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THE POLYMERASE CHAIN REACTION (PCR) The Polymerase chain reaction is basically a different and an innovative technology which is used for amplifying a single piece of DNA for different orders of magnitude resulting in creating millions of copies of one single DNA sequence…
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Download file to see previous pages All these process are important for the thermal cycler which is an instrument which is used for automatically controlling and it is also important for alternative temperatures for programmed periods of time which are for the PCR cycles. For the initial step which is denaturation by heat and for these initial step things gets separated by double standards of DNA which are linked with two single strands known as denaturation. It is possible if there is presence of hydrogen bonds which links bases to one another which are not very strong. Second step is annealing primer to target sequence and the goal of this is not to replicate the entire strand of DNA but to target sequence which is of around 100 to 600 base pairs which are very unique for the studied organisms. The last and a very important step is extension in which one the primers are annealed to the DNA sequence which is complementary and the temperature is raised to 72 degree Celsius which will also end up in tagging DNA polymerase enzyme. DNA polymerase here is linked with recombinant DNA thermostable which has thermus aquaticus and also they also include normal polymerase enzyme which is active during high temperatures (EELES, R. A., & STAMPS, A. C. 1993).  There are several variants of PCR, and one of the most important one is Multiple PCR, which uses several pairs of annealing primers which are linked and associated with different target sequencing. By using this, simultaneous analysis is permitted which is associated with multiple targets being present in a single sample. An example of this can be taken from in testing take place of the genetic mutations along with combinations of six or even more amplifications. When a standard protocol for DNA fingerprinting is taken into account, the targets which are assayed are linked and amplified in a 3 or even 4 groups formation. In the same position when multiple ligation dependent probe amplification is permitted for targeting multiple targets who are associated with amplified and usage of single primers pair and avoiding resolutions which can limit PCR multiplex. Microsatellites and also SNP’s also properly and in detail analyzed through multiple PCR (NEWTON, C. R., & GRAHAM, A. 1997).  Another important variant of PCR is variable number of tandem repeats which helps in targeting the areas of genome which will exhibit the length variation and also help in the analysis of genotypes linked and associated with sample which is involved usually in sizing of the amplification products linked and associated with gel electrophoresis. Short Tandem repeats is basically the analysis associated with smaller VNTR segments and also is also linked with important basis for CODIS which are important for DNA fingerprinting databases (MCPHERSON, M. J., & MOLLER, S. G. 2000).  Ethidium Bromide is an important material which allows the gel electrophoresis which allows DNA to be visualized. Ethidium Bromide is mixed with agarose power and it makes the EDTA buffer and also water to form the gel matrix before the process of electrophoresis. The major result of this is ethidum bromide molecules uniformly dispersing throughout the matrix and once the gel is filled well, the DNA samples and tracking dyes making the voltage being applied for drawing the polar compounds slowly throughout the matrix. Then Methylene blue comes into play and UV is not something which becomes visible making the technicians to render DNA and ...Download file to see next pagesRead More
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