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

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

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Title: Differential strand separation at critical temperature: A minimally disruptive enrichment method for low-abundance unknown DNA mutations
Author: Guha, Minakshi; Castellanos-Rizaldos, Elena; Liu, Pingfang; Mamon, Harvey Jay; Makrigiorgos, Gerassimos M.

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Citation: Guha, Minakshi, Elena Castellanos-Rizaldos, Pingfang Liu, Harvey Mamon, and G. Mike Makrigiorgos. 2012. Differential strand separation at critical temperature: a minimally disruptive enrichment method for low-abundance unknown DNA mutations. Nucleic Acids Research 41(3): e50.
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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.
Published Version: doi:10.1093/nar/gks1250
Other Sources: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3561944/pdf/
Terms of Use: This article is made available under the terms and conditions applicable to Other Posted Material, as set forth at http://nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of-use#LAA
Citable link to this page: http://nrs.harvard.edu/urn-3:HUL.InstRepos:11180395
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