BLOTTING TECHNIQUES: BLOTTING TECHNIQUES BY DR BRIJESH MUKHERJEE What is blotting?: What is blotting? Blots 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. TYPES OF BLOTTING TECHNIQUES : TYPES OF BLOTTING TECHNIQUES SOUTHERN BLOTTING: SOUTHERN BLOTTING Professor Sir Edwin Southern, Professor of Biochemistry and Fellow of Trinity developed this method in 1975. Southern won the Lasker Award for Clinical Medical Research prize for the method of finding specific DNA sequences he developed this procedure at Edinburgh University more than 30 years ago. The technique is known as DNA transfer or 'Southern blotting' Professor Sir Edwin Southern Cont….: Cont…. This method Involves separation, transfer and hybridization. It is a method routinely used in molecular biology for detection of a specific DNA sequence in DNA samples. The DNA detected can be a single gene, or it can be part of a larger piece of DNA such as a viral genome. Cont….: Cont…. Southern blotting combines agarose gel electrophoresis for size separation of DNA with methods to transfer the size separated DNA to a filter membrane for probe hybridization. The key to this method is Hybridization. Hybridization - Process of forming a double-stranded DNA molecule between a single-stranded DNA probe and a single-stranded target patient DNA. PRINCIPLE: PRINCIPLE 1. The mixture of molecules is separated. 2. The molecules are immobilized on a matrix. 3. The probe is added to the matrix to bind to the molecules. 4. Any unbound probes are then removed. 5. The place where the probe is connected corresponds to the location of the immobilized target molecule. APPARATUS: APPARATUS PowerPoint Presentation: Whatman 3MM paper nitrocellulose membrane PowerPoint Presentation: Capillary plotting apparatus Steps in southern blotting: Steps in southern blotting 1. Digest the DNA with an appropriate restriction enzyme. 2.The complex mixture of fragments is subjected to gel electrophoresis to separate the fragments according to size. Cont….: Cont…. 3.The restriction fragments present in the gel are denatured with alkali and transferred onto 4. a nitrocellulose filter or nylon membrane by blotting. This procedure preserves the distribution of the fragments in the gel, creating a replica of the gel on the filter. Cont….: Cont…. 5.The filter is incubated under hybridization conditions with a specific radiolabeled DNA probe. The probe hybridizes to the complementary DNA restriction fragment. Cont….: Cont…. 6. Excess probe is washed away and the probe bound to the filter is detected by autoradiography, which reveals the DNA fragment to which the probe hybridized. Step 1. Restriction Enzyme Digestion: Step 1. Restriction Enzyme Digestion Eco R I Eco R I Eco R I Eco R I Step 1. Restriction Enzyme Digestion: Step 1. Restriction Enzyme Digestion Step 2. Gel Electrophoresis: Step 2. Gel Electrophoresis _ + Step 2. Gel Electrophoresis: Step 2. Gel Electrophoresis _ + Step 2. Gel Electrophoresis: Step 2. Gel Electrophoresis Step 3. DNA Denaturation: T G A A T C A C A T T G Step 3. DNA Denaturation Eliminate hydrogen bonds with sodium hydroxide ( NaOH ) Step 4. Transfer DNA to Membrane: Step 4. Transfer DNA to Membrane Two methods for transferring DNA to a membrane capillary electrophoretic Step 5. Making a Probe: Step 5. Making a Probe A probe is a small (25-2000 bp ) length of DNA or RNA Complementary to the sequence (gene) of interest Labeled for subsequent detection procedures Step 5. Making a Probe: Step 5. Making a Probe Gene X from Arabidopsis Partial or full-length probes by PCR Step 5. Making a Probe: Step 5. Making a Probe Gene X from Arabidopsis Partial probes by random-priming Step 5. Making a Probe: Step 5. Making a Probe Denature template with heat Step 5. Making a Probe: Step 5. Making a Probe Add random primers Step 5. Making a Probe: Step 5. Making a Probe Extend random primers with polymerase Step 5. Making a Probe: Step 5. Making a Probe A probe complementary to the sequence (Gene X) of interest! Step 5. Making a Probe: Step 5. Making a Probe How do we detect the probe? Radioactivity ( 32 P) Step 6. Pre-hybridization: Step 6. Pre-hybridization Prehybridization buffers contain ‘blocking reagents’ that occupy available binding sites on the membrane Step 7. Hybridization: Step 7. Hybridization Step 7. Hybridization: Step 7. Hybridization Step 8. Washes: Step 8. Washes Step 9. Anti-DIG: Step 9. Anti-DIG Step 9. Anti-DIG: Step 9. Anti-DIG Step 10. Washes: Step 10. Washes Step 11. CSPD: Step 11. CSPD APPLICATIONS: APPLICATIONS Southern blots are used in gene discovery , mapping, evolution and development studies, diagnostics and forensics (It is used for DNA fingerprinting, preparation of RFLP maps) identification of the transferred genes in transgenic individuals, etc. APPLICATIONS: APPLICATIONS Southern blots allow investigators to determine the molecular weight of a restriction fragment and to measure relative amounts in different samples. Southern blot is used to detect the presence of a particular bit of DNA in a sample analyze the genetic patterns which appear in a person's DNA. Northern Blotting: Northern Blotting Northern blotting is a technique for detection of specific RNA sequences. Northern blotting was developed by James Alwine and George Stark at Stanford University (1979) and was named such by analogy to Southern blotting Steps involved in Northern blotting: Steps involved in Northern blotting 1. RNA is isolated from several biological samples (e.g. various tissues, various developmental stages of same tissue etc.) * RNA is more susceptible to degradation than DNA. Cont……: Cont…… 2. Sample’s are loaded on gel and the RNA samples are separated according to their size on an agarose gel . The resulting gel following after the electrophoresis run. Cont……: Cont…… 3. The gel is then blotted on a nylon membrane or a nitrocellulose filter paper by creating the sandwich arrangement. Cont……: Cont…… 4. The membrane is placed in a dish containing hybridization buffer with a labeled probe. Thus, it will hybridize to the RNA on the blot that corresponds to the sequence of interest. 5. The membrane is washed to remove unbound probe. Cont……: Cont…… 6. The labeled probe is detected via autoradiography or via a chemiluminescence reaction (if a chemically labeled probe is used). In both cases this results in the formation of a dark band on an X-ray film. Now the expression patterns of the sequence of interest in the different samples can be compared. APPLICATIONS: APPLICATIONS 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 Study RNA half-life Often used to confirm and check transgenic / knockout mice (animals) Disadvantage of Northern plotting: Disadvantage of Northern plotting 1.The standard northern blot method is relatively less sensitive than nuclease protection assays and RT-PCR 2. Detection with multiple probes is a problem 3. If RNA samples are even slightly degraded by RNases , the quality of the data and quantitation of expression is quite negatively affected. Western blotting: Western blotting Western blotting (1981) is an Immunoblotting technique which rely on the specificity of binding between a protein of interest and a probe (antibody raised against that particular protein) to allow detection of the protein of interest in a mixture of many other similar molecules. The SDS PAGE technique is a prerequisite for Western blotting . Steps in western blotting: Steps in western blotting A protein sample is subjected to electrophoresis on an SDS- polyacrylamide gel. Electroblotting transfers the separated proteins from the gel to the surface of a nitrocellulose membrane. Cont…: Cont… 3. The blot is incubated with a generic protein (such as milk proteins or BSA) which binds to any remaining sticky places on the nitrocellulose. 4. An antibody that is specific for the protein of interest (the primary antibody - Ab1) is added to the nitrocellulose sheet and reacts with the antigen. Only the band containing the protein of interest binds the antibody, forming a layer of antibody molecules . Cont…: Cont… 5. After washing for removal of non-specifically bound Ab1, second antibody (Ab2)is added, which specifically recognizes the Fc domain of the primary antibody and binds it. Ab2 is radioactively labeled, or is covalently linked to a reporter enzyme, which allows to visualize the protein-Ab1-Ab2 complex. Application: Application 1. The confirmatory HIV test 2. Western blot is also used as the definitive test for Bovine spongiform encephalopathy (BSE( 3. Some forms of Lyme disease testing employ Western blotting.