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Polymerase chain reaction practical, Genetics - Essay Example

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Invention of Polymerase chain reaction (PCR) by Kery Mullis in 1983(1) has revolutionized the way DNA was looked upon. PCR as name suggests is an enzyme based in vitro technique by which one can generate millions of copy of given DNA in short time…
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Polymerase chain reaction practical, Genetics
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Download file to see previous pages Currently, there are hundreds of different types of PCR are being used in different laboratories but the basic principle remains same. The overall process of PCR can be summarized as follows. 1) Mixture of all constituents of PCR like dNTPs, Primers (forward and reverse), Buffer and template DNA will be mixed in desired concentration. 2) The first step is amplification, where the temperature is set at 94C for 2-5 minutes for denaturation of double stranded DNA, a process called initial denaturation, 3) After initial denaturation, sample is kept for 30 sec at 94C for further denaturation, 4) After denaturation temperature is brought down to 55-60C for 30sec to allow annealing of primer with specific DNA site called annealing temperature. 5) The temperature is now brought up to the 72C for polymerase to start new DNA synthesis using primer as starting material. 6) After extension, the cycles are repeated for almost 30 times to get 230 copy of initial DNA template. Finally, after 3 cycle extension is be performed at 72C for 5 min to complete any unamplified reaction. Figure 1 shows steps involves in PCR.
Development of PCR and identification of DNA as signature molecules for individual leads to introduction of DNA based technique for establishment of parental relation and subsequently for crime and criminal detection. Based on DNA sequence of humans it was found that there are many places in entire DNA that are conserved nucleotide repeats and based on size or length of these sequences they are termed as micro and mini satellite (4). It was found that number of repeat in these sequences varies from person to person and inherited from parents to child makes it ideal choice for criminal identification. Later, this process was termed as DNA fingerprinting. Moreover, development of PCR made this technique more powerful and realistic compared to any other technique for criminal identification, since most of the time, the specimen obtained in the crime site is always in less quantity. This small part of body or body fluids like blood, sperm, saliva or even hair is sufficient to isolate DNA and then amplification with PCR make it possible to do different analysis on it.
Here the aim of this experiment was to understand fundamental principle and use of polymerase chain reaction and based on that to understand how PCR is used in DNA fingerprinting based crime investigation and criminal identification
2. Methods:
2.1 Buccal DNA extraction: Ten ml, 0.9% saline solution was rubbed vigorously against the cheeks for 10 seconds. The sample (extract from the bucaal cavity) was then transferred into 15 ml centrifuge tube and centrifuged at 2000 g for 10 minutes for the pellet. Thereafter, 500 l of chelex beads were added into the pellet and resuspended with the chelex by pipetting in and out various times such that there are no visible clumps of cells. Five hundred microliter of the aliquot was transferred into 1.5ml microfuge tube and was boiled into a hot block at 100C for 10 mins. The sample was then spin for 30 secs top speed to spin down chelex. Fifty microliter of the fresh supernatant was transferred ...Download file to see next pagesRead More
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