COLD-PCR-enhanced high-resolution melting enables rapid and selective identification of low-level unknown mutations.
COLD-PCR-enhanced high-resolution melting enables rapid and selective identification of low-level unknown mutations.
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DOI：
10.1373/clinchem.2009.131029
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发表时间：
2009-12
影响因子：
9.3
通讯作者：
Makrigiorgos GM
中科院分区：
文献类型：
--
作者：
Milbury CA;Li J;Makrigiorgos GM
临床样品的分析通常需要鉴定野生型DNA中的低水平体细胞突变;然而，该方法的选择性和灵敏度通常是有限的。COLD-PCR（在较低变性温度下的共扩增-PCR）是一种新形式的PCR，其将含有突变的扩增子富集到足以直接测序的浓度;然而，测序本身仍然是一种昂贵的突变筛选方法。相反，高分辨率熔解（HRM）是一种快速，廉价的扫描方法，但它不能特异性地识别检测到的突变。为了能够富集、快速扫描和鉴定低水平的未知突变，我们将COLD-PCR与HRM突变扫描相结合，然后对阳性样品进行测序。将含有突变的细胞系DNA连续稀释成野生型DNA和来自含有低水平突变的人肺腺癌的DNA样品，通过COLD-PCR和通过常规PCR扩增TP 53（肿瘤蛋白p53）外显子6-8，并比较2种方法。HRM分析用于筛选扩增子的突变;对突变阳性扩增子进行测序。稀释实验表明COLD-PCR/HRM的选择性提高了约6至20倍。常规PCR/HRM显示突变检测限约为2%至10%，而COLD-PCR/HRM显示突变与野生型比率的限制约为0.1%至1%。HRM分析肺腺癌样本后，我们检测到7个突变的外显子7的两种PCR方法，然而，在外显子8中，我们检测到9个突变的COLD-PCR扩增子，相比之下，只有6个突变的常规PCR扩增子。此外，94%的HRM检测到的突变与COLD-PCR扩增子成功测序，相比之下，50%与传统的PCR扩增子。COLD-PCR/HRM提高了HRM的突变扫描能力，并将高选择性、方便性和低成本与对临床样品中未知低水平突变进行测序的能力相结合。
Analysis of clinical samples often necessitates identification of low-level somatic mutations within wild-type DNA; however, the selectivity and sensitivity of the methods are often limiting. COLD-PCR (coamplification at lower denaturation temperature–PCR) is a new form of PCR that enriches mutation-containing amplicons to concentrations sufficient for direct sequencing; nevertheless, sequencing itself remains an expensive mutation-screening approach. Conversely, high-resolution melting (HRM) is a rapid, inexpensive scanning method, but it cannot specifically identify the detected mutation. To enable enrichment, quick scanning, and identification of low-level unknown mutations, we combined COLD-PCR with HRM mutation scanning, followed by sequencing of positive samples. Mutation-containing cell-line DNA serially diluted into wild-type DNA and DNA samples from human lung adenocarcinomas containing low-level mutations were amplified via COLD-PCR and via conventional PCR for TP53 (tumor protein p53) exons 6–8, and the 2 approaches were compared. HRM analysis was used to screen amplicons for mutations; mutation-positive amplicons were sequenced. Dilution experiments indicated an approximate 6- to 20-fold improvement in selectivity with COLD-PCR/HRM. Conventional PCR/HRM exhibited mutation-detection limits of approximately 2% to 10%, whereas COLD-PCR/HRM exhibited limits from approximately 0.1% to 1% mutant-to-wild-type ratio. After HRM analysis of lung adenocarcinoma samples, we detected 7 mutations by both PCR methods in exon 7; however, in exon 8 we detected 9 mutations in COLD-PCR amplicons, compared with only 6 mutations in conventional-PCR amplicons. Furthermore, 94% of the HRM-detected mutations were successfully sequenced with COLD-PCR amplicons, compared with 50% with conventional-PCR amplicons. COLD-PCR/HRM improves the mutation-scanning capabilities of HRM and combines high selectivity, convenience, and low cost with the ability to sequence unknown 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：
9.3
作者：
Milbury CA;Li J;Makrigiorgos GM
通讯作者：
Makrigiorgos GM
高分辨率熔化用于TP53外显子5-8的突变扫描。
影响因子：
3.8
作者：
Krypuy, Michael;Ahmed, Ahmed Ashour;Etemadmoghadam, Dariush;Hyland, Sarah J.;deFazio, Anna;Fox, Stephen B.;Brenton, James D.;Bowtell, David D.;Dobrovic, Alexander
通讯作者：
Dobrovic, Alexander
影响因子：
11.5
作者：
Fukui, Tomoya;Ohe, Yuichiro;Tamura, Tomohide
通讯作者：
Tamura, Tomohide
影响因子：
7.5
作者：
Delaney, David;Diss, Tim C.;Flanagan, Adrienne M.
通讯作者：
Flanagan, Adrienne M.