Several techniques are employed to identify the isolated clones using a specific molecular probe.
Some of these techniques are described below:
1. Polyacrylamide gel electrophoresis:
When the genomic DNA extracted from a particular source is digested by a specific restriction enzyme it is broken into several fragments. These fragments of different sizes can be separated through a technique called gel electrophoresis.
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This separation is necessary to identify a specific gene that is required for use. After these are separated a specific molecular probe can be used to detect the segments which have sequences similar to those in the probe.
For instance if a gene with a specific nucleotide sequence is required to be separated from a mixture a probe is prepared with nucleotide sequence complementary to the gene. These (molecular probes) are then allowed to hybridize and the particular gene to be identified.
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In gel electrophoresis the DNA molecules migrate from one end of the gel to the other. The gel is usually a cylindrical structure or occasionally even may be a slab.
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The rate of movement of fragments of DNA on the gel is inversely proportional to the size of the molecules.
Heavier fragments will remain at the beginning while lighter fragments will move away. Fragments of different sizes appear as discrete bands on the gel and can be isolated for further study.
Separation of DNA Molecules Using PFGE (Pulsed Field Gel Electrophoresis):
Gel electrophoresis is suitable for only small fragments of DNA which can easily get separated. Large size molecules of DNA however cannot be separated using this technique.
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A new technique called PFGE is used for the separation of large sized DNA fragments, sometimes representing even whole chromosomes.
Using the PFGE technique separation of DNA molecules belonging to each of the sixteen chromosomes of yeast has been made possible.
In this technique short pulses of electric current are used in two different directions and the DNA is embedded in Agarose plugs to avoid fragmentation. Using this technique genomes of several fungi have been resolved into chromosomal bands.
2. Blotting techniques:
Gel electrophoresis helps in the separation of fragments of DNA or RNA. The separated bands can be stained and absorbed on the gel.
This technique however will only help in the separation of fragments of DNA but will not tell as to which fragment contains the desired gene.
For this purpose a molecular probe (which is a DNA fragment) consisting of nucleotides complimentary to the gene are used for hybridization and the required gene is identified. For easy identification and detection, the probe is radioactively labeled.
To facilitate hybridization the bands of the genome are usually transferred to a nitrocellulose membrane in a technique called blotting. Blotting technique was first invented by E.M. Southern and is known as the Southern blotting technique.
Two other blotting techniques discovered subsequently are named Northern blotting technique and Western blotting technique. These are described briefly below.
Southern blotting technique the DNA sample is first cleaved with a restriction enzyme and the sample is loaded on to gel electrophoresis for separation. The DNA bands that appear in the gel are denatured into single strands using an alkali solution.
Later the gel is laid on top of a buffer saturated filter paper placed on a glass plate with its two edges immerged in the buffer. A sheet of nitrocellulose membrane is placed on top of the gel and a stack of many paper towels along with a weight are placed on this membrane (see figure).
The buffer solution is drawn up by the filter paper folds and passes through the gel and to the nitrocellulose membrane.
Finally buffer moves up to the paper towels. While passing through the gel the buffer carries with it the fragments of single stranded DNA and binds them on to the nitrocellulose membrane as it passes through it (membrane) to the paper towels above.
For the complete migration of the buffer it may take a few hours. Subsequently the paper towels are discarded, the nitrocellulose membrane with the single stranded fragments of DNA attached to it in different positions is baked at 80°C to fix the strands permanently on it.
This membrane now has the fragments of single stranded DNA fixed on to it in the same position in which they were on the gel in other words the nitrocellulose membrane is the replica of the gel with its attached DNA fragments.
The nitrocellulose membrane is now dipped in a solution containing labeled gene that is present on the membrane. The radio labeled probe hybridizes with the complimentary fragment of DNA on the membrane.
The filter subsequently is removed from the solution and washed thoroughly to remove any unbound fragments of DNA.
Later the filter is placed against a photographic film. Wherever the probe has hybridized with the DNA fragments there will be markings like black dots.
This can now be compared with the original gel and the corresponding DNA band can be identified as the required gene.
Northern blotting technique this is used to identify the required RNA bands. The technique of southern blotting used for DNA transfer from the gel to the nitrocellulose membrane could not be used for transfer of RNA because RNA would not bind with the nitrocellulose membrane.
Alwin et al (1979) devised a technique in which the mRNA bands from the gel were blot transferred to a chemically reactive paper prepared by diazotization of aminobenzyloxymethyl paper.
RNA fragments can bind themselves on to this paper and the paper can be used for hybridization with radio labeled DNA probes. Subsequently by autoradiography the hybridized bands can be identified.
Some of the researches of Thomas (1980) however have shown that mRNA bands can also be blotted directly on to nitrocellulose membrane using special techniques.
Western blotting technique this technique is used to detect proteins of a particular specialty. Discovered by Towbin etal( 1979) in this technique newly coded proteins by a transformed cell can be identified. In this method however nucleic acid probes are not used.
This technique involves the following steps:
(a) Electrophoreses separation of the required protein on a polyacry lamide gel
(b) Transferring of the separated peptide chains on to a nitrocellulose membrane
(c) Using radio labeled antibodies as probes the required peptide chain may be hybridized. (The probe consists of antibodies which bind to the antigenic protein)
(d) Auto radiographic identification of the required hybridized protein.