: In this technique, RNA are separated by gel electrophoresis, the RNA bands are transferred onto a suitable membrane, e.g. diazobenzyleoxymethyl (DBM) paper or nylon membranes, and immobilized; the bands are hybridized with radioactive single stranded DNA probes and the bands showing hybridization are detected by autoradiography.
Northern blotting hybridization is simply an extension of Southern blotting technique. It shows following differences from Southern Blotting:
• In Southern hybridization DNA is separated by gel electrophoresis, while in northern blotting RNAs are separated.
• In Southern hybridization DNA has to be denatured before blotting, while this step is not needed in northern hybridization.
• Nitrocellulose membrane is generally not used for northern while it is often used for Southern hybridization.
• Hybridization with the probe produces DNA: DNA hybrid molecules in Southern but RNA: DNA molecules in northern hybridization.
Initially specially prepared paper (diazobenzylomethyl, DBM, paper prepared by diazotization of aminobenzylomethyl paper) was used for northern blotting since RNA did not bind to nitrocellulose membrane. RNA becomes covalently bound to DBM paper due to which these blot transfers are reusable. DBM is also equally effective in binding to denatured DNA, and is more efficient than nitrocellulose in binding to small DNA fragments. Recently developed nylon membranes have superceded the use of DBM paper as they are robust, reusable and bind (by cross linking) to RNA on a brief exposure to UV light.
Uses: This technique is useful in the identification and separation of the RNA that is complementary to a specific DNA probe. This is a sensitive test for the detection of transcription of a DNA sequence that is used as probe.
What is Northern Hybridization and its effects
Author: Dr. Leena Kansal
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