胶体金标记/银染信号放大法在寡核苷酸芯片检测人HCM突变中的应用

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。

Detection of known mutations in hypertrophic cardiomyopathy using oligonucleotide microarrays with nanogold particles/silver-stained targets

Objective To identify 7 known hypertrophic cardiomyopathy-related mutations in a low-density oligonucleotide microarrays assisted by nanogold particle/silver labelling. Methods The hybridized targets, amplified from 11 plasmids containing wild type or mutation sequences of MYH7 and genomic DNA of a healthy person, were prepared by single-step biotin labeled asymmetric PCR. The signal generated by this method resulted from the precipitation of silver onto nanogold particles was bound to streptavidin. The latter was used for detecting biotinylated targets. Results A total of 6 single base substitutions and a trinucleotide deletion could be identified unambiguously, and the average discrimination ratio (Q mut ) for artificial heterozygous samples was as high as 16.2. Conclusion The sensitivity of this method by microscopy is comparable to detection in fluorescence using a confocal scanner. This method is less expensive and versatile.