Li Jin, Makrigiorgos G Mike
Department of Radiation Oncology, Division of Genomic Stability and Division of DNA Repair and Medical Physics and Biophysics, Dana Farber Cancer Institute and Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.
Biochem Soc Trans. 2009 Apr;37(Pt 2):427-32. doi: 10.1042/BST0370427.
PCR is widely employed as the initial DNA amplification step for genetic testing and cancer biomarker detection. However, a key limitation of PCR-based methods, including real-time PCR, is the inability to selectively amplify low levels of variant alleles in a wild-type allele background. As a result, downstream assays are limited in their ability to identify subtle genetic changes that can have a profound impact on clinical decision-making and outcome or that can serve as cancer biomarkers. We developed COLD-PCR (co-amplification at lower denaturation temperature-PCR) [Li, Wang, Mamon, Kulke, Berbeco and Makrigiorgos (2008) Nat. Med. 14, 579-584], 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. Consequently, COLD-PCR amplification from genomic DNA yields PCR products containing high-prevalence variant alleles that can be detected. Since PCR constitutes a ubiquitous initial step for almost all genetic analysis, COLD-PCR provides a general platform to improve the sensitivity of essentially all DNA-variation detection technologies including Sanger sequencing, pyrosequencing, single molecule sequencing, mutation scanning, mutation genotyping or methylation assays. COLD-PCR combined with real-time PCR provides a new approach to boost the capabilities of existing real-time mutation detection methods. We replaced regular PCR with COLD-PCR before sequencing or real-time mutation detection assays to improve mutation detection-sensitivity by up to 100-fold and identified novel p53/Kras/EGFR (epidermal growth factor receptor) mutations in heterogeneous cancer samples that were missed by all existing methods. For clinically relevant micro-deletions, COLD-PCR enabled exclusive amplification and isolation of the mutants. COLD-PCR is expected to have diverse applications in the fields of biomarker identification and tracing, genomic instability, infectious diseases, DNA methylation testing 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和癌症生物标志物检测的初始DNA扩增步骤。然而，包括实时PCR在内的基于PCR的方法的一个关键局限性在于，无法在野生型等位基因背景下选择性地扩增低水平的变异等位基因。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或可作为癌症生物标志物的细微基因变化方面的能力有限。我们开发了冷循环PCR（低温变性温度下的共扩增PCR）[李、王、马蒙、库尔克、贝贝科和马克里乔戈斯（2008年）《自然医学》14卷，579 - 584页]，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，而不考虑突变类型或在序列上的位置。因此，从基因组DNA进行冷循环PCR扩增可产生包含高流行率变异等位基因的PCR产物，这些产物能够被检测到。由于PCR几乎是所有基因分析的普遍初始步骤，冷循环PCR为提高包括桑格测序、焦磷酸测序、单分子测序、突变扫描、突变基因分型或甲基化检测在内的几乎所有DNA变异检测技术的灵敏度提供了一个通用平台。冷循环PCR与实时PCR相结合为提高现有实时突变检测方法的能力提供了一种新方法。我们在测序或实时突变检测分析之前用冷循环PCR取代常规PCR，将突变检测灵敏度提高了多达100倍，并在所有现有方法均遗漏的异质性癌症样本中鉴定出了新的p53/Kras/表皮生长因子受体（EGFR）突变。对于临床相关的微缺失，冷循环PCR能够实现突变体的特异性扩增和分离。冷循环PCR有望在生物标志物识别与追踪、基因组不稳定性、传染病、DNA甲基化检测以及母血中胎儿等位基因的产前鉴定等领域有多种应用。
