Advertisements
Advertisements
Question
Suggest and describe a technique to obtain multiple copies of a gene of interest in vitro.
Advertisements
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:
-
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).
-
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.
-
This cycle is repeated several times to generate up to one billion identical copies of DNA.
APPEARS IN
RELATED QUESTIONS
Name two commonly used bioreactors.
From what you have learnt, can you tell whether enzymes are bigger or DNA is bigger in molecular size? How did you know?
Describe briefly the following:
Downstream processing
Explain briefly:
PCR
______ plays an important role in blood clotting.
PCR and restriction fragment length polymorphism are the methods for ______.
Which of the following contributed in popularising the PCR (polymerase chain reactions) technique?
Which of the following steps are catalysed by Taq DNA polymerase in a PCR reaction?
How is copy number of the plasmid vector related to yield of recombinant protein?
Assertion (A): Synthetic oligonucleotide polymers are used during Annealing in a PCR.
Reason (R): The primers bind to the double stranded DNA at their complementary regions.
