fragmentation.

9. The method of claim 1 wherein the first oligonucleotide hybridized to the first common sequence forms a single base 3′

flap.

10. The method of claim 1 wherein the first oligonucleotide hybridized to the first common sequence forms a single base 3′

flap and the single base 3′

flap is the same as the 3′

-end base of the 5′

flap.

11. The method of claim 1 wherein the 5′

flap nuclease is a 5′

to 3′

exonuclease or endonuclease domain of a eubacterial DNA polymerase.

12. The method of claim 1 wherein the 5′

flap nuclease is E. coli DNA polymerase, Tag DNA polymerase, archeal flap endonuclease 1, eukaryotic flap endonuclease 1, murine flap endonuclease 1, yeast flap endonuclease 1, human flap endonuclease 1, P. horikoshii Pho flap endonuclease 1, or Pfu flap endonuclease 1.

13. The method of claim 1 wherein the 3′

flap nuclease is human xeroderma pigmentosa complementation group F, archeal P. furiosus helicase-associated endonuclease, archeal I xeroderma pigmentosa complementation group F, or Nar71.

14. The method of claim 1 wherein the 3′

flap nuclease creates a gap between the target region and the second common sequence and a DNA polymerase and dNTPs are added to fill the gap.

15. The method of claim 1 wherein the 5′

flap nuclease is added to cleave the 5′

flap structure and a 3′

to 5′

ssDNA exonuclease is added to remove the 3′

flap structure.

16. The method of claim 1 wherein a 5′

to 3′

ssDNA exonuclease is used to shorten the 5′

flap before being cleaved by the 5′

flap nuclease.

17. The method of claim 1 wherein the 5′

or 3′

end of the first common sequence or the 5′

or 3′

end of the second common sequence include one or more phosphorothioate bonds.