Li Jin, Wang Lilin, Mamon Harvey, Kulke Matthew H, Berbeco Ross, Makrigiorgos G Mike
Department of Radiation Oncology, Divisions of Genomic Stability and DNA Repair, Physics and Radiation Therapy, Room JF514, 44 Binney Street, Boston, Massachusetts 02115, USA.
Nat Med. 2008 May;14(5):579-84. doi: 10.1038/nm1708. Epub 2008 Apr 13.
PCR is widely employed as the initial DNA amplification step for genetic testing. However, a key limitation of PCR-based methods is the inability to selectively amplify low levels of mutations in a wild-type background. As a result, downstream assays are limited in their ability to identify subtle genetic changes that can have a profound impact in clinical decision-making and outcome. Here we describe co-amplification at lower denaturation temperature PCR (COLD-PCR), a novel form of PCR that amplifies minority alleles selectively from mixtures of wild-type and mutation-containing sequences irrespective of the mutation type or position on the sequence. We replaced regular PCR with COLD-PCR before sequencing or genotyping assays to improve mutation detection sensitivity by up to 100-fold and identified new mutations in the genes encoding p53, KRAS and epidermal growth factor in heterogeneous cancer samples that had been missed by the currently used methods. For clinically relevant microdeletions, COLD-PCR enabled exclusive amplification and isolation of the mutants. COLD-PCR will transform the capabilities of PCR-based genetic testing, including applications in cancer, infectious diseases and prenatal identification of fetal alleles in maternal blood.
聚合酶链反应（PCR）被广泛用作基因检测的初始DNA扩增步骤。然而，基于PCR的方法的一个关键局限性是无法在野生型背景下选择性地扩增低水平突变。因此，下游检测在识别可能对临床决策和结果产生深远影响的细微基因变化方面能力有限。在此，我们描述了低温变性温度下的共扩增PCR（COLD-PCR），这是一种新型的PCR形式，可从野生型和含突变序列的混合物中选择性地扩增少数等位基因，而不管突变类型或在序列上的位置如何。在测序或基因分型检测之前，我们用COLD-PCR取代常规PCR，将突变检测灵敏度提高了多达100倍，并在异质性癌症样本中鉴定出了目前使用的方法遗漏的p53、KRAS和表皮生长因子编码基因中的新突变。对于临床相关的微缺失，COLD-PCR能够实现突变体的特异性扩增和分离。COLD-PCR将改变基于PCR的基因检测能力，包括在癌症、传染病以及母血中胎儿等位基因的产前鉴定等方面的应用。
