Summary auto-generated
Subtractive hybridisation (SH) and suppression subtractive hybridisation (SSH) are molecular techniques that identify DNA sequences present in one bacterial strain but absent from another, particularly useful for studying virulence differences between pathogenic and less virulent strains. By comparing genomic DNA from virulent strains with closely related avirulent strains, researchers can isolate regions often associated with pathogenicity islands (PIs)—genomic elements that contribute to disease-causing ability. The technique involves hybridizing DNA from a 'tester' strain with excess DNA from a 'driver' strain, then isolating and amplifying sequences unique to the tester through adaptor-based PCR. SSH, available as a commercial kit, has become particularly accessible. Applications of these methods include identifying strain-specific diagnostic markers, detecting mobile genetic elements like insertion sequences, characterizing virulence-related genomic islands, and comparing gene expression patterns between strains or under different growth conditions. SH has successfully identified virulence factors in numerous pathogens including Escherichia coli, Salmonella, Burkholderia pseudomallei, and Mycobacterium tuberculosis. While SH is effective at detecting regions with atypical GC content characteristic of horizontally-acquired sequences, some variations may be missed, necessitating complementary approaches for comprehensive genomic analysis.
Key findings
- SH and SSH techniques isolate DNA sequences unique to virulent bacterial strains by hybridizing tester DNA with excess driver DNA, followed by selective amplification using adaptor-based PCR
- Applications include identification of pathogenicity islands, mobile genetic elements, strain-specific diagnostic markers, and virulence-associated genes across diverse bacterial pathogens
- SH is particularly effective at identifying genomic regions with atypical, lower GC content typical of horizontally-acquired genes, though may miss variations with typical GC content
- Commercial SSH kits have made the technique more accessible; successful identification of known virulence regions validates the approach
- Gene expression studies using cDNA allow comparison between virulent and avirulent strains or same strains under different growth conditions
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Abstract
Comparison of DNA from virulent strains of bacterial pathogens with DNA from less virulent or avirulent close relatives allows the identification of those genomic regions that are present only in virulent strains. Such regions are often associated with pathogenicity islands (PIs) and their characterisation can lead to a greater understanding of the pathogenesis of infectious diseases. There is now a large database of bacterial genomic sequences that provides useful reference information with which to compare the genomes of strains that exhibit variations in virulence or host preferences. Subtractive hybridisation (SH) and its sister method, suppression subtractive hybridisation (SSH), are techniques designed to identify those regions present in one genome but absent from another. The application of these techniques has led to the identification of PIs, mobile genetic elements and variations in virulence gene expression in a range of bacterial pathogens.