While the discovery of the DNA brought about excitement and significant change in the twentieth century, the invention of the various DNA analyse technique even brought about more excitement in the field of molecular biology and genetics. One of such techniques that has revolutionized and shaped the modern era of molecular biology, genetics and biotechnology is the polymerase chain reaction (PCR). Kary Mullis invented the polymerase chain reaction (PCR) technique in 1983 of which he won a Nobel peace price for chemistry in 1993 for the invention, though a controversial one. (Rabinow; 1997)
According to Enger et al (2009); Polymerase chain reaction is a laboratory technique for copying selected segments of DNA from larger DNA molecules. It's like a 'DNA photocopier'. Polymerase chain reaction functions like the cellular DNA replication process that occur in living cells, but however a highly complex process occur in the cell which involves many different replicates of protein. In the PCR process few basic laboratory components are used in making large numbers of copies of a specific DNA fragment in a test tube. The PCR process happens in three different cycling phase namely denaturing, annealing and synthesis or extension. The entire PCR process requires a DNA polymerase the enzyme responsible for the replication and also nucleotides for the new strand that is formed. A repetition of the three stages constitutes one circle, and doubles the amount of DNA sample present (fig 1) the repeated duplication of the target DNA makes PCR a chain reaction. Each of the phases is controlled by temperature change which enables the reactants to be mixed in the reaction tube. In the denaturing stage the temperature is raised to about 950C the heat brought about by the high temperature break the hydrogen bond between the amino acid of each of strand of DNA double helix which causes the helix to unzip and separate into two strands. At the next stage annealing the temperature is reduced to about (50 to 600C) this makes it possible for the primers ( short strands of DNA segments that bind complementarily to the strand ) to attach to the template, the primers attach to particular area of the start DNA strand according to base pairing rule in that A will always pair T and G always pair C. To complete the circle the extension stage is done at this stage the temperature is increased to about 720C the primers are extended by the DNA polymerase adding a complementary nucleotides to form a second strand. At the completion of the circle the net result is two strand instead of the initial one started with, following the key concept of PCR if the temperature is raised to 950C to start another circle, the total number of DNA strands doubles from two to four. After 20 circles there should be about a million copies of the original molecule going by the theory, A typical PCR protocol is carried out in 30 cycles. The temperature is control by a programmable microprocessor machine know as a thermal cycler. (Brown T; 2000) The polymerase used for the process is gotten from a thermophilic bacteria such as Thermus aquaticus a species that is found in hot springs, it is used in place of the DNA polymerase (protein) because it can tolerate the high temperature and work effectively. (Hogg S;2005)
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A typical PCR program is the optimal PCR, this is used in the amplification of specific locus without any unspecific by-products, for this program annealing is done at a higher temperature so that only the perfect DNA-DNA matches can occur in the reaction. PCR programs are selected based on the (GC makeup) of the primer composition and also on the expected length of the PCR product. (Designing PCR programs; http://www.med.yale.edu/genetics/ward/tavi/p08.html). Another type of PCR program is the Real time PCR this is used in the analysis of single nucleotide polymorphisms (SNPs) (Edwards et al.,2009)
According to Rabinow (1997) 'PCR's distinctive characteristics is unquestionably its extraordinary versatility. That versatility is more than its "applicability" to many different situations. PCR is a tool that has the power to create new situations for its use and those required to use it.' PCR opened new front in the field of molecular biology through vastly extending the capacity to replicate, identify and manipulate genetic materials and widely used in fields such as; bio security, GM food testing , detection of mutations, disease screening, and in DNA finger printing and forensics etc.
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In detecting genetically modified food PCR is use to detect for abnormality in the DNA sequencing of the sample resulting from genetic modification. Firstly DNA is extracted from the sample to be tested and isolated, then using PCR the DNA is amplified using primers designed to amplify DNA from the promoter component of gene cassette (CaMV35S) if present in the sample, further visualisation of the amplified DNA is done using agarose gel electrophoresis, the product of the amplification of this promoter are DNA strands of about 195 base in length. A positive test for this promoter indicates that the sample is genetically modified since all GM foods contain the CaMV35S promoter. (Brandne: 2002)
For the detection of mutations Real-Time PCR is deployed; mutations are changes that occur in the DNA bases these include large re-arrangements such as translocations, inversion and gene insertion or deletions, a hybridisation based method of Real-Time PCR is mostly used for the detection of such mutations. This method is a specific, inexpensive, sensitive and rapid (can be carried out in about 30mins)
Polymerase chain reaction was an invention that changed certain fields of life from science to the social aspects of our daily life, it was a vital tool in for the successful accomplishment of the human genome project, it has also had major impact in the medical diagnosis practice, similarly it has been an effective tool used in crime detection and in court cases likewise in the study of animal behaviour.
Brown, T. 2000; Essential molecular biology, practical approach. New York: Oxford university press
Edward, K. Logan, J. Saunders ,N. 2009; Real-Time PCR current technology and application, Essex: Caister academic press
Enger, D. Ross, F. Bailey, D. 2009; Concepts in Biology (13th edition). New York: McGraw-Hill
Hogg' S.2005; Essential microbiology. Sussex: John Wiley and sons ltd
Rabinow, P. 1997; Making PCR, a story in biotechnology. London: The university of Chicago press
Designing PCR programs; 03/09/09 http://www.med.yale.edu/genetics/ward/tavi/p08.html. (Date accessed 12/02/11)
Brandne, D. L 2002; PCR-Based Detection of Genetically Modified Foods
http://ucbiotech.org/resources/methods/GMO_detection.pdf (Date accessed 13/02/11)