Predictive modelling of fluorescent AFLP: a new approach to the molecular epidemiology of E. coli

Abstract

Amplified fragment length polymorphism (AFLP) permits simultaneous sampling of multiple loci distributed throughout a genome, using restriction site/adaptor-specific primers under stringent conditions. Fluorescent detection instrumentation further refines this methodology, permitting internal size standards and accurate, reproducible sizing of amplified fragments. We have evaluated the potential of fluorescent AFLP (FAFLP) as a potentially definitive genotyping method for bacteria, by comparing MseI/EcoRI fragments derived experimentally from the Escherichia coli K12 MG1655 genome with those predicted by analysis of its published sequence. In silico, MseI/EcoRI digestion of this sequence produced 1200 fragments from 36 and 2151 base pairs (bp) in size. Fragment subsets which would be amplified by seven different selective (1-2 bases added to the 3' end of the core primer sequence) primer combinations were modelled. Depending on the primer pair, three to 54 fragments (range 70-400 bp) were predicted, while all seven primer pair combinations together generated 121 predicted fragments. When genomic DNA of strain MG1655 was subjected to experimental FAFLP with these seven primers, 111 correctly sized fragments were observed (+/- 1 bp) out of the 121 predicted (92% accuracy). Twenty-five unpredicted fragments were obtained; an average of four per primer pair. The size and number of fragments in FAFLP, and their gel distribution, were dictated by the choice of restriction endonucleases and the degree of primer selectivity. Our data show that FAFLP is accurate, discriminatory, reproducible and capable of standardisation. Under agreed conditions, this method shows considerable promise as a generally applicable standardised bacterial genotyping method. The fragments predicted in silico to result from amplification of MseI/EcoRI-digested DNA with the seven primer pairs described are here used to define a prototypic FAFLP analysis of E. coli.