RNA Blotting Techniques Quiz

Blotting Techniques

• Western blotting is used to transfer proteins from acrylamide gels to membranes.
• Northern blotting is a technique for determining the number of genes (through mRNA) present on a given DNA.
• Southern blotting is a procedure in which RNA bands are blot transferred from the gel onto chemically reactive paper, where they are bound covalently.
• Dot blotting is a modification of Southern and Northern blotting techniques.

Southern Blotting Procedure

• Genomic DNA is isolated from cells/tissues and digested with one or more restriction enzymes.
• The mixture is loaded into a well in an agarose or polyacrylamide gel and then subjected to electrophoresis.
• DNA, being negatively charged, migrates towards the anode; smaller DNA fragments move faster.
• The separated DNA molecules are denatured by exposure to a mild alkali and transferred to nitrocellulose or nylon paper.

Transfer of DNA Molecules

• A nitrocellulose or nylon paper is sandwiched between the gel and a stack of paper towels, which draws the transfer buffer through the gel by capillary action.
• The DNA molecules are carried out of the gel by the buffer flow and immobilized on the membrane.

Hybridization and Autoradiography

• The nitrocellulose or nylon paper is exposed to labeled cDNA probes, which hybridize with complementary DNA molecules on the paper.
• The paper, after thorough washing, is exposed to X-ray film to develop autoradiograph, revealing specific bands corresponding to the DNA fragments recognized by the probe.

Variants of Blotting Techniques

• Eastern blotting is a variant of Southern blotting used for specific analysis.
• Dot blotting is a variant of Southern and Northern blotting techniques.