Ice-COLD-PCR enables rapid amplification and robust enrichment for low-abundance unknown DNA mutations.
Ice-COLD-PCR enables rapid amplification and robust enrichment for low-abundance unknown DNA mutations.
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DOI：
10.1093/nar/gkq899
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发表时间：
2011-01
影响因子：
14.9
通讯作者：
Makrigiorgos GM
中科院分区：
文献类型：
--
作者：
Milbury CA;Li J;Makrigiorgos GM
识别野生型DNA中的低丰度突变在几个医学领域非常重要，包括癌症、产前诊断和传染病。然而，利用罕见突变的临床和诊断潜力受到所用分子技术的敏感性的限制，特别是在突变的类型和位置未知的情况下。我们开发了一种新的平台，将合成的参考序列整合到聚合酶链式反应(PCR)反应中，旨在增强冷-PCR(较低变性温度下的共扩增)中未知突变序列的扩增。这一新平台实现了对所有突变类型的改进和完整的浓缩(ICE-COLD-PCR)，并消除了以前的COLD-PCR格式的缺点。我们在系列稀释的突变体和野生型DNA混合物中评估了冰冷-聚合酶链式反应在TP53区域的富集性。常规-聚合酶链式反应、冷-聚合酶链式反应和冰冷-聚合酶链式反应同时进行扩增和测序，以确定代表所有可能的单碱基变化的一系列突变的最终突变丰度。冰冷-聚合酶链式反应扩增丰富了所有突变类型，Sanger测序和焦磷酸测序的突变丰度分别降至1%和0.1%，超过了其他形式的聚合酶链式反应。ICE-COLD-PCR将有助于阐明低丰度突变的临床意义，以及我们对癌症的起源、进化、复发风险和治疗诊断的理解。
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.
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影响因子：
9.3
作者：
Milbury CA;Li J;Makrigiorgos GM
通讯作者：
Makrigiorgos GM
影响因子：
3.9
作者：
Li, Jin;Milbury, Coren A.;Li, Cheng;Makrigiorgos, G. Mike
通讯作者：
Makrigiorgos, G. Mike
影响因子：
7.5
作者：
Delaney, David;Diss, Tim C.;Flanagan, Adrienne M.
通讯作者：
Flanagan, Adrienne M.
影响因子：
11.5
作者：
Fukui, Tomoya;Ohe, Yuichiro;Tamura, Tomohide
通讯作者：
Tamura, Tomohide
影响因子：
158.5
作者：
Kobayashi, S;Boggon, TJ;Halmos, B
通讯作者：
Halmos, B