A novel type of cloning vectors for ultrarapid chemical degradation sequencing of DNA

The construction and evaluation of a novel type of chemical sequencing vectors (pCSV) is described. These small plasmids bear a unique asymmetric restriction site suitable for direct single-end labeling by filling-in polymerization with a selected radiolabeled nucleoside triphosphate. Thus, a secondary cleavage reaction or segregation step is rendered superfluous. Sequencing gels can be read unambiguously starting from the 3′ penultimate nucleotide. pCSV03 and pCSV27 are prototypes of such sequencing plasmids based on Bst EII as the labeling site. In its proximity, restriction sites are present that allow subcloning of the DNA fragments to be sequenced. Approaches to random and progressive sequencing using these vectors are discussed. pCSV31 has two Tth 111I sites that allow single-end labeling of inserts on either strand by the use of different labeled nucleoside triphosphates. Thus, bidirectional sequencing of larger inserts (>500 bp) is possible.