Ice-COLD-PCR Enables Rapid Amplification and Robust Enrichment for Low-Abundance Unknown DNA Mutations

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Ice-COLD-PCR Enables Rapid Amplification and Robust Enrichment for Low-Abundance Unknown DNA Mutations

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dc.contributor.author Milbury, Coren Audrey
dc.contributor.author Makrigiorgos, Gerassimos M.
dc.contributor.author Li, Jin
dc.date.accessioned 2011-11-16T16:43:12Z
dc.date.issued 2010
dc.identifier.citation Milbury, Coren A., Jin Li, and G. Mike Makrigiorgos. 2011. Ice-COLD-PCR enables rapid amplification and robust enrichment for low-abundance unknown DNA mutations. Nucleic Acids Research 39(1): e2. en_US
dc.identifier.issn 0305-1048 en_US
dc.identifier.uri http://nrs.harvard.edu/urn-3:HUL.InstRepos:5346648
dc.description.abstract Identifying low-abundance mutations within wild-type DNA is important in several fields of medicine, including cancer, prenatal diagnosis and infectious diseases. However, utilizing the clinical and diagnostic potential of rare mutations is limited by sensitivity of the molecular techniques employed, especially when the type and position of mutations are unknown. We have developed a novel platform that incorporates a synthetic reference sequence within a polymerase chain reaction (PCR) reaction, designed to enhance amplification of unknown mutant sequences during COLD-PCR (CO-amplification at Lower Denaturation temperature). This new platform enables an Improved and Complete Enrichment (ice-COLD-PCR) for all mutation types and eliminates shortcomings of previous formats of COLD-PCR. We evaluated ice-COLD-PCR enrichment in regions of TP53 in serially diluted mutant and wild-type DNA mixtures. Conventional-PCR, COLD-PCR and ice-COLD-PCR amplicons were run in parallel and sequenced to determine final mutation abundance for a range of mutations representing all possible single base changes. Amplification by ice-COLD-PCR enriched all mutation types and allowed identification of mutation abundances down to 1%, and 0.1% by Sanger sequencing or pyrosequencing, respectively, surpassing the capabilities of other forms of PCR. Ice-COLD-PCR will help elucidate the clinical significance of low-abundance mutations and our understanding of cancer origin, evolution, recurrence-risk and treatment diagnostics. en_US
dc.language.iso en_US en_US
dc.publisher Oxford University Press en_US
dc.relation.isversionof doi://10.1093/nar/gkq899 en_US
dc.relation.hasversion http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3017621/pdf/ en_US
dash.license LAA
dc.title Ice-COLD-PCR Enables Rapid Amplification and Robust Enrichment for Low-Abundance Unknown DNA Mutations en_US
dc.type Journal Article en_US
dc.description.version Version of Record en_US
dc.relation.journal Nucleic Acids Research en_US
dash.depositing.author Li, Jin
dc.date.available 2011-11-16T16:43:12Z
dash.affiliation.other HMS^Radiation Oncology-BWH-DFCI-CH en_US
dash.affiliation.other HMS^Radiation Oncology-BWH-DFCI-CH en_US

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