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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,F-DDF检测出16例突变标本(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对较低,这两种方法都能检测基因突变,DHPLC的缺点是费用昂贵,操作较复杂,而F-DDF优势在于不需要特殊的仪器及操作简便,可作为临床筛查基因突变的方法。
Objective: To detect p53 gene mutation in sporadic ovarian cancer by denaturing high performance liquid chromatography (DHPLC) and fluorescent dideoxy fingerprinting (F-DDF). Methods: Fifty cases of sporadic ovarian cancer DNA were extracted. The exon 5-8 mutations of p53 gene were detected by DHPLC and F-DDF. The mutation of exon 5-8 in 18 cases of p53 gene was confirmed by sequencing, and the mutation of ddGTP And ddTTP amplification, Cy5 labeled primers, DHPLC using the United States Transgenomic’s WAVE system. RESULTS: All 18 mutations were detected by DHPLC (100% sensitivity), no false positives and false negatives, and 16 of them were detected by F-DDF (sensitivity 78%). In terms of tumor heterogeneity (unknown mutant / wild-type DNA ratios), DHPLC can still detect mutations even if the mutant DNA is diluted with wild-type DNA, indicating the high sensitivity of this method, based on the loss of alleles (LOH), even if only 5% of the mutant DNA, DHPLC still found abnormal, but this anomaly is difficult to confirm by sequencing. Conclusions: For heterogeneous tumors, DHPLC detection of mutations is reliable and F-DDF has relatively low sensitivity and specificity. Both of these methods are capable of detecting gene mutations. The disadvantage of DHPLC is its high cost and complicated operation , While the advantage of F-DDF is that it does not require special equipment and is easy to operate. It can be used as a clinical screening method for gene mutation.