Polymerase Chain Reaction

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In order to perform polymerase chain reaction, the following cocktail was made: DNA template, deoxynucleotide triphosphates (dNTP), Taq polymerase, two primers, and a buffer solution. Before starting the PCR, first four samples were collected from the prime suspects and 19 uL “cocktail” was added. 1 uL of DNA from each suspect was placed into separate tubes, then 89 uL dH20, 6 uL dNTP, 3ul of forward primer, 3 uL of reverse primer, 1 uL Taq polymerase, and 12 uL 10X PCR buffer was added into each eppendorf tube. After thoroughly mixing, the samples were placed into the PCR tubes and then placed into the thermocyler.

After the PCR cycle was preformed 2 uL of tracking dye was added to each sample and then they each were loaded into 7% acrylamide gel in 1X TBE solution for gel electrophoresis. The gel was run for approximately 1 1/2 hours at 240 volts, and then the gel was placed in an ethidium bromide solution for 10 minutes to help stain the DNA. Then the TA photographed it under a UV light.

Results:

After the gel electrophoresis procedure was complete, we were able to generate the PCR result

The figure on the picture found in the appendix depicts the results that were obtained from the gel electrophoresis done for the PCR. The lanes were numbered from left to right as Cs (crime scene), 4, 3, 2, and 1.

The banding patterns were clear for all five lanes. The lane that contained the DNA from the crime scene had a total of three bands near the well of the gel one band at 16 mm (millimeter), one at 18 mm, and a faint band at 38 mm. The bands that contained the DNA from suspects 4, 3, and 2 contained one band that was located further down form the well. That lane that contained DNA from suspect 1 contained three bands that were near the well of the gel which was a band at 16 millimeters and another band at 18 mm. A faint band was seen at 38 mm. This band pattern matched the band pattern of the crime scene, which indicated that suspect 1 was possibly the criminal. (The appendix has the total summery of all the bands)

Discussion:

DNA fingerprint is based on Polymorphism, which is when genes are variable between individuals. There are three specific procedures that occur during PCR. These include denaturation of the DNA, annealing, and extension. During denaturation, the temperature would be increased to 94C so the hydrogen bonds between the DNA would break forming two DNA templates, then the temperature would be decreased to 55C and the two primers would join with their specific bases and then the temperature would be increased slightly to 72C so the Tap polymerase would extend the DNA template by pairing free bases with the unpaired bases on the DNA template. This cycle would be repeated about 40 times until enough new DNA was performed.

There are four possible suspects for the murder of a man named Kanye West. These suspects included 50 Cent (sample 1), Jay-Z (sample 2) Ludacris (sample 3), and T.I (sample 4). Each of these suspects had access to Kanye because there were all backstage waiting to go on stage, and they were all jealous because Kanye West was the Headliner of the tour. Even though each of the suspects was found near the murder clean, there was foreign blood identified at the site along with Kanye’s blood. All the suspects claimed their innocence, saying that they would never commit such a horrible crime. In order to find the culprit, a PCR experiment was conducted. According to the PCR results, the band pattern from the crime scene was a close match the band pattern for suspect one, which meant that 50 Cent (sample 1) was the prime suspect.

The lane that contained his DNA had two identical bands that matched those from the crime scene which were 16mm and 38mm, therefore everyone has the potential to be the murder. When confronted with the compelling evidence, 50 Cent was shocked and immediately called for his lawyer. Due to the fact that the first two bands are very light and line up exactly with the crime scene. Though the third band does not line up with the crime scene DNA bands there is a possibility that the gel was not stable, the temperature in each step was not accurate, I would perform another PCR for suspect 1 along with suspect 2 and the crime scene sample to compare them again. In this new experiment I would leave one empty well in between in order to make sure that there is no leakage of one well into the next one.

In circumstances such as above, PCR results help in identifying murders through forensics, rapists, parents, and etc. in the Supreme Court System and everyday life. However, it is very easy to falter and there experiment with errors during the procedures. For example, the wells can be overloaded or loaded incorrectly, which would contaminate the samples, resulting in inaccurate readings. However, when PCR method is conducted correctly, it is very useful.

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