Laboratoire de Microbiologie, Hôpital Salvator, 249 bld Sainte Marguerite, 13009, Marseille, France.
World J Microbiol Biotechnol. 1995 Sep;11(5):478-80. doi: 10.1007/BF00286355.
The direct sequencing of the products of polymerase chain reactions (PCR) still presents difficulties and often requires special manipulations, such as the generation of excess single-stranded DNA using asymmetric PCR. Several alternative methods involve cloning PCR products into vector DNA suitable for sequencing analysis. Three of these methods have been compared in the present study. The two direct cloning methods, TA/cloning and the PCR-script system, initially gave large numbers of false positives (60% and 55%, respectively) but the number of false positives was reduced (to 35% and 31%, respectively) by modifying the protocols used. However, ligation of the termini of the digested PCR product in the corresponding digested vector was the most efficient and consequently the most reliable method for routine cloning.
聚合酶链反应（PCR）产物的直接测序仍然存在困难，通常需要特殊的操作，例如使用不对称 PCR 产生过量的单链 DNA。几种替代方法涉及将 PCR 产物克隆到适合测序分析的载体 DNA 中。本研究比较了这三种方法。最初，两种直接克隆方法，TA 克隆和 PCR-script 系统，产生了大量的假阳性（分别为 60%和 55%），但通过修改使用的方案，假阳性的数量减少了（分别为 35%和 31%）。然而，在相应的酶切载体中连接消化的 PCR 产物的末端是最有效的方法，因此也是常规克隆最可靠的方法。
