Wiemann S, Rupp T, Zimmermann J, Voss H, Schwager C, Ansorge W
European Molecular Biology Laboratory Heidelberg, Germany.
Biotechniques. 1995 Apr;18(4):688-97.
We have identified additional criteria for the walking primer design that improve the success rate of automated fluorescent DNA sequencing using the internal labeling technique and T7 DNA polymerase. These criteria resulted from the evaluation of over 220 sequences generated with walking primers and fluorescein-15-dATP as internal label in the course of the European Community (EC) yeast genome sequencing project. In this project primers were designed using standard commercial software. Intensities of sequencing signals varied over a broad range from very strong to very weak, depending on the primers used. This led us to evaluate primer performance relative to (i) the template sequence immediately downstream of the primer binding site and (ii) the primer sequence itself. Our experiments show that the position of the first labeled dATP to be incorporated downstream of the primer into the growing strand is substantial for the signal intensity of the sequence. The closer to the primer that the first 'A' is incorporated, the stronger the peak intensities are. An additional feature of sequencing with native T7 DNA polymerase is its ability to remove a 3'-terminal 'A' of the primer by the 3'-->5' exonuclease activity and to exchange the nucleotide with a labeled dATP by the polymerase activity.
我们已经确定了用于步移引物设计的额外标准，这些标准提高了使用内部标记技术和T7 DNA聚合酶进行自动化荧光DNA测序的成功率。这些标准源于在欧洲共同体（EC）酵母基因组测序项目中，对使用步移引物和荧光素-15-dATP作为内部标记生成的220多个序列的评估。在该项目中，引物是使用标准商业软件设计的。测序信号的强度在很宽的范围内变化，从非常强到非常弱，这取决于所使用的引物。这使我们评估引物性能与（i）引物结合位点下游紧邻的模板序列以及（ii）引物序列本身的关系。我们的实验表明，第一个被掺入引物下游延伸链中的标记dATP的位置对于序列的信号强度至关重要。第一个“A”掺入的位置离引物越近，峰强度就越强。使用天然T7 DNA聚合酶进行测序的另一个特点是其能够通过3'→5'核酸外切酶活性去除引物的3'末端“A”，并通过聚合酶活性用标记的dATP交换该核苷酸。
