[1]谢锋,朱蕾,尚广东,等.DNA克隆载体的“三片段克隆法”改造策略[J].南京师大学报(自然科学版),2007,30(04):80-83.
　Xie Feng,Zhu Lei,Shang Guangdong.A General Method for Direct DNA Vector Modification[J].Journal of Nanjing Normal University(Natural Science Edition),2007,30(04):80-83.
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DNA克隆载体的“三片段克隆法”改造策略()
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《南京师大学报（自然科学版）》[ISSN:1001-4616/CN:32-1239/N]

卷:
第30卷
期数:
2007年04期
页码:
80-83
栏目:
生命科学
出版日期:
2007-12-30

文章信息/Info

Title:
A General Method for Direct DNA Vector Modification
作者:
谢锋;朱蕾;尚广东;
江苏省生物多样性和生物技术重点实验室,江苏南京210046
Author(s):
Xie Feng; Zhu Lei; Shang Guangdong
Jiangsu Key Laboratory for Biodiversity and Biotechnology,Nanjing 210046,China
关键词:
重组工程;  三片段克隆法;  结合转移载体
Keywords:
recombineering;  three p ieces cloning;  conjugal transfer vector
分类号:
Q78
摘要:
将一个或多个DNA片段克隆至载体是分子生物学研究中最常用的技术之一.本研究使用新颖的Red/ETDNA重组克隆技术原理一步法将结合转移片段oriT,阿普霉素抗性基因Am和链霉素抗性基因(rpsL)克隆至常用的克隆载体pBluescriptKS(-),同时去除载体的氨苄青霉素抗性基因(bla).此"三片段克隆法"简便,快速,避免了经典的克隆策略常常难以寻找合适的限制性内切酶,长片段PCR、暴露于紫外线和凝胶回收等步骤所可能引入的碱基突变等等难以克服的问题,有可能成为通用的克隆策略.所得到的结合转移载体在通过结合转移将外源片段从大肠杆菌等转移至放线菌方面将有着广泛的用途.
Abstract:
Cloning one ormore DNA fragments into a vector is the one ofmost commonly used techniques in molecular biology. Classic cloning strategy, when dealt with comp licated cloning step s, like multip le fragments insertion, selective marker exchange, are often troubled by the choice of restriction enzymes, the base mutations caused by long fragment PCR, UV exposure and gel purification. We made use of the Red /ET technology to clone two fragments into the vector while remove the unnecessary vector part in a single step. This three p ieces cloningmanipulation is straightforward and convenient, with the potential to be a general cloning strategy for vector engineering.

参考文献/References:

[ 1 ] 　Zhang Y, Buchholz F, Muyrers J P, et al. A new logic for DNA engineering using recombination in Escherichia coli [ J ].Nat Genet, 1998, 20 (2) : 123-128.
[ 2 ] 　Glaser S, Anastassiadis K, StewartA F. Current issues in mouse genome engineering[ J ]. Nat Genet, 2005, 37 (1) : 1 187-1193.
[ 3 ] 　SarovM, Schneider S, PozniakovskiA. A recombineering p ipeline for functional genomics app lied to Caenorhabditis elegans[ J ]. NatMethods, 2006, 3 (10) : 839-844.
[ 4 ] 　Wang J , SarovM, Rientjes J, et al. An imp roved recombineering app roach by adding RecA to lambda Red recombination[ J ]. Mol Biotechnol, 2006, 32 (1) : 43-53.
[ 5 ] 　BiermanM, Logan R, O’Brien K, et al. Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia coli to Strep tomyces spp [ J ]. Gene, 1992, 116 (1) : 43-49.
[ 6 ] 　Hosted T J , Baltz R H. Use of rp sL for dominance selection and gene rep lacement in trep tomyces roseosporus[ J ]. J Bacteriol, 1997, 179 (1) : 180-186.
[ 7 ] 　Sambrook J, Fritsch E F, Maniatis T. Molecular Cloning:A LaboratoryManual[M ]. 2nd ed. New York: Cold Sp ring Harbor Laboratory Press, 1989.
[ 8 ] 　Kieser T, BibbM J , ButtnerM J. Practical Strep tomyces Genetics[M ]. Norwich: The John Innes Foundation, 2000: 247-248.

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备注/Memo

备注/Memo:
基金项目：“十一五”863国家高技术研究发展计划(2006AA02Z159)资助项目.
作者简介:谢锋(1982—) ,硕士研究生,主要从事重组工程在次级代谢产物来源的药物中的学习与研究. E-mail: XF - 821026@163. com
通讯联系人:尚广东(1969—) ,副教授,主要从事微生物药物的教学与研发. E-mail: shanggd@hotmail. com
更新日期/Last Update: 2013-05-05