用生物素标记DNA探针检测Southern印迹上人类单拷贝基因

DETECTION OF HUMAN SINGLE COPY GENE BY SOUTHERN BLOT HYBRIDIZATION WITH BIOTINYLATED PROBES

本文用缺口转移进行生物素标记探针的Southern印迹杂交,以检测人类基因组单拷贝序列的方法学研究。结果表明,可以检出474fg实际λDNA量,取2.5μg人基因组DNA经Southern杂交后,可以检出人类基因组中单拷贝基因,其检出的实际DNA量仅为5.83pg。并将该方法初步用于肌营养不良症的分子诊断。

DNA probes were labelled with biotin-11-dUTP by standard nick translation protocol and hybridized to target DNA sequences on Southern blot. the biotinylated probes were detected by a two-step procedure with streptavidin and biotinylated calf intestinal alkaline phosphatase. the results showed that, 1. In out experimental conditions, the detectabl(?) concentration of labelled probes was as low as 0.53pg/μl. 2. 474fg of λ phage DNA would be detected by Southern Hy. bridization. 3. 5.83pg of human single copy DNA could be detected on Southern blot hybridization when the loading sample was 2.5μg genomic DNA; the hybridization bands were clearly distinquished. 4. As compared with nitrocel lulose filter the nylon membrane (Hybond-N) could reach higher sensitivity. The results also demonstrated the sensitvity of the biotin-labelled probes and their detection system to detect the single copy gene of human DNA as exemplified by the preliminary application to the case of human X-linked progressive muscular dystrophy (DMD/BMD) .