COLD-PCR-enhanced high-resolution melting enables rapid and selective identification of low-level unknown mutations.

ABSTRACT:

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.

SUBMITTER: Milbury CA 

PROVIDER: S-EPMC2828872 | biostudies-literature | 2009 Dec

REPOSITORIES: biostudies-literature