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目的 验证胶体金标记 /银染信号放大法在寡核苷酸芯片检测人肥厚型心肌病 (HCM )基因突变中对单碱基错配的灵敏性和特异性。方法 不对称PCR扩增、生物素标记待检测的人 β 肌凝蛋白重链基因片断 ,与构建的低密度HCM寡核苷酸芯片杂交 ,胶体金标记 /银染放大法检测 7种HCM恶性突变位点 ,观察胶体金标记 /银染信号放大法对单碱基错配形式的判别率。结果 胶体金标记 /银染信号放大法在寡核苷酸芯片中的单硝基突变的平均判别率为 16 2 ,可以鉴别所有的单碱基错配和三碱基缺失。结论 胶体金标记 /银染信号放大法代替荧光标记法用于寡核苷酸芯片靶标分子的标记和结果检测 ,可以区分所有的单碱基错配形式 ,实现了芯片杂交结果的普通光学显微镜检测 ,提高了DNA芯片的实用性。
Objective To verify the sensitivity and specificity of colloidal gold labeling / silver staining signal amplification for single base mismatch in the detection of human hypertrophic cardiomyopathy (HCM) gene mutation by oligonucleotide microarray. Methods Asymmetric PCR amplification, biotin labeling of human β-myosin heavy chain gene fragment to be detected, hybridization with the constructed low density HCM oligonucleotide chip, colloidal gold labeling / silver staining amplification method detected seven kinds of HCM malignant mutations Site, observe colloidal gold mark / silver staining signal amplification of the single base mismatch form of discrimination. Results The average discrimination of single-nitro mutagenesis in colloidal gold / silver staining was 16 2, which could identify all single-base mismatches and triple-base deletions. Conclusion Colloidal gold labeling / silver staining signal amplification method instead of fluorescent labeling method for oligonucleotide chip target molecule labeling and detection of results, you can distinguish all single base mismatch forms, to achieve the chip hybridization results of ordinary optical microscopy , Improve the practicality of DNA chip.