Estimating mutant microsatellite allele frequencies in somatic cells by small-pool PCR
Estimating mutant microsatellite allele frequencies in somatic cells by small-pool PCR
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DOI：
10.1016/j.ygeno.2004.03.007
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发表时间：
2004-08-01
期刊：
影响因子：
4.4
通讯作者：
Brown, BW
中科院分区：
文献类型：
--
作者：
Coolbaugh-Murphy, M;Maleki, A;Brown, BW
通过将DNA分成仅含有单个基因组量的DNA的多个小池来鉴定微卫星不稳定性（MSI）导致将祖基因和低频突变等位基因捕获到池中，在池中它们可以在PCR后被鉴定和计数。统计方法确定这些泊松分布的等位基因的频率和频率之间的显着差异。结果表明，标准PCR方法不可能达到一定的灵敏度和定量水平。使用来自肿瘤中具有高水平MSI的结肠癌患者的材料，我们还提出了进行此类研究的分子和机器人方法。验证实验表明突变体可在高于背景>0.03的频率下检出。在肿瘤组织中获得的频率（>0.25）符合该方法的预期。在携带用于错配修复的生殖系突变的患者的组成性组织中检测到显著水平的MSI，这表明该程序的机械和临床应用。(C)2004年爱思唯尔公司All rights reserved.
Identifying microsatellite instability (MSI) by partitioning DNA into multiple small pools containing only single genome amounts of DNA results in trapping both progenitor and low-frequency mutant alleles into pools where they can be identified and counted following PCR. Statistical approaches determining both the frequencies and the significant differences between frequencies of these Poisson-distributed alleles are presented. Results indicate a level of sensitivity and quantification not possible by standard PCR methods. Using material from colon cancer patients with high levels of MSI in their tumors, we also present the molecular and robotic methods for carrying out such studies. Validation experiments indicated mutants detectable at frequencies >0.03 above background. Frequencies obtained in tumor tissue (>0.25) met the expectations of the approach. Significant levels of MSI were detected in the constitutive tissue of the patient carrying a germ-line mutation for mismatch repair, suggesting both mechanistic and clinical applications of the procedure. (C) 2004 Elsevier Inc. All rights reserved.