DNA Extraction and Characterization of Salmonella‟s Lytic Bacteriophage Genome

Abstract

Bacteriophages are ubiquitous in nature and are known to proliferate wherever their bacterial hosts exist. Their virion particles can exist independently outside the host, however all phages are need their host to propagate. Several phages are highly specific to host cell surface receptors and any slight changes in structure results in little or no interaction between the phage and its host. Salmonella‟s phage isolated from sewage water where their bacteria exist. Three isolated of phages (phages 15, 19 and 38) were used in this research that concentrated using three types of methods. PEG/ NaCl method was firstly used to concentrate the phages based on their molecular weight to precipitate the phage particles during centrifugation. The second method was Scraping Plaque method. In this method, overlay plaques were scraped and collected in SM buffer to get phage concentrates. Enrichment method was the last method used to concentrate the phages. Phage genome isolation was done using two methods i.e. EDTA/SDS and Proteinase K methods. The first method was done using DNase I to eliminate bacterial DNA followed by SDS/EDTA treatment to release phages DNA and phenol-chloroform-isoamyl alcohol to extract the DNA. DNA was collected by isopropanol extraction. While in Proteinase K method, bacterial DNA was eliminated by DNase I and Proteinase K/SDS was used to degrade phages capsids and ethanol for DNA precipitation. The result showed that PEG/NaCl method resulted the highest phage concentration for phage 15 and phage 19, while scrapping method was the highest for phage 38. The quantity and purity of extracted DNA were low for PEG/NaCl and Scraping methods, while Enrichment method was higher indicated higher purity. Run on agarose gel electrophoresis of extracted DNA confirmed that Enrichment method results were the best while scraping and PEG/NaCl method was appeared smear background. Based on DNA genome size determination, the bacteriphages of Salmonella sp were comparable with other study of Salmonella phages. Phage P15 with genome size of 39,5 kb was close to phage epsilon15. Phage P19 with genome size of 41 kb was close to phage ST160. And phage P38 with genome size of 48,5 kb was close to phage Gifsy-1.