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Plasmid DNA from strains CP24-03 aNCTC8131131, and cut by EcoRI or HindIII showed different restriction patterns (data not shown), suggesting that they are not identical.
Fragments producing different restriction patterns were sequenced.
The amplified fragment from microorganisms that presented different restriction patterns was selected for sequencing.
The resulting fragments were resolved on a 2% agarose gel to identify different restriction patterns.
Further analysis showed different restriction patterns between the two KPC-carrying plasmids.
To minimize redundancy we choose overlapping clones but with different restriction patterns.
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However, this 2 bp difference did change a HinfI restriction site resulting in a different restriction pattern.
For the five fragments, a total of 114 clones out of 750 colonies in total showed a different restriction pattern.
Further, hsp60 specific amplicons from 23 out of 25 patients gave identical restriction pattern, while 6 fliI and 1 ureC specific amplicon produced different restriction pattern.
The amplified fliI gene segment, which had different restriction pattern from previous strain were purified from salts and primers using HiPura silica kit for DNA isolation (HiMedia).
All transcripts with a different restriction pattern were sequenced (primer sequences available upon request) using the Thermo Sequenase fluorescent labelled primer cycle sequencing kit (Amersham Biosciences) and analysed on an ALF-express automated DNA sequencer (Amersham Biosciences).
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