New vectors for rapid sequencing of DNA fragments by chemical degradation

Abstract

Nucleotide sequencing by the chemical degradation method requires the labeling of a single nucleotide at the 3′ or 5′ terminus of the fragment to be sequenced. In the present report, I described the construction of two new sequencing plasmids, pSP64CS and pSP65CS, which are ideally suited for use when sequencing a nested set of deletion fragments by the chemical degradation method. Plasmids pSP64CS and pSP65CS contain the sequencing element derived from pGV403 required for specific labeling of individual 3′ ends, and the polylinker site derived from pSP64 or pSP65, respectively. The plasmids also retain the SP6 promoter element which allows for the efficient in vitro synthesis of complementary RNA. Any DNA fragment having a blunt end and a second end which is any of the following restriction sites, HindIII, PstI, SalI, AccI, XbaI, BamHI, SacI, BstEII or EcoRI, can be directionally sequenced in these plasmids. Fragments having two blunt ends can also be sequenced. pSP64CS and pSP65CS differ only in the orientation of the polylinker/sequencing element relative to the SP6 promoter.