Received on June 24, 2008
Accepted on October 14, 2008

Molecular Diagnostics and Genetics

Coamplification at Lower Denaturation Temperature–PCR Increases Mutation-Detection Selectivity of TaqMan-Based Real-Time PCR

¹ Department of Radiation Oncology, Divisions of Genomic Stability and DNA Repair, and Medical Physics, Dana Farber Cancer Institute, Harvard Medical School, Boston, MA
² Department of Medical Oncology, Lowe Center for Thoracic Oncology, Dana Farber Cancer Institute, Harvard Medical School, Boston, MA

^* To whom correspondence should be addressed. E-mail: mmakrigiorgos{at}partners.org.

BACKGROUND: DNA genotyping with mutation-specific TaqMan® probes (Applied Biosystems) is broadly used in detection of single-nucleotide polymorphisms but is less so for somatic mutations because of its limited selectivity for low-level mutations. We recently described coamplification at lower denaturation temperature–PCR (COLD-PCR), a method that amplifies minority alleles selectively from mixtures of wild-type and mutation-containing sequences during the PCR. We demonstrate that combining COLD-PCR with TaqMan technology provides TaqMan genotyping with the selectivity needed to detect low-level somatic mutations.

METHODS: Minor-groove binder–based or common TaqMan probes were designed to contain a nucleotide that matches the desired mutation approximately in the middle of the probe. The critical denaturation temperature (T_c) of each amplicon was then experimentally determined. COLD-PCR/TaqMan genotyping was performed in 2 steps: denaturation at the T_c, followed by annealing and extension at a single temperature (fast COLD-PCR). The threshold cycle was used to identify mutations on the basis of serial dilutions of mutant DNA into wild-type DNA and to identify TP53 (tumor protein p53) and EGFR [epidermal growth factor receptor (erythroblastic leukemia viral (v-erb-b) oncogene homolog, avian)] mutations in tumors.

RESULTS: COLD-PCR/TaqMan genotyping identified G>A mutations within TP53 exon 8 (codon 273 mutation hot spot) and C>T mutations within the EGFR gene (drug-resistance mutation T790M) with a selectivity improvement of 15- to 30-fold over regular PCR/TaqMan genotyping. A second round of COLD-PCR/TaqMan genotyping improved the selectivity by another 15- to 30-fold and enabled detection of 1 mutant in 2000 wild-type alleles. Use of COLD-PCR/TaqMan genotyping allowed quantitative identification of low-level TP53 and T790 mutations in colon tumor samples and in non–small-cell lung cancer cell lines treated with kinase inhibitors.

CONCLUSIONS: The major improvement in selectivity provided by COLD-PCR enables the popular TaqMan genotyping method to become a powerful tool for detecting low-level mutations in clinical samples.