Blotting Technique Defination, Purpose, Types, Applications, Procedure Blotting Compass Northern (astm ot 1975) Northwestern ‘dame ot 1904) Protein:RNA Far-Western Western Middle DF aft. Eaear (emote otake ome) (Tomei Protein: "sry" Protein Protein rosehnes star etorece o oes " "Themen ctals acon) Mevtbare. 1980) ‘Translational NN Modification Far-Eastern onan ae Southwestern E feorernea tne) Protein:DNA Lipid:PTM Southern Electrophoretic — Mobility Shift Assay ‘Supe stem natePharmaceutical biotechnology Principle and application of blotting techniques. By:— Drx Jayesh.M. Rajput 1. Norther blotting Norther bloting or Northern tytridzalion Ia widely used technique in molecular bilogy to oterine the maleculer weight of mRNA and fo measure relalve amounts of mRNA prosont in ‘iferont somplne and for Wanting stemalvely spliced Vareeripts and multgene family members. Northen Bloting.invelves sopsration of RNA sampies according to size by agarose gel lectrophocesis and detacton wih hybridization probe compiementay to part of o the entre target soqvonce. Norham Blot refers to capilary Vanster of RNA from the electrophoresis gel 10 the toting membranes. Principle:- Norther bloting is @ commonly used method 1 study gene expression by detection of RNA (of taolated mRNA) in samples. Northern blot technique was developed by James Anvine and George ‘Sarck and was named such by analogy to Soutnem bioting. In Nonnem Bloting the total RNA oF "MRNA Is 'sdiated ‘rom an organism of inerest, and then elecrophoresed on danaluring agarose ge, "wnich separates the fragments on he basis of size. The next stop isto transfer fragments rom the {21 onto nitrocallose fiter or nylon membrane. This can be performed by the sinpie capillary ‘Method. The transfer ora subsequent treatment results in immablization of the RNA fragments, 30 the membrane carries a Semi permanent reproduction of te banding pattern of the gel. The FINA is. ‘bound irreversibly o the membrane by baking at high temperature (80°C)or by UV crosslinking. For the deiecion ofa spectic RNA sequence, a hybridvaton probe is used. A hybridization probe is a short (102-5000p), single standed nudeic acid either DNA or RNA probe that will bind to @ ‘complementary piace of RNA. Hybridization protes are labeled with a marker (radioactve of non radioactive) so that they can be detected after hybridization. In non-radioactive detection te probe is labeled wth biotin or ioxigerin. The membrane is washed lo remove non-specically bound prebe ‘nd the hybridized probe is detected by treating he membrane wih a conjugated enzyme, folowed ‘by incubation with the chromegenic substrata solution, As a result a vsible band can be sean on the ‘membrane where the probe is bound tothe RNA sample Applications: jes/Applicatior2. Southern blotting else St ee teeteti, Souther === [ Blotting ‘Te fist of these techniques developed was tne Souther blot, named ater Dr. Eowin Southem who developed it 0 identify specific ONA sequences. Southern blotting is 2 detection technique used to ind the target DNA sequences in the ONA sample ie the fed of molecalr biology. The process starts from lectrophoress of DNA molecules which are ybridted Ina biotirg membrane followed by a transfer ‘step where ONA from go is tranterrad onto the ating membrane Principie:— Restriction endonucleases, which I an enzyme, i used to break the DNA into small fragments. These ‘fragments are thon separated using electraphoress. The fragments achieved i then slaseed according to the sie (Da). Thus, DNA fragments are transferees to the bloting paper where kf Incabated with probes. Probes used inthe Southem blotting can be highly selective. They can selectively bind with a resolutien of Lina millon andthe characterise: to ind to the Intended target fragments Applications:— Southera blotting is used in a number of applications. The primary usage of Souther biotting to identify » specific DNA in a DNA sample. It is mostly used in the identification of viral fection and certain bacterial infections. In rDNA technology, The Southem blcting technique is used to Solute a particular DNA. It is also useful in the study of mutation and gene his property is used to diagnose neonatl disease ard genetic disease. Duc tothe ation this technique is used in phylogenetic studies, paternity & rmatemiy analysis, foersic studies and personal iemtfication, Southern blotting can be applied in studying structure of a gere or to elucidate restriction ‘enzyme mape. Particularly, Souther blotting can be used inthe identification of the methylated sites present in case of some genes in particulr, Applying restriction nucleases such as ‘Mspl and Hpall, which can both identify and cleave among the identical sequence this can be implemented. The discovery of RFLP s by southem blotting has helped in the mapping of several ‘Benomes that were enucial to be mapped. In the arena of immunclogy the clonal rearrangements Of the immunoglobulins as well as the T Cell receptor genes that play an important role in cliciting an immune response can be analysed by Souter blowing8. Dot blotting ‘A dot bet (or sot bot is echrique in molecular biology wed to detest proteins. It represents «simplification ofthe western bat method, withthe exeeption tht the proteins to be detected arene fst separated by clectsophorsis.Iastad, the stmple is applied deatly ona menbrane ina single pot, and the bloting procedare is performed. ‘The technique offers significant savings in time, as chromatography or gel clsctrophoresis. and the complex blotting procedures fr the gel are not required. However. it offers no information ‘onthe size ofthe target prot Principle:— Dot biot isa simple way totes fr the presence ofa protein of inerest (PON) ina sample. The technique is actually very similar to the Western blot, but dot blot, for reasons we'll cover later, isa faster, cheaper, and casier technique. As an aside, tne dot blot can also be used for detection ‘oF mucicic acids, but for the sake of simplicity, we'llretrstthisto a discussion of protcia. Application: 11s used in the detection of dea, ma, and proteins. 4. Colony hybridization Colony hybrtatzation is » method of sclecting tactcrial colonics with desived gonsa! This mated as discovered by Michasl Grusisin and David S, Hogness. Colony hybridization begins with culturing sparsely populated bacterial colonies on a nutrient agar plate, Those cobbnies are symmetialy replicated on «nitroselulse ier by direc contact afer whih the cols on the fier membrane are laed and the DNA is denatured, allowing io biad to the filter. These DNA clusters are then hybridized to a desired radiontively-abeled RNA or DNA probe (chosen specifically beforehand) and screened by autoradiography. DNA clusters that exhibit a desired gene are then matched up to the corresponding (living) bacterial colonics, which can be isolated for further growth and experimentation. Principle:— 11s arapid method of isolating a colony containing a plasmid harboring a particular sequence or ‘a gene ffoma mixed population. The colonies to be screened are fist replica-plated on to a nitrocellulose filter disc that has been placed on the surfuce ofan agar plate prior to inoculation, Master plate is retained for reference set of colonies. The filter bearing the colonies is removed ‘and treated with alkali so that the bacterial colonies are lysed ard the DNA they cont ‘denatured. The filer is then trated with proteinase K to remove protein and leave denatured DNA bound tothe nitrocellulose. The DNA is fied fiemly by baking the fiker at 80°C. A. lateled probe is hybridized to this DNA which it monitored by autoradiography. A colony whose DNA print gives a positive auto radiographic result on X-ray film ean then be picked from the eforence plate. Colony hybridization can be used to screen plasmid of cosmid based librariesApplications: Hybridisation Recombinant DNA: Screening + Cotony nyoriatsation 1s used to ‘dantity acter olonles containing the Gene of interes 5. Plaque hybridization. ‘Plaque hybridization a technique usedin Molecular biology forthe identficatin of recombinant ‘hanes t'The pocedure can also be wed forthe detection ef diferenvialy represented repettve ONA. ‘Tre techrique (simi te colony habrdizaton) involves tybridisngiclated phage DNA to alabel probe {or the gene of study. Thsis followed by autoradlogranhy to detect the postion ofthe label. The aque nyoridization procedure nas some advantages aver colony tyeriduation due tothe smaller and ‘well detined area f the iter to which the DNA bres Principle:— ‘A screening methed used in the identifeation of recombinant phages first developed by Benton and devin 1977 involved in the identification of phoge libraries cane lifted several ‘mas lees background signal,