Patent ID: 8329405

Claim:
A method for detecting a presence or an absence of a mutant DNA sequence in a sample, the method comprising the steps of: a) contacting the sample with an oligonucleotide system under hybridization conditions so as to form a reaction mixture, said oligonucleotide system including a reverse primer and a mutagenic primer, wherein i) said reverse primer comprises: a 5′ end region comprising a recognition sequence being able to be cut by a high temperature resistant restriction endonuclease; said 5′ end region having covalently linked at its extremities a coupled detection system so that when the recognition sequence is cleaved by said high temperature resistant restriction endonuclease, a signal is generated; a 3′ end region able to hybridise to a complementary region downstream of the putative mutant DNA sequence; and ii) said mutagenic primer comprises: a 5′ end region comprising a first recognition sequence being able to be cut by a high temperature resistant restriction endonuclease; a 3′ end region able to hybridise to a complementary region of the opposite extremity of the other strand of the sample DNA sequence, said mutagenic primer has a sequence so that a second recognition sequence being able to be cut by the high temperature resistant restriction endonuclease is created, only when said primer is extended on the wild type sequence so that the restriction endonuclease digests the amplicon and suppresses the amplification reaction; and said second recognition sequence being able to be cut by high temperature resistant restriction endonuclease is not polymerised when the mutant sequence is present in the DNA sample; b) adding with appropriate substrates and cofactors, at a suitable ionic and pH environment, both a temperature resistant DNA polymerase and said high temperature resistant restriction endonuclease to said reaction mixture under predetermined reaction conditions, such that, if the mutant DNA sequence is present in the sample, said reverse primer and said mutagenic primer hybridize to the same and prime the DNA polymerase reaction to obtain a first specific amplified product; c) cycling the hybridization of said oligonucleotide system so that a second specific amplified product is extended comprising the 5′ end region of the reverse primer forming a double stranded recognition site for said restriction endonuclease so that the high temperature resistant restriction endonuclease cuts specifically at the recognition sequence and induces the generation of a signal by the coupled detection system; and d) detecting the generated signal.