A reliable amplification technique for the characterization of genomic DNA sequences flanking insertion sequences


Corresponding author. Tel.: +33 (1) 45 68 88 77; Fax: +33 (1) 45 68 88 43; E-mail: cguilhot@pasteur.fr


A simple and efficient ligation-mediated PCR (LMPCR) is described for amplifying DNA adjacent to known sequences. The method uses one primer specific for the known sequence and a second specific for a synthetic linker ligated to restricted genomic DNA. Perkin-Elmer AmpliTaq Gold polymerase is used to minimize non-specific primer annealing and amplification. This LMPCR method was successfully applied to isolate DNA sequences flanking mobile elements present in mycobacterial mutants generated by transposon mutagenesis.