Received on May 23, 2009
Accepted on September 17, 2009

Molecular Diagnostics and Genetics

COLD-PCR–Enhanced High-Resolution Melting Enables Rapid and Selective Identification of Low-Level Unknown Mutations

¹ Department of Radiation Oncology, Division of Medical Physics and Biophysics Division of Genome Stability and DNA Repair, Dana-Farber/Brigham and Women's Cancer Center, Harvard Medical School, Boston, MA

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

BACKGROUND: Analysis of clinical samples often necessitates identification of low-level somatic mutations within wild-type DNA; however, the selectivity and sensitivity of the methods are often limiting. COLD-PCR (coamplification at lower denaturation temperature–PCR) is a new form of PCR that enriches mutation-containing amplicons to concentrations sufficient for direct sequencing; nevertheless, sequencing itself remains an expensive mutation-screening approach. Conversely, high-resolution melting (HRM) is a rapid, inexpensive scanning method, but it cannot specifically identify the detected mutation. To enable enrichment, quick scanning, and identification of low-level unknown mutations, we combined COLD-PCR with HRM mutation scanning, followed by sequencing of positive samples.

METHODS: Mutation-containing cell-line DNA serially diluted into wild-type DNA and DNA samples from human lung adenocarcinomas containing low-level mutations were amplified via COLD-PCR and via conventional PCR for TP53 (tumor protein p53) exons 6–8, and the 2 approaches were compared. HRM analysis was used to screen amplicons for mutations; mutation-positive amplicons were sequenced.

RESULTS: Dilution experiments indicated an approximate 6- to 20-fold improvement in selectivity with COLD-PCR/HRM. Conventional PCR/HRM exhibited mutation-detection limits of 2%–10% mutant in mixtures with wild-type DNA, whereas COLD-PCR/HRM exhibited detection limits of 0.1%–1%. After HRM analysis of lung adenocarcinoma samples, we detected 7 mutations by both PCR methods in exon 7; however, in exon 8 we detected 9 mutations in COLD-PCR amplicons, compared with only 6 mutations in conventional-PCR amplicons. Furthermore, 94% of the HRM-detected mutations were successfully sequenced with COLD-PCR amplicons, compared with 50% with conventional-PCR amplicons.

CONCLUSIONS: COLD-PCR/HRM improves the mutation-scanning capabilities of HRM and combines high selectivity, convenience, and low cost with the ability to sequence unknown low-level mutations in clinical samples.