Iwahana H, Yoshimoto K, Tsujisawa T, Itakura M
Otsuka Department of Clinical and Molecular Nutrition, School of Medicine, University of Tokushima, Japan.
PCR Methods Appl. 1994 Feb;3(4):219-24. doi: 10.1101/gr.3.4.219.
We describe a method to ligate a PCR-amplified DNA fragment with T-protruding cassettes, which have multiple sites for endonuclease, promoter sequences of T3 and T7, and annealing sites for the universal M13 forward and reverse primers. This method, which we named T-cassette ligation, substantially facilitated direct sequencing and subcloning of PCR products. Two T-cassettes with a protruding T at their 3' ends were obtained by annealing adaptor oligomers to XbaI- or XhoI-digested pBluescript. A DNA fragment amplified by the first PCR was ligated separately to one of the two T-cassettes. The second PCR was performed using a primer complementary to one of the two T-cassettes and one of the two primers used for the first PCR to amplify the ligation product in low abundance. The resultant two DNA fragments had the T3 and T7 promoter, and an annealing site for the universal M13 forward and reverse primer, respectively. These DNA fragments were applicable to direct sequencing. For the purpose of subcloning, the third PCR was carried out with two primers each complementary to each of two T-cassettes and two PCR-amplified DNAs by the second PCR, as templates. The PCR product of the third PCR had multiple sites of the pBluescript polycloning site at both ends, facilitating its subcloning into pBluescript.
我们描述了一种将PCR扩增的DNA片段与T突出盒连接的方法，该T突出盒具有多个内切酶位点、T3和T7启动子序列以及通用M13正向和反向引物的退火位点。我们将此方法命名为T盒连接，它极大地促进了PCR产物的直接测序和亚克隆。通过将衔接子寡聚物与经XbaI或XhoI消化的pBluescript退火，获得了两个在其3'端具有突出T的T盒。通过第一次PCR扩增的DNA片段分别与两个T盒之一连接。使用与两个T盒之一互补的引物和用于第一次PCR的两个引物之一进行第二次PCR，以扩增低丰度的连接产物。所得的两个DNA片段分别具有T3和T7启动子以及通用M13正向和反向引物的退火位点。这些DNA片段适用于直接测序。为了进行亚克隆，以第二次PCR扩增的两个DNA为模板，用分别与两个T盒之一互补的两个引物进行第三次PCR。第三次PCR的产物在两端具有pBluescript多克隆位点的多个位点，便于将其亚克隆到pBluescript中。
