Coamplification at lower denaturation temperature-PCR increases mutation-detection selectivity of TaqMan-based real-time PCR.
Coamplification at lower denaturation temperature-PCR increases mutation-detection selectivity of TaqMan-based real-time PCR.
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DOI：
10.1373/clinchem.2008.113381
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发表时间：
2009-04
影响因子：
9.3
通讯作者：
Makrigiorgos GM
中科院分区：
文献类型：
--
作者：
Li J;Wang L;Jänne PA;Makrigiorgos GM
使用突变特异性TaqMan®探针（Applied Biosystems）的DNA基因分型广泛用于检测单核苷酸多态性，但由于其对低水平突变的选择性有限，因此用于体细胞突变的情况较少。我们最近描述了在较低变性温度下的共扩增-PCR（COLD-PCR），这是一种在PCR过程中从野生型和含突变序列的混合物中选择性扩增少数等位基因的方法。我们证明，COLD-PCR与TaqMan技术相结合，提供TaqMan基因分型与检测低水平体细胞突变所需的选择性。基于小沟结合物的或常见的TaqMan探针被设计为在探针的大约中间含有与所需突变匹配的核苷酸。然后通过实验确定每个扩增子的临界变性温度（Tc）。COLD-PCR/TaqMan基因分型分2步进行：Tc变性，然后在单一温度下退火和延伸（快速COLD-PCR）。阈值循环用于基于突变体DNA到野生型DNA的系列稀释鉴定突变，并鉴定肿瘤中的TP 53（肿瘤蛋白p53）和EGFR [表皮生长因子受体（成红细胞白血病病毒（v-er B-B）致癌基因同源物，禽类）]突变。COLD-PCR/TaqMan基因分型识别出TP 53外显子8内的G>A突变（密码子273突变热点）和EGFR基因内的C>T突变（耐药突变T790 M），选择性比常规PCR/TaqMan基因分型提高了15至30倍。第二轮COLD-PCR/TaqMan基因分型将选择性提高了15- 30倍，并能够在2000个野生型等位基因中检测到1个突变体。使用COLD-PCR/TaqMan基因分型允许定量鉴定结肠肿瘤样品和用激酶抑制剂处理的非小细胞肺癌细胞系中的低水平TP 53和T790突变。COLD-PCR提供的选择性的重大改进使流行的TaqMan基因分型方法成为检测临床样品中低水平突变的有力工具。
DNA genotyping with mutation-specific TaqMan® probes (Applied Biosystems) is broadly used in detection of single-nucleotide polymorphisms but is less so for somatic mutations because of its limited selectivity for low-level mutations. We recently described coamplification at lower denaturation temperature–PCR (COLD-PCR), a method that amplifies minority alleles selectively from mixtures of wild-type and mutation-containing sequences during the PCR. We demonstrate that combining COLD-PCR with TaqMan technology provides TaqMan genotyping with the selectivity needed to detect low-level somatic mutations. Minor-groove binder–based or common TaqMan probes were designed to contain a nucleotide that matches the desired mutation approximately in the middle of the probe. The critical denaturation temperature (Tc) of each amplicon was then experimentally determined. COLD-PCR/TaqMan genotyping was performed in 2 steps: denaturation at the Tc, followed by annealing and extension at a single temperature (fast COLD-PCR). The threshold cycle was used to identify mutations on the basis of serial dilutions of mutant DNA into wild-type DNA and to identify TP53 (tumor protein p53) and EGFR [epidermal growth factor receptor (erythroblastic leukemia viral (v-erb-b) oncogene homolog, avian)] mutations in tumors. COLD-PCR/TaqMan genotyping identified G>A mutations within TP53 exon 8 (codon 273 mutation hot spot) and C>T mutations within the EGFR gene (drug-resistance mutation T790M) with a selectivity improvement of 15- to 30-fold over regular PCR/TaqMan genotyping. A second round of COLD-PCR/TaqMan genotyping improved the selectivity by another 15- to 30-fold and enabled detection of 1 mutant in 2000 wild-type alleles. Use of COLD-PCR/TaqMan genotyping allowed quantitative identification of low-level TP53 and T790 mutations in colon tumor samples and in non–small-cell lung cancer cell lines treated with kinase inhibitors. The major improvement in selectivity provided by COLD-PCR enables the popular TaqMan genotyping method to become a powerful tool for detecting low-level mutations in clinical samples.
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影响因子：
3.9
作者：
Olivier, M;Eeles, R;Hainaut, P
通讯作者：
Hainaut, P
影响因子：
11.2
作者：
Ogino, Atsuko;Kitao, Hiroyuki;Tanimoto, Mitsune
通讯作者：
Tanimoto, Mitsune
影响因子：
7
作者：
Heid, CA;Stevens, J;Williams, PM
通讯作者：
Williams, PM
影响因子：
11.2
作者：
Nagai, Y;Miyazawa, H;Hagiwara, K
通讯作者：
Hagiwara, K
DOI：
10.1073/pnas.0502860102
发表时间：
2005-05-24
影响因子：
11.1
作者：
Kwak, EL;Sordella, R;Haber, DA
通讯作者：
Haber, DA