Blotting Techniques PDF

zu BLOTTING TECHNIQUES by Dr.Ezat Mersal Explain southern blotting Procedures Discuss principles of northern blot procedure Describe techniques of western blot Explain dot blot procedures 1. SOUTHERN BLOTTING DNA 2. NORTHERN BLOTTING RNA 3. WESTERN B...

zu BLOTTING TECHNIQUES by Dr.Ezat Mersal Explain southern blotting Procedures Discuss principles of northern blot procedure Describe techniques of western blot Explain dot blot procedures 1. SOUTHERN BLOTTING DNA 2. NORTHERN BLOTTING RNA 3. WESTERN BLOTTING protine 4. DOT BLOT TECHNIQUE 5 3  DNA  Southern blotting is the first blotting technique which made analysis and recording easy.  InSouthern blotting ,a DNA fragment containing a specific sequence can be identified by electrophoresis , transferring them into nitrocellulose & hybridizing with a 32p labelled single stranded DNA probe complementary to the sequence. have k  Thefragment containing the sequence then visualized by autoradiography 4  For Southern blotting , DNA sample is first digested with a restriction enzyme and digested sample is electrophoresed.  The DNA bands in the gel are denatured into single strands with the help of an alkali solution. (to break bonds and then DNA become single strand)  Subsequently the gel is laid on top of a buffer saturated filter paper placed on a solid support (eg. glass plate),with its two edges immersed in the buffer.  A sheet of nitrocellulose filter membrane is placed on top of the gel and a stack of many papers on top of this membrane 5  Capillary action pulls the buffer from the bottom filter through the gel, to the transfer medium and up through the paper towel stack.  While passing through the gel, the buffer carries with its single stranded DNA, which binds on to the nitrocellulose membrane, when the buffer passes through it to the paper towels.  The nitrocellulose membrane with single stranded DNA bands blotted on to it, is baked at 80c for 2-3 hours to fix the DNA permanently on the membrane.  DNA fragments on membrane can then be probed for sequence of interest by hybridization between them.  Membrane is washed to remove the unbound DNA.  X-ray film is then exposed to the membrane to get autoradiographs. 6 7  Southern blotting is extremely sensitive and can be applied to mapping restriction sites around a single copy gene sequence in a complex genome such as that of man.  When mini-satellite probe is used the technique can be also used for forensic purpose for identification of minute amount of DNA.  Theuse of southern blot technique has also been done for analyzing the role of DNA methylation in gene expression. 8  Blotting analysis useful in detection of mutated genes in genetic disorders.  Restriction analysis of genes studied with southern blot helps in this respect.  RFLPmapping.(restriction fragment length polymorphisms) For  DNA fingerD.D. of hereditary and familial diseases printing. 9  RNA  The Northern blot procedure essentially identical to that of southern blotting except that here RNAs are separated by gel electrophoresis.  Initially southern blotting could not be used directly to blot transfer RNA from gel to nitrocellulose membrane because RNA did not bind to cellulose nitrate.  Alwine , Kemp and Stark(1979)developed a procedure for blot transfer of RNA.  He used a chemically reactive paper (diazotization of amino benzyl oxymethyl paper which is prepared from Whatsman 540 papers). 10  RNA species are separated on the basis of size by electrophoresis through agarose gel containing formaldehyde or glyoxal and dimethyl sulfoxide. (this for denaturation because RNA present in secondary structure) Sprimary  The separated RNA bands are then blotted on chemically reactive filter paper.  RNA species after blotting are hybridized to radio- labelled DNA probe.  Auto radiography is carried onto locate RNA bands that are complementary to the probe. 11 DNA probes 12  Used for detection and quantitative estimation of hybridized mRNA.  Study RNA degradation  Study RNA half life  Study RNA splicing  It is useful in the studies of gene expression. 13  ies protine  This technique is used to detect the proteins of a particular specificity.  When a transferred gene expresses in transformed cells,  The translated product in the form of proteins can be identified by this technique. 14  DNA protiea antibo st  First we isolate the protein or extract the protein.  The extracted proteins are subjected to PAGE(Poly Acrylamide Gel Electrophoresis) and are then transferred onto nitro cellulose to which they bind.  Then radio-labelled specific antibody is added on such membrane and it binds only to specific complementary protein.  The antibody is labelled with 125 I and the signal is detected again with autoradiography.  If radio active label is not used, bound antibody may be detected by a second antibody tagged with an enzyme. 15 probes 16  it  The confirmatory HIV test employs a western blot to detect anti- HIV antibody in a human serum sample. Proteins from known HIV-infected cells are separated and blotted on a membrane as above. Then, the serum to be tested is applied in the primary antibody incubation step; free antibody is washed away, and a secondary anti-human antibody linked to an enzyme signal is added. The stained bands then indicate the proteins to which the patient's serum contains antibody.  A western blot is also used as the definitive test for Bovine spongiform encephalopathy (BSE, commonly referred to as mad cow disease').  Western blot can also be used as a confirmatory test for Hepatitis B infection. 17  This technique is used to detect the presence of a given sequence of DNA/RNA in the non fractionated (not subjected to electrophoresis) DNA sample.  DNA from many samples can be tested in a single test. A Dot blot (or Slot blot) is a technique used to detect biomolecules.  Itrepresents a simplification of the northern blot, Southern blot, or western blot methods. In a dot blot the biomolecules to be detected are not first separated by electrophoresis. 18  Instead, a mixture containing the molecule to be detected is applied directly on a membrane as a dot.  Then is spotted through circular templates directly onto the membrane or paper substrate.  Then followed by detection by either nucleotide probes (for a northern blot and Southern blot) or antibodies (for a western blot). 19  Sample DNA/RNA from different individuals are transferred to a nitrocellulose filter paper in the form of dots. Several samples are blotted on to a single filter paper.  The DNA is first denatured and then the filter is baked at 80c to fix DNA firmly on to the filter.  The filter is treated with the appropriate radio active single stranded probe under conditions favoring hybridization. The filter is washed to remove the unhybridized probes.  The dots with hybridized probes are detected by autoradiography intensity of the dots corresponds fairly with the extent to which DNA/RNA is represented in the sample. 20 21  Thedot blot test can be used to detect the Chlamydia trachomatis infection and other sexually transmitted diseases.  Dotblot is used to detect Anti diacyltrehalose Antibodies in Tuberculosis patients and Typhoid Fever. 22  Comparison DNA RNA protine 23  Basic genetics : a human approach / BSCS. Dubuque, IA, Kendall/Hunt Pub. Co., c1999. 147 p. QH431.B305 1999  Genes, ethnicity, and ageing. Edited by Lincoln H. Schmitt, Leonard Freedman, Rayma Pervan. Nedlands, Australia, Centre for Human Biology, University of Western Australia ; Singapore, River Edge, NJ, World Scientific, c1995. 100 p.QH455.G45 1995  Genetic polymorphisms and susceptibility to disease. Edited by M. S. Miller and M. T. Cronin. New York, Taylor & Francis, 2000. 266 p.  GENETIC ANALYSIS AN INTEGRATED APPROACH Mark F. Sanders , John L. Bowman Second edition 2015 ISBN 978 0-321-94890-8 (student edition) www.pearsonhighered.com