METHOD FOR AMPLIFICATION OF TARGET NUCLEIC ACID

摘要

The present invention provides a method for amplifying at least one target nucleic acid in a nucleic acid sample, comprising:(a) fragmenting the nucleic acid sample to create at least one target fragment comprising said target nucleic acid and comprising two probe-complementary portions, wherein at least one of said two probe-complementary portions is located at an end of the target fragment; (b) contacting said fragmented nucleic acid sample with at least one probe which is provided with an immobilisation moiety and optionally is immobilised on a solid phase by means of said moiety, and which comprises two target fragment-complementary portions which are complementary in sequence to the probe- complementary portions of the target fragment, wherein said portions of the probe may be adjacent, or separated by an intervening non-target fragment-complementary portion; (c) rendering the fragmented nucleic acid sample single- stranded, wherein the step may occur before, contemporaneously with or after step (b); (d) allowing the probe-complementary portions of the target fragment to hybridise with the target-fragment complementary portions of the probe; (e) if the probe provided in step (b) is not immobilised, immobilising the probe-target fragment hybrid on a solid phase by means of said immobilisation moiety; (f) separating non-immobilised nucleic acid fragments from the solid phase; (g) contacting the solid phase with a ligase to ligate, directly or indirectly, ligatable 5' and 3' ends of the target fragment whereby the target fragment is circularised, wherein where one of said probe-complementary portions of the target fragment is not located at an end of the target fragment, the ligatable end, other than that at which a probe-complementary portion is located, is created by the action of a flap endonuclease or an exonuclease; and (h) amplifying said circularised target fragment. Kits for use in the method of the invention are also provided.