A reliable amplification technique for the characterization of genomic DNA sequences flanking insertion sequences |
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| Authors: | Guy Prod'hom,Bé atrice Lagier,Vladimir Pelicic,Allan J Hance,Brigitte Gicquel,Christophe Guilhot |
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| Affiliation: | Centre national de référence des mycobactéries, Institut Pasteur, Paris, France;Unitéde Génétique Mycobactérienne, Institut Pasteur, 25 rue du Dr. Roux, 75724 Paris Cedex 15, France;INSERM U82, FacultéXavier Bichat, Paris, France |
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| Abstract: | 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. |
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| Keywords: | Mycobacterium Cloning Insertion sequence Transposon mutagenesis |
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