Changes of 5＇ Terminal Nucleotides of PCR Primers Causing Variable T-A Cloning Efficiency
Changes of 5＇ Terminal Nucleotides of PCR Primers Causing Variable T-A Cloning Efficiency作者机构：College of Biological Sciences and Biotechnology Beijing Forestry University Beijing 100083 China
出 版 物：《Journal of Integrative Plant Biology》 (植物学报（英文版）)
年 卷 期：2007年第49卷第3期
页 面：382-385页
核心收录：
学科分类：0710[理学-生物学] 07[理学] 08[工学] 09[农学] 071007[理学-遗传学] 0901[农学-作物学] 0836[工学-生物工程] 090102[农学-作物遗传育种]
基 金：Supported by the National Natural Science Foundation of China (30671476 and 30371143) Fok Ying Tung Education Foundation (71030) and Beijing Science and Technology Project (H020720110190)
主 题：efficiency polymerase chain reaction primer T-A cloning terminal nucleotide
摘 要：T-A cloning takes advantage of the unpaired adenosyl residue added to the 3＇ terminus of amplified DNAs by Taq and other thermostable DNA polymerase and uses a Ilnearlzed plasmld vector with a protruding 3＇ thymldylate residue at each of Its 3＇ termini to clone polymerase chain reaction （PCR）-derived DNA fragments. It Is a simple, reliable, and efficient Ilgatlon-dependent cloning method for PCR products, but the drawback of variable cloning efficiency occurs during application. In the present work, the relationship between variable T-A cloning efficiency and the different 5＇ end nucleotlde base of primers used In PCR amplification was studied. The results showed that different cloning efficiency was obtained with different primer pairs containing A, T, C and G at the 5＇ terminus respectively. The data shows that when the 5＇ end base of primer pair was adenosyl, more white colonies could be obtained In cloning the corresponding PCR product In comparison with other bases. And the least white colonies were formed when using the primer pair with 5＇ cytldylate end. The gluanylate end primers resulted In almost the same cloning efficiency In the white colonies amount as the thymldylate end primer did, and this efficiency was much lower than that of adenosyl end primers. This presumably is a consequence of variability In 3＇dA addition to PCR products mediated by Taq polymerase. Our results offer instructions for primer design for researchers who choose T-A cloning to clone PCR products.
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