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 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.