title: Kinetic hairpin oligonucleotide blockers for selective amplification of rare mutations.
abstract: Detection of rare mutant alleles in an excess of wild type alleles is increasingly important in cancer diagnosis. Several methods for selective amplification of a mutant allele via the polymerase chain reaction (PCR) have been reported, but each of these methods has its own limitations. A common problem is that Taq DNA polymerase errors early during amplification generate false positive mutations which also accumulate exponentially. In this paper, we described a novel method using hairpin oligonucleotide blockers that can selectively inhibit the amplification of wild type DNA during LATE-PCR amplification. LATE-PCR generates double-stranded DNA exponentially followed by linear amplification of single-stranded DNA. The efficiency of the blocker is optimized by adjusting the LATE-PCR temperature cycling profile. We also demonstrate that it is possible to minimize false positive signals caused by Taq DNA polymerase errors by using a mismatched excess primer plus a modified PCR profile to preferentially enrich for mutant target sequences prior to the start of the exponential phase of LATE-PCR amplification. In combination these procedures permit amplification of specific KRAS mutations in the presence of more than 10,000 fold excess of wild type DNA without false positive signals.
authors.affiliation: 1] Department of Biology, Brandeis University, Waltham, MA 02453, USA [2] State Key Laboratory of Analog and Mixed-Signal VLSI, University of Macau, Macau, China.
authors.forename: Yanwei
authors.initials: Y
authors.lastname: Jia
authors.affiliation: Department of Biology, Brandeis University, Waltham, MA 02453, USA.
authors.forename: J Aquiles
authors.initials: JA
authors.lastname: Sanchez
authors.affiliation: Department of Biology, Brandeis University, Waltham, MA 02453, USA.
authors.forename: Lawrence J
authors.initials: LJ
authors.lastname: Wangh
mesh_terms: D004252:DNA Mutational Analysis / Q000379:methods*; D017931:DNA Primers; D006801:Humans; D055029:Inverted Repeat Sequences; D007700:Kinetics; D009154:Mutation; D016133:Polymerase Chain Reaction; D011518:Proto-Oncogene Proteins / Q000235:genetics; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins / Q000235:genetics
chemical_list: D017931:DNA Primers; C117307:KRAS protein, human; D011518:Proto-Oncogene Proteins; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins
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