DHPLC和DDF检测散发性卵巢癌p53基因突变的应用

目的：借助变性高效液相色谱仪（DHPLC）和荧光双脱氧指纹印迹法（F-DDF）检测散发性卵巢癌p53基因突变。方法：提取50例散发性卵巢癌标本DNA，使用DHPLC和F-DDF检测p53基因外显子5～8突变，其中测序证实18例p53基因外显子5～8有突变，F-DDF使用ddGTP和ddTTP扩增，Cy5标记其引物，DHPLC使用美国Transgenomic公司的WAVE系统。结果：18例突变都可以使用DHPLC的方法发现（敏感性100％），无假阳性和假阴性，FDDF检测出16例突变标拳（敏感性78％）。就肿瘤的异质性而言（未知的突变型／野生型DNA比率），即使突变性DNA被野生性DNA稀释，DHPLC仍然能发现突变，说明这一方法的高度敏感性，根据等位基因丢失（LOH）判断，即使只有5％的突变DNA，DHPLC仍能发现异常，但这一异常难以被测序证实。结论：对于异质性肿瘤而言，DHPLC检测突变是可靠的，F-DDF的敏感性和特异性相时较低，这两种方法都能检测基因突变，DHPI上的缺点是费用昂贵，操作较复杂，而F-DDF优势在于不需要特辣的仪器及操作简便。可作为临床筛查基因突变的方法．

Validation of Two New Methods for Mutation Detection in Sporadic Ovarian Cancer

Objective: Two lately developed methods for mutation detection, DHPLC (denaturing high-performance liquid chromatography) and F-DDF (fluorescent dideoxy fingerprinting) have been tested for their suitability by analysing heterogeneous tumor material. Methods:For comparison of both techniques 50 sporadic ovarian cancers (OC) including 18 known p53 mutations (blind study) were examined analysing exons 5-8 by DHPLC and by F-DDF (total 200 PCR-fragments). The conventional DDF was adapted to an ALF express?DNA sequencing system using Cy5-labeled primers for single-strand sequencing reaction with ddGTP or ddTTP. For DHPLC analyses the WAVE system (Transgenomic) was used. Results:All 18 mutations could be detected by DHPLC analysis (sensitivity: 100%) without any false positive/negative results whereas 4 mutations were missed by F-DDF (sensitivity: 78%). Interpretation of F-DDF results is more difficult therefore a higher number of false positive results were obtained. Conclusion:DHPLC is a reliable high throughput technique for mutation detection in heterogeneous tumor material. Mutation detection by F-DDF revealed only a sensitivity of 78% and a lower specificity. However in contrast to DHPLC analysis no additional equipment is necessary for F-DDF analysis.