Use of the lac repressor in constructing sequential deletions and a new sequencing vector.
Large sequencing projects require an efficient strategy to generate a series of overlapping clones. This can be accomplished by protecting one end of a linear DNA molecule while sequential deletions are introduced into the other end by exonuclease digestion. We demonstrate that the lac repressor can protect the ends of linear nucleotide sequences from digestion by exonuclease if these ends contain the lac operator sequence. To exploit this, we have inserted the lac operator sequence between the primer-binding site and multiple cloning site of an M13 sequencing vector. Linearizing the replicative form and binding lac repressor protein protects the end next to the vector sequences. Sequential deletions are then introduced into the insert by digesting with exonuclease III or BAL 31. Because the rate and time of digestion are readily controlled, the region brought next to the sequencing primer site, after religation, can be selected in a timed series of reactions. This minimizes the screening needed to isolate an overlapping series of clones and facilitates sequencing of long regions.
Johnson
D F
DF
Department of Microbiology, University of California, Los Angeles 90024.
Nierlich
D P
DP
Lusis
A J
AJ
D001483:Base Sequence; D002872:Chromosome Deletion; D003001:Cloning, Molecular / Q000379:methods; D004251:DNA Transposable Elements; D004926:Escherichia coli / Q000235:genetics*; D005818:Genetic Engineering / Q000379:methods; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D010957:Plasmids; D012097:Repressor Proteins / Q000378:metabolism*; D015183:Restriction Mapping
D004251:DNA Transposable Elements; D012097:Repressor Proteins