Arnold W, Pühler A
Lehrstuhl für Genetik, Fakultät für Biologie, Universität Bielefeld, F.R.G.
Gene. 1988 Oct 15;70(1):171-9. doi: 10.1016/0378-1119(88)90115-1.
A set of plasmid vectors was developed which allows fast sequencing by the chemical degradation method. These high-copy-number vectors are derivatives of the plasmid pUC8 containing different multiple-purpose cloning sites flanked by unique recognition sequences for the restriction enzymes BstEII, Tth111I and Eco81I as sites for end-labelling DNA. Due to their partially asymmetric recognition sequences, each of these three restriction sites can be singly end-labelled by a filling-in reaction with selected nucleotides. This allows easy single end-labelling of any cloned DNA fragment for sequencing by the chemical degradation method without any isolation and purification step after the labelling reaction. In addition, the nucleotide sequence of the complementary strand from the same end can be determined by the dideoxy chain termination procedure using the universal M13 primers. In most of the new vectors, the reading frame of the lacZ' gene is retained, allowing identification of cloned fragments.
开发了一组质粒载体，可通过化学降解法进行快速测序。这些高拷贝数载体是质粒pUC8的衍生物，含有不同的多用途克隆位点，两侧是限制性内切酶BstEII、Tth111I和Eco81I的独特识别序列，作为DNA末端标记位点。由于它们部分不对称的识别序列，这三个限制性位点中的每一个都可以通过与选定核苷酸的填充反应进行单末端标记。这使得任何克隆的DNA片段都能轻松地进行单末端标记，以便通过化学降解法进行测序，标记反应后无需任何分离和纯化步骤。此外，使用通用M13引物，通过双脱氧链终止法可以确定同一末端互补链的核苷酸序列。在大多数新载体中，lacZ'基因的阅读框得以保留，从而能够鉴定克隆片段。
