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Blotting Techniques .pptx
- 2. ● Mutant genes and alleles can be determined by this analysis ● We can determined many genetic diseases ● Detection of large duplication and large deletion
- 3. Methods Westren Blotting Southren Blotting Northren Blotting Detect DNA sequence in Blood Detect RNA from mixture of RNA Detect protein molecule from protein mixture
- 4. Southern Blotting detection of a specific DNA sequence in DNA samples 01
- 5. Southern Blotting Extract & purify DNA from cell Eislectrophorasis Denature of DNA Autoradiograph Restrict DNA with Enzyme Transfer& Hybradize 1 2 1 2 3 4 5 6
- 6. Cell lysis to breakdown membrane of cell Lysis freez protein &DNA Protein damedge by protenase Organic & non- organic extraction remove protein DNA purify by Alcohal precipitation Visible DNA fibers remove Suspended in buffer
- 7. Restriction Enzyme • It act as a molecular seisor to cut the DNA. • Cut the DNA into different sizez using restriction endonuclease. • i.e E-coli Electrophorasis • Technique use in lab to separate charge molecule like DNA acording to size. • Solid agarose gel is used in this method . • Stained with ethidium bromide for visulization Denature of DNA • The restriction fragments present in gel denature with alkali. • This cause double standard become single • DNA neutralize with NaCL prevent re- hybradization brfore adding probe .
- 8. Hybradization • Process of forming double standard DNA and RNA with single standard probe. • Probe added to membrane in buffer incubate for several hours allow probe molecule to find target. • Probe : small piece of labeled DNA use to find complementary DNA fragments. Autoradiograph • For radioactive probe, X-ray filam is placed over the membrane. • After development there will be dark band on the filam where the probe found.
- 9. ● To identify specific DNA in a DNA sample ● To isolate Desire DNA for making rDNA ● Diagnosis of HIV-1 and infectious disease ● IN DNA fingerprinting; I. Criminal identification II. personal identification
- 10. Northern Blotting Detect RNA from mixture of RNA 02
- 12. STEPS 01 02 03 04 05 06 Samples are loaded on gel saparate by electrophorasis The labeled probe detected by autoradiograph DNA fragments are treated by formaldihyde. Gel is transfer on nylon membrane by creating sandwitch arrangement Membrane place in dish containing hybradization buffer with labeled probe It will hybradize RNA on blot that correspond sequence of intrest.
- 13. ● Use to study gene of expression ● Study RNA half life ● Used for detection of viral micro RNA that play key role in viral infection ● Study RNA degradation ● Study RNA splicing
- 14. Western Blotting Detect protein molecule from protein mixture 03
- 17. ● Western blot can also be used as confirmatory test for Hepatitis B infection. ● Use in biochemistary to detect presence of specific proteins ● Senstivity and specificity ● Use to detect carbohydrate epitopes