A rapid method to identify cosmids containing rare restriction sites.
A procedure for identifying specific cosmid clones containing recognition sites for "rare cutting" restriction enzymes has been developed. Cosmid clones containing human inserts were selected by hybridisation to human repetitive DNA. An oligonucleotide corresponding to the NotI recognition site, eight bases long, was labelled and used to probe DNA samples from one hundred cosmids. By optimising the difference in melting characteristics between eight-base perfect match and six-base match/two base mismatch hybrids, we were able to detect the cosmids containing either NotI (8 bp match) or XmaIII/EagI (6 bp match) sites. The generation of a map for rare cutter sites along a human chromosome, or a chromosome region, should be simplified using this approach, which will enable the identification of a set of "milestones" at intervals of several hundred kilobases (kb) along the DNA sequence.
D001483:Base Sequence; D003001:Cloning, Molecular; D003360:Cosmids; D004262:DNA Restriction Enzymes / Q000378:metabolism; D006801:Humans; D009693:Nucleic Acid Hybridization; D013379:Substrate Specificity
D004262:DNA Restriction Enzymes