Multiplex targeted amplification using flap nuclease

摘要

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.

主权项

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.