Molecular biology is the branch of science which deals with the structure and function of macromolecules. It has various techniques which are also applied in other branches of biology.
Polymerase Chain Reaction:-
Polymerase chain reaction is a technique of molecular biology which makes multiple copies of DNA from a single fragment. The process of polymerase chain reaction takes place as follows:
1) First the DNA molecule is denatured by heating under the temperature of 94 to 98 degree centigrade.
2) When the strands are separated, they are bound with DNA primers with the temperature 50 to 65 degree Celsius.
3) After annealing, the elongation of the separated DNA strands take place by using heat stable DNA polymerases like Taq (Thermus aquaticus).
There is another method of PCR named reverse transcription PCR which is used to amplify the RNA molecules, while real time PCR is used to measure the DNA o RNA molecule quantitatively.
Cloning is the most useful technique of molecular biology because it is used to study the function of the proteins. This technique works in the way that it clones the DNA coding which makes protein, using the process of polymerase chain reaction or restriction enzymes and made multiple copies of that coding. The cloning converts the DNA samples into plasmid vectors. The plasmid either has promoter elements which make possible the production of protein of interest or they have the antibiotic resistant markers.
The plasmid produced from cloning can be inserted in the bacterial cell or the animal cell depending on the mode of experiment. If it is inserted in the bacterial cell, then three processes are involved which make possible the insertion of plasmid into the bacterial cell. These processes are transformation, conjugation and transduction. When the insertion of the cloned plasmid in the animal cell is concerned then the process of transfection is involved. Transfection techniques are of various types for example, microinjection, calcium phosphate transfection and liposome transfection. But it is also possible that the plasmid can be inserted in the animal cell by bacteria or virus. If bacteria are involved for insertion then it is called as bactofention and mainly the specie of Agrobacterium tumafeciens is used for this purpose.
Plasmid can stay in the eukaryotic cell in two forms; either it mixes up with the genome of the host cell which results in the stable transfection or it stays independently in the cell by transient transfection. In both the cases, the protein of interest is in the cell and can be expressed. If the cell contains specific cell signaling factors or inducible promoters then protein is expressed efficiently than without these factors.
Gel electrophoresis is the basic technique of molecular biology which is used to separate DNA, RNA or protein molecules. The method involved is that, the molecules are placed in the immobilized gel and an electric current is passed through it. This current separates the molecules according to the size of the strands. Similarly proteins are also separated in the gel by using an SDS-PAGE gel or it can also be separated according to their size and the charge which they carry.
Allele Specific Oligonucleotide:-
Allele specific technique is used to detect the single base mutations in the genes. It does not need any gel electrophoresis or polymerase chain reaction. In this technique, short probes are used usually 20 to 25 nucleotides long and they are also labeled. The labeled probes are exposed to the non-fragment target DNA; as a result hybridization takes place due to the short length probes. If there is only a single change in the nucleotide base, then the hybridization will stop. After this step, the target DNA is washed and those probes are removed which did not hybridize. Either radiation or florescence detects the presence of probe on the DNA fragment.
This is the technique of molecular biology which uses DNA arrays in the form of spots which are attached to the solid support. This solid support is usually microscopic slide which contains the single stranded DNA oligonucleotide fragment present on each spot. This technique is very feasible because it contains large amount of very short spots with DNA fragments on each slide. The spot is so small that it contains a single DNA fragment. This fragment is complementary to a single DNA sequence. The process starts with the extraction of RNA molecule from a tissue which is converted in the form of cDNA. The DNA fragments present on the spots in the slide are then hybridized with these cDNAs. They can be visualized after hybridization.
This technique is useful in the gene expression data as many arrays can be made with the same position of the DNA fragments on the spots, so that the gene expression of healthy and diseases genes can be compared. For example common yeast consists of 7000 genes. With the microarray, it is possible to observe the expression of each gene qualitatively. It is also possible to view the changes in the expression of these genes due to temperature. There are also other methods of construction of arrays instead of suing DNA fragments. For example an antibody array can be used to determine the presence f proteins or bacteria in the sample of blood.
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