40 - A Rapid Single-Stranded Cloning, Sequencing, Insertion, and Deletion Strategy

Abstract

This chapter discusses a rapid single-stranded cloning, sequencing, insertion, and deletion strategy. A new procedure for producing a sequential series of overlapping clones for use in DNA sequencing uses single-stranded DNA and complementary DNA oligomers to form specific cleavage and ligation substrates. A synthetic oligomer, which hybridizes to a portion of the gene of interest at a restriction site, is used to create a double-stranded region within the molecule. This should present little difficulty because there are many restriction enzymes available, and the only possible site for cleavage will be in the gene fragment. The RNA can then be used for in vitro translation or injection into Xenopus oocytes, so that one can readily study structure/function relationships of cloned and sequenced genes using this overall approach.