突变敏感性分子开关对乳腺癌DBC2基因7776C〉T突变的快速检测

Mutation-sensitive molecular switch in rapidly detecting the mutation of the DBC2 gene 7776C 〉 T

目的应用硫化磷酸修饰的碱基特异性引物和高保真DNA聚合酶所构成的突变敏感性分子开关技术结合琼脂糖凝胶电泳，快速检测乳腺癌及乳腺纤维腺瘤中DBC2基因7776C〉T突变频率。方法设计2对分别与DBC2基因7776位点突变碱基（C）和野生碱基（T）配对的硫化磷酸修饰的引物，硫化磷酸修饰等位基因位点特异性引物与组织DNA样本完全配对时，能被高保真聚合酶延伸，而硫化磷酸修饰等位基因位点特异性引物与组织DNA样本不完全配对时，不能被高保真聚合酶延伸。用此突变敏感性分子开关技术对85例乳腺癌组织DNA样本及10例乳腺纤维腺瘤组织DNA样本进行PCR检测，并利用凝胶成像系统对PCR产物进行分析。结果利用分子开关技术在85例乳腺癌组织DNA样本中检测出DBC2基因7776C〉T突变率为2．4％（2／85），10例乳腺纤维腺瘤未发现该突变。结论该突变敏感性分子开关技术结合琼脂糖凝胶电泳可快速检测乳腺癌DBC2基因7776C〉T突变。突变敏感性分子开关技术可在单碱基水平对相关基因突变进行特异性检测，提示其在乳腺癌等基因突变检测中具有广阔的应用前景。

Objective To rapidly detect the mutation frequgncy of DBC2 gene 7776C 〉 T in breast canc- er and breast fibroadenoma by applying the mutation-sensitive molecular switch （ comprised of high-fidelity DNA polymerase and phosphorothioate-modified allele-specific primers ） and agarose get electrophoresis. Methods Allelic specific primers targeting mutation type and wild type were designed with the primers＇3＇terminal phospho- rothioate modification. When the primers matched with the tissue DNA, the primers could be extended with high- fidelity polymerase; when they mismatched with the tissue DNA, the primers could not be extended. DNA sam- ples from 85 cases of breast cancer and 10 cases of breast fibroadenoma tissues were chosen and analyzed by PCR amplifications mediated by high-fidelity DNA polymerase. Gel imaging system was employed to make analysis of PCT products. Results The mutation-sensitive molecular switch system showed that the mutation rate of 7776C 〉 T was 2.4% （2/85）in the 85 cases of breast cancer, and no mutation was found in the 10 cases of breast fibro- adenoma. Conclusions The mutation-sensitive molecular switch combined with agarose gel electrophoresis can rapidly detect the mutation of breast cancer DBC2 gene 7776C 〉 T. It is applicable in single nucleotide polymor- phisms assay and has enormous application value in detecting gene mutation.