Supercoil Sequencing: A Fast and Simple Method for Sequencing Plasmid DNA

Abstract

A method for obtaining sequence information directly from plasmid DNA is presented. The procedure involves the rapid preparation of clean supercoiled plasmid DNA from small bacterial cultures, its complete denaturation by alkali, and sequence determination using oligodeoxyribonucleotide-primed enzymatic DNA synthesis in the presence of dideoxynucleoside triphosphates. The advantages of the method include speed, simplicity, avoidance of additional cloning steps into single-stranded phage M13 vectors, and hence applicability to sequencing large numbers of samples.