Barnard R, Futo V, Pecheniuk N, Slattery M, Walsh T
Queensland University of Technology, Gardens Point, Brisbane, Australia.
Biotechniques. 1998 Oct;25(4):684-91. doi: 10.2144/98254dt08.
PCR-based cancer diagnosis requires detection of rare mutations in k-ras, p53 or other genes. The assumption has been that mutant and wild-type sequences amplify with near equal efficiency, so that they are eventually present in proportions representative of the starting material. Work on factor IX suggests that this assumption is invalid for one case of near-sequence identity. To test the generality of this phenomenon and its relevance to cancer diagnosis, primers distant from point mutations in p53 and k-ras were used to amplify wild-type and mutant sequences from these genes. A substantial bias against PCR amplification of mutants was observed for two regions of the p53 gene and one region of k-ras. For k-ras and p53, bias was observed when the wild-type and mutant sequences were amplified separately or when mixed in equal proportions before PCR. Bias was present with proofreading and non-proofreading polymerases. Mutant and wild-type segments of the factor V, cystic fibrosis transmembrane conductance regulator and prothrombin genes were amplified and did not exhibit PCR bias. Therefore, the assumption of equal PCR efficiency for point mutant and wild-type sequences is invalid in several systems. Quantitative or diagnostic PCR will require validation for each locus, and enrichment strategies may be needed to optimize detection of mutants.
基于聚合酶链反应（PCR）的癌症诊断需要检测k-ras、p53或其他基因中的罕见突变。一直以来的假设是，突变型和野生型序列的扩增效率几乎相等，因此它们最终呈现出与起始材料成比例的比例。关于凝血因子IX的研究表明，对于一种序列相似度极高的情况，这一假设是无效的。为了测试这种现象的普遍性及其与癌症诊断的相关性，使用了远离p53和k-ras基因点突变的引物来扩增这些基因的野生型和突变型序列。在p53基因的两个区域和k-ras基因的一个区域观察到对突变体PCR扩增存在显著偏差。对于k-ras和p53，当野生型和突变型序列分别扩增或在PCR之前以相等比例混合时，均观察到偏差。在校读和非校读聚合酶存在的情况下均存在偏差。凝血因子V、囊性纤维化跨膜传导调节因子和凝血酶原基因的突变型和野生型片段被扩增，且未表现出PCR偏差。因此，点突变型和野生型序列PCR效率相等的假设在多个系统中是无效的。定量或诊断性PCR需要对每个位点进行验证，可能需要富集策略来优化突变体的检测。
