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| 应用改良PCR法构建高GC含量启动子多位点定点突变载体 | |
| 引用本文： | 姜鸾,邵蕾,胡旸,董凌月. 应用改良PCR法构建高GC含量启动子多位点定点突变载体[J]. 首都医科大学学报, 2010, 31(1): 88-92 |
| 作者姓名： | 姜鸾 邵蕾 胡旸 董凌月 |
| 作者单位： | 姜鸾(首都医科大学2005级临床医学专业七年制3班);邵蕾(首都医科大学2005级临床医学专业五年制3班);胡旸(首都医科大学2005级临床医学专业五年制4班);董凌月(首都医科大学基础医学院细胞生物学系) |
| 摘 要： | 目的利用StrataGene公司的QuickChangeTM定点突变试剂盒对高GC含量的人肝刺激因子启动子进行多位点定点突变。方法采用含有多位点突变的引物扩增启动子片段,通过加入不同浓度的DMSO,降低高GC含量模板及引物的解链温度,利用乙醇沉淀提高酶切产物的浓度。结果经测序鉴定成功构建5个含有不同突变位点的hHSS启动子突变载体。结论这种改良的PCR方法提高了对高GC含量启动子进行扩增的效率,具有快速、简便、经济、成功率高的特点,是值得推广的多位点定点突变载体构建方法。 |
| 关 键 词： | 定点突变 PCR DMSO 人肝刺激因子 |
Improvement of a PCR-based Method for Vector Construction of Multi-site-directed Mutations in GC-rich Promoter |
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| JIANG Luan,SHAO Lei,HU Yang,DONG Ling-yue. Improvement of a PCR-based Method for Vector Construction of Multi-site-directed Mutations in GC-rich Promoter[J]. Journal of Capital Medical University, 2010, 31(1): 88-92 | |
| Authors: | JIANG Luan SHAO Lei HU Yang DONG Ling-yue |
| Affiliation: | JIANG Luan1,SHAO Lei2,HU Yang3,DONG Ling-yue4(1.Class No.3 of Grade 2005 in Department of 7-Year Clinical Medicine,Capital Medical University,2.Class No.3 of Grade 2005 in Department of 5-Year Clinical Medicine,3.Class No.4 of Grade 2005 in 5 Year' s Clinical Medicine,4.Department of Cell Biology,Basic Medical Sciences,School of Basic Medical Sciences,Capital Medical University) |
| Abstract: | Objective To construct several vectors in which multi-site mutation occurs within a GC-rich hHSS promoter.Methods An interested genomic region of human hepatic stimulator substance(hHSS) was multi-site-directly mutated and amplified.During amplification,the melting temperature was reduced by adding certain concentration of DMSO and the concentration of the enzyme-digested products was increased through ethanol precipitation.Results The results showed that five different hHSS promoter vectors containing mult... |
| Keywords: | PCR DMSO |
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