Hybridization Technique; Southern Hybridization Technique probe

1. SOUTHERN BLOTING
Hybridization Technique-
Hybridization isatechnique inwhichsingle strandednucleicacidsare allowedtointeracttoform
complexes.
Or, hybridswithsufficientlysimilar complementarysequences.
Thistechnique allowsthe detectionof specificsequencesormaybe usedto assessthe degree of
sequence identity.
Hybridizationmaybe carriedinsolutionormore commonlyona solid-phase support.
Eg. Nitrocellulose paper.
Southern Blotting
In 1975, Edward M. Southernintroducedthistechnique.
A technique of DNA separationandhybridization.
Southerndevelopedthistechnique.Therefore,thisisknownasSouthermblottingorSouthern
hybridizationtechnique.
A specificDNA fragmentcanbe separatedandidentifiedinaheterogenouspopulationof DNA
molecules.
On the basisof bindingof DNA probe.
The genomicDNA isisolatedfromthe clone anddigestedwithrestrictionenzyme.
The DNA fragmentsare separatedbyagarose gel electrophoresis.
DifferentDNA bandsare formedonagarose gel whichrepresentDNA fragmentsof varyingsizes.
These fragmentsare transferredfromgel tonylonornitrocellulose membrane.
Now,thisputin a containerhavingNaOHsolution.
NaOH denaturesDNA and resultsinformationof single strandedDNA.
DNA fragments are transferredfromgel tomembrane bycapillaryaction.
DNA fragmentscan alsobe transferredbyvaccum blottingandcetrifugation.
The DNA fragmentsare fixedtoa membrane byusingUV radiationor bakingat80 degree celcius.

2. SOUTHERN BLOTING
The patternof DNA bands onmembrane correspondingtothe positionof DNA ongel.
The membrane isputin solutioncontainingradiolabeledDNA probe andincubatedforsometime.
DNA probe hybridizescomplementaryDNA fragmentsfixedonmembrane.
It isgentlywashedat12 degree celciusanddried.
The membrane isexposedthroughaphotographicfilm.
DNA bands formedonphotographicfilmcorrespondingtothe original positionof DNA fragments
presentonagarose gel.

3. SOUTHERN BLOTING
Application-
Useful inDNA separationandhybridization.
Specificfragmentscanbe identifiedeasily.
DNA Probes
It isa radio-labelled32
Psmall fragments.
These usesradioactive isotopicelements,
Isotopicsulphurorfluorescentmoleculesmayalsobe used.
It recognize A,T,GorC nucleotidesandcombineswiththe complementarysequencesof the targetDNA.
A-Tor G-C
Or, A DNA probe is a fragment of DNAthat contains a nucleotide sequence specific for the
gene or chromosomal region of interest. DNA probes employ nucleic acid hybridization with
specifically labeled sequences to rapidly detect complementary sequences in the test sample.
Application-
In searchingthe functional targetDNA isgiveninnucleicacidhybridizationtechnique.