【摘要】 针对临床标本基因组DNA的提取方法缺乏广泛适用性,提取步骤繁琐,需要进行离心操作,并且提取过程中会用到苯酚、氯仿等有机试剂,会对操作人员有一定的危害性等不足,本研究拟建立一种适用于临床标本的基因组DNA快速提取方法。在传统的基因组DNA提取试剂和方法的基础上,本研究采用二氧化硅修饰的超顺磁珠设计了一种基因组DNA快速提取方法;探讨磁珠用量、裂解液p H、盐酸胍浓度等因素对基因组DNA提取效率的影响并采用凝胶电泳实验进行验证。当磁珠用量在50~100μg/100 mg样品,裂解液p H约为6,盐酸胍的浓度为6 mol/L时基因组DNA提取效果好、效率高,且磁珠的用量与吸附表面积成正比,但达到一定用量后不会增加提取量,对于100 mg肺组织,适宜的磁珠用量为80μg。此基因组DNA提取方法高效省时,简便快捷,性价比高,适用临床大量样本基因组DNA提取。更多还原

【Abstract】 The method of extracting genomic DNA from clinical specimens lacks wide applicability and often uses organic reagents such as phenol and chloroform are used in the extraction process. The extraction process is complicated and cumbersome,and the samples need to be centrifuged. This study aims to establish a rapid extraction method of genomic DNA suitable for clinical specimens. Based on traditional extraction reagents and extraction methods of genomic DNA,a rapid extraction method of genomic DNA was designed by using super paramagnetic beads modified with silica. Our study focused on the effects of magnetic beads concentration,pH of lysate and concentration of guanidine hydrochloride on the extraction efficiency of genomic DNA. The optimized method was verified by gel electrophoresis experiments. The results showed that the genomic DNA extraction effect was best and the efficiency was highest when the amount of magnetic beads was 50-100 μg/100 mg sample,the pH of the lysate was around 6,and the concentration of guanidine hydrochloride was 6 mol/L. The amount of magnetic beads was proportional to the adsorption surface area,but the amount of extracted DNA was no longer increased when the magnetic beads were excessive. For 100 mg of lung tissue,the appropriate amount of magnetic beads is 80 μg. This extraction method of genomic DNA is efficient,time-saving,simple,fast,and cost-effective,and is suitable for genomic DNA extraction from a large number of clinical specimens.更多还原