DNA-DNA Hybridisation

This is the technique used in the bacterial systematics and taxonomy

1. Presented by Nishanth S 2016601106 DNA–DNA HYBRIDIZATION TAMIL NADU AGRICULTURAL UNIVERSITY

2. The first attempt to elucidate taxonomic relationships based on single-stranded DNA reassociation conducted by Schildkraut et al. (1961) was a breakthrough for microbial systematics and for the construction of the current microbial classification system . Ramon Rossello -Mora (2006)

3. The Structure of DNA The strands of DNA are linked together by “WEAK HYDROGEN BOND”

4. PCR - Principle Denaturation Annealing Extension

5. “DNA Hybridization is the process of establishing a non-covalent, sequence-specific interaction between two or more complementary strands of nucleic acids into a single hybrid, which in the case of two strands is referred to as a duplex.” DNA-DNA hybridization values have been used by bacterial taxonomists since 1960s To determine relatedness between strains Delineation of bacterial species DNA hybridization

6. Hybridisation in Bacterial Taxonomy DNA PROBE HYBRID ISATION DETERMINE THE DEGREE OF GENETIC SIMILARITY

7. S ingle stranded DNA molecules of defined sequence (PROBE), can base-pair to a second DNA molecule that contains a complementary sequence (TARGET). Shearing the gDNA of the reference into small fragments of 600-800 bp Heating the mixture of DNA fragments from both strains to dissociate the DNA double strands Subsequently decreasing the temperature until the fragments reanneal Principle of DNA-DNA hybridization

8. Hybrid stability is expressed as the melting temperature (Tm) Rate of hybridisation depends on: DNA concentration & Time DNA strand length GC content The stability of the hybridisation product  “ to what extent the base pairing has occurred” Rate of hybridisation depends on: DNA concentration & Time DNA strand length GC content

9. Access melting profile of hybridized DNA ds DNA bound to the column The mixture is heated The mixture is heated in small steps Each step – column washed Sequence melt – ss DNA washed off Temperatures at which labelled DNA comes of the column reflects the a mount of similarity between sequences Combined results – Determine the degree o f genetic similarity between organisms

10. PROBE TARGET

11. Illustration

12. Use of DDH in modern taxonomy

13. DNA microarray in DNA-DNA hybridisation DNA fragmented by bead beating – to ensure randomness Agarose gel  eluted and purify DNA PCR amplified with T3-T7 promoter primer Resuspende d in amino silane -coated slides Fragments were spotted after processed with succinic anhydride blocking method BACTERIAL STRAINS AND DNA EXTRACTION MICROARRAY FABRICATION Pseudomonas fluorescens ATCC 13525T P. fluorescens ATCC 17397 P. fluorescens ATCC 17400 P. fluorescens ATCC 17467 P. fluorescens ATCC 33512 P. marginalis LMG 5039 P. chlororaphis ATCC 9447 P. chlororaphis ATCC 17811 P. aureofaciens ATCC 13985T P. putida ATCC 12633T P. aeruginosa ATCC 15692 P. aeruginosa ATCC 17429

14. Labeled with FluoroLink Cy3-dCTP by random priming Reference strains – FluoroLink Cy5-dCTP Prehybridisation for 20 min Hybridisation @ 65C overnight GENOMIC DNA LABELING & HYBRIDISATION SCANNING AND DATA PROCESSING Scan – GenePix 4000 laser scanner Laser lights of wavelength @ 532 and 6335 nm excite Cy3 & Cy5 respectively Image capture as analysed GenePix 3.0 software Hybridisation signal ratios were calculated

15. Microarray - plate

16. Similarity dendrogram (UPGMA) of microarray hybridization profiles

17. Goris , J et al., 2007. DNA–DNA hybridization values and their relationship to whole-genome sequence similarities. International Journal of Systematic and Evolutionary Microbiology, 57: 81–91. Ramon Rossell´o -Mora. 2006. DNA–DNA reassociation methods applied to microbial taxonomy and their critical evaluation. Molecular Identification, Systematics , and Population Structure of Prokaryotes. E. Stackebrandt (Ed.) C Springer- verlag Berlin Heidelberg Chang Cho, J and Tiedje , J M. 2001. Bacterial Species Determination from DNA-DNA Hybridization by using Genome Fragments and DNA Microarrays. Applied and Environmental Microbiology, Vol. 67 , No. 8 p. 3677–3682 Auch A F et al., 2010. Digital DNA-DNA hybridization for microbial species delineation by means of genome-to-genome sequence comparison. Standards in Genomic Sciences. 2: 117-134 References

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