Ice -COLD-PCR enables rapid amplification and robust enrichment for low-abundance unknown DNA mutations

摘要: 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 diagnostic potential rare limited by sensitivity molecular techniques employed, especially when type position are unknown. We have developed a novel platform that incorporates synthetic reference sequence polymerase chain reaction (PCR) reaction, designed to enhance amplification unknown mutant sequences during COLD-PCR (CO-amplification at Lower Denaturation temperature). This new enables an Improved Complete Enrichment (ice-COLD-PCR) for all mutation types eliminates shortcomings previous formats COLD-PCR. evaluated ice-COLD-PCR enrichment regions TP53 serially diluted mixtures. Conventional-PCR, amplicons were run parallel sequenced determine final abundance range representing possible single base changes. Amplification enriched allowed identification abundances down 1%, 0.1% Sanger sequencing or pyrosequencing, respectively, surpassing capabilities other forms PCR. Ice-COLD-PCR will help elucidate significance our understanding cancer origin, evolution, recurrence-risk treatment diagnostics.