Patent ID: 8623603

Claim:
A method for enriching a target sequence in an amplification mixture, said method comprising: a) preparing an amplification reaction mixture comprising at least the following constituents: a nucleic acid sample comprising a reference sequence and also suspected of having one or more target sequences that are at least 50% homologous to said reference sequence and are also amplifiable by the same primer pair as said reference sequence, and a molar excess of reference blocking sequence relative to the amount of reference sequence, the reference sequence being fully complementary with at least a portion of the sequence of one of the strands of the reference sequence between its primer pair binding sites; b) increasing the temperature of the reaction mixture suspected of having said target sequence to a first denaturing temperature that is above the melting temperature (T m ) of the double stranded reference sequence and above the melting temperature (T m ) of the double stranded target sequence so as to form denatured reference strands and denatured target strands; c) reducing the temperature of the reaction mixture so as to permit formation of duplexes of the reference blocking sequence and the complementary reference strand and heteroduplexes of the reference blocking sequence and target strands; d) increasing the temperature of said reaction mixture to a critical temperature (T c ) sufficient to permit preferential denaturation of said heteroduplexes of the reference blocking sequence and target strands in preference to denaturation of the duplexes of the reference blocking sequence and reference strands; e) reducing the temperature of the reaction mixture so as to permit said primer pair to anneal to denatured target strands and any denatured reference strands in the reaction mixture; f) extending said primers annealed to the denatured target strands and denatured reference strands in the reaction mixture; and g) repeating steps b) through f) in order for two or more cycles so as to enrich said target sequence relative to said reference sequence.