To verify that the method detects the presence or absence of a single template copy, 48 copies of genomic BRAF mutant DNA were distributed across the wells of a 96-well plate and amplified. The melting curves clearly clustered into 2 groups with 43 positive and 53 negative results (data not shown). The ability of melting analysis to distinguish the presence or absence of single-copy templates without probes has been previously reported (32). Similar results have been reported for digital PCR with labeled probes, where the complexity of the genomic background is limited (33). However, specific amplification becomes more difficult as the concentration of background wild-type DNA increases. Nevertheless, when 10 copies of BRAF mutant DNA were admixed with 10 µg wild-type DNA (3.4 x 106 copies) and amplified in 20 PCR capillaries, 7 out of 20 reactions were positive by melting analysis. Even in the presence of >105 copies of wild-type DNA, single mutant DNA molecules were easily distinguished from the complete absence of mutant DNA. The sensitivity may even be greater, but testing is difficult because of the high DNA concentrations and/or PCR volumes required. If a mutant copy is present in the sample, it will be detected without the risk of false-positive reactions.
Increased sensitivity was demonstrated on 91 clinical thyroid samples previously tested for BRAF c.T1799A (43 positive, 48 negative) by dual-hybridization probes (30). These samples were blinded and reanalyzed by the unlabeled probe method described above. All samples were concordant except for an additional four samples that were positive by the allele-specific method. Detection of BRAF c.T1799A increased in these clinical samples by 9.3% when the allele-specific, competitive blocking method with melting was used.
The enrichment method used here is similar to allele-specific competitive blocker PCR that is typically analyzed on agarose gels (13) or by internal hydrolysis probes (15). However, the blocker oligonucleotide serves a dual purpose: both blocking wild-type DNA and indicating the presence of mutant alleles by melting analysis. Asymmetric PCR is employed to increase allele-specific amplification and augment detection of the melting signal. Detection of the mutant allele by melting prevents false-positive results, even with >105-fold excess of wild-type DNA. Unlabeled, dual hybridization, and molecular beacon probes can all function as blockers with similar sensitivities. Unlabeled probes are low in cost, while dual hybridization and molecular beacon probes can be multiplexed by color. The method is fast, performed in a closed tube, and should work on any thermocycler with melting analysis capability. The combination of allele-specific priming, competitive probe blocking of wild-type amplification, asymmetric PCR, and probe melting analysis allows single copy detection of variant alleles down to a sensitivity of ≥0.001% mutant to wild-type DNA.
This work was supported by the National Institutes of Health (NIH; grant no. R43GM0882116). This paper is subject to the NIH Public Access Policy.
Aspects of melting analysis, unlabeled probes and dual-hybridization probes are licensed from the University of Utah to Idaho Technology. C.T.W. has equity interest in Idaho Technology.
Address correspondence to Carl T. Wittwer, 15 N Medical Drive, Department of Pathology, University of Utah Medical School, Salt Lake City, UT 84132 USA. e-mail: [email protected]
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