Simple Methods for Concentration, DNA Extraction and Agarose Gel Electrophoresis of Single-Stranded DNA Somatic Coliphages
DNA fingerprinting of waterways using bacteriophages common to human sewage is a potential way to monitor the health of an aquatic ecosystem. Somatic coliphages, a single-stranded DNA (ssDNA) coliphage that infect Escherichia coli (E. coli), are a common component of wastewater and are a potential indicator of human faecal pollution. Current methods for ssDNA extraction from bacteriophage use the replicative form (RF) (double-stranded DNA) of the phage DNA. The methods to produce RF DNA are time-consuming and require an ultracentrifuge. Alternative methods for the concentration, DNA extraction and agarose gel electrophoresis of coliphage ssDNA were investigated. E. coli C liquid host culture proved to be unsatisfactory for extracting coliphage DNA, as genomic DNA from the host interfered with the electrophoresis of the coliphage DNA. Plaques from coliphage assays of samples were placed in buffer and concentrated using centrifugal ultrafiltration. The concentration step increased the number of virus particles/mL (VLPs/mL) twenty-fold and there was no genomic DNA contamination. Five plaques were sufficient to produce clear bands on an agarose gel. Heat treatment and proteinase K proved successful in releasing the DNA from the phage concentrate and produced brighter bands on agarose gels than samples that were not treated. The addition of 300mN sodium hydroxide (NaOH) to the dsDNA markers for electrophoresis denatured the markers to ssDNA. The DNA samples for electrophoresis were resuspended in alkaline dissolution buffer. The loading dye for the samples was alkaline with the addition of 300 mN NaOH to retain the ssDNA in a denatured form. The DNA markers containing 300 mN NaOH matched the migration rate of the positive control. The agarose gels were stained with SYBR Gold, a stain that can bind to ssDNA, RNA and dsDNA. This dye provided highly visible bands under ultraviolet light. These alternative methods were successfully optimized for ssDNA bacteriophage.
ASM 2005 Canberra
HISTORY AND ARCHAEOLOGY