BME103:T130 Group 1
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(→Research and Development)
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Revision as of 16:55, 3 November 2012
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Lab Write-Up 1
Lab Write-Up 2
Lab Write-Up 3
Course Logistics For Instructors
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LAB 1 WRITE-UP
Initial Machine Testing
The OpenPCR runs from your computer and connects through your USB port. You can add and delete steps, edit temperatures, create thermocycler protocols, from your computer.
- When the PCB board of LCD is unlplugged from the Circuit Board, the LCD goes off.
- When the white wire connecting the circuit board to the heat plate, the LCD showed an incorrect reading of the temperature.
(Write the date you first tested Open PCR and your experience(s) with the machine)
Polymerase Chain Reaction
Polymerase Chain Reaction is the process of rapid duplication of a strand of DNA. The process first requires a DNA template, which contains the strand of DNA that is intended to be copied multiple times. The template strand is then been heated in order to separate the double stranded DNA and DNA Polymerase is added in order to create a strand of DNA that is complimentary to the original intended DNA strand, which creates a primer. Then the temperature is lowered so that the nucleotides will bind together in complementary to the primers in order to create a copy of the targeted DNA strand. The heating and cooling process will be repeated over a period of approximately 2 hours in order to create a large amount of DNA strand copies.
(Add your work from Week 3, Part 2 here)
Research and Development
Specific Cancer Marker Detection - The Underlying Technology
There are many factors that contribute to a PCR reaction. First, a sample of DNA from a patient must be extracted. This sample is called template DNA. During the reaction, primers, or artifically synthesized bits of DNA, bind to the target sequence if it is present in the DNA. The enzyme taq polymerase's job is to regenerate the DNA strand that was melted away. Magnesium chloride (MgCl2) binds to the taq polymerase protein as a cofactor to help it function properly. In addition, there are dNTP's floating around in the solution of DNA. These deoxynucleotidetriphosphates are the bases (A,T,C,G) ready to be bound by the taq polymerase enzyme.
During a PCR reaction, the solution of DNA is first heated to a temperature of 95 degrees Celsius to essentially melt the hydrogen bonds between the bases on the double strand of DNA. Next, the solution is cooled down to 57 degrees Celsius so the primers can bind to the target sequence of the DNA if it is present. Finally, the solution is heated up to 72 degrees Celsius, where polymerization occurs. This process of heating and cooling is repeated for a total of 34 cycles. This will ensure that the DNA has been amplified enough so it is able to be seen when a flourescent solution is added.
Since the primers will not bind to the DNA if the target sequence is not present, a non-cancer patient will produce a negative result. This is because instead of having millions of double stranded DNA in the solution, the solution will only contain single strands.
The specific cancer-associated gene sequence being analyzed is the r17879961 SNP. The mutation is present at position number 29,121,087 in the DNA. The "ATT" sequence in a non-cancer human is replaced by the "ACT" sequence. Therefore, during a PCR reaction, primers will bind to the sequence, "ACT", since it is the cancer gene.
(Your group will add the results of your Fluorimeter measurements from Week 4 here)