The new LM-PCR/shifter method for the genotyping of microorganisms based on the use of a class IIS restriction enzyme and ligation mediated PCR.
This study details and examines a novel ligation-mediated polymerase chain reaction (LM-PCR) method. Named the LM-PCR/Shifter, it relies on the use of a Class IIS restriction enzyme giving restriction fragments with different 4-base, 5' overhangs, this being the Shifter, and the ligation of appropriate oligonucleotide adapters. A sequence of 4-base, 5' overhangs of the adapter and a 4- base sequence of the 3' end of the primer(s) determine a subset of the genomic restriction fragments, which are amplified by PCR. The method permits the differentiation of bacterial species strains on the basis of the different DNA band patterns obtained after electrophoresis in polyacrylamide gels stained with ethidium bromide and visualized in UV light. The usefulness of the LM-PCR/ Shifter method for genotyping is analyzed by a comparison with the restriction endonuclease analysis of chromosomal DNA by the pulsed-field gel electrophoresis (REA-PFGE) and PCR melting profile (PCR MP) methods for isolates of clinical origin. The clustering of the LM-PCR/Shifter fingerprinting data matched those of the REA-PFGE and PCR MP methods. We found that the LM-PCR/Shifter is rapid, and offers good discriminatory power and excellent reproducibility, making it a method that may be effectively applied in epidemiological studies.
Gdansk University of Technology, Chemical Faculty, Department of Microbiology, ul. Narutowicza 11/12, 80-233 Gdansk, Poland. beakrawc@pg.gda.pl
Beata
B
Krawczyk
Justyna
J
Leibner-Ciszak
Karolina
K
Stojowska
Józef
J
Kur
D015373:Bacterial Typing Techniques / Q000379:methods; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D004926:Escherichia coli / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification*; D004927:Escherichia coli Infections / Q000382:microbiology*; D005838:Genotype; D006801:Humans; D022521:Ligase Chain Reaction / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D000091402:Prohibitins; D015183:Restriction Mapping / Q000379:methods*
C000717331:PHB2 protein, human; D000091402:Prohibitins; D015252:Deoxyribonucleases, Type II Site-Specific