Biotechnology : Principles And Process Revision Notes For NEET

BIOTECHNOLOGY PRINCIPLES AND PROCESSES

        Biotechnology can be defined as the use of microorganisms, plants or animal cells or their components to produce products and processes useful to humans. The traditional biotechnology was dependent upon the natural capabilities of microorganisms. The modern biotechnology on the other hands depends upon maintenance of sterile ambience and genetic engineering.

        Genetic engineering is a process of manipulation of genetic material by man in vitro. It includes recombinant DNA technology.

        Tool of recombinant DNA technology involves enzymes, vectors, gene of interest, genetic markers, probes, etc.

        Restriction enzymes are used for cutting DNA at specific locations. These enzymes are isolated from bacteria. They are named on their specific bacterial genus, species and strain from which they are obtained. These enzymes recognise a 4-8 nucleotide long recognition site which is palindromic in nature, e.g. Eco RI, Hind II, etc.

        Vectors are vehicle DNA which carry gene of interest to host cells. A good vector should have an origin of replication, selectable marker, small size, high copy number and recognition sites for several enzymes. Plasmids, bacteriophage, cosmids, phagemids, Ti plasmids are used as vectors.

        Passenger DNA is the gene of the interest or DNA fragment to be cloned or expressed. We can get it artificially synthesised or in the form of cDNA or complementary DNA.

        DNA probes are used to identify and label DNA fragments. These are single stranded DNA or RNA labelled radioactively or fluorescently.

        Genetic markers are tools used to identify host cells that have successfully taken up a cloning vector, e.g. R-plasmid.

        Some other tools used in RDT are shot gun and antisense genes.

        Some interesting name use in biotechnology are

‣ Gene taxi = plasmid,

‣ Molecular scissor = restriction endonuclease,

‣ Molecular glue = ligases,

‣ Natural genetic engineer = Agrobacterium tumefaciens and

‣ Passenger DNA = foreign DNA.

        Technique used in RDT are gene synthesis method, cDNA synthesis, electrophoresis, PCR, etc.

        Artificial gene synthesis is a technique of artificially synthesising gene of interest. It can be synthesised either chemically in the form of oligonucleotides or as complementary DNA with the help of reverse transcriptase.

        Gel electrophoresis is the method used to separate DNA fragments on the basis of their size. Agarose is most commonly used in matrix in this method. Separated fragments can be visualised after staining with Ethidium Bromide (EtBr) followed by exposure to UV light.

        PCR is a method developed by Kary Mullis in 1985, which have the ability to amplify the DNA fragment by using template, primers and Taq polymerase. This technique can be carried out in three steps including denaturation, annealing and extension. PCR is used to detect pathogens, mutations, in palaentology, etc.

        Replica plating is a simple technique used to identify the transformed cells. It works by making an exact copy of an agar plate.

        Southern blotting was developed by EM southern to detect DNA by blotting it onto a nitrocellulose membrane.

        Northern blotting, developed by James Alwin, is a technique to detect RNA. Similarly Western blotting detects proteins.

        Recombinant DNA technology involves various steps in specific sequence, which are as follows: Isolation of genetic material cutting of DNA at specific location Amplification/copying of gene of interest using PCR Insertion of DNA into vector Insertion of rDNA into host cell Selection and screening of transformed cell.

        The transfer of rDNA into host cell is known as gene transfer. It may occur directly, i.e. direct gene transfer or indirectly, i.e. indirect gene transfer. Indirect gene transfer may occur by tumour causing plasmid (Ti plasmid) or viruses such as adenovirus, retrovirus, etc.

        Direct gene transfer may occur by physical methods or chemical methods. Physical methods include heat shock, electroporation, gene gun/biolistics, microinjection or liposome mediated transfer. The chemical methods include treatment with polyethylene glycol and calcium-phosphate coprecipitation.

        Selection of transformed cells may take place via immunological method, i.e. use of labelled immunoglobins with replica plating nucleic acid hybridisation, i.e. detection via molecular probes. Blue-white screening, i.e. Recombinants are differentiated from non-recombinants on the basis of their ability to produce colour in the presence of a chromogenic substrate.

        Genomic library is the collection of entire genome of an organism in the form of plasmid clone or phage lysates. It is constructed via shotgun sequencing and used for retrieving DNA clones.

        A biochip is a desirable collection of gene fragments on a stamp-sized chip, which is used to screen the presence of a particular gene variants.

        DNA fingerprinting is a technique to identify a person on the basis of genetic material. It is dependent upon the presence of VNTRs or minisatellites in genetic material. It is used in forensics, phylogeny, etc.

        Cloning is the production of organisms, which are genetically identical to their parents. In this technique the nucleus from an egg is replaced by nucleus of another cell. It can be either cell cloning, gene cloning or organisms cloning.

        Dolly was the first cloned animal.

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