Nucleic Acid Hybridization, Nucleic Acid.pptx

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1. Nucleic Acid Hybridization Rabia Amin M. phil. Biochem

2. What is Hybridization? Hybridization is a process of establishing noncovalent, sequence specific interaction between two or more complementary strands of nucleic acids into a single hybrid

3. Principle NAH is used to identify how close DNA molecules are. Factors to consider when performing a NAH assay between a probe and a target molecule are - strand length - base composition - chemical environment Basic Procedure Single stranded target DNA is bound to a membrane support. DNA probe labeled with detector substance is added DNA probe pairs with the complementary target DNA Sequence of nucleotide in the target DNA can be identified

4. DNA probe/Gene probe Synthetic single stranded DNA molecule that can recognize and specifically bind to a target DNA by complementary base pairing in a mixture of bio molecule. Detector system in DNA hybridization Radioactive Detector system Non-Radioactive Detector system

5. Types of hybridization Southern : electrophoresed DNA Northern : electrophoresed RNA Western : electrophoresed protein Dot blot : unfractionated target Colony : bacterial genome Plaque : virus genome

6. Northern Blotting Isolate RNA & treat with formaldehyde Electrophorese RNA in agarose gel Transfer single-stranded RNA to nitrocellulose or nylon membrane. Covalently link RNA to membrane. Incubate membrane with labeled DNA or RNA probe with target sequence Visualize RNA in gel using Ethidium bromide stain and photograph

7. Western Blotting The identification of specific antibodies is possible after the separation and blotting of proteins. Additionally, unspecific binding pockets can be blocked before the addition of specific antibodies. Primary antibodies are usually applied first, which are then recognized by a secondary antibody The secondary antibody is conjugated with color, radioactivity or an enzyme for detection

8. Dot Blot: The sample DNA or RNA from different individuals are loaded on to a nitrocellulose filter in the form of dots. The DNA is then denatured and the filter is baked at 80ºc to fix the DNA firmly to the filter. The filter is pre-treated to prevent non-specific binding of the probe to the filter. The filter is then treated with appropriate radioactive single stranded DNA probe under condition favoring hybridization. Filter is then washed repeatedly washed to remove free probe. The hybridized probes are detected by autoradiography

9. COLONY and PLAQUE BLOTTING Used for identification and purification of colonies Procedure Bacteria are grown as colonies on an agar plate Nitrocellulose filter paper over laid on the agar plate permanently fixed by heat Then treat with alkali the cell lyses occur and get de natured NDA When DNA prints are exposed to specific probes , hybridization occur Hybridization complex can be localized and detected by autoradiography

10. References Gösseringer R., Küster, E., Galinier A., Deutscher J. and Hillen W. (1997) Cooperative adn noncooperative DNA binding modes of catabolic control protein CcpA from Bacillus megaterium result from sesing two different signals. J. Mol. Biol., 266, 665-676. Grundy F.J., Waters D.A., George Allen S.H. and Henkin T.M. (1993) Identification of genes involved in utilization of acetate and acetoin in Bacillus subtilis Mol. Microbiol . 10, 259-271 Kolbert C.P., Persing D.H. (1999) Ribosomal DNA sequencing as a tool for identification of bacterial pathogens, Curr.Opin . Microbiol ., 2, 299-305. Kurtzman C.P. (1992) rRNA comparisions for assesing phylogenetic relationships among yeasts, Int. J. of Syst. Bacteriol ., 42, 1-6