The document discusses DNA-DNA hybridization, a technique used in bacterial taxonomy since the 1960s. It involves breaking genomic DNA into fragments, denaturing the strands, and allowing complementary strands to reanneal. The stability of the hybridization product, measured by melting temperature, indicates genetic relatedness between strains and is used to delineate bacterial species. More recently, DNA microarrays have been used to conduct high-throughput DNA-DNA hybridization comparisons to construct similarity dendrograms and classify bacterial relationships.

1. Presented by
Nishanth S
2016601106

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.

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

4. PCR - Principle
1. Denaturation
2. Annealing
3. 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
1. To determine relatedness between strains
2. Delineation of bacterial species
DNA hybridization

6. Hybridisation in Bacterial Taxonomy

7. Single stranded DNA molecules of defined sequence
(PROBE), can base-pair to a second DNA molecule that contains a
complementary sequence (TARGET).
1. Shearing the gDNA of the reference into small fragments of
600-800 bp
2. Heating the mixture of DNA fragments from both strains to
dissociate the DNA double strands
3. 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:
1. DNA concentration & Time
2. DNA strand length
3. GC content
The stability of the hybridisation product
“to what extent the base pairing has occurred”
Rate of hybridisation depends on:
1. DNA concentration & Time
2. DNA strand length
3. GC content

9. Access melting profile of hybridized DNA

12. Illustration

13. Use of DDH in modern taxonomy

14. DNA microarray in DNA-DNA hybridisation
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

15. GENOMIC DNA LABELING & HYBRIDISATION
SCANNING AND DATA PROCESSING

16. Microarray - plate

17. Similarity dendrogram (UPGMA) of microarray hybridization profiles

18. 1. 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.
2. 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
3. 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
4. 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