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Question
Suggest and describe a technique to obtain multiple copies of a gene of interest in vitro.
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Solution
For the PCR, two sets of primers (chemically synthesised oligonucleotides that are complementary to a region of DNA), the enzyme DNA polymerase and deoxynucleotides are added.
The PCR consists of three steps:
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Denaturation: Double helical DNA is denatured at a high temperature. DNA polymerase does not get degraded at this temperature, as the DNA polymerase used in this reaction is thermostable. It is isolated from the thermophilic bacterium Thermus aquaticus (Taq).
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Annealing: It is the step in which primers are annealed to single-stranded DNA templates. Two sets of primers (chemically synthesised small oligonucleotides that are
complementary to the regions of DNA) are used. The temperature of the reaction mixture is lowered to 50–65 °C for some seconds to allow annealing of the primers. DNA polymerase extends the primer in the 5' to 3' direction. -
Extension: Replication of DNA occurs in vitro.
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This cycle is repeated several times to generate up to one billion identical copies of DNA.
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Name two commonly used bioreactors.
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PCR
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| Enzyme Taq polymerase, is extracted from a eubacterial microorganism Thermus aquaticus from Yellowstone National Park in Montana, USA and isolated by Chien et al. (1976). Taq polymerase successfully replaced the DNA polymerase from E.coli that was being used in PCR earlier and this shift revolutionised the PCR technique. |
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