Northern & Southern Hybridization. Avalon Garcia 02-20-04. Introduction. Concept: reannealing nucleic acids to identify sequence of interest. Separates DNA/RNA in an agarose gel, then detects specific bands using probe and hybridization.
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Developed by Edwin Southern.
Uses gel electrophoresis together with hybridization probes to characterize restriction fragments of genomic DNA (or DNA from other sources, such as plasmids).
Identifies DNA with a specific base sequence.
Can be done to detect specific genes present in cells.
1. DNA to be analyzed is digested to completion with a restriction endonuclease.
2. Electrophoresis to maximally separate restriction fragments in the expected size range. A set of standards of known size is run in one lane of the gel.
3. Blot fragments onto a nitrocellulose membrane.
4. Hybridize with the 32P probe.
Soak gel in 0.5 M NaOH
1. Isolate RNA & treat with formaldehyde.
2. Electrophorese RNA in denaturing agarose gel (has formaldehyde). Visualize RNA in gel using Ethidium bromide stain and photograph.
3. Transfer single-stranded RNA to nitrocellulose or nylon membrane. Covalently link RNA to membrane.
4. Incubate membrane (RNA immobilized on membrane) with labeled DNA or RNA probe with target sequence.
-To detect rare mRNA, isolate the poly A+ mRNA.
-RNA is both biologically and chemically more labile than DNA. Thus eliminate RNases.
-Performed in formaldehyde agarose gel to prevent RNA from folding on itself.
-Stain with EtBr to visualize the RNA bands.
-Transfer single-stranded RNA to nitrocellulose or nylon membrane:
Traditionally, a nitrocellulose membrane is used, although nylon or a positively charged nylon membrane may be used.
Nitrocellulose typically has a binding capacity of about 100µg/cm, while nylon has a binding capacity of about 500 µg/cm. Many scientists feel nylon is better since it binds more and is less fragile.
-Covalently link RNA to membrane:
UV cross linking is more effective in binding RNA to the membrane than baking at 80C.
-Prehybridize before hybridization:
Blocks non-specific sites to prevent the single-stranded probe from binding just anywhere on the membrane.
-Incubate membrane with labeled DNA or RNA probe with target sequence:
Probe could be 32P, biotin/streptavidin or a bioluminescent probe.
Place membrane over X-ray film.
X-ray film darkens where the fragments are complementary to the radioactive probes.
Sobel, R., Dubitsky, A., Brickman, Y. (2002) Genetic Engineering News 23, 2.
When trying to learn about the function of a certain protein, it is sometimes useful to purify mRNA from many different tissues or cell types and then prepare a Northern blot of those mRNAs, using a cDNA clone of the protein of interest as the probe.
Only mRNA from the cell types that are synthesizing the protein will hybridize to the probe.
DNA on membrane.
Convert dsDNA to ssDNA.
Probe with DNA or RNA.
RNA on membrane.
No need to digest DNA.
Denature “folded” RNA with formaldehyde.
Probe with DNA or RNA.Summary
Kornberg, A., Baker, T. A., DNA Replication. (2nd ed.) pp 15, W. H. Freeman & Co. (1992)
Sobel, R., Dubitsky, A., Brickman, Y. (2002), Genetic Engineering News, 23, 2.
Southern, E. M., (1975) J. Mol. Biol. 98:503-517.
Voet, D., Voet, J. Biochemistry. (vol. 1, 3rd ed.), pp 110-112, John Wiley & Sons Inc. (2004).