Step-up method for COLD-PCR enrichment

摘要

Methods of using polymerase chain reactions to enrich a target sequence in a sample containing reference sequences and target sequences having high homology and amplifiable by the same primer pair are provided herein. In particular the methods provide a robust means to improve the fold enrichment of the target sequence and minimize reaction-to-reaction, well-to-well and run-to-run variations in the enrichment methods.

主权项

1. A step-up method for enriching a target sequence in an amplification reaction mixture, said method comprising:
a) preparing an amplification reaction mixture including at least the following constituents:
a primer pair capable of amplifying a reference sequence and a nucleic acid sample having the 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 a reference blocking sequence relative to the amount of reference sequence, the reference blocking sequence is fully complementary with at least a portion of the sequence of one of the strands of the reference sequence between or overlapping its primer binding sites, and wherein the target sequence is less prevalent than the reference sequence; b) selecting a critical temperature (Tc) sufficient to permit preferential denaturation of heteroduplexes of the reference blocking sequence and target strands as compared to denaturation duplexes of the reference blocking sequence and reference strands; c) selecting second denaturing temperatures (Td2) for multiple sets of amplification cycles the second denaturing temperature (Td2) for a first set of amplification cycles being less than the selected critical temperature (Tc) and the second denaturing temperature (Td2) for subsequent sets of amplification cycles being progressively higher than the previous set of amplification cycles; d) for each set of amplification cycles, cycling through the following steps to enrich the target sequence in the amplification reaction mixture:
1) increasing the temperature of the amplification reaction mixture to a first denaturing temperature (Td1) that is above the melting temperature (Tm) of the double-stranded reference sequence and above the melting temperature (Tm) of the double-stranded target sequence to form reference strands and target strands;2) reducing the temperature of the amplification reaction mixture to permit formation of duplexes of the reference blocking sequence and the complementary reference strand and heteroduplexes of the reference blocking sequence and the target strands;3) increasing the temperature of the amplification reaction mixture to the selected second denaturing temperature (Td2);4) reducing the temperature of the amplification reaction mixture to permit said primer pair to anneal to target strands and reference strands in the amplification reaction mixture; and5) extending said primers annealed to the free target strands and free reference strands in the reaction mixture to enrich said target sequence relative to said reference sequence; and e) repeating step (d) for each set of amplification cycles using said selected second denaturing temperatures (Td2) for the respective set of amplification cycles.