Analysis of the allele-specific PCR method for the detection of neoplastic disease
Analysis of the allele-specific PCR method for the detection of neoplastic disease
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DOI：
10.1097/00019606-199702000-00008
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发表时间：
1997-02-01
影响因子：
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通讯作者：
Sorenson, GD
中科院分区：
文献类型：
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作者：
Rhodes, CH;Honsinger, C;Sorenson, GD
PCR 检测是否存在突变 K-ras 或 p53 序列可能可用作肿瘤诊断的敏感测试。技术挑战是设计足够灵敏的检测方法，以检测一些突变 DNA 分子，同时又足够特异，以保证 10(5) 或 10(6) 倍过量的正常 DNA 不会产生假阳性信号。我们使用两个不同 DNA 序列环境（K-ras 密码子 12 和 p53 密码子 273）中所有 12 种可能的错配，将等位基因特异性 PCR (ASA) 的检测限确定为特定错配的函数。根据错配的身份，错配模板的扩增量比完美匹配模板少 10(2)-10(4) 倍。换句话说，如果突变等位基因占该位点总 DNA 的 1-0.01% 以上，则 ASA 可以检测到该突变等位基因。肽核酸 (PNA) 钳夹用于改进 K-ras ASA 测定。使用与正常序列互补的 PNA 来抑制野生型 DNA 的扩增，从而实现突变序列的选择性扩增。 PNA 钳夹和 ASA 显着提高了灵敏度和特异性，允许检测用正常人类 DNA 300,000 倍过量稀释的肿瘤 DNA。
PCR assays for the presence of mutant K-ras or p53 sequences are potentially useful as sensitive tests for tumor diagnosis. The technical challenge is to design assays sensitive enough to detect a few molecules of mutant DNA yet sufficiently specific that a false positive signal is not produced by a 10(5)- or 10(6)-fold excess of normal DNA. We determined the detection limit of allele-specific PCR (ASA) as a function of the particular mismatch involved using all 12 possible mismatches in two different DNA sequence contexts (K-ras codon 12 and p53 codon 273). Depending on the identity of the mismatch, mismatched template was amplified 10(2)-10(4)-fold less than perfectly matched template. In other words, a mutant allele could be detected by ASA if it represented >1-0.01% of the total DNA from that locus. Peptide nucleic acid (PNA) clamping was used to improve the K-ras ASA assay. Selective amplification of mutant sequences was achieved using a PNA complementary to the normal sequence to inhibit the amplification of wild-type DNA. PNA clamping followed by ASA resulted in significant improvement in sensitivity and specificity, permitting the detection of tumor DNA diluted with a 300,000-fold excess of normal human DNA.