Nicholas Chang

A Standardized Protocol for the Rapid Preparation of Bacterial DNA for Pulsed-Field Gel Electrophoresis

A rapid method for the preparation of bacterial DNA for pulsed-field gel electrophoresis was developed for Gram-positive and Gram-negative bacteria. This method was accomplished by reducing the time for the cell lysis reaction, restriction endonuclease digestion, and electrophoresis to 1, 1.5, and 18 h, respectively. The whole procedure from the initial bacterial culture plate to the final analysis of restriction fragments can be completed within 24 h. This rapid method was successfully achieved for Staphylococcus aureus, Enterococcus faecalis, Neisseria gonorrhoeae, Salmonella typhimurium, Serratia marcescens, and Stenotrophomonas maltophilia.

Immunoblot and enzyme-linked immunosorbent assays of Campylobacter major outer-membrane protein and application to the differentiation of Campylobacter species

The major outer-membrane protein (MOMP) from Campylobacter jejuni NCTC 11168 was purified by solubilization in Triton X-100. Whole-cell proteins of Campylobacter species and the MOMP were subjected to electrophoresis on sodium dodecyl sulfate-polyacrylamide gels, immunoblotting on nitrocellulose paper and enzyme-linked immunoblot assay (ELISA) analyses using polyclonal antiserum to the C. jejuni MOMP. Purified MOMP from C. jejuni NCTC 11168 contained a single major protein of 46 kDa. Whole-cell preparation of C. jejuni NCTC 11168 also showed a major band of 46 kDa which was recognized in immunoblots by the anti-MOMP serum. Other C. jejuni strains, as well as C. coli and C. laridis strains showed similar MOMP bands at 45-46 kDa. Other Campylobacter species (C. fetus ss. fetus, C. hyointestinalis and C. pylori (new nomenclature] did not react in immunoblots with antiserum to the C. jejuni MOMP. ELISAs showed that antiserum raised against the C. jejuni MOMP reacted with C. jejuni, C. coli and C. laridis strains. Other Campylobacter species displayed only a very low degree of cross-reactivity. The distinct antigenic relationship demonstrated between the MOMP of C. jejuni, C. coli and C. laridis is consistent with the close degree of relatedness of these species as determined by DNA homology studies. The anti-MOMP serum appeared to be useful in the rapid differentiation of C. jejuni, C. coli and C. laridis from other Campylobacter species.

Genome map of Campylobacter fetus subsp. fetus ATCC 27374

A physical map of the chromosome of Campylobacter fetus subsp. fetus was constructed by using pulsed-field gel electrophoresis of restriction fragments generated by SalI, SmaI and NotI. Digestion of the type strain ATCC 27374 with these restriction endonucleases resulted in generating 4-14 fragments. The order of the fragments was deduced from hybridization of these restriction fragments to Southern blots of pulsed-field gel electrophoresis gels generated by the other two enzymes. The estimated genome size was 1160 kb. The position of several homologous and heterologous genes was determined on the circular map. These included the 2.8-kb sapA gene, encoding the 97-kDa surface array protein. Three copies of ribosomal RNA genes for which the 16S, 23S and 5S rRNA appeared to be located in close proximity in each of the three regions. The RNA polymerase genes rpoA, rpoB, and rpoD were mapped and appeared to be situated close together in one region. The flagellin genes (flaAB) of C. jejuni and the gyrase genes gyrA and gyrB of C. perfringens and Bacillus subtilis, respectively, were used to identify the locations of flaAB, the gyrA and the gyrB genes on the ATCC 27374 chromosome.

Construction of a Genomic Map of H. pylori by Pulsed-Field Gel Electrophoresis (PFGE)

The inability of conventional gel electrophoresis to separate DNA molecules exceeding 50 kb in size led to the development of pulsed-field gel electrophoresis (PFGE) by Schwartz et al, (1)in 1982. He introduced the concept of applying two alternating electric fields (i.e., pulsed-field) to separate DNA molecules greater than 50 kb embedded in an agarose gel matrix. Since then, many instruments based on this principle have been developed. For a discussion of various pulsed-field applications, see review articles by Lai et al. (2) and Crété et al. (3). It was shown that, under the influence of an electric field, a DNA molecule embedded in a gel matrix undergoes reorientation, elongation, and migration along the field toward the anode. When a second field is applied in an alternate direction, the DNA molecule must reorientate, elongate, and migrate along the direction of the new field. Larger DNA molecules will take longer to reorientate than smaller molecules; therefore the larger ones spend less time migrating down the gel than the smaller per pulse time. Consequently, larger DNA molecules will appear near the origin while the smaller molecules will migrate furthest. This principle becomes important when different fragment sizes of DNA are to be separated. PFGE has become an important tool for determining genome sizes, physical mapping of the chromosome, and localization of genes on the chromosome of prokaryotic micro-organisms.