摘要
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 196 bp长的 PCR片段用毛细管电泳可在 2 5 min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 。
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.
出处
《中华医学遗传学杂志》
EI
CAS
CSCD
2001年第4期317-321,共5页
Chinese Journal of Medical Genetics
基金
国家自然科学基金 (3970 0 1 71 )&&
