Patent ID: 6555317
Filing Date: 2003-04-29
Classification: C12Q,Y10S

Abstract:
A method of detecting a mutation within a target nucleic acid sequence, said method comprising:(a) amplification of said target sequence by polymerase chain reaction, using primers P1 and P2 to produce an amplicon AA, wherein one of said primers P1 and P2 comprises a label and wherein said primer P1 is comprised of a 3â€²-end portion Pa that can hybridize with said target sequence and 5-end portion B1 that cannot hybridize with said target sequence, (b) extending a primer P3 by chain extension along one strand of amplicon AA to produce a tailed target partial duplex Aâ€², wherein said primer P3 is comprised of said 3â€²-end portion Pa and a 5â€²-end portion A1 that cannot hybridize to said target sequence or its complement, (c) amplification of a reference nucleic acid sequence, using said primer P2 and said primer P3, by polymerase chain reaction to produce amplicon BB, said reference sequence being identical to said target sequence but lacking a possible mutation, wherein said primer P2 comprises a label when said primer P2 in step (a) above comprises a label and said primer P3 comprises a label when said primer P1 in step (a) above comprises a label, (d) extending said primer P1 by chain extension along one strand of amplicon BB to produce a tailed reference partial duplex Bâ€², (e) allowing said tailed target partial duplex Aâ€² to bind to said tailed reference partial duplex Bâ€², and (f) detecting the binding of one of said labels to another of said labels as a result of the formation of a complex between said tailed partial duplexes, the binding thereof being directly related to the presence of said mutation.