基于水凝胶的基因组DNA固定法研究  

Gel immobilization of human genome

作　　者：潘映秋[1] 张伟[2] 陈枢青[2] 

机构地区：[1]温州医学院附属浙江省台州医院中心实验室,浙江临海317000 [2]浙江大学药学院药理毒理与生化药学研究所,浙江杭州310058

出　　处：《浙江大学学报（医学版）》2013年第1期6-13,共8页Journal of Zhejiang University(Medical Sciences)

基　　金：国家自然科学基金资助项目(No.81273578);台州市科技计划资助项目(No.091KY04)

摘　　要：目的:通过对人基因组DNA固定化的研究,探讨特异性高的单点共价固定方法,实现对人基因组DNA的反复利用。方法:选取Mirzabekov等发明的DNA-水凝胶共聚化学法作为基因组DNA固定方法,使用甲基丙烯酰胺凝胶作为固相支持载体,以(CH2)6-NH2氨基末端修饰的PCR产物及甲酸随机断裂修饰的pGEM-T-HLA-G质粒为模板,使用聚丙烯酰胺凝胶电泳检测其固定效率,并通过PCR法检测该固定方法的热稳定性。将该法初步应用于人类基因组DNA的固定和反复利用,对基因组DNA扩增的片段进行测序,排除突变可能。结果:使用甲基丙烯酰胺凝胶作为固相支持载体,实现了对质粒DNA以及人基因组DNA的固定和反复使用,该方法可以经受16轮的PCR扩增(每轮36个循环)。测序结果证实基因组DNA的扩增片段未发生突变。结论:甲基丙烯酰胺凝胶法固定DNA的热稳定性好,特异性高,可用于全基因组测序及SNP检测。Objective： To develop a solid phase PCR method by covalent single point immobilization for recycle utilization of human genome. Methods： Polymethacrylamide gel was selected as a solid PCR carrier based on DNA-hydrogel copolymer chemistry presented by Mirzabekov. （CH2 ）6 NH2 aminomodified PCR product and randomly fractured formic acid-modified plasmid pGEM-T-HLA-G were used as templates. The specificity of the attachment chemistry was characterized by acrylamide gel electrophoresis, and the thermal stability of method was demonstrated by PCR. This method was applied for the recycle utilization of human genome. Sequencing was used to exclude the possibility of introduced mutations during modification and immobilization procedures. Results： The PCR detections of plasmid DNA and human genome DNA immobilized by polymethacrylamide gel was successful. The thermal stability of method was successfully demonstrated by performing PCR after 16 rounds of standard 36 PCR cycles. And the sequencing was found no mutation. Conclusion： The DNA immobilization method with polymethacrylamide gel as a solid phase carrier is stable and specific, which can be a possible approach for realizing recycle utilization of human genome for whole-genome sequencing and SNP detection.

关 键 词：DNA 分析 基因组 聚合酶链反应 基因扩增 水凝胶 电泳 聚丙烯酰氨凝胶 

分 类 号：Q78[生物学—分子生物学]