直接测序法检测结肠癌K-ras基因的灵敏度确认及评估

目的探讨直接测序法检测K-ras基因的灵敏度及可行性。方法用已知K-ras基因突变型及野生型细胞株DNA按比例混合,用直接测序法检测K-ras基因突变情况,对实验敏感度进行评估。结果正反向测序结果均显示,含突变型K-ras基因DNA 5%的5个复孔中,均未能明确检出突变,而含突变型基因DNA≥10%的所有样本及其复孔都检测出了突变;突变型的基因碱基T峰与野生型的对应碱基G峰的比例,各组间有明显差异（P均〈0.05）,且峰高随突变型DNA的质量浓度增加而增加,二者存在明确相关性（相关系数r=-1.00,相伴概率〈0.05）。结论直接测序法检测突变型DNA,当敏感度为10%时,特异性达到100%,且电泳图上突变峰与野生峰的高低比例可对细胞比例进行半定量评估,可以满足临床检测与研究。

Sensitivity confirmation and assessment of the direct sequencing for the K-RAS gene in colorectal cancers

Objective To investigate the sensitivity and feasibility of K-ras gene detected by direct sequencing.Methods Using the certified cell colons containing wild and mutant type K-ras gene,the gradient concentrations were got,the mutation of samples were tested by the direct sequencing method,the sensitivity of the experiment was estimated.Results Both the forword and reverse sequencing showed that each of the five tubes which had the 5% mutant DNA showed no K-ras mutation,and all the tubes which contain 10% mut...