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

摘要

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.

主权项

1. A method for enrichment during amplification of a target nucleic acid sequence in a mixture of target and homologous nucleic acid sequences, the method comprising:
a) providing an amplification reaction mixture having a target nucleic acid sequence and at least one other nucleic acid sequence that is homologous to the target nucleic acid sequence but differs therefrom in at least one nucleotide position, at least one cleavage-directing oligonucleotide sequence, the respective binding sites of which to said target and to said homologous nucleic acid sequences include the at least one nucleotide position having the nucleotide sequence difference, a DNA polymerase activity, a FEN1 activity, one or more amplification primers, deoxynucleoside 5′-triphosphates and other reagents suitable to support the amplification of both target and homologous nucleic acid sequences; and b) amplifying the nucleic acids under reaction conditions suitable to support hybridization of the cleavage-directing oligonucleotide sequence to the homologous nucleic acid sequence providing for FEN1-mediated cleavage of the homologous nucleic acid and its amplification products, to provide for preferential amplification and increase in amount of the target nucleic acid sequence relative to that of the homologous nucleic acid sequence.