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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Title: Ice-COLD-PCR Enables Rapid Amplification and Robust Enrichment for Low-Abundance Unknown DNA Mutations
Author: Milbury, Coren Audrey; Makrigiorgos, Gerassimos M.; Li, Jin

Note: Order does not necessarily reflect citation order of authors.

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.
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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.
Published Version: doi://10.1093/nar/gkq899
Other Sources: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3017621/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:5346648

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