质粒制备和反应温度对嵌套缺失技术的影响  

Influence of Plasmid Preparation and Temperature on Nested Deletions

作　　者：王子良[1] 许丽艳[2] 李恩民[1] 

机构地区：[1]汕头大学医学院生化与分子生物学教研室,广东汕头515041 [2]汕头大学医学院肿瘤病理研究室,广东汕头515041

出　　处：《癌变．畸变．突变》2005年第2期79-82,共4页Carcinogenesis,Teratogenesis & Mutagenesis

基　　金：国家自然科学基金面上项目(No.39900069 ;No.30170428 ;No.30370641);广东省自然科学基金重点项目(No.37788) ;广东省自然科学基金面上项目(990799;010431)

摘　　要：背景与目的 :探讨影响嵌套缺失反应的重要因素 ,找出相应的解决方案。材料与方法 :以构建好的中性粒细胞明胶酶相关脂质运载蛋白(neutrophilgelatinaseassociatedlipocalin,NGAL)基因5’端调控区质粒pGLB_G6为研究对象,采用碱裂解法、聚二乙醇纯化法及QIAGEN试剂盒等3种不同方法制备该质粒 ,分别研究ExoⅢ对它们进行非特异性切割的敏感性 ,选出对ExoⅢ不敏感的质粒制备方法 ;在22℃和37℃分别进行缺失反应 ,琼脂糖凝胶电泳监控 ,以确定缺失反应是否已经发生 ;应用PCR技术与限制性内切酶酶切相结合的方法鉴定缺失子。 结果 :QIAGEN试剂盒制备的质粒对ExoⅢ的非特异性切割最不敏感 ,可用于进行下一步的缺失反应 ;在22℃进行缺失反应时 ,电泳鉴定发现DNA片段的长度没有明显的变短 ,而在37℃时 ,同一反应体系的DNA片段长度明显缩短 ;进一步用限制性内切酶酶切可筛除PCR中的假阳性 ,获得准确的缺失子。 结论 :质粒的质量是确保进行特异性缺失反应的关键因素 ,通过QIAGEN试剂盒制备的质粒质量较好 ;设立一个37℃缺失对照管可作为判断低温条件下是否发生缺失的标准 ;PCR技术与限制性内切酶酶切相结合的方法鉴定缺失子 ,可确保获得特异的缺失子。BACKGROUND&AIM: To explore the influencing factors of nested deletions and find some methods to resolve interrelated problems. MATERIAL AND METHODS: Alkaline lysis,purification by polyethylene glycol(PEG)and QIAprep Spin Miniprep Kit were used to prepare the plasmid pGLB-G6carrying the5'flanking region of NGAL gene to study the nonspecific degradation of Exonuclease(ExoⅢ),the best method of preparing the plasmid,not susceptible to ExoⅢ,would be screened.ExoⅢdeletion reaction was generated at22℃and37℃respectively and the agarose gel was run to determine the extent of digestion.PCR amplification and restriction endonuclease digestion was used to identify deleted DNA fragments. RESULTS: The plasmid extracted by QIAprep Spin Miniprep Kit is insusceptible to ExoⅢand suitable for deletion reaction.The agarose gel electrophoresis showed that DNA fragments had not obviously shortened at22℃,but at37℃.Restriction endonuclease digestion is needed for further the identification of deleted DNA fragments. CONCLUSION: The quality of plasmid DNA is a crucial factor to generate specific deletion reactions of Exo III,the plamid extracted by QIAprep Spin Miniprep Kit is satisfactory;setting a control of37℃may ensure deletion reactions have happened and by PCR amplification and restriction endonuclease digestion,real deleted DNA can be attained.

关 键 词：嵌套缺失技术 影响因素 NGAL基因 

分 类 号：Q784[生物学—分子生物学]