Full Cold-PCR Enrichment with Reference Blocking Sequence

A method for enriching a target nucleic acid sequence in an amplification reaction mixture, said method comprising:

• a. preparing an amplification reaction mixture including at least the following constituents:
• a nucleic acid sample having 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
• an excess amount of reference blocking nucleic acid sequence which is fully complementary with at least a portion of the sequence of one of the strands of the reference sequence between its primer sites;
• b. subjecting the reaction mixture suspected of having said target sequence to a first denaturing temperature that is above the melting temperature (Tm) of the reference 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 heteroduplexes of the reference blocking sequence and the complementary reference strand and heteroduplexes of the reference blocking sequence and target strands;
• d. subjecting said reaction mixture to a critical temperature (Tc) sufficient to permit preferential denaturation of said heteroduplexes of the reference blocking sequence and target strands, yet insufficient to denature heteroduplexes of the reference blocking sequence and the complementary reference strand;
• e. reducing the temperature of the reaction mixture so as to permit said primer pair to anneal to free target strands and any free reference strands in the reaction mixture;
• and
• f. extending said primer pair so as to enrich said target sequence relative to said reference sequence.