Multiplex targeted amplification using flap nuclease
View Patent ↗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.
1. A method for amplifying target sequences within a nucleic acid sample, the method comprising:
fragmenting the nucleic acid sample with a restriction enzyme, thereby producing a fragmented nucleic acid sample comprising one or more target sequences;
adding circularization probes to the fragmented nucleic acid sample, wherein each circularization probe comprises a first probe region and a second probe region, wherein the first and second probe regions are respectively complementary to a first target region and a second target region of one of the target sequences, and wherein the circularization probes hybridize with target sequences to form probe-target structures in which each target sequence includes a flap of two or more bases at the 5′ end and a flap of one base at the 3′ end;
removing the 5′ flaps with a 5′ flap nuclease, thereby generating probe-target structures in which each target sequence includes a 5′ end juxtaposed with the 3′ end, with the 5′ end and the 3′ end separated by a nick;
ligating the 5′ ends to the juxtaposed 3′ ends of the target sequences to circularize the target sequences; and
amplifying the circularized target sequences.
2. The method of claim 1 , wherein the 5′ flap nuclease is selected from the group consisting of E. coli DNA polymerase, Taq DNA polymerase and flap endonuclease 1.
3. The method of claim 1 , wherein each 5′ flap includes a 3′ end base that is identical to the one base of the 3′ flap of the same target sequence.
4. A method for amplifying target sequences within a nucleic acid sample, the method comprising:
fragmenting the nucleic acid sample with a restriction enzyme, thereby producing a fragmented nucleic acid sample comprising one or more target sequences;
adding circularization probes to the fragmented nucleic acid sample, wherein each circularization probe comprises a first probe region and a second probe region, wherein the first and second probe regions are respectively complementary to a first target region and a second target region of one of the target sequences, and wherein the circularization probes hybridize with target sequences to form probe-target structures in which the target sequences include 3′ flaps of two or more bases and 5′ ends perfectly complementary to a corresponding first probe region,
removing the 3′ flaps with a 3′ flap nuclease, wherein removing the 3′ flap forms a 3′ end of the target sequence that is separated from the 5′ end of the target sequence by a gap;
extending the 3′ ends of the target sequences with DNA polymerase and dNTPs to fill in the gaps, wherein the dNTPs comprise only two of dATP, dCTP, dGTP and dTTP:
ligating the 3′ ends to the 5′ ends of the same target sequences to circularize the target sequences; and
amplifying the circularized target sequences.
5. The method of claim 4 , wherein the 3′ flap nuclease is selected from the group consisting of Xeroderma pigmentosa complementation group F, P. furiosus helicase associated endonuclease, S. solfataricus XPF and Nar71.
6. The method of claim 4 , wherein the dNTPs comprise only dATP and dGTP.
7. The method of claim 1 , wherein the 5′ flap nuclease is a 5′ flap endonuclease, and wherein removing the 5′ flaps with a 5′ flap nuclease additionally comprises:
shortening the 5′ flaps with a ssDNA exonuclease, wherein the ssDNA exonuclease has 5′ to 3′ exonuclease activity.
8. The method of claim 7 , wherein the 5′ flaps are more than 50 bases in length.
9. The method of claim 7 , wherein the ssDNA exonuclease is selected from the group consisting of RecJ and exonuclease VII.
10. The method of claim 1 , further comprising digesting non-circularized nucleic acids with an exonuclease, wherein said digesting is performed after circularizing the target sequences but before amplifying the circularized target sequences.
11. The method of claim 10 , wherein the exonuclease is selected from the group consisting of exonuclease I, exonuclease VII, exonuclease III, and T7 exonuclease.
12. The method of claim 1 , wherein amplifying the circularized target sequences does not involve PCR amplification.
13. The method of claim 1 , wherein amplifying the circularized target sequences is performed by rolling circle amplification.
14. The method of claim 1 , wherein the fragmented nucleic acid sample includes 100 to 50000 different target sequences.
15. The method of claim 1 , wherein the circularization probes are 6 to 60 bases in length.
16. The method of claim 15 , wherein the circularization probes are about 12 to about 40 bases in length.
17. The method of claim 1 , wherein the circularization probes are about 40 bases in length.
18. The method of claim 1 , wherein each circularization probe consists of a first probe region and a second probe region.
19. The method of claim 4 , wherein each circularization probe consists of a first probe region and a second probe region.