Subtracted Restriction Fingerprinting—A Tool for Bacterial Genome Typing

Abstract

Reproducible, discriminative, high-throughput methods are required for the identification of bacterial strains and isolates in a clinical environment. A new molecular typing method for bacteria was developed and tested on Salmonella and E. coli species. The technique is called subtracted restriction fingerprinting and is based on double restriction enzyme digestion of genomic DNA followed by end labeling. The detection enzyme produces TTAA overhangs that are filled in with digoxigenated nucleotides for subsequent detection, while the subtraction enzyme produces GCGC overhangs that are filled in with biotinylated nucleotides that permit the removal of this subset of fragments with either streptavidin-coated magnetic particles or AffiniTipTM streptavidin columns. The two restriction enzymes are selected to produce a fragment size profile suitable for a specific analytical system. In this demonstration of the principle of subtracted restriction fingerprinting, analysis of Salmonella enterica subsp. enterica serovar Dublin and E. coli on a 30-cm 1.2% agarose gel revealed up to 50 sharp evenly spaced bands, which were sufficient for the discrimination between various isolates and substrains. The restriction enzyme combinations suitable for the analysis of Salmonella and E. coli are presented. The method requires fewer enzymatic steps than amplified fragment length polymorphism, does not need the specialized DNA preparation essential for pulsed field gel electrophoresis, and has a higher reproducibility than PCR-based methods.

Subtracted restriction fingerprinting – a new typing technique using magnetic capture of tagged restriction fragments

“…45 fragments) and Eco RI– Cfr 10I (ca. 22 fragments) [12]. This observation corresponded well with the cutting frequencies of these subtraction enzymes (Table 1).…”

“…The development of SRF [12] was initiated in response to the demand for a new typing method that is easy to perform, is highly reproducible and universally applicable, has high discriminatory power and does not require expensive equipment. SRF should combine the advantages of AFLP analysis and macrorestriction analysis (large number of resolved bands, high reproducibility) with that of restriction fragment end labelling (equal band intensity).…”

“…2). In our application of SRF for bacterial genome analysis the amount of DNA ranged from 100 to 500 ng for 5 μl (50 μg) of magnetic particles which is at least 2‐fold excess in absorption capacity [12].…”

“…Examination of different pairs of detection and subtraction enzymes allowed us to find suitable combinations for E. coli and Salmonella strains. The best choice for E. coli was Eco RI– Cfr I enzymes while Salmonella required Eco RI– Pau I enzymes [12].…”

“…The aim of this minireview is to present a background for the development of a recently published new typing method called subtracted restriction fingerprinting (SRF), which combines the advantages of existing bacteria typing techniques, and to provide examples of its application for the typing of Salmonella isolates [12,13].…”