(a) PCR: 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 original double stranded molecules by synthesizing new strands in presence of enzyme DNA polymerase. Thus a double stranded molecule of DNA is duplicated and 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 nucelotide primers usually 20 nucleotides long and DNA polymerase which is stable at high temperature (usually) Taq polymerase)
Working mechanism of PCR is as follows:
(i) First of all, the target DNA (DNA segment to be amplified) is heated to high temperature (94°C). Heating results in the separation of two strands of DNA. Each of the two strand of the target DNA now act as template for synthesis of new DNA strand. This step is called denaturation.
(ii) Denaturation is followed by annealing (anneal = join). During this step, two oligonucleotide primers hybridize to each of single stranded template DNA in presence of excess of synthetic oligonucleotides. Annealing is carried out at lower temperature (40° - 60°C).
(iii) Third and final step is 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 aquaticus, is used in most of the cases. The two primers extend towards each other in order to copy the DNA segment lying between the two primers. This step requires presence of deoxynucleoside triphosphates (dNTPs) and and occurs at .
The above mentioned three steps complete the first cycle of PCR. The second cycle begins with denaturation of extension step, product of 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 to approximately billion times, i.e., one billion copies of desired DNA segment are made.
Applications of PCR:
(i) Diagnosis of pathogens
(ii) Diagnosis of specific mutations
(iii) DNA fingerprinting
(iv) In prenatal diagnosis
(v) In gene therapy.
(b) Restriction enzymes and DNA: These enzymes are used to break DNA molecules. They belong to a larger class of enzymes called nucleases which are of several types.
Exonucleases remove nucleotides from the terminal ends (either 5' or 3') of DNA in one strand of duplex.
Endonucleases make cuts at specific position within the DNA. These enzymes do not cleave the ends and involve only one strand of the DNA duplex.
Restriction endonucleases were found by Arber in 1963 in bacteria. They act as "molecular scissors" or chemical scalpels. They recognize the base sequence at palindrome sites in 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 recognize and cut within specific DNA sequence typically consisting of 4 to 8 nucleotides.
(c) Chitinase: Chitinase in an enzyme which dissolves the fungal cell wall. It results in the release of DNA along with several other macromolecules from the cell.
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