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. mixing the fragmented nucleic acid sample with a plurality of dU probes to the fragmented nucleic acid sample, wherein there is a dU probe for each target sequence in the plurality and wherein each dU probe comprises:
i. a central target region that is perfectly complementary to a target region in a target fragment in the nucleic acid sample, wherein the target fragment comprises a target sequence and a 5′ non-target sequence and wherein the target fragment hybridizes to the dU probe so that the target sequence is hybridized to the dU probe and the 5′ non-target sequences forms a single stranded 5′ flap structure;ii. a 5′ first common sequence; andiii. a 3′ second common sequence; c. mixing the fragmented nucleic acid sample with a first oligonucleotide that is complementary to said first common sequence and a second oligonucleotide that is complementary to said second common sequence; d. adding a 5′ flap nuclease to the mixture from (c) to cleave the 5′ flap from the target fragment to generate a new 5′ end in the target; e. adding a ligase to the reaction to ligate the first oligonucleotide to the new 5′ end in the target and the second oligonucleotide to the 3′ end of the target; f. cleaving the dU probes; and g. amplifying the target sequences using primers for said first and second common sequences.