A cloning vehicle suitable for strand separation

Abstract

A new plasmid has been constructed which contains a poly(dA):poly(dT) duplex segment of length approx. 100 base pairs (bp) inserted into the PvuII site of pBR322. This plasmid, pKH47, has all the other restriction sites of pBR322 available for insertion of foreign DNA, and has the same drug resistance genes as does the parental plasmid. The complementary strands of the linearized denatured plasmid DNA can be separated rapidly and efficiently by affinity chromatography with oligo(dA)- and oligo(dT)-cellulose columns m series. More than 90% of the input DNA is recovered as separated strands which can be annealed to form full length double-stranded molecules. One of the applications of the plasmid is to prepare separated complementary strands for sequencing by the chain-terminator technique using DNA primers. This application is illustrated by a sequencing example for a Drosophila DNA insert carrying a tRNA gene. © 1980, All rights reserved.