SELECTIVE AMPLIFICATION OF MINORITY MUTATIONS USING PRIMER BLOCKING HIGH-AFFINITY OLIGONUCLEOTIDES

摘要

In certain embodiments this invention pertains to methods of detecting and/or quantifying rare mutant nucleic acids in populations of nucleic acids in which the wild-type nucleic acids are in substantially greater abundance than the rare mutants. In various embodiments the methods utilize short high affinity oligonucleotides targeted to the wild type rather than the minority or mutant sequence. Rather than directly detecting mutant DNA, these probes block detection of wild type DNA. These “blocker” probes can be used in combination with longer “detection” probes or PCR primers to amplify and/or identify the minority mutation in, e.g., clinical specimens. The combination of short high affinity blocker probes and longer, lower affinity detection probes eliminates the single base specificity/complexity tradeoff in the design of nucleic acid probes.