Notes-Part-1-Class-12-Biology-Chapter-12-Biotechnology-Maharashtra Board

(i) Genetic engineering : Manipulation of genetic material towards a desired end and in a directed and predetermined way, using in vitro process.

• Definition of genetic engineering (By Smith) : ‘The formation of new combination of heritable material by the insertion of nucleic acid molecule produced by whatever means outside the cells, into any virus, bacterial plasmid or other vector system so as to allow their incorporation into a host organism in which they do not occur naturally but in which they are capable of continued propagation’.

• Genetic engineering is also called recombinant DNA technology or gene cloning, as it involves alterations in DNA.

(ii) Chemical engineering: Maintaining sterile environment for manufacturing of useful products like vaccines, antibodies, enzymes, organic acids, vitamins, therapeutics, etc.

Three essential steps in PCR : Denaturation, annealing of primer and extension of primer.

• The DNA segment and excess of two primer molecules, four types of dNTPs, the thermo stable DNA polymerase are mixed together in ‘eppendorf tube’.

One PCR cycle is of 3-4 minutes duration and it involves following steps :

• Denaturation : The reaction mixture is heated at 90—98°C. Due to this hydrogen bonds in the DNA break and two strands of DNA separate. This is called denaturation.

• Annealing of primer : When the reaction mixture is cooled to 40—60°C, the primer pairs with its complementary sequences in ssDNA. This is called annealing.
• Extension of primer : In this step, the temperature is increased to 70-75°C. At this temperature thermostable Taq DNA polymerase adds nucleotides to 3’end of primer using single-stranded DNA as template. This is called primer extension. Duration of this step is about two minutes.
• In an automatic thermal cycler, the above three steps are automatically repeated
• 20-30 times.
• Thus, at the end of ‘n’ cycles 2ⁿ copies of DNA segments, get synthesized.

Plasmids as cloning vectors :

• Plasmids are small, extra-chromosomal, double stranded circular forms of DNA that replicate autonomously. They are seen in bacterial cells, yeast and animal cell.

• Plasmids are considered as replicons as they are capable of autonomous replication in suitable host.
• The most commonly used vectors in r-DNA technology are plasmids as they replicate in E. coli.
• Plasmid as a cloning vector should have a replication origin, a marker gene for antibiotic resistance, control elements like promoter, operator, ribosome binding site, etc. and a region where foreign DNA can be inserted. Naturally plasmids do not have all these features. Hence, they are constructed by inserting gene for antibiotic resistance. pBR 322, pBR320, paCYC177 are the constructed plasmids.
• T1 plasmid (for tumor-inducing) of Agrobacterium tumefaciens is an important vector for carrying new DNA in many plants. It contains a transposon, called T DNA, which inserts copies of itself into the chromosomes of infected plant cells. The transposon, with the new DNA, can be inserted into the host cell’s chromosomes. A plant cell containing this DNA, can then be grown in culture or induced to form a new, transgenic plant.

The steps involved in gene cloning are as follows :

• Isolation of DNA (gene) from the donor organism.
• Insertion of desired foreign gene into a cloning vector (vehicle DNA).
• Transfer of r-DNA into suitable competent host or cloning organism.
• Selection of the transformed host cell.
• Multiplication of transformed host cell.
• Expression of the gene to obtain desired product.

(i) Isolation of DNA (gene) from the donor organism :

• The desire gene to be cloned has to be obtained from the source organism (donor).The cells of the donor organism are sheared with the blender and treated with suitable detergent. Genetic material from the donor is removed isolated and purified by using several techniques. Isolated DNA can be spooled on to a glass rod.
• Isolated purified DNA is then cleaved using restriction Endonucleases.
• Restriction fragment containing desired gene is isolated and selected for cloning. This is now called foreign DNA or passenger DNA.
• A desired gene can also be obtained directly from genomic library or c-DNA library.

(ii) Insertion of desired foreign gene into a cloning vector (vehicle DNA) :

• The foreign DNA or passanger DNA is inserted into a cloning vector (vehicle DNA) like bacterial plasmids and the bacteriophages like lamda phage and M13. The most commonly used plasmid is pBR 322.
• Plasmids are isolated from the bacteria and are cleaved by using same RE which is used in the isolation of the desired gene from the donor.
• Enzyme DNA ligase is used to join foreign DNA and the plasmid DNA.
• Plasmid DNA containing foreign DNA is called recombinant DNA (r-DNA) or chimeric DNA.

(iii) Transfer of r-DNA into suitable competent host or cloning organism :

• The r-DNA is introduced into a competent host cell, which is mostly a bacterium.
• Host cell takes up naked r-DNA by process of ‘transformation’ and incorporates it into its own chromosomal DNA which finally expresses the trait controlled by passenger DNA.
• The transfer of r-DNA into a bacterial cell is  assisted by divalent Ca⁺⁺
• The cloning organisms are E.coli and  Agrobacterium tumifaciens.
• The competent host cells which have taken up r-DNA are called transformed cells.
• By using techniques like electroporation, microinjection, lipofection, shot gun, ultrasonification, biolistic method, etc.
• Foreign DNA can also be transferred directly into the naked cell or protoplast of the competent host cell, without using vector.
• In plant biotechnology the transformation is through Ti plasmids of A.tumifaciens.

(iv) Selection of the transformed host cell :

• For isolation of recombinant cell from non-recombinant cell, marker gene of plasmid vector is employed.
• For example, pBR322 plasmid vector contains different marker genes like ampicillin resistant gene and tetracycline resistant gene. When pst1 RE is used. It knocks out ampicillin resistant gene from the plasmid, so that the recombinant cells become sensitive to ampicillin.

(v) Multiplication of transformed host cell :

• The transformed host cells are introduced into fresh culture media where they divide.
• The recombinant DNA carried by them also multiplies.

(vi) Expression of gene to obtain desired product :

• Then desired products like enzymes, antibiotiocs etc. separated and purified through downstream processing using bioreactors.