Hultman T, Bergh S, Moks T, Uhlén M
Department of Biochemistry, Royal Institute of Technology, Stockholm, Sweden.
Biotechniques. 1991 Jan;10(1):84-93.
A solid-phase approach is described for manual and automated sequencing of plasmid DNA obtained directly from bacterial colonies through the polymerase chain reaction. The DNA fragment is selectively immobilized to magnetic beads and after strand-specific elution, the eluted strand, as well as the remaining immobilized strand, is used for bidirectional dideoxy sequencing. The solid-phase approach ensures that the amplification and the sequencing reactions can be performed under optimal conditions. The approach is exemplified by fluorescent sequencing of a cloned Streptomyces curacoi gene having a G + C content of more than 70%.
本文描述了一种固相方法，用于对通过聚合酶链反应直接从细菌菌落获得的质粒DNA进行手动和自动测序。DNA片段被选择性地固定在磁珠上，经过链特异性洗脱后，洗脱的链以及剩余的固定链用于双向双脱氧测序。固相方法确保扩增和测序反应能够在最佳条件下进行。以对一个G + C含量超过70%的克隆的库拉索链霉菌基因进行荧光测序为例说明了该方法。
