Patent ID: 7862999

Claim:
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 5â€² target region that is perfectly complementary to a first region in a complementary target sequence, (ii) a 3â€² target region that is perfectly complementary to a second region in the complementary target sequence and (iii) a central region immediately 3â€² of the 5â€² target region and immediately 5â€² of the 3â€² target 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 region is immediately 3â€² of the second region and said fourth region is immediately 5â€² of said first region and wherein said third and fourth regions are distinct regions but share a common sequence of at least 2 contiguous bases; (c) adding a 3â€² to 5â€² exonuclease to the mixture; (d) adding a 5â€² 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.