Rhodes C H, Honsinger C, Porter D M, Sorenson G D
Department of Pathology, Dartmouth Hitchcock Medical Center, Lebanon, New Hampshire 03756, USA.
Diagn Mol Pathol. 1997 Feb;6(1):49-57. doi: 10.1097/00019606-199702000-00008.
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.
用于检测突变型K-ras或p53序列的聚合酶链反应（PCR）检测方法，作为肿瘤诊断的敏感检测手段具有潜在用途。技术上的挑战在于设计出足够灵敏的检测方法，既能检测到少量的突变DNA分子，又要具有足够的特异性，以避免在存在10⁵或10⁶倍过量正常DNA的情况下产生假阳性信号。我们使用两种不同DNA序列背景（K-ras密码子12和p53密码子273）中的所有12种可能错配情况，确定了等位基因特异性PCR（ASA）的检测限与特定错配之间的函数关系。根据错配的性质，错配模板的扩增倍数比完全匹配模板少10²至10⁴倍。换句话说，如果突变等位基因占该基因座总DNA的比例大于1%至0.01%，则可通过ASA检测到。肽核酸（PNA）钳夹技术被用于改进K-ras ASA检测方法。通过使用与正常序列互补的PNA抑制野生型DNA的扩增，实现了突变序列的选择性扩增。PNA钳夹后再进行ASA，显著提高了灵敏度和特异性，能够检测出被300,000倍过量正常人DNA稀释的肿瘤DNA。
