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2.7 Polymerase Chain Reaction (PCR)

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2.7 Polymerase Chain Reaction (PCR) In 1971, Dr. Har Gobind Khorana’s group described the idea to amplify a DNA (Templeton, 1992). Later on in 1985, Kary B. Mullis invented the Polymerase Chain Reaction (PCR) which is used today in different fields, such as scientific research, clinical diagnostics and criminal investigations. PCR is a molecular biology tool that is used to amplify a fragment of DNA to generate thousands to millions of copies. This technique is based on an enzymatic reaction which is controlled by thermal cycling, where every cycle consists of heating and cooling steps. The generated DNA fragments after every cycle are used as templates for the next cycle. This reaction consists of 5 major components: DNA template, two primers that are complementary to the 3’ ends of each strand of the DNA template, DNA polymerase, desoxynucleoside …show more content…

Cells were disrupted using RLT buffer and the lysate was homogenized by centrifugation through a QIAshradder spin column for 2 min at full speed. The flowthrough was mixed with 70% ethanol (equal volume as the RLT buffer used), transferred into an RNeasy spin column and centrifuged at 17000 g for 1 min. after having the flowthrough discard, RW1 buffer was added to the column, centrifuged at 17000 g for 1 min and the flowthrough was discard. The spin column was washed twice with RPE buffer under at 17000 g centrifugation for 2 min. The column was centrifuged empty at full speed for 1 min to remove any residual ethanol. The RNA was eluted by adding nuclease-free water into the column and centrifugation of 1 min at full speed. The yielded RNA was checked by nanodrop (Thermo) and the integrity of the RNA was assessed in the Experion automated electrophoresis station

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After multiple cycles of ligation, detection and tail cleavages, the extended chain reached the end of the template. Then the whole extension chain is removed and a new starting primer switching down 1 nucleotide position binds onto the template for another cycle of reaction. Totally, five round of primer binding cycles are performed to complete the sequencing of each fragment. 3. Pitfalls and limitations of NGS Errors could be introduced in any step of the sequencing process, including library

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They apply different temperatures which results in melting of the DNA. After this process the double helix will separate and enzymatic replication will occur. Complementary primers are added resulting in multiplying of the sample. Therefore this is how Forensics investigators successfully tested the blood stain on the suspect jacket and they were able to say if Dobson was guilty for Stephen Lawrence death. DNA was first used in UK for an emigration case.

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Then helicase opens the helix of DNA, proteins bind to the single strand to stabilize it. As DNA unwinds, topoisomerase are released. This replication requires primers; DNA polymerase does synthesizing of DNA. As the leading strand is continuously synthesized and the lagging strand is synthesize

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The lab focuses on DNA fingerprinting which is usually done in forensics to catch criminals by the analysis of DNA fragments of different sizes by a method called electrophoresis. Electrophoresis is a separation technique that uses an electric field to separate and distinguish biological molecules (this including DNA) in the experiment, gel is used to as a way to apply friction in electrophoresis which can help you easily distinguish between the DNA because of different sizes in the DNA confirmations. The goal of the experiment was to identify between two separate suspects DNA with restriction enzymes that help tell apart the suspects and who was actually at the crime scene. The methods used were the use of a gel made from agarose placed in a gel tray connected to an electrical current because of the DNA being negative and being able to be pulled apart to the positive side and therefore getting a clear spread of the DNA that was cut with restriction enzymes. The conclusion of this experiment was that suspect two was the criminal because of the crime scene DNA that was found matched the DNA of the second suspect when cut with

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