基因组DNA快速提取方法的探讨与应用

Discussion and Application about Rapidly Extracting of Genome DNA

目的探讨以硫氰酸胍作为组织细胞裂解液的可行性，并建立基因组DNA快速提取方法。方法利用硫氰酸胍等作为裂解消化液，分别从组织、细胞和全血中提取基因组DNA。采用紫外分光光度仪检测基因组DNA含量及纯度，琼脂糖凝胶电泳检测基因组DNA的完整性，以提取的基因组DNA作为模板进行PCR扩增GAPDH基因，并用该方法检测凋亡细胞DNA ladder。结果基因组DNA提取时间短（耗时30～50min），基因组DNA含量85pg／mg（组织），52μg／1×10^7细胞，27μg／ml（血液）。A260nm／A280nm比值在1．8～1．9之间，电泳显示基因组DNA完整性好，无蛋白质和RNA污染，基因组DNA长度大于48kb。PCR扩增出306bpGAPDH目的基因，肿瘤细胞经抗癌基因诱导凋亡后琼脂糖凝胶电泳可见180-200bp典型的凋亡带。结论硫氰酸胍可作为基因组DNA提取裂解消化液，该方法具有简便、快捷、稳定性好等特点。

Objective To explore the feasibility of using guanidinium thiocyanate as histiocyte cell lysate,and found the way of rapidly extracting genome DNA. Methods Separately extracted genome DNA from organizations ,cells and whole blood using guanidinium thiocyanate and others as alimentary juice. Detected the contect and purity of genome DNA by means of UV Spectrophotometer,detected its integrity by means of agarose gel electrophoresis,amplifact GAPDH gene by PCR using the extracted genome DNA as template,and detected apoptotic cells DNA ladder. Results Extracting of genome DNA quickly（time-consuming 30-50 min）,the content of genome DNA was 85 9g/mg（organizations）,52μg/l × 10^7（cells） and 27 μg/ml （whole blood）. The ratio of A260nm/A280nm was between 1.8 and 1.9. Electrophoretic displaied that integrity of genome DNA was good ,without pollution of protein and RNA ,its length exceed 48 kb. Amplification 306 bp GAPDH purpose gene by PCR. Topical apoptosis zone of 180-200bp could be seen in agarose gel electrophoresis 48 hours after the tumor cells transfection of anti-oncogene. Conclusion Guanldinium thlocyanate can be used as lysate in extracting of genome gene,and the method is simple and quick with good stability.