Polymerase Chain Reaction, or PCR Applications.
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PCR Applications Polymerase Chain Reaction, or PCR, is a procedure that was invented in the late 1980's and is widely used in the selective amplification of a chosen region of a DNA molecule. This chosen region can be from any part of the DNA molecule, however PCR will only work if the base sequences of the 3' and 5' ends are known. This is to ensure that two short oligonucleotides, acting as primers, can anneal onto the DNA strands and initiate DNA synthesis reactions. The process of PCR is dependant on the thermostable DNA pol I enzyme from the bacterium Thermus aquaticus The basic procedure of PCR is several cycles of denaturation, hybridisation and synthesis resulting in the eventual synthesis of several hundred million copies of the amplified DNA fragment (see Fig.1 for the overall stages of PCR). Following its completion, a sample of the mixture is analysed by agarose gel electrophoresis, where it is visible as a discrete band by staining with Ethidium Bromide.
Genetic profiling is used to identify differences in the human genome in different people. An example is identifying kinship, where each individual carry different versions of short tandem repeats known as microsatellites. They can be passed onto offspring, hence allowing scientists to solve problems where there are paternal uncertainties. PCR can be used when carrying out prenatal screening of fetus to determine if it is carrying any genetic diseases. One example is to identify whether the fetus is carrying the cystic fibrosis gene. Cystic fibrosis is caused by a 3bp deletion that leads to a protein, which lacks a critical phenylalanine amino acid. PCR primers have been developed that can distinguish normal gene from mutant gene. With these primers, a 154bp product is produced from a normal individual and a 151bp product is amplified from DNA of an individual with the disease.
This PCR can be used to check if the Mst I restriction site is present; this is simply done by adding the Mst I restriction enzyme prior to PCR. If two bands appear it indicates that the restriction site is present and if one band appears then the restriction site is not present. PCR can surprisingly be applied to RNA, its specific name being reverse transcriptase-PCR (PCR on RNA via cDNA). This measures the amounts of an mRNA in different tissues or in the same tissue at different times. Its amounts in the cell reflect the activity of the parent gene; therefore quantification of mRNA enables changes in gene expression to be monitored. The above examples are only a few of the diverse applications of PCR and it is hard to exaggerate the impact of the polymerase chain reaction. Generally PCR is a quick and easy method for generating unlimited scientific development. Basically PCR is without doubt the major scientific development of the last quarter century.
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