Patent ID: 8828688
Filing Date: 2014-09-09
Classification: C12N,C12Q

Abstract:
1. A method for amplifying a plurality of target nucleic acids comprising: (a) obtaining a plurality of capture probes comprising a capture probe for each target nucleic acid in the plurality of target nucleic acids, wherein each capture probe comprises a first 3′ region that is complementary to a target nucleic acid in the plurality of target nucleic acids, a second region that has a first priming sequence, and optionally a third region between said first and second regions that comprises a tag sequence; (b) mixing the capture probes with a nucleic acid sample that includes the target nucleic acids under conditions that allow hybridization of the capture probes to form duplexes between the capture probes and the target nucleic acids; (c) extending the capture probes from the 3′ end with a polymerase using the target nucleic acids as template to form extended capture probes that comprise a newly added region that is complementary to the target nucleic acid; (d) hybridizing a splint probe, comprising a target complementary region and a 5′ overhang region, to a portion of the newly added region to form a duplex between the target complementary region of the splint probe and a portion of the newly added region leaving a 3′ region of the extended capture probe single stranded, wherein the 3′ region includes the 3′ end of the extended capture probe; (e) degrading the 3′ region of the extended capture probes with a single strand specific 3′ endonuclease; (f) forming a duplex between the 5′ overhang region of the splint probe and a ligatable sequence comprising a region that is complementary to a region of the splint probe and a second priming sequence, and ligating said ligatable sequence to the 3′ end of the extended capture probe to form a ligated product; and (g) amplifying the ligated product using the first and second priming regions.