Bacteriophage genotyping using BOXA repetitive-PCR
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METHODOLOGY ARTICLE
Open Access
Bacteriophage genotyping using BOXA repetitive-PCR Dragica Damnjanovic, Xabier Vázquez-Campos, Daniel L. Winter, Melissa Harvey and Wallace J. Bridge*
Abstract Background: Repetitive-PCR (rep-PCR) using BOXA1R and BOXA2R as single primers was investigated for its potential to genotype bacteriophage. Previously, this technique has been primarily used for the discrimination of bacterial strains. Reproducible DNA fingerprint patterns for various phage types were generated using either of the two primers. Results: The similarity index of replicates ranged from 89.4–100% for BOXA2R-PCR, and from 90 to 100% for BOXA1R-PCR. The method of DNA isolation (p = 0.08) and the phage propagation conditions at two different temperatures (p = 0.527) had no significant influence on generated patterns. Rep-PCR amplification products were generated from different templates including purified phage DNA, phage lysates and phage plaques. The use of this method enabled comparisons of phage genetic profiles to establish their similarity to related or unrelated phages and their bacterial hosts. Conclusion: The findings suggest that repetitive-PCR could be used as a rapid and inexpensive method to preliminary screen phage isolates prior to their selection for more comprehensive studies. The adoption of this rapid, simple and reproducible technique could facilitate preliminary characterisation of a large number of phage isolates and the investigation of genetic relationship between phage genotypes. Keywords: Bacteriophage, Phage genotyping, Repetitive-PCR
Background Repetitive DNA sequences constitute a substantial component of both eukaryotic and prokaryotic genomes. In some higher plant species, they can account for up to 90% of the genomic DNA [1], while in humans DNA repeats comprise nearly half of the genome [2]. The presence or absence of certain types of repeats, diversity in their nucleotide sequences, their size, location and copy number per genome characterize various bacterial species, even those with the smallest genomes [3]. Interspersed repeats play a significant role in genomic rearrangements, such as inversions, deletions, duplications and translocations [3]. The proposed functional roles of repetitive sequences involve the regulation of coding * Correspondence: [email protected] School of Biotechnology and Biomolecular Sciences, Faculty of Science, UNSW Sydney, Kensington, Australia
sequence expression and the formatting necessary for genome packaging; DNA repair and restructuring; genome replication and transmission to progeny cells; formation of nucleoprotein complexes; and formation of a characteristic genome system organization that allows for evolutionary significant changes without altering coding sequences [4]. Specific families of interspersed DNA elements have been observed in many bacterial and archaeal genomes [3, 5], while bacteriophages are considered to carry few repetitive elements [5]. The BOX family of repetitive DNA elements, consisting of different combinations of th
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