The New LM-PCR/Shifter Method for the Genotyping of Microorganism

Beata Krawczyk; Karolina Stojowska; Józef Kur

The New LM-PCR/Shifter Method for the Genotyping of Microorganism

Abstract

Techniques relies on the ligation of appropriates adapters (LM-PCR) as AFLP, PCR MP and ADSRRS are successfully used for epidemiological studies for prokaryotic and eukaryotic microorganisms. In this study we propose a new method, called the LM-PCR/Shifter, based on the use of a Class IIS restriction enzyme giving restriction fragments with different 4 base 5' overhangs (Shifter) and the ligation of appropriate oligonucleotide adapters corresponding to cohesive ends of DNA fragments, and selective PCR amplification of the ligation products (LM-PCR). A sequence of 4-base 5' overhangs of the adapter and 4-base sequence of 3' end of primer determine the number of fragments which are amplified by PCR.The usefulness of the LM-PCR/Shifter method for microorganism strain differentiation was also demonstrated using reference and clinical strains of Escherichia coli, Serratia marcescens, Enterococcus faecium, Enterococcus faecalis, Klebsiella pneumoniae, Klebsiella oxytoca, Acinetobacter sp., and Candida albicans which were tested previously in our laboratory using REA-PFGE and/or PCR MP or ADSRRS techniques.The LM-PCR/Shifter procedure is cost-effective because it utilizes a relatively small set of oligonucleotides, one restriction enzyme and can be easily standardized to allow rapid and parallel amplification of genomic DNA fragments for high through-put genomic analysis in various species. Considering the low cost and relatively high discriminatory power of the LM-PCR/Shifter method, we propose that this is a convenient alternative genotyping method for epidemiological studies.

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Category:: Other
Type:: supllement, wydanie specjalne, dodatek
Published in:: Acta Biochimica Polonica no. 58,
ISSN: 0001-527X
Title of issue:: Acta Biochimica Polonica.
Language:: English
Publication year:: 2011
Bibliographic description:: Krawczyk B., Stojowska K., Kur J.: The New LM-PCR/Shifter Method for the Genotyping of Microorganism// Acta Biochimica Polonica. -Vol. 58., nr. suppl. 4 (2011),
Verified by:: Gdańsk University of Technology

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