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 from a complex mixture of nucleic acid comprising:
(a) fragmenting the nucleic acid to obtain a fragmented nucleic acid sample; (b) adding a plurality of circularization probes to the fragmented nucleic acid sample to form a mixture, wherein there is a circularization probe for each target sequence in the plurality and wherein each circularization probe comprises:
(i) a 5′ target region that is perfectly complementary to a first region in a complementary target sequence,(ii) a 3′ target region that is perfectly complementary to a second region in the complementary target sequence and(ii) a central region that is perfectly complementary to a third region in the complementary target sequence and also perfectly complementary to a fourth region in the complementary target sequence wherein said third and fourth regions are distinct regions but share a common sequence of at least 2 contiguous bases; (c) adding a 3′ to 5′ exonuclease to the mixture; (d) adding a 5′ flap nuclease to the mixture; (e) adding a ligase to the mixture, following step (d), to generate circularized target sequences; and (f) amplifying the circularized target sequences by rolling circle amplification.