Bidirectional solid-phase sequencing of in vitro-amplified plasmid DNA

Abstract

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%.