The Polymerase Chain Reaction (PCR)
The polymerase chain reaction (PCR) provides a simple and ingenious method for exponentially amplification of specific DNA sequences by in vitro
DNA synthesis. This technique was developed by Kary Mullis at Cetus Corporation in Emery Ville, California in 1985. Kary Mqllis shared the Nobel prize for chemistry in 1993. This technique has made it possible to synthesize large quantities of a DNA fragment without cloning it. It is ideally suited where the quantity of .biological specimen available is very low such as a single hair strand or a tiny blood stain left at the site of a crime. The details of PCR techniques and its mechanism are described by Erlich (1989) in his edited book TCR Technology'. The PCR technique has now been automated and is carried out by a specially designed machine.
Amplification of DNA
The PCR includes the following three essential steps to amplify a specific DNA sequence (Fig. 2.15).
Fig. 2.15. The working system of PCR. Cycle two follows the steps of cycle one.
Melting of target DNA. The target DNA containing sequence (between 100 and 5,000 base) to be amplified is heat denatured (around 94°C for 15 second) to separate its complementary strands (step 1). This process is called melting of target DNA.
Annealing of primers. The second step is the annealing of two oligonucleotide primers to the denatured DNA strands. Primers are added in excess and the temperature lowered to about 68°C for 60 seconds consequently the primers form hydrogen bonds i.e.
anneal to the DNA on both sides of the DNA sequence (step 2).
Primer extension. Finally, nucleoside triphosphate (dATP, dGTP, dCTP, dTTP) and a thermostable DNA polymerase are added to the reaction mixture. The DNA polymerase is added to the reaction mixture. The DNA polymerase accelerates die polymerization process of primers and, therefore, extends the primers (at 68°C) resulting in synthesis of copies of target DNA sequence (step 3). Only those DNA polymerases which are thermostable i.e.
function at the high temperature are employed in PCR technique. For this purpose the two popular enzymes, Taq polymerase (of a thermophilic bacterium, Thermus aquaticus)
and the vent polymerase (from Thermococcus litoralis)
are used in PCR technology. These enzymes exhibit relative stability at DNA-melting replenishment after each cycle of synthesis. Also it reduces the cost of PCR and allows automated thermal cycling.
However, after completion of step 3 (of one cycle) the targeted sequences on both strands are copied and four strands are produced. Now, the three step cycle (first cycle) is repeated which yields 8 copies from four strands.
Similarly, the third cycle produces 16 strands. This cycle is repeated about 50 times. Theoretically, 20 cycles (each of three steps) will produce about one million copies of the target DNA sequence, and 30 cycles will produce about one billion copies. In each, cycle the newly synthesized DNA strands serve as targets for subsequent DNA synthesis as the three steps are repeated upto 50 times. For the working of PCR about 10-100 picomoles of primers are required the concentration of target DNA can be about 10-20 to 10-15 M (or 1 to 105 DNA copies per ml). The PCR machine can carry out 25 cycles and amplify DNA 105
times in 75 minutes.
The PCR technology has been improved in recent years. RNA can also be efficiently used in PCR technology. The rTth DNA polymerase is used instead of the Taq polymerase. The rTth polymerase will transcribe RNA to DNA, thereafter amplify the DNA. Therefore, cellular UNA and RNA viruses may be studied when they are present in small quantities.
Application of PCR Technology
The PCR technology is extensively applied in the following areas (of molecular biology, medicines and biotechnology):
||Amplification of DNA and RNA
||Determination of orientation and location of restriction fragments relative to one another.
||Diagnosis of diseases and causal microorganisms. For example, PCR-based diagnostic tests for AIDS, Chlamydia, tuberculosis, hepatitis, human papilloma virus, and other infectious agents and diseases are being developed. The tests are rapid, sensitive and specific.
||The PCR is important in detection of genetic diseases such as sickle cell anaemia, phenylketonuria and muscular dystrophy.
||It is most applicable in forensic science where it is being used in search of criminals through DNA fingerprinting technology. However, the feasibility of fingerprinting is now being challenged in court of law. In these cases only small samples of biological materials are required.
||It is also applied in diagnosis of plant diseases. A large number of plant pathogens in various hosts or environmental samples are detected by using PCR, for example, viroids (associated with hops, apple, pear, grape, citrus, etc), viruses (such as TMV, cauliflower mosaic virus, bean yellow mosaic-virus, plum pox virus, polyviruses), mycoplasmas, bacteria (Agrobacterium tumifaciens, Pseudomonas solanacearum, Rhizobium leguminosarum, Xanthomonas compestris, etc), fungi (e.g. Colletotrichum gloeosporioides, Glomus spp; Laccaria spp., Phytophthora spp, Verticillium spp), and nematodes (e.g. Meloidogyne incoginta, M. javanica, etc) (Henson and French, 1993; Chawla, 1998).
||PCR is finding considerable and unique use in archaeology; it is doubtful whether scientists will be able to resurrect wooly mammoth and dinosaurs from the remains of ancient animals as recently epitomized in Michael Crighton's Jurassic Park.