Question
Explain the different steps in the construction of recombinant DNA.
✓
Answer
Isolation of the genetic material (DNA):
- RNA is removed by treatment with ribonuclease and proteins are removed by treatment with protease.
- After several treatments, the purified DNA is precipitated by adding chilled ethanol.
- The bacterial/ plant/ animal cell is broken down by enzymes to release DNA, along with RNA, proteins, polysaccharides and lipids.
- Bacterial cell is treated with enzyme lysozyme.
- Plant cell is treated with enzyme cellulase.
- Fungal cell is treated with chitinase.
- The DNA is cut using restriction enzymes.
- The purified DNA is incubated, with the specific restriction enzyme at conditions optimum for the enzyme to act. Isolation of desired DNA fragment
- Using agarose gel electrophoresis, the activity of the restriction enzymes can be checked.
- Since the DNA is negatively charged, it moves towards the positive electrode or anode and in the process, DNA fragments separate out based on their sizes.
- The desired DNA fragment is eluted out.
- The Polymerase Chain Reaction (PCR) is a reaction in which amplification of specific DNA sequences is carried out in vitro.
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