Gene Amplification Testing hela Level
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Added on 2020-05-28
Gene Amplification Testing hela Level
Added on 2020-05-28
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GENE AMPLIFICATION & TESTING HeLa LEVEL CHANGESINTRODUCTIONAgarose gel electrophoresis is a very elementary and indispensable technique in molecularbiology. It is usually used for scrutiny of PCR products, plasmid DNA, and products obtainedvia restriction enzyme digestion. It is considered to be the initial step in analysing specificDNA and RNA fragments. It used northern and southern blots [ CITATION Wes16 \l 16393 ].The molecular modus operandi called PCR is in vitro augmentation of a specific segment ofDNA using a thermostable enzyme.The applications of PCR are vast and used in many research applications [ CITATION Erl15 \l16393 ]. PCR id used for Direct Cloning of DNA and without the use of bacteria. Using PCR,we can create DNA fingerprinting for forensic uses. We can diagnose genetic diseasesprenatally. Using PCR, evolutionary analysis is possible. We can detect allelic sequencevariations. Detection of chromosomal rearrangements is possible using PCR. PCR is alsoused for DNA sequencing processes. We can detect viral and/or bacterial infections with thehelp of PCR. There are certain requirements or conditions that need to be fulfilled for PCR. Following areneeded for PCR [ CITATION Erl15 \l 16393 ]:dsDNAdNTP nucleotides (dATP, dGTP, dCTP, dTTP)Magnesium ionForward PrimerReverse Primer
Tag polymerase (extracted from Thermus aquaticus)BufferUsing the above a cycle is created and in each cycle the gene is doubled in number. Since,there is doubling of number, it is observed that more than a million copies of genes aregenerated from a single DNA molecule in just 20 cycles.METHODOLOGYThere were four main reactions that were needed to be performed [ CITATION Far151 \l 16393].PCR Reaction 1: HeLa control (H – C) DNA sample. It was extracted from HeLa cells usedas template in PCR reactions. The reaction was with HPV18/E6 primers.PCR Reaction 2: The sample extracted from ATO 2uM reacts with HeLa cells (H – T1). Itwas used as template in PCR reactions. The reaction occurred with HPV18/E6 primers.PCR Reaction 3: The extracted sample was treated with HeLa cells (HT2). This was used as atemplate in PCR reaction with HPV18/E6 primers.PCR Reaction 4: This was the last reaction. It is known as NTC. This is control reaction.Here there was no template but the reaction takes place with HPV18/E6 primers.RESULTSThe DNA gel electrophoresis is used to investigate effect of ATO on HPV. This is done onviral E6 gene. In lane 1 the results with HeLa DNA are shown. In lane 2 results of treatmentwith HeLa DNA (H – T1). In lane 3 the results with HeLa DNA (H – T2) are shown. In lane4, the DNA is treated with control GAPDH, and HeLa is untreated. In lane 5, treatment withboth GAPDH and HeLa 2uM. In lane 6, treatment is with both GAPDH and HeLa, but the
concentration is more, that is, 5uM. In lane 7, there is no template control. The figure belowshows the ideal result.The result obtained by me while performing the experiment is shown below.DISCUSSIONAgarose gel electrophoresis is a useful process of determining if restraint digest procedurehas been successful. The said DNA ladder is used to determine the size of fragment of DNAin electrophoresis gel [ CITATION Sah14 \l 16393 ].
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