DNA,RNA and protein are separated by blotting techniques. These are described below:
1. Southern blotting techniques
A method developed by molecular biologist E.M. Southern (1975) for analysing the foreign gene in a DNA restriction fragment is called southern blotting technique. Southern blots can be easily provide a physical map of restriction sites within a gene located normally on chromosome, and reveal the number of copy of gene in the genome, and the degree of similarity of the gene when compared with the other complimentary genes.
Fig: procedure of sodium blotting technique.
DNA isolated from a given sample is digested with one or many restriction enzymes (Fig.1). consequently, DNA fragments of unequal lengths are produced. This preparation is passed through agarose gel electrophoresis which result in separation of DNA fragment based on their size. DNA restriction fragment present in gel are denatured by alkali treatment. Gel is then put on the top of the buffer saturated filter paper. The dry filter paper draws the buffer through the gel. Buffer contain single stranded DNA. Nitrocellulose filter binds DNA fragments strongly then come in contact of it. After baking in at 80°C, DNA fragments are permanently fixed to the nitrocellulose filter. Then the filter is placed in the solution containing radio-labelled RNA or denatured DNA probe of known sequences. These are complimentary in sequence to blot-transferred DNA.
The radiolabelled nucleic acid probe hybridized the complimentary DNA on nitrocellulose filter. The filter is thoroughly washed to remove the probe. The hybridized region are detected autoradiographically by placing the nitrocellulose filter in contact with photographic film. The image shows the hybridized DNA molecule. This, the sequence of DNA are recognized following the sequence of probe.
2. Northern blotting techniques
Southern blotting technique could not applied directly to the blot transfer of mRNA separated by gel electrophoresis, because RNA was found not to bind with nitrocellulose filter. J.C.Alwine and co-workers in 1979 devised a technique in which RNA was blot transferred from the gel into chemically reactive paper. In aminobenzyloxymethyl cellulose paper, prepared from whatman filter paper No. 540 after a series of uncomplicated reactions, is diazotised and rendered into the reactive paper and, therefore, available for hybridization with radiolabelled DNA probe. The hybridized bands are found out by autoradiography. Thus, Alwine’s method extends that of southern’s method and for this reason it has been given the jargone term ‘northern blotting’. Like southern, there is nothing northern or western.
These blot transfer are reusable because of the firm covalent bonding of RNA to the reactive paper. The chemically paper is equally effective in binding the denatured DNA as well. Small fragment of DNA can more effectively be transferred to the diazotised paper derivative than to nitrocellulose. These techniques were more being advanced and more recently have been demonstrated that mRNA bands can also be blotted directly on nitrocellulose paper under appropriate condition. In this method preparation of reactive paper is not required. The mRNA is isolated from the transformed cell and electrophoresed under such conditions that don not permit the development of secondary structures. The mRNA separated on the gel are transferred on nitrocellulose filter which are than hybridized to single stranded probe (RNA or DNA ). Thereafter, hybrid are treated with SI nuclease and RNAase which digest the single stranded RNA/DNA probe. It does not affect the double stranded nucleic acid formed due to hybridization of RNA by the complimentary sequences of nucleic acid probe. Structure of mRNA is revealed to the extent to which mRNA protect the nucleic acid probe.
3. Western blotting techniques (protein blotting or electroblotting technique)
In 1979, H.Towbin and co-workers developed the western blotting technique to findout the newly encoded protein by a transformed cell. Its working principle lies on antigen-antibody reaction; hence, it is an immuno detection technique. In this method radiolabelled nucleic acid probe are not used. This technique follow the following steps:
(1) Extraction of protein from transformed cells.
(2) Separation of protein by using SDS-PAGE (sodium dodicyl sulphate polyacrylamide gel electrophoresis) where SDS acts as solvent for electrophoresis.
(3) Transfer of electrophoresed cell in a buffer at low temperature (40°C) for half an hour.
(4) Blotting of protein onto nitrocellulose filter paper.
(5) Soaking of nitrocellulose filter, Whatman filter and coarse filter in transfer buffer.
(6) Placing of whatman filter paper on a cathode plate followed by stalk of coarse filter, whatman filter, electrophoresed gel, nitrocellulose filter, whatman filter paper, coarse filter stack, whatman filter and anode plate (fig.2).
Fig: Western blotting by using radiolabelled antibodies and detecting the bands through autoradiography.
(7) Putting the complete set up in a transfer tank containing sufficient transfer buffer.
(8) Application of an electric field (30 V overnight for 5 hours) to cause the migration of protein from the gel to nitrocellulose filter and binding on its surface. (The nitrocellulose filter has exact image of pattern of proteins as present in the gel. This type of blotting is called western blotting.)
(9) Hybridization of protein by using radiolabelled antibodies (I123- antibodies) of known structure, isolated from the rabbit.
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