Speek M, Ilves H, Lind A
Anal Biochem. 1986 Nov 1;158(2):242-9. doi: 10.1016/0003-2697(86)90544-0.
An improved strategy for the dideoxy chain termination sequencing of M13 recombinant DNA using enzymatically generated primers is presented. It involves synchronous extension of the universal primer with the Klenow fragment of DNA polymerase I at an average rate of 200 deoxynucleoside triphosphates per minute to the immediate downstream region of a preselected restriction enzyme site. Subsequent cleavage with the restriction enzyme generates the short primers with homogeneous 5' ends which can be used for further sequencing. The next restriction sites are selected in the newly sequenced regions of DNA by means of a microcomputer. By repeating this primer extension-cleavage-sequencing strategy sequences altogether about 6 kb long from several recombinant single-stranded M13 DNAs have been determined by using twenty restriction enzymes with different specificities.
本文提出了一种改进策略，用于使用酶促生成的引物对M13重组DNA进行双脱氧链终止测序。该策略包括用DNA聚合酶I的Klenow片段以每分钟平均200个脱氧核苷三磷酸的速率同步延伸通用引物至预选限制性酶切位点的紧邻下游区域。随后用限制性酶切割产生具有均一5'端的短引物，可用于进一步测序。通过微型计算机在DNA新测序区域中选择下一个限制性酶切位点。通过重复这种引物延伸-切割-测序策略，使用20种具有不同特异性的限制性酶已确定了来自几个重组单链M13 DNA的总长约6 kb的序列。
