Souther and northern blotting systems

Souther and northern blotting systems

1. Southern Blotting and Northern Blotting Presented by : Chaudhary Ankit R. Submitted to : Dr. Kapil K Tiwari Department of Genetics & Plant Breeding C .P . College of Agriculture Sardarkrushinagar , Dantiwada Reg. No. : 04-AGRMA-01571-2017

2. Blotting are techniques for transferring DNA, RNA and proteins onto a carrier so they can be separated, and often follows the use of a gel electrophoresis. The Southern blot is used for transferring DNA, the Northern blot for RNA and the western blot for Protein . These powerful techniques allow us to identify and characterize specific molecules in a complex mixture of related molecules What is blotting?

3. The key to these methods is Hybridization. Hybridization : It is the process of forming a double stranded DNA molecule between a single-stranded DNA probe or a single-stranded target DNA. There are 2 important features of hybridization: The reactions are specific-the probes will only bind to targets with a complementary sequence. The probe can find one molecule of target in a mixture of millions of related but non-complementary molecules. PRINCIPLE

4. Hybridization technique Southern Blot It is used to detect DNA. Northern Blot It is used to detect RNA. Western blot It is used to detect protein. Dot Blot used to detect sexually transmitted diseases In Situ Hybridization Used to detect Location of specific DNA sequences.

5. Nitrocellulose membrane Poly vinylidene difluoride (PVDF) membrane Nylon membrane Diazo benzyloxy methyl(DBM) and Diazo phenyl thioether (DPT) Blotting membranes

6. A Southern blot is a method used in molecular biology for detection of a specific DNA sequence in DNA samples. Southern blotting combines transfer of electrophoresis - separated DNA fragments to a filter membrane and subsequent fragment detection by probe hybridization. The method is named after its inventor, the British biologist Edwin Mellor Southern. Southern blot

7. 1. Extract & purify DNA from cells 2. DNA is restricted with enzymes 3. Separated by electrophoresis 4. Denature DNA 5. Transfer to nitrocellulose paper (blotting) 6. Add labeled probe for hybridization 7. Wash off unbound probe 8. Autoradiograph Steps in Southern Blotting

8. Step 1 :DNA purification Isolate the DNA in question from the rest of the cellular material in the nucleus. Incubate specimen with detergent to promote cell lysis, cell lysis frees cellular protein and DNA. Proteins are enzymatically degraded by incubation with proteinase. DNA is purified from solution by alcohol precipitation. Visible DNA fibers are removed and suspended in buffer. PROCEDURE

9. Step 2:RESTRICTION DIGEESTION Cut the DNA into different sized fragments using restriction endonucleases (RE).

10. Step 3 : Gel electrophoresis Nucleic acid has a net negative charge and will move from the left to right. The larger molecules are held up while the smaller ones move faster. This results in a separation by size. Gels are Agarose or polyacrylamide With microscopic pores. Gel is soaked in a buffer which Controls the size of the pores.

11. Gels can be stained with ethidium bromide, this cause DNA to fluoresce under UV light which permits photography of the gel. This will help us to know the exact migration of DNA strands and the quality of the RE digestion of the test DNA.

12. Cover gel with nitrocellulose paper…then… Cover nitrocellulose paper with thick layer of paper towels. Compress apparatus with heavy weight. ssDNA binds to nitrocellulose at same position it had on the gel. Vacuum dry nitrocellulose at 80 C to permanently fix DNA in place. Step 3. Nitrocellulose Blot

13. Incubate nitrocellulose sheet with a minimal quantity of solution containing 32P-labeled ssDNA probe. Probe sequence is complementary to the DNA of interest. Incubate for several hours at suitable temperature that will permit probe to anneal to its target sequence(s). Wash & dry nitrocellulose sheet. Step 4. Hybridization

14. Place nitrocellulose sheet over X-ray film. X-ray film darkens where the fragments are Complementary to the radioactive probes. Step 5. Autoradiography

15. General Scheme for Southern Blot

16. To identify specific DNA in a DNA sample. To isolate desired DNA for construction of rDNA . Identify mutations , deletions ,and gene rearrangements. Diagnosis for HIV and infectious disease. APPLICATIONS

17. Modern Blotting Techniques Using nylon instead of nitrocellulose membrane is speed up the movements of DNA. (membrane = 18h, nylone = 2h) Electro blotting using electrophoresis between gel to nylon speed up the DNA movements. Vacum blotting

18. Advantages Effective way to detect a specific DNA sequence in a large, complex sample of DNA. Can be used to quantify amount of the present DNA. Cheaper than DNA sequencing Disadvantages More expensive than most other tests. Complex and labour intensive. Time consuming and cumbersome.

19. Technique was developed in 1977 by James Alwine , David Kemp and George Stark at Stanford University. No need to digest RNA with REs. SS RNA can form the secondary structure during running. Use formaldehyde to break H-bonds and denature RNA because single-stranded RNA will form intramolecular base pairs and "fold“ on itself. Northern Blot: RNA

20. Isolate RNA & treat with formaldehyde Gel electrophoresis Transfer single-stranded RNA to nitrocellulose or nylon membrane Incubate membrane with labeled DNA or RNA probe Autoradiography Steps

21. Isolate RNA : To detect rare mRNA, isolate the polyA + mRNA. RNA is both biologically and chemically more labile than DNA. Thus eliminate RNases . Step 1 Electrophoresis: Performed in formaldehyde agarose gel to prevent RNA from folding on itself. Stain with EtBr to visualize the RNA bands. PAGE can be used with Urea. Step 2

22. Transfer ssRNA to nitrocellulose or nylon membrane: Transfer buffer contain formamide ( it lowers the annealing temperature of the probe-RNA interaction) Nitrocellulose typically has a binding capacity is lesser (of about 100 μ g/cm) than nylon (has a binding capacity of about 500 μ g/cm.) Covalently link RNA to membrane: UV cross linking is more effective in binding RNA to the membrane than baking at 80 C Step 3

23. Pre-hybridize 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. Autoradiography : Place membrane over X-ray film. X-ray film darkens where the fragments are complementary to the radioactive probes Step 4 & 5

24. To determine the conditions under which specific genes are being expressed (mRNA level). A standard for the study of gene expression at the level of mRNA (messenger RNA transcripts) Detection of mRNA transcript size Study RNA degradation Study RNA splicing Application

25. Advantages Simple method Highly specific Quality and quantity of gene can be measured Disadvantages Detect small number of genes at a time Use of ethidium bromide and UV light needs special training Detection with multiple probes is difficult