(a) Polymerase chain reaction (PCR) is a technique of synthesizing multiple copies of the desired gene (DNA) in vitro. This technique was developed by Kary Mullis in 1985. It is based on the principle that a DNA molecule, when subjected to high temperature, splits into two strands due to denaturation. These single-stranded molecules are then converted to double-stranded molecules by synthesising new strands in presence of enzyme DNA polymerase. Thus, multiple copies of the original DNA sequence can be generated by repeating the process several times. The basic requirements of PCR are, DNA template, two nucleotide primers usually 20 nucleotides long, and enzyme DNA polymerase which is stable at high temperature (usually Taq polymerase):

The working mechanism of PCR is as follows:

First of all, the target DNA (DNA segment to be amplified) is heated to high temperature (94 to 96° C). Heating results in the separation of two strands of DNA. Each of the two strands of the target DNA now acts as a template for the synthesis of a new DNA strand. This step is called denaturation.

Denaturation is followed by annealing. During this step, two oligonucleotide primers hybridise to each single-stranded template DNA in presence of excess synthetic oligonucleotides. Annealing is carried out at lower temperatures (40° – 60°C).

The third and final step is an extension. During this step, the enzyme DNA polymerase synthesizes the DNA segment between the primers. Usually, Taq DNA polymerase, isolated from a thermophilic bacterium Thermus aquatics, is used in most cases. The two primers extend towards each other in order to copy the DNA segment lying between the two primers. This step requires the presence of deoxynucleoside triphosphates (dNTPs) and Mg2+ and occurs at 72°C. The above-mentioned three steps complete the first cycle of PCR. The second cycle begins with denaturation of the extension product of the first cycle and after completing the extension step, two cycles are completed. If these cycles are repeated many times, the DNA segment can be amplified approximately a billion times, i.e., one billion copies of desired DNA segment are made.

(b) Restriction enzymes are used to break DNA molecules. They belong to a larger class of enzymes called nucleases. Restriction enzymes are of three types – exonucleases, endonucleases, and restriction endonucleases.

(i) Exonucleases: They remove nucleotides from the terminal ends (either 5′ or 3′) of DNA in one strand of the duplex.

(ii) Endonucleases: They make cuts at specific positions within the DNA. These enzymes do not cleave the ends and involve only one strand of the DNA duplex.

(iii) Restriction endonucleases: These were found by Arber in 1963 in bacteria. They act as “molecular scissors” or chemical scalpels. They recognise the base sequence at palindrome sites in the DNA duplex and cut its strands. Three main types of restriction endonucleases are type I, type II, and Type III. Out of the three types, only type II restriction enzymes are used in recombinant DNA technology because they can be used in vitro to recognise and cut within specific DNA sequences typically consisting of 4 to 8 nucleotides.

(c) Chitinase is a lysing enzyme that dissolves the fungal cell wall. It results in the release of DNA along with several other macromolecules.