An improvement of restriction analysis of bacteriophage DNA using capillary electrophoresis in agarose solution

Abstract

Seven representatives of the serogroup B Staphylococcus aureus bacteriophages, 29, 53, 55, 83A, 85, phi 11 and 80 alpha, were examined by capillary electrophoresis (CE) for genomic homology using DNA restriction analysis. Genomic DNA of individual bacteriophages was cleaved by HindIII restriction endonuclease, and the resulting restriction fragments were separated by standard horizontal agarose slab gel electrophoresis (SGE) as well as by CE in low-melting-point agarose solutions. The number and size of restriction fragments identified by both methods were compared. The high separation power of CE makes it possible to extend the restriction fragment patterns. In most of the restriction patterns, some additional restriction fragments as small as 150 bp, not identified by SGE, were detected. With respect to speed, high separation efficiency, low sample consumption and automation, CE offers a simple procedure for processing of multiple samples cost-effectively in a reasonable time. The comparison of the complemented restriction patterns of the different phage strains and the subsequent identification of their common fragments leads to a deeper understanding of their phylogenetic relationships. The genome homologies expressed for individual phage pairs in terms of coefficient F values ranged from 15 to 69%. These values are in good accordance with the degree of DNA homology of these phages as determined by DNA hybridization studies and thermal denaturation analysis of DNA by other authors. The total size of each phage genome was estimated by adding the sizes of individual restriction fragments.

Principles of DNA separation with capillary electrophoresis

“…For separating DNA in CE, a number of different polymers have been used so far (see also [8] for review). Most of the polymers used are modified polysaccharides: agarose and its derivatives with low gelling temperature [7,9] and various cellulose derivatives such as methylcellulose…”

Capillary electrophoresis in sieving matrices: Selectivity per base, mobility slope, and inflection slope

“…The possibility of replacing the sieving matrix after each electrophoretic run made this a popular tool for separation of biopolymers. Many natural and synthetic polymers have been successfully used to achieve separation, including hydroxyethyl cellulose [10±14], galactomannan [15], dextran [16], linear polyacrylamide [17±24], polyvinylalcohol [25], polydimethylacrylamide [26], polyacryloylethoxyethanol [27,28], polyethylene oxide [29±31], agarose [32], polyacryloylaminopropanol [33], and others.…”

Quantitative analysis of DNA methylation in the promoter region of the methylguanine‐O6‐DNA‐methyltransferase gene by COBRA and subsequent native capillary gel electrophoresis

“…GE represents a standard procedure for quantification of DNA . Therefore, two artificial series (series 1, series 2) and two normal series of DNA fragments (series D, series E) were analyzed using GE (sample preparation described in chapter 2.3.).…”