Ligation mediated PCR performed at low denaturation temperatures--PCR melting profiles.
We show that using low denaturation temperatures (80-88 degrees C) during ligation mediated PCR (LM PCR) of bacterial DNA leads to the amplification of limited sets of the less stable DNA fragments. A set of electrophoretic patterns of such fragments obtained at different denaturation temperatures forms the PCR melting profile (PCR MP). A single pattern obtained for a given temperature and a set of patterns arising after application of several denaturation temperatures (PCR MP) are very specific for the given bacterial genome and may be used for strain characterisation and differentiation. The method may also be used for amplification and isolation of the less stable DNA fragments in a genome.
Masny
Aleksander
A
Institute of Biotechnology and Antibiotics, Staroscinska 5, 02-516 Warsaw, Poland. masnya@iba.waw.pl
Płucienniczak
Andrzej
A
D004269:DNA, Bacterial / Q000737:chemistry* / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D041121:Klebsiella oxytoca / Q000235:genetics; D007711:Klebsiella pneumoniae / Q000235:genetics; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D013045:Species Specificity; D013696:Temperature
D004269:DNA, Bacterial