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Nucleic Acids Research, 1983, Vol. 11, No. 2 349-368
© 1983

MOLECULAR BIOLOGY

Kilo-sequencing: an ordered strategy for rapid DNA sequence data acquisition

Wayne M. Barnes and Michael Bevan

Department of Biological Chemistry, Washington University School of Medicine Saint Louis MO 63110, USA

Received September 29, 1982. Revised December 10, 1982. Accepted December 10, 1982.

A strategy for rapid DNA sequence acquisition in an ordered, nonrandom manner, while retaining all of the conveniences of the dideoxy method with M13 transducing phage DNA template, is described. Target DNA 3 to 14 kb in size can be stably carried by our M13 vectors. Suitable targets are stretches of DNA which lack an enzyme recognition site which is unique on our cloning vectors and adjacent to the sequencing primer; current sites that are so useful when lacking are Pst, Xba, HindIII, BglII, EcoRI. By an in vitroprocedure, we cut RF DNA once randomly and once specifically, to create thousands of deletions which start at the unique restriction site adjacent to the dideoxy sequencing primer and extend various distances across the target DNA. Phage carrying a desired size of deletions, whose DNA as template will give rise to DNA sequence data in a desired location along the target DNA , may be purified by electophoresis alive on agarose gels . Phage running in the same location on the agarose gel thus conveniently give rise to nucleotide sequence data from the same kilobase of target DNA.


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