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

  • US 9,487,824 B2
  • Filed: 06/28/2012
  • Issued: 11/08/2016
  • Est. Priority Date: 06/28/2011
  • Status: Active Grant
First Claim
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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.

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