Fluorescent amplified fragment length polymorphism genotyping of bacterial species

High-resolution and reproducible whole genome methodologies are needed as tools for rapid and cost-effective analysis of genetic diversity within bacterial genomes. These should be useful for a broad range of applications such as identification and subtyping of microorganisms from clinical samples, for identification of outbreak genotypes, for studies of micro- and macrovariation, and for population genetics. Fluorescent amplified fragment length polymorphism analysis is one such technique that has been used successfully for studying several bacterial genera. It combines the principle of restriction fragment length polymorphism with the capacity to sample bacterial genomes by selective amplification of a subset of DNA fragments generated by restriction endonucleases, thereby sampling multiple loci distributed throughout the genome. Typically, the genomic DNA is digested with two restriction endonucleases, followed by ligation of double-stranded oligonucleotide adaptors to ends of restriction fragments. Subsets of these fragments are amplified by PCR using fluorescently labeled primers complementary to the adaptor sequences. Amplified fragments are resolved by electrophoresis on an automated DNA sequencer and precisely sized using internal size standards in each sample.