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Sequential cloning by a vector walking along the chromosome

Sequential cloning by a vector walking along the chromosome

Gene (Amsterdam) 104(1): 11-18

A procedure was devised for sequential cloning of chromosmal DNA by cyclical integration and exicision of a plasmid vector so that slightly overlapping chromosomal segments are successively cloned. The method depends on circular integration of the vector into the chromosome of a host nonpermissive for its replication, and on excision and reduction of a recombinant plasmid by use of an appropriately designed set of restruction enzyme sites in the vector. A vector suitable for cloning in Escherichai coli was constructed by combining a segment of pBR322 with a gene encoding chloramphenicol resistance expressible in many species. Sequential cloning was demonstrated in Streptococcus pneumoniae by extending a previously cloned segment of the region of the chromosome encoding maltosaccharide utilizaiton by 8 kb in three cycles of cloning. Accuracy of the method was confirmed by hybridization of cloned DNA with chromosomal restriction fragments. It is pointed out that the similarity of the requisite genetic processes in bacteria and yeasts should allow use of the method for sequential cloning of yeast chromosomal DNA and of human or other mammalian DNA in artificial chromosomes of yeast.

Accession: 002219145

Download citation: RISBibTeXText

PMID: 1655574

DOI: 10.1016/0378-1119(91)90458-N

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