I know people don't use it these days, but i always feel its good to learn how people overcame the challenges of little resource and knowledge in those days and shaped the way for us future biologists! So here it is mates, #SouthernBlotting i.e. detection of specific DNA fragments by blotting!
Principle
The principle here is detection of #DNA by radioactive #DNAProbes by means of #hybridization.
Protocol
For detecting specific #DNAFragments or #genes, the #GenomicDNA is cut with a specific #RestrictionEndonuclease into a pool of #DNA. This pool of fragment is separated on #AgaroseGel. As many different fragments may be of the same size or differing in few #bases they are not properly separated.
The fragments are next denatured with mild #alkali treatment (eg. dilute #NaOH solution) to single-stranded form, without affecting their original distribution over the gel.
The gel is then laid upon a buffer-saturated #FilterPaper, placed on a solid support of a glass plate with two of its edges touching the buffer.
A piece of #NitrocelluloseMembrane with a charged surface is placed over the gel and a bunch of paper towels weighing about 500g are placed atop the #membrane.
The #buffer solution is drawn up by the paper towel stack and while passing through the gel and membrane it carries and transfers the #ssDNA to the membrane tenaciously.
After leaving the setup for few hours to overnight, the paper towels are removed and the membrane is peeled off the gel. Peeling is done after baking the gel for 80C for 2-3 hours (Hot vacuum drying) to anneal and fix the #ssDNA fragments permanently on the gel.
The sheet containing the bound DNA is placed inside a ziplock bag with a buffered salt solution containing P32-labelled #cDNAProbes.
The moistened membrane is then incubated for few hours at suitable temperature for #hybridization with probes and #renaturation.
Lastly the membrane is washed with buffer to remove unbound probes and #autoradiographed to reveal sizes of target #DNA fragments.
Merits
The method is #efficient as a specific gene can be detected and isolated from even a 5ug genomic DNA sample.
Demerits
Radioactive hazard
Ethidium bromide health hazard
Long procedure involving at least 2 days.
Applications
#SouthernBlotting allows a comparison between the restriction map of cloned #DNAs. This comparison is necessary to be certain that no rearrangements have occurred during the cloning procedure.
It is also used to map the restriction sites in genomic #DNA samples.
It is useful in determining how many copies of a gene are there in a given tissue or whether there are any gross alterations in a gene. Rarely with abolition of a restriction site, #PointMutation can also be detected.
It can detect few genetic disorders like #transition or #transversion like #SickleCellAnemia and few #thalassemias.
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