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GENETIC ENGINEERING

The technology of production of recombinant DNA by inserting an isolated or artificially synthesized fragment of DNA at a desired place into a vector and then introduction of this recombinant DNA into a host cell is known as genetic engineering (recombinant DNA technology), e.g., transfer of human insulin gene into Escherichia coli where it expressed to produce the insulin protein that is used for the treatment of diabetes. Thus, a single copy of a gene can be cloned into an indefinite number of copies, all being identical, by multiplication of the bacterium or phage containing that particular gene in the usual manner. This technique is also known as gene cloning. Genetic engineering ill eukaryotes requires new methods and tools as, unlike in prokaryotes, the chromosomes are found in the nucleus and, moreover, many genes are split genes (exons interrupted by introns) that cannot be correctly expressed when cloned in prokaryotes (prokaryotes do not have machinery for the processing of HnRNA, and when cloned as such in prokaryotes, the entire HnRNA is translated into respective protein).

Now a days, polymerase chain reaction (PCR) discovered by Carry Mullis (1985), involving Taq DNA polymerase (that is obtained from a thermophilic bacterium Thermus aquaticus found growing in hot springs) is used to obtain millions of copies of DNA segment of choice. In this technique of DNA amplification, the two strands of the desired DNA fragment act as templates that are separated (denatured) by increasing the temperature for 90-98°C in an automatic PCR machine. After adding the two primers (artificially synthesized Short segments of single-stranded DNA) that are complementary to the 3’ends of the two templates, the temperature is lowered to 40-60°C to allow the base pairing of the primers with their respective complementary sequences in the template DNAs. Now, the addition of Taq DNA polymerase, dNTPs (all the four nucleotides) and other requirements lead to the synthesis of the complementary sequences using the primers to produce double-stranded DNA molecules. This cycle is repeated as per requirement (20-30 times) where the temperature is alternately increased (for denaturation of DNA) and decreased (for renaturation of templates with primer and polymerization) to get millions or billions of copies of the desired DNA fragment, that can be purified by the gel-electrophoresis. Whenever the temperature is increased for the denaturation of DNA, Taq DNA polymerase remains active and is not destroyed, thus, allowing automation of the process; whereas, the ordinary DNA polymerase obtained from the mesophilic microorganisms gets denatured at higher temperatures of 90-98° C required for denaturation (Fig. 12.1 and 12.2). The PCR technique is not only used for the DNA amplification (in gene amplification, DNA fingerprinting, etc.) but, is also used for RAPID (Randomly Amplified Polymorphic DNA that is used as molecular marker in genome mapping). site-directed mutagenesis (by inducing mutation at the desired location in the primers), etc.

Fig-12.1

Fig. 12.1 PCR machine (left) and PCR tubues (right).

Fig-12.2

Fig-12.2

Fig-12.2

Fig-12.2