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प्रश्न
A cell-free method of amplifying DNA first developed in the mid 1980's revolutionised the field of biotechnology, Name the method and explain the basic steps of the technique involved.
उत्तर
In molecular biology, the Polymerase Chain Reaction (PCR) method is used to multiply a gene or a portion of DNA. It is heavily utilised during the gene-editing process. A primer, a template strand, and a thermostable DNA polymerase enzyme from the bacterium Thermus aquaticus are used in the in-vitro synthesis of sequences. The DNA segment is multiplied by a constant rate of DNA replication to a maximum of 1 billion copies.
- Denaturation: The two strands of the double-stranded DNA molecules are split into a single strand by heating them to a high temperature (94° C). Denaturation is the term for this action. The DNA synthesis process uses each strand as a template.
- Annealing: Since the sequences of the two oligonucleotide primers are complementary to the 3' ends of the template DNA, they anneal (hybridise) to each of the single-stranded DNA templates in this stage. Depending on the length and order of the primers, this step is performed at a lower temperature. The original DNA molecule is duplicated as a result.
- Extension of primers: The primers are extended by DNA polymerase (Taq polymerase) using the nucleotides present in the process. 72°C is the ideal temperature for this polymerization phase. To acquire many copies of the rDNA segment, this procedure is performed several times. The amplified region can be ligated to a vector and used for further cloning. As a result, recombinant DNA is produced (rDNA).
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