Polymerase+Chain+Reaction+(PCR)

__**Polymerase Chain Reaction (PCR) **__


 * Basic Description **

Polymerase Chain Reaction (PCR) is a common technique used in cellular biology and biochemistry. It involves making many copies of small DNA segments of interest several times during a small time frame. It is a fast and convenient process that results in the amplification of a desired segment of DNA. For PCR, a DNA segment is required, along with DNA polymerase, two primers (upstream and downstream) that flank the DNA segment of interest, and dNTPs. The DNA sample is heated to separate the double helix into two single stands. It is then cooled, and the primers anneal to the two segments at its complementary site. Because of an excess of dNTPs, the DNA polymerase begins its work and adds on to the new DNA strand that began with the primers, so that two DNA strands are now present. The cycle is then repeated, by heating and denaturing the DNA strands and then cooling for the four new strands to be flanked by primers. Every cycle of PCR yields 2n new strands of DNA. There is no need to add new enzyme after every cycle, even though most enzymes would be denatured at such a high temperature, because the common DNA polymerase used is isolated from Thermus aquaticus which is a bacterium that lives in very high temperatures.PCR has the ability to generate 100 billion replications of an original sample in a short amount of time[2]


 * Purpose of Technique **

DNA amplification is very important as it facilitates the study of genetics, the detection of mutations, as well as the diagnosis of infectious diseases. Another amazing benefit of PCR is that is does not require DNA purification. It also greatly helps expedite cloning procedures.


 * Origin and History **

Kari Mullis is the individiaul identified with the creation of PCR in 1983. In the Pre-PCR days, scientists were only able to synthesize short chains of DNA. It was a very tedious process because when the process was introduced to heat the enxymes broke down. It is said that Mulls was in California driving though the hot springs where he realozed how ho could amplify any DNA region through repeated cycles of duplication. DNA polymerase was already discovered but the enxymes found in the hot springs would have the ability to withstand high temperatures [2]. Kari Mullis was awarded the Nobel Prize for the invention of this technique in 1985.


 * Recent Research **

Camila Carlos et al., authors of “Use of E.coli BOX-PCR fingerprints to identify sources of fecal contamination of water bodies in the State of São Paulo, Brazil” use PCR as a means to identify the animal sources of fecal contamination in water. This is important to know as it helps detect a potential risk for those who use the water supply for their own use, as fecal matter can contain quite a number of pathogens. The PCR in this experiment were used to identify BOX elements in DNA. After an analysis of a large range of samples, it was concluded that BOX PCR is a useful and low-cost technique that can accurately identify the sources of water contamination in developing countries.

Another use for PCR is amplification of mitochondrial DNA extracted from Neandertals. One problem with this is the fact that the Neandertal remains are often contaminated with modern DNA, and it is difficult to obtain pure Neandertal DNA. Therefore, the PCR was modified to preferentially bind to and therefore block the amplification of modern DNA, thereby enabling pure Neandertal DNA to be amplified.

The third paper, written by Rowley et al., describes a different version of PCR, called Allele-specific polymerase chain reaction (ASPCR) that was used to detect drug resistant viruses when present in the serum of humans at a surprising less than 1%. Traditional assays used for the detection of HIV drug resistance normal requires a presence of 20% or higher.

From the study "a simple and rapid method for the preparation of plant genomic," the polymerase chain reaction has allowed for rapid analysis of mammalian genomic DNA. Since PCR hadn’t been around too long when this paper was written, scientists believed that PCR was less useful in plant DNA because it difficult extracting nucleic acids [1].

During a study performed by Welsch and McMlelland, they used PCR and arbitrary primers, were a simple and reproducible fingerprints of complex genomes weas able to be generated. This method was performed using two cycles of low stringency amplification which was then followed by PCR at higher stringency [4].

In the last study, the real time PCR procedure measured PCR product accumulated using a dual-labeled fluorogenic probe. This allowed for very accurate and reproducible quantitation of the gene copies. This method also cut down on sample handling and the possibility of comtamination [3]. Therefore, PCR is a very versatile and reliable way of DNA amplification and is a very basic technique used in cellular biology.


 * References **

<span style="font-family: 'Times New Roman',Times,serif; font-size: 140%;">Alberts, B. et al. 2008. Molecular biology of the cell, 5th edition. Garland Science. New York, NY. <span style="font-family: 'Times New Roman',Times,serif; font-size: 140%;">Carlos, C., et al. 2012. Use of Escherichia coli BOX-PCR fingerprints to identify sources of fecal contamination of water bodies of the state of São Paulo, Brazil. Journal of Environmental Management 93: 38-43. <span style="font-family: 'Times New Roman',Times,serif; font-size: 140%;">Gigli, E., et al. 2009. An improved PCR method for endogenous DNA retrieval in contaminated Neandertal samples based on the use of blocking primers. Journal of Archaelogical Science 36: 2676-2679. <span style="font-family: 'Times New Roman',Times,serif; font-size: 140%;">Rowley, C., et al .2008. Improvement in allele-specific PCR assay with the use of polymorphism-specific primers for the analysis of minor variant drug resistance in HIV-1 subtype C. Journal of Virological Methods. 149: 69-75. <span style="font-family: 'Times New Roman',Times,serif; font-size: 140%;">Voet, D. et al. 2008. Fundamentals of biochemistry: life at the molecular level. 2008. John Wiley & Sons, Inc. Hoboken, NJ.

<span style="font-family: 'Times New Roman',Times,serif; font-size: 140%;">[1] Edwards K, Johnstone C, Thompson C (1991) A simple and rapid method for the preparation of plant genomic DNA for PCR analysis. Nucleic Acids Res 19: 1349 <span style="font-family: 'Times New Roman',Times,serif; font-size: 140%;">[2] Mullis KB (1990) The unusual origin of the polymerase chain reaction. Sci Am 56-65 <span style="font-family: 'Times New Roman',Times,serif; font-size: 140%;">[3] Heid CA, Steven J, Livak KJ, Williams PM (1996) Real time quantitative PCR. Genome Res 6: 986-994 <span style="font-family: 'Times New Roman',Times,serif; font-size: 140%;">[4] Welsh J, McClelland M (1990) Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acids Res 18: 7213-7218