毛细管电泳快速PCR-SSCP分析方法研究

目的：建立一种快速、简便、敏感检测人类基因组DNA点突变的方法。方法：用人工诱发的带有单个碱基改变的PCR片段作目的基因，借助高效毛细管电泳技术对PCR片段作SSCP分析。电泳采用中性毛细管涂层柱和4％线性聚丙烯酰胺凝胶电泳缓冲液，紫外检测仪检测。结果：196bp长的PCR片段用毛细管电泳可在25min内分离出双链及单链峰，仅有一个碱基差异的两条单链DNA也能得到较好地分离。结论：毛细管电泳技术可用于作PCR－SSCP分析，具有快速、灵敏、准确、重复性好等特点，为筛选基因点突变提供了一种十分有效的方法。

A rapid and simple detection of DNA fragment with point mutation by capillary electrophoresis

OBJECTIVE: To establish a rapid and simple method with high efficiency in detecting point mutation of genomic DNA. METHODS: Four DNA fragments that were different from each other in only one based were amplified by using primers with artificial point mutation based on the sequence of exon 7 of p53 gene, and then were separated by capillary electrophoresis(CE). The neutral coated capillary and 4% linear polyacrylamide gel buffer were used, and the wave length of ultraviolet detector was 254 nm. RESULTS: A homozygous 196 bp DNA fragment can be separated into one dsDNA peak and two ssDNA peaks within 25 minutes, and the heterozygous 196 bp DNA fragments that were made with mixed wild type and mutated DNA can be separated into one dsDNA peak and three ssDNA peaks. The three ssDNA fragments that differ in one nucleotide can be easily separated with good resolution. CONCLUSION: CE technique is rapid, sensitive, accurate and well reproducible. It is an efficient and reliable method for rapidly screening point mutation.