POLYMERASE CHAIN REACTION (PCR)

POLYMERASE CHAIN REACTION (PCR)

The polymerase chain reaction (PCR) provides a simple and ingeneous method for exponentially amplification of specific DNA sequences by in vitro DNA synthesis. This technique was developed by Katy Mullis at Cetus Corporation in Emery Ville, California in 1985. Katy Mullis shared the Nobel prize for chemistry in 1993. This technique has made it possible to synthesize large quantities of a DNA fragment without its cloning. It is ideally suited where the quantity of biological specimen available is very low such as a single fragment of hair 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 'PCR Technology'. The PCR technique has now been automated and is carried out by a specially designed machine.

Mechanism of PCR

STEP 1: Denaturation (Melting of Target DNA)

The polymerase chain reaction is a method for making many copies of a specific segment of DNA, starting with small amount. The DNA to be amplified is mixed with deoxyribonucleotides, Taq polymerase (a thermal stable DNA polymerase enzyme) and DNA primers (the DNA primers hydrizide to the end of the gene to be amplified & provide a starting point of Taq polymerase)

STEP 2: Primer Annealing

The mixture is heated to break down the hydrogen bond in the DNA Forming single-stranded molecules. The mixture cooled to allow the DNA primer to anneal to each end of the segment to be copied. The Taq polymerase then synthesized the complementary stands of DNA, using the primer as starting point.

STEP 3: Primer Extension (Polymerization) 

The temp. raised again to separate the Dna stands and then lower the temperature to allow the primers to attached. Taq polymerase now synthesis another set of new complementary stands. This process is repeated until enough DNA has been produced to be identified or used for further research.  After 21 cycles, one molecule of DNA can be amplified to over a one million copies. This amount of amplification can be achived by running the reaction in overnight a thermal cycler, and the instrument that automatically raised and lower the temp. appropriate time interval.

 Fig: POLYMERASE CHAIN REACTION (PCR)

Application of PCR

After the discovery of PCR, the modern biology has been revolutionized in each and every aspect. Some of the areas of application of PCR have briefly been discussed herewith.

01. Diagnosis of Pathogens: There are several pathogens that grow slowly. Therefore, their cells are found less in number in the infected cells/tissues. It is difficult to culture them on artificial medium. Hence, for their diagnosis, PCR-based assays have been developed. These detect the presence of certain specific sequences of the pathogens present in the infected cells/tissues. Besides, it is useful in detection of viral infection before they cause symptoms or serious diseases.

02. Diagnosis of Specific Mutation: In humans there are thousands of genetic diseases. Mutations are also related to genetic diseases. Presence of a faulty DNA sequence can be detected before establishment of disease. By using PCR sickle cell anaemia, phenylketonuria and muscular distrophy can also be detected. The other diseases can also be diagnosed by using PCR. For example PCR­based diagnostic tests for AIDS, Chlamydia, tuberculosis, hepatitis, human pappiloma virus, and other infectious agents and diseases are being developed. The tests are rapid, sensitive and specific.

03. In Prenatal Diagnosis: It is useful in prenatal diagnosis of several genetic diseases. If the genetic diseases are not curable, it is recommended to go for abortion.

04. DNA Fingerprinting: In recent years DNA fingerprinting is more successfully used in forensic science to search out criminals, rapists, solving disputed parentage and uniting the lost parentage       uniting children to their parents or relatives by confirming their identity. This is done through making link between the DNA recovered from samples of blood, semen, hairs, etc. at the spot of crime and the DNA of suspected individuals or between child and his/her parents/relatives.

05. In Research: In addition, DNA fingerprinting of new microorganisms isolated from various extreme environment (soil, water, sediments, air, extreme habitats, etc.) is also carried out to confirm their identity by comparin^g with the DNA sequences of known microorganisms. Their DNA and RNA can be amplified. Besides, it is also useful to determine the orientation and location or restriction fragments relative to one another.

06. In Molecular Archaeology (Palaentology): PCR has been used to clone the DNA fragments from the mummified remains of humans and extinct animals such as the woolly mammoth and dinosaurs from the remains of ancient animals as recently epitomized in Michel Crighton's Jurassic Park. DNA from buried humans has been amplified and used to trace the human migration that occurred in ancient time.

07. Diagnosis of Plant Pathogens: 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, potyviruses), mycoplasmas bacteria (Agrobacterium tumifticiens, P,Yeudotiioticis,vol(in(iceartini, Rhizobium legunfinosaritin, Xanthol"onas compestris, etc), fungi (e.g., collectotrichum gloeosporioides, Glomus spp., Laccaria spp., Phvtophthora spp., Verticillium spp), and nematodes (e.g. Meloido,^,,^,vne incoginta, M. javanica, etc) (Henson and French, 1993; Chawla, 1998).