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Nucleic Acids Res. 2013 Feb 1;41(3):e50. doi: 10.1093/nar/gks1250. Epub 2012 Dec 20.

Differential strand separation at critical temperature: a minimally disruptive enrichment method for low-abundance unknown DNA mutations.

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  • 1Division of DNA Repair and Genome Stability, Department of Radiation Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA.

Abstract

Detection of low-level DNA variations in the presence of wild-type DNA is important in several fields of medicine, including cancer, prenatal diagnosis and infectious diseases. PCR-based methods to enrich mutations during amplification have limited multiplexing capability, are mostly restricted to known mutations and are prone to polymerase or mis-priming errors. Here, we present Differential Strand Separation at Critical Temperature (DISSECT), a method that enriches unknown mutations of targeted DNA sequences purely based on thermal denaturation of DNA heteroduplexes without the need for enzymatic reactions. Target DNA is pre-amplified in a multiplex reaction and hybridized onto complementary probes immobilized on magnetic beads that correspond to wild-type DNA sequences. Presence of any mutation on the target DNA forms heteroduplexes that are subsequently denatured from the beads at a critical temperature and selectively separated from wild-type DNA. We demonstrate multiplexed enrichment by 100- to 400-fold for KRAS and TP53 mutations at multiple positions of the targeted sequence using two to four successive cycles of DISSECT. Cancer and plasma-circulating DNA samples containing traces of mutations undergo mutation enrichment allowing detection via Sanger sequencing or high-resolution melting. The simplicity, scalability and reliability of DISSECT make it a powerful method for mutation enrichment that integrates well with existing downstream detection methods.

PMID:
23258702
[PubMed - indexed for MEDLINE]
PMCID:
PMC3561944
Free PMC Article
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