BME100 f2016:Group4 W1030AM L4
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LAB 4 WRITE-UP
PCR Reaction Sample List
DNA Sample Set-up Procedure
Initial Step: 95°C for 2 minutes
Number of Cycles: 25 Denature at 95°C for 30 seconds, Anneal at 57°C for 30 seconds, and Extend at 72°C for 2 minutes
Final Hold: 4°C
Research and Development
PCR - The Underlying Technology
The 4 components of a PCR reaction are Template DNA, Primers, Taq Polymerase, and Deoxyribonucleotides (dNTP's). The template DNA will be tested for a specific sequence. The Primers will attach to the beginning and end of the desired DNA sequence form the DNA template. Taq Polymerase will replicate DNA by adding complimentary nucleotides, called dNTP's, to the strand.
During the initial step of thermal cycling, the DNA strands separate and uncurl from their double helix structure. This separation continues into the Denature step of thermal cycling. Next, during the Anneal step, the temperature drops low enough where the primers are activated. They bind to the targeted sequences of DNA. The next step is the Extend step. Here, Taq Polymerase begins to add complimentary dNTP's starting at the primers and continues until the strand ends. During the Final Step, all the DNA is extended and then stored at a very low temperature in the Final Hold.
Adenine(A) - Thymine
Thymine(T) - Adenine
Cytosine(C) - Guanine
Guanine(G) - Cytosine
The first step of base-pairing in thermal cycling occurs during the Anneal stage. The primer base pairs to a specific site of the targeted DNA sequence. The second is the Extend step. Here, base pairs are added individually by Taq Polymerase from the Primer to the end of the strand.
SNP Information & Primer Design
Background: About the Disease SNP
The SNP disease first and foremost is a genetic disease that is cause by a single point mutation. The SNP diseases' point mutation lies in an area of the genome that codes for the protein Ankyrin 2 a member of the ankyrin family of proteins. These proteins are critical in a few cell functions such as cell motility, activation, proliferation, contact and the maintenance of specialized membrane domains. With this SNP point mutation occurring all of those functions would be hindered and not occur. In addition,SNP disease point mutations of this gene causes long QT syndrome 4 and cardiac arrhythmia in patients.
Primer Design and Testing
When the non-diseased forward and reverse primers were included in the experiment, a 220 base pair sequence result was amplified. In this case, the experiment was a success. This shows that non diseased DNA was included in the DNA sample. When the diseased forward primer was included in the experiment, no viable sections of DNA was amplified. This result suggests that the mutated ANK2 gene was not present in the DNA sample.