Methods and compositions for enrichment of nucleic acids in mixtures of highly homologous sequences

Abstract

Provided are methods of enrichment and detection of target nucleic acids during target amplification in the presence of excess amounts of highly homologous sequences, the methods having substantial diagnostic utility (e.g., cancer diagnostics). Provided are amplification reaction mixtures having at least one cleavage-directing oligonucleotide, the respective binding sites of which, for the target and homologous sequences, include one or more nucleotide positions differing in sequence between the target homologous sequences. During target sequence amplification in the presence of DNA polymerase activity, a FEN1 activity, one or more amplification primers, deoxynucleoside 5'-triphosphates and other reagents suitable to support amplification of both target and homologous nucleic acid sequences, the cleavage directing oligonucleotide hybridizes to the homologous sequence and its amplification products resulting in a FEN1-mediated cleavage of these sequences providing for increased amounts of the target nucleic acid sequence relative to that of the homologous nucleic acid sequences.