Digitized fluorescent RFLP analysis (fRFLP) as a universal method for comparing genomes of culturable dsDNA viruses: application to bacteriophages

Identification and classification of bacteriophages remains a cumbersome process even with the use of genotypic approaches, due to the lack of genes present in all phages. Restriction fragment length polymorphism analysis (RFLP) of the viral genome is a universal approach, but RFLP fingerprints obtained on agarose gels remain difficult to compare between laboratories. Here we describe the digitization of RFLP of viral genomes by amplification of all restriction fragments – after ligation of adapters – using primers complementary to the adapters only. Since one of the primers is fluorescently labelled, the restriction fragments become visible to a fluorescent capillary electrophoresis system (ABI310) and their lengths can be digitized immediately. The digitized fluorescent RFLP (fRFLP) fingerprint can be stored as an entry in a library. Dendrogram construction of the fRFLP fingerprints obtained for a total of 69 Caudovirales (tailed bacteriophages) showed that genomically and/or serologically closely related phages clustered, whereas host range was not completely in correspondence with genotype. fRFLP might be a tool for quickly establishing the relationship of newly isolated phages to previously isolated ones and for constructing an fRFLP library electronically accessible on the internet, to which fRFLP patterns of new phages can be compared.