Molecular Biology Techniques
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[edit] Molecular Biology Techniques
In Molecular Biology,various Techniques are used for various purposes.
There are few.
[edit] Cloning
Cloning is a process of creating identical copies of DNA fragments,cell or even organism from a simple template DNA. The steps involved in Cloning are:-
1. DNA is extracted from Blood.
2.Restriction enzyme like EcoRI,HIND III are used for cutting the DNA into small pieces.Different DNA pieces cut with the same enzyme can join or recombine.
3.On the other hand, Bacterial Plasmid are cutted with the same Restriction Enzyme.
4.A Chunk of DNA is inserted into the plasmid DNA to form Recombinant.
5.Recombinant Plasmid is mixed with E.Coli bacteria.
6. Transformation is allowed to occur.
7.Transformed Cell and Non-Transformed cell are formed.The Transformed Cell is given the Nutrient and growth is allowed to occur.Finally, A resistant Colonies are formed.
[edit] Significance of Cloning
1.A particular gene can be isolated and its nucleotide sequence can be determined.
2.Protein/Enzyme/RNA function can be investigated.
3.Mutation can be identified.Example-Gene defects related to specific disease.
4.Organism can be engineered for specific purpose. Example-Insulin production.
[edit] Polymerase Chain Reaction(PCR)
Polymerase Chain Reaction, usually known as PCR is the artifical way of DNA replication.It is a DNA amplification procedure in which numerous copies of DNA are generated from minor or trace or initial amount.Thermus aquaticus,is the bacterium from which the heat stable DNA polymerase is essential to PCR is obtained. Mullis discovered the PCR procedure in 1993.
[edit] How to do PCR?
1.DNA is heated to separate th strandsof double helix at 95 degree celsius for one minute.
2.Nucleotides are added to PCR reaction,mix along with short DNA pieces of length 15-20 nucleotide called as primers.
3.The primers match the original DNA sequence which is to be amplified.
4.Heat stable DNA polymerase(Taq.DNA polymerase) is added adn mix is subjected to several cycle of replication in which new DNA strands are made by adding nucleotide to the primers.
5.DNA helices made of each cycle are separated by heating to serve as template from making more new DNA.
6.Billions of copies of original DNA can be generated in few hours.
[edit] Mass Spectrometry
Mass Spectrometry is analytical technique used to measure mass to charge ratio to ions.It is used to find the composition of a physical sampling by generating a mass spectrum representing the masses of sample components.The mass spectrum is measured by mass spectrometer.
[edit] Importance of Mass Spectrometer
1.Structural information of compound can be generated.
2.Useful for peptide and oligonucleotide sequencing.
3.Identification of individual compounds in complex mixture.
4.Isotopic composition of elements can be determined.
5.It can also detect the Post translation modification(PTM).
[edit] Blotting
Blotting is a technique for visualizing a particular subset of macromolecules — proteins, or fragments of DNA or RNA — initially present in a complex mixture.
Steps involved in Blotting
1.Digest the DNA with an appropriate restriction enzyme.
2.Run the digest on a agrose gel.
3.Denature the DNA with NAOH.
4.Transfer the denatured DNA to a nitrocellulose membrane.
5.Probe the membrane with labelled ssDNA, probing can be done with P-32 also.
6.Visualise the radioactivity if it is labelled with P-32.
There are different types of Blotting
1.Western Blotting It is method to detect a specific protein in a given sample of tissue homogenate or extract.It uses gel electrophoresis to separate native or denatured protein by the length of polypeptide(denaturing conditions) or by the 3-D structure of the protein(native/non-denaturing condition).The protein are then tranformed to a membrane(typically nitrocellulose or PVDF,Polyvinylidene Difluroide) where they are probed detected using antibodies specific to the target protein.
2.Southern Blotting It is technique for the detection of DNA. Southern blotting combines agarose gel electrophoresis for size separation of DNA with method to transfer the size-separated DNA to a filter membrane for probe hybredization.This technique was named after the name of Edwin Southern.
3.Northern Blotting This technique is used for detection of RNA.
