The interaction between dam methylation sites and Xba1 restriction digest sites in Escherichia coli O157:H7 EDL933.
OBJECTIVE
The aim of this study was to gain a better understanding of the reason for the predicted pulsed-field gel electrophoresis (PFGE) pattern for the sequenced Escherichia coli O157:H7 EDL933 (EDL933) being different from that observed in practice, using the restriction enzyme Xba1.


RESULTS
Primers were designed that flanked either side of each of the predicted Xba1 restriction sites, and the resultant PCR products were sequenced. No sequencing errors were found in the published genome. The distribution of dam methylation sites within the genome was investigated, and a new PFGE pattern was predicted by assuming that any Xba1 restriction site that coincided with a dam methylation site would not be cut. The estimated mean band sizes were obtained from six replicate gels. It was found that the observed and predicted PFGE patterns were in good agreement.


CONCLUSIONS
The difference between the observed and the predicted PFGE patterns for EDL933, using Xba1, could be accounted for by assuming that the methylated restriction sites were not cut.


CONCLUSIONS
PFGE is commonly used as a subtyping method. This study provides additional information about the basic technique that could enhance the interpretation of PFGE patterns in comparative studies of the E. coli isolates.
Sales
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J
Biomathematics & Statistics Scotland, Edinburgh, UK. jill@bioss.ac.uk
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Hoyle
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Yates
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Amyes
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McKendrick
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D015373:Bacterial Typing Techniques / Q000379:methods; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D016521:Electrophoresis, Gel, Pulsed-Field / Q000379:methods; D019453:Escherichia coli O157 / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D008745:Methylation; D016133:Polymerase Chain Reaction / Q000379:methods; D017422:Sequence Analysis, DNA / Q000379:methods; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000235:genetics* / Q000378:metabolism
D017931:DNA Primers; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); C499294:dam protein, E coli; C025580:endodeoxyribonuclease XBAI; D015252:Deoxyribonucleases, Type II Site-Specific