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Southern Blotting - There is often need to determine which particular fragment of DNA is complementary to another fragment.

Restriction fragments of cloned or total genomic DNA can be mapped by hybridization to a probe.

The technique known as "Southern blotting (Southern, 1975) allows this to be done. It is sensitive enough to map restriction sites around single copy genes in total genomic DNA, if a suitable probe is available (Jeffreys, 1979).

Restriction fragments of the DNA to be tested are prepared and separated by agarose gel electrophoresis.

These fragments are transferred, or blotted, to a nitrocellulose filter and then fixed firmly in place by baking at 80ºC.

The filter is subsequently incubated with the radiolabeled RNA or DNA to be used as the probe and hybridization occurs between complementary sequences. After washing, hybridization sequences are detected by autoradiography. Development of this powerful technique made this type of analysis much easier and obviated the need for lengthy procedures such as eluting nucleic acids from gel slices.

Southern blotting is simpler to carry out and gives much higher resolution. In plant molecular biology it has been used extensively to map chloroplast genomes using probes from a different species.

This method utilizes the hybridization of complementary DNA fragments to identify integrated foreign DNA. For example, the probe can be labeled with 32p.

The probes specifically bind to cDNA present on the filter and can be visualized on an autoradiograph of the filter.

    
 
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