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Polymerase Chain Reaction
Introduction
The polymerase chain reaction (PCR) is an in vitro technique which allows the amplification of a specific deoxyribonucleic acid (DNA) region that lies between two regions of known DNA sequences. Before going into PCR technique, we double helix. This helix comprises of two single strands of DNA running antiparallel to each other and held together non-covalently by hydrogen bonds. The hydrogen bonds form between the complementary bases, i.e., adenine (A) with thymine (T) and guanine (G) with cytosine (C). The bases are each attached to a sugar molecule, deoxyribose and each sugar molecule is joined to the adjacent sugar molecule and a base and is known as nucleotide; sugar and a base is known as nucleoside.
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The numbers, for the sugar carbons each have a ‘prime’, e.g. 5’ and 3’. It is the 5’ and 3’ carbons of adjacent sugars that are linked via phosphate groups, hence the single-stranded DNA and the related molecule, ribonucleic acid (RNA) will have a 5’ and a 3’ end. The 5’ end of one strand of double-stranded DNA is complementary to the 3’ end of the other strand. RNA has a base uracil (U) instead of thymine (T), and has a hydroxyl group at its sugar moiety (ribose) at the 2’ position. In RNA, uracil (U) can base-pair with adenine (A).
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The PCR amplification of DNA is achieved by using oligonucleotide primers, also known as amplimers. These are short, single-stranded DNA molecules which are complementary to a defined sequence of DNA template. The primers are extensions of a single-stranded DNA (template) denatured by a DNA polymerase, in the presence of deoxynucleoside triphosphates (dNTPs) under suitable reaction conditions. This results in the synthesis of new DNA strands complementary to the template strands.
These strands exist at this stage as double-stranded DNA molecules. Strand synthesis can be repeated by heat denaturation o f the dsDNA, annealing of primers by cooling the mixture and primer extension by DNA polymerase at a temperature suitable for the enzyme reaction. Each repetition of strand synthesis comprises a cycle of amplification. Each new DNA strand synthesized becomes template for any further cycle of amplification and so the amplified target DNA sequence is selectively amplified, cycle after cycle.
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The first extension products result from DNA synthesis on the original template and these do not have a distinct length as the DNA polymerase will continue to synthesis new DNA until it either stops or is interrupted by the start of the next cycle. The second cycle extension products are also of intermediate length, at the 3rd cycle, fragments of “target” sequence are synthesized which are of defined length corresponding to the position of the primers on the original template. From the 4th cycle onwards, the target sequence is amplified exponentially (Figure 12.1). This amplification, as a final number of copies of the target sequence, is expressed by the formula, (2n – 2n) X, where
n = number of cycles
2n = first product obtained after cycle 1 and second product obtained after cycle 2 with undefined length
X = number of copies of the original template
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Potentially after 20 cycles of PCR, there will be 220-fold amplification, assuming 100% efficiency during each cycle. However, in practice, only 20-30% efficiency is achieved in PCR methods.
PCR was invented by Kary Mullis in 1983 while working for Cetus Corporation in California. In 1993, the Nobel Prize for Chemistry was awarded to Dr. Mullis for having invented PCR.
The original PCR protocols used Klenow fragment of E. coli DNA polymerase I to catalyse the oligonucleotide extension. However, this enzyme is thermally inactivated during the denaturation step of a PCR cycle and so the researchers had to add a fresh aliquot of enzyme at each cycle, to the amplification process. In later years, a thermostable polymerase like Taq polymerase was discovered for use in PCR methods.
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Nucleic acid amplification techniques are most useful for the detection and characterization of viruses for which cell culture and serological methods are difficult, expensive or unavailable. Especially, the very high level of sensitivity provided by DNA amplification makes PCR the method of choice to detect viral DNA directly in clinical samples. The most obvious advantage of PCR for virus diagnosis is the selective amplification of extremely small number of viral DNA or RNA molecules in clinical samples to amounts sufficient for detection by simple methods. The high sensitivity of PCR makes this technique especially appropriate for diagnosis of viral infections where viral antigens or virus-specific antibodies of infection. This is particularly true for latent virus infections including herpesvirus, retrovirus or papilloma virus infections.
PCR is most useful to detect “non-cultivable” viruses such as certain enteric adenoviruses, papilloma viruses, astrovirus or rotaviruses, that are difficult, impossible and/or tedious to cultivate or viruses that grow without visible CPE such as respiratory syncytial virus, mucosal disease virus, coronavirus or certain gamma herpesviruses.
PCR can be used for the rapid detection of those pathogens whose in vitro cultivation is difficult, time-consuming or unavailable. RFLP patterns using PCR-amplified DNA is an excellent method for bacterial typing and has already been used for the identification of the bacterial strains involved in human food-borne outbreaks (Hill, 1996). Parasitic infestations will probably be the last field of veterinary clinical diagnosis to incorporate PCR techniques.
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