改良盐析法提取全血基因组DNA的影响因素研究  

Influencing factors on Genomic DNA extraction from whole blood using modified salting-out method

作　　者：张帅[1] 徐锐[1] 张强[1] 邓勇[1] 刘阳[1] 赖翼[1] 

机构地区：[1]成都医学院检验医学院,四川成都610083

出　　处：《中国现代医学杂志》2012年第35期42-46,共5页China Journal of Modern Medicine

基　　金：成都医学院自然基金(No:CYZ08-015;06Z2006-001)

摘　　要：目的观察红细胞裂解时间、DNA纯化方式以及DNA干燥时间对改良盐析法提取全血基因组DNA的影响。方法改良盐析法提取全血基因组DNA时,分别采用不同的红细胞裂解时间、DNA纯化方式以及DNA干燥时间。使用流式细胞术检测红细胞裂解后的细胞总数及死细胞比例,紫外分光光度计检测DNA的浓度和纯度,琼脂糖凝胶电泳检测PCR扩增产物。结果红细胞裂解时间从10 min延长至120 min,死细胞比例随时间延长而增加,但细胞总数、DNA产量与纯度无明显变化;采用异丙醇沉淀法纯化DNA得到的DNA产量与纯度均显著高于使用DNA分离柱纯化的DNA;DNA干燥时间从120 min缩短至30 min,既不影响DNA的产量与纯度,也不影响后续PCR扩增实验。结论改良盐析法提取全血基因组DNA时,红细胞裂解时间可延长至120 min,DNA干燥时间可缩短至30 min,异丙醇沉淀法优于DNA分离柱纯化的DNA。[Objective] To observe the effects of erythrocyte lysis time, DNA purification method and DNA drying time on genomic DNA extraction from whole blood using modified salting-out method. [Meth- ods] Different erythrocyte lysis time, DNA purification method and DNA drying time were used for genomic DNA extraction from whole blood using modified salting-out method. Then, the cell counts and percentage of dead cells were observed by flow cytometry after erythrocyte lysis. The DNA concentration and OD260 / OD280 were observed by ultraviolet spectrophotometer. Finally, PCR products were detected by agarose gel electrophoresis. [Results] The percentage of dead cells increased but the cell counts, the DNA production and OD260 / OD280 had no significant change when erythrocyte lysis time used from 10 rain to 120 rain. The DNA production and OD260 / OD280 purified by isopropanol were greater than purified by silica membrane column. DNA drying time from 30min to 120 min had no influence on DNA production, OD260 / OD280 and PCR. [Conclusions] When modified salting-out method was used for genomic DNA extraction from whole blood, erythrocyte lysis time could be increased to 120 min, DNA drying time could be decreased to 30 min, and DNA purified by isopropanol was better than purified by silica membrane column.

关 键 词：全血基因组DNA提取 改良盐析法 红细胞裂解时间 DNA纯化方式 DNA干燥时间 

分 类 号：Q789[生物学—分子生物学]