Question

d. During the isolation of TOL plasmid from Pseudomonas putida, it was observed that the concentration of the plasmid DNA per 50 mL of sample was extremely low. Describe a technique you could employ to increase the concentration of the TOL plasmid.

Answer 1

The concentration of the TOL plasmid can be increased using the polymerase chain reaction (PCR). In PCR, the target DNA is mixed with two primers (in excess), a thermostable DNA polymerase, and four deoxynucleotides. The PCR process involves three cycles; denaturation, renaturation, and synthesis of DNA.

First, the reaction mixture is placed in a high temperature of about 95 ^oC for one minute so the double-stranded DNA molecule can denature and form two single-stranded molecules. Then the temperature is reduced to allow for DNA annealing or renaturation. In this stage, the single DNA molecule will complementary pair with the primer. Lastly, initiation of DNA synthesis occurs at 3'-OH end of each primer.