Construction of a Zero-background T-vector for Cloning PCR Products
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摘要: 为解决平末端加T法构建的T载体，在PCR产物克隆时连接效率低、阳性克隆筛选困难的问题，本研究在利用Taq DNA聚合酶末端转移酶活性对线性化平末端pBluescript SK（+）载体3'-端进行加T反应后，使用T4 DNA连接酶处理加T后pBS载体，使3'-端未加T平末端载体重新形成超螺旋状态，而3'-端加T载体则保持线性化状态，在琼脂糖电泳时切胶回收3 000 bp处DNA片段，成为pBS-T载体。使用构建的pBS-T载体分别克隆500 bp和1 000 bp PCR片段，pBS-T载体克隆效率达到100%，实现了对PCR产物的零背景克隆。Abstract: This experiment aimed to improve the shortcomings of low ligation efficiency and weak positive selection in cloning PCR products with T-vector.The approach called for an addition of T-overhang to a blunt-end plasmid. Firstly, a 3'-T overhang was attached to the blunt-end pBluescript SK (+) plasmid utilizing a terminal transferase, Taq DNA polymerase. Secondly, thepBS vector was ligated by T4 DNA ligase to form thesupercoiled blunt-end pBS plasmid without a 3'-T overhang, whereas the plasmidwith a 3'-T overhang remained linear in appearance. Finally, the DNA fragment of 3 000 bp, which was pBST-vector for cloning PCR products, could be purified byagarose gel electrophoresis followed by usinga gel extraction kit. The 500 bp and 1 000 bp DNA fragments were cloned into pBS T-vector with a100% cloning efficiency. It appeared that the newly developed procedures provided a valid zero-background T-vector for cloning PCR products.
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Key words:
- cloning PCR products /
- zero-background /
- T-vector
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图 1 pBS质粒酶切电泳
注：M为250 bp DNA Ladder；1为未酶切pBS质粒；2为酶切后pBS质粒。
Figure 1. Agarose gel electrophoretic patterns of pBS plasmid digested by Eco RV
图 2 pBS载体加T效率检测和菌落PCR验证
注：M为DL2000 DNA Marker；1~5为白斑菌落PCR验证结果；6~10为蓝斑菌落PCR验证结果。
Figure 2. Cloning efficiency of pBS vector and identification by colony PCR
图 3 回收后pBS-T载体电泳
注：M为250 bp DNA Ladder；1为T4 DNA连接酶处理后pBS载体；2为切胶回收后pBS-T载体。
Figure 3. Agarose gel electrophoretic patterns of purified pBST-vector
图 4 pBS-T载体克隆效率检测和菌落PCR验证
注：M为DL2000 DNA Marker；1~10为白斑菌落PCR验证结果。
Figure 4. Cloning efficiency of 500 bp DNA fragments inserted into pBST-vector and identification by colony PCR
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[1] HOLTON T A, GRAHAM M W. A simple and efficient method for direct cloning of PCR products using ddT-tailed vectors[J]. Nucleic Acids Res, 1991, 19(5):1156. doi: 10.1093/nar/19.5.1156 [2] JO C, JO S A. A simple method to construct T-vectors using Xcm Ⅰ cassettes amplified by nonspecific PCR[J]. Plasmid, 2001, 45(1):37-40. doi: 10.1006/plas.2000.1500 [3] MEAD D A, PEY N K, HERRNSTADT C, et al. A universal method for the direct cloning of PCR amplified nucleic acid[J]. Biotechnology (N Y), 1991, 9(7):657-663. doi: 10.1038/nbt0791-657 [4] HADJEB N, BERKOWITZ G A. Preparation of T-over-hang vectors with high PCR product cloning efficiency[J]. Biotechniques, 1996, 20(1):20-22. https://www.ncbi.nlm.nih.gov/pubmed/8770397 [5] 李新波, 赵小松, 田德志, 等.一种PCR产物克隆的新方法--T-A克隆法[J].生物化学与生物物理进展, 1999, (2):90-92. http://www.cnki.com.cn/Article/CJFDTOTAL-SHSW902.027.htm [6] 杨鹏, 陈喜文, 陈德富.不同二价阳离子对T-载体生成的影响[J].科技通报, 2006, (2):169-172. http://www.cnki.com.cn/Article/CJFDTOTAL-KJTB200602006.htm [7] MARCHUK D, DRUMM M, SAULINO A, et al. Construction of T-vectors, a rapid and general system for direct cloning of unmodified PCR products[J]. Nucleic Acids Res, 1991, 19(5):1154. doi: 10.1093/nar/19.5.1154 [8] CHEN S B, SONGKUMARN P, LIU J L, et al. A versatile zero background T-vector system for gene cloning and functional genomics[J]. Plant physiology, 2009, 150(3):1111-1121. doi: 10.1104/pp.109.137125 [9] HARRISON J, MOLLOY P L, CLARK S J. Direct cloning of polymerase chain reaction products in an Xcm Ⅰ T-vector[J]. Analytical biochemistry, 1994, 216(1):235-236. doi: 10.1006/abio.1994.1033 [10] GU J S, YE C J. pYEMF, a pUC18-derived XcmⅠ T-vector for efficient cloning of PCR products[J]. Molecular biotechnology, 2011, 47(3):229-233. doi: 10.1007/s12033-010-9333-y [11] 吴国球, 沈子龙.利用XcmⅠ构建T载体[J].东南大学学报:医学版, 2002, 21(4):293-295. http://www.cnki.com.cn/Article/CJFDTOTAL-DSDX200105059.htm [12] 夏志辉, 董军美, 罗越华.利用限制性内切酶Xcm Ⅰ构建T载体[J].热带生物学报, 2010, 1(3):206-209. http://www.cnki.com.cn/Article/CJFDTOTAL-HNNX201003005.htm [13] ARASHI-HEESE N, MIWA M, SHIBATA H. XcmⅠ site-containing vector for direct cloning and in vitro transcription of PCR product[J]. Molecular biotechnology, 1999, 12(3):281-283. doi: 10.1385/MB:12:3 [14] ARANISHI F, OKIMOTO T. Engineered Xcm Ⅰ cassette-containing vector for PCR-based phylogenetic analyses[J]. J Genet, 2004, 83(1):33-34. doi: 10.1007/BF02715826 [15] ICHIHARA Y, KUROSAWA Y. Construction of new T vectors for direct cloning of PCR products[J]. Gene, 1993, 130(1):153-154. doi: 10.1016/0378-1119(93)90361-6 -
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