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Process of recombinant DNA technology
Recombinant DNA technology involves the following stages:
1. Isolation of genetic material
2. Cutting of DNA at specific locations
3. Amplification of gene of interest
4. Preparation and insertion of recombinant DNA into the host cell/organism
5. Obtaining the foreign gene product
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DNA isolation
1. DNA isolation is an extraction process of DNA from various sources
2. It is then used for scientific purposes for various applications (gene cloning), medicinal purposes or forensic science
3. Many different methods and technologies are available for isolating DNA but, all of the methods involve:
a. Disruption and lyses of the cell
b. Removal of proteins and other contaminants using various enzymes
c. Recovery of the DNA
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Restriction endonucleases
Types of restriction endonuclease
1. They are of three types: type 1, type 2 and type 3.
Nomenclature of restriction endonuclease
1. The first letter of the name of genus in which a given enzyme is discovered is written in capital.
2. Strain or type identification is depicted as subscript, e.g., EcoK; if the enzyme is encoded by a plasmid, the plasmid name is written as a subscript, e.g., EcoRI.
3. This is followed by the first two letters of species name of the organism. These three letters are generally written in italics. e.g., Eco from Escherichia Coli, Hin from Haemophilus influenzae, Hpa from Haemophilus parainfluenzae, etc.Functioning of restriction endonuclease
1. A restriction enzyme identifies the introduced foreign DNA and cuts into pieces called restriction endonucleases.
2. A modification enzyme adds a methyl group to one or two bases usually within the sequence reorganized by the restriction enzyme.
Palindromic nucleotide sequence
1. The restriction endonuclease inspects the length of a DNA sequence.
2. Once, it recognizes specific sequence, it binds to the DNA and cuts each of the two strands of the double helix at specific points in their sugar-phosphate backbone.
3. Special sequence in the DNA recognized by restriction endonucleases is called the palindromic nucleotide sequence.
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Gel electrophoresis
1. Gel electrophoresis is a method for separation and analysis of macromolecules (DNA, RNA and proteins) and their fragments, based on their size and charge.
2. Electrophoresis involves running a current through a gel containing the molecules of interest.
Based on their size and charge, the molecules will travel through the gel in different directions or at different speeds, allowing them to be separated from one another.
3. All DNA molecules have the same amount of charge per mass, therefore, gel electrophoresis of DNA fragments separates them based on size only.
4. DNA fragments can be visualized by the use of staining dyes like Ethidium bromide (visualized under UV).
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Cloning vector
1. A cloning vector is a small piece of DNA, taken from a virus, a plasmid, or the cell of a higher organism, that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for cloning purposes.
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Features required to facilitate cloning into vector
1. Origin of replication
2. Selectable marker
3. Cloning sites
4. Vectors for cloning genes in plants and animals
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Working mechanism of PCR
A single PCR involves three basic steps. They are
1. Denaturation
2. Annealing
3. Extension
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Competent host
It can be done by two ways:
1. DNA mediated gene transfer
2. Direct or vector mediated gene transfer (microinjection, electroporation, chemical method and biolistic method)
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Bioreactors
1. Bioreactors are vessels in which raw materials are biologically converted into specific products by microbes, plants and animal cells and/or their enzymes.
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Obtaining foreign gene product
1. When recombinant DNA is transferred into a bacterial, plant or animal cell, the foreign DNA is multiplied.
2. The cells can be multiplied in a continuous culture system where the used medium is passed out from one side and fresh medium is added from another side to maintain the cells in their physiologically most active log/exponential phase.
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