BME100 f2013:W1200 Group5 L4

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OUR TEAM Name: Dominick Cocciola Role: Protocol Planning Name: Hany Arafa Role: Protocol Planning Name: Wendy Gray Role: Open PCR Machine Testing Name: Kristina Roscher Role: Research and Development Name: Estefania Meza Role: Open PCR Machine Testing LAB 1 WRITE-UP Initial Machine Testing The Original Design This is the PCR machine that we tested. The Open PCR machine is a machine that uses heat cycles to replicate specific DNA strands. DNA is placed in PCR tubes, which are then placed in the PCR block. The PCR machine, that is hooked up to a computer, is programmed to cycle through different temperatures. Each temperature change designates a different step in the replicating of DNA cycle. The heated lid is set to 100°C and the machine is initially set to 95°C for three minutes. The machine then goes through 35 cycles of 95°C for 30 seconds (which splits the DNA into two strands), 57°C for 30 seconds (which is when primers bind to the target sequences and begin to replicate the strand) and then 72°C for 30 seconds (which by the end has created double the number of DNA strands each cycle had started with). It is then set at 72°C for three minutes and then a final hold temperature of 4°C. Experimenting With the Connections When we unplugged part 3 the screen from the mother board, the screen on the machine turned off. When we unplugged the white wire that connects the mother board to the PCR block, the temperature reading on the machine lowered which caused the reading to be incorrect and read that it was at -40°C. Test Run On October 23, 2013 we tested the Open PCR machine and found that it functioned well. The temperatures fluctuated somewhat when it went through the heat cycles, but usually only by about .1°-.2°. There was somewhat of a lag between the readings on the machine and the readings on the computer as well. As for the timing of the cycles, the machine worked correctly and properly and cycled through the temperatures at the correct time. We started at 12:55pm and at 1:40 pm our machine had gone through 15 cycles. Protocols Thermal Cycler Program PCR Protocol: The PCR machine begins by warming up to 100 degrees Celsius to begin the process. Then, the initial step takes place for three minutes at 95 degrees Celsius. This is done to heat up all the DNA strands, serving as a pre-denaturation step. Then the next three steps are repeated for a total of 35 cycles. The first step in this cycle is the denaturation cycle, and it is done at 95 degrees Celsius for 30 seconds. This is done to separate the double stranded DNA to single strands, forcing all reactions from enzymes to stop. The second step of the cycle is the annealing process, and it is done at 57 degrees Celsius for 30 seconds. This is when the polymerase attaches and begins to copy the DNA template. The last step of the cycle is the extending process, and it is done at 72 degrees Celsius for 30 seconds. This is when the polymerase couples to the primer on the 3' side, adding the bases that are complementary to the DNA template. The denaturation, annealing, and extension cycles are repeated for a total of 35 cycles. Then, the final step at 72 degrees Celsius for three minutes ensures that all the DNA goes through the extension process before the PCR machine cools down again. Finally, the PCR machine cools down to 4 degrees Celsius to ensure that all the single strands of DNA bond into double strands once again. DNA Sample Set-up Positive Control: Tube A Patient 1 ID:40197 Tube: A1 Patient 1 ID: 40197 Tube: A2 Patient 1 ID: 40197 Tube: A3 Negative Control Tube: J Patient 2 ID: 90029 Tube: J1 Patient 2 ID: 90029 Tube: J2 Patient 2 ID: 90029 Tube: J3 DNA Sample Set-up Procedure Step 1: Label each tube with its corresponding sample letter and number. Step 2: Pipette 50 μL of the PCR reaction mix into each of the six tubes. Step 3: Pipette 50 μL of the DNA/primer mix into each of the six tubes. Replace the micro-pipette tip after inserting the DNA in each tube to decrease the chances of sample contamination. Step 4: Insert each tube into the PCR thermocycler and run the program with same specifications as the one outlined in the "PCR Protocol". PCR Reaction Mix The PCR Reaction mix is 50 μL containing Taq DNA, MgCl2, and dNTP. DNA/ primer mix The DNA Primer mix is 50 μL containing a unique DNA template, and all have the same forward and reverse primer. Research and Development PCR - The Underlying Technology Components of a PCR Reaction A PCR reaction needs many components to synthesize a strand of DNA including, template DNA, primers, Taq Polymerase, Magnesium Chloride (MgCl2), and Deoxyribonucleotides. The template strand of DNA is the original strand of DNA that contains the target sequence you wish to amplify. DNA is made of up the nucleotides Adenine (A), Thymine(T), Cytosine (C), and Guanine(G) which pair in a A-T or C-G format. Therefore, to amplify the target strand of DNA, you need to have these nucleotides in a deoxyribonucleotide (dNTP) solution to pair to the existing strands of DNA. However, having the dNTP's is not enough. Primers are used as customizable units of complementary base pairs or nucleotides which can "seek out" and bind to the target sequence. This effectively creates starting point for the rest of the synthesis on the DNA strand. It is from this point that Taq Polymerase (a heat tolerant form of DNA polymerase) can bind and pull dNTP's out of the surrounding solution to match the target strand of DNA. However, Magnesium Chloride needs to be present in the solution to act as a catalyst for the reaction that Taq Polymerase carries out. PCR: The Process of Thermal Cycling The template strand containing the target sequence. (http://learn.genetics.utah.edu/content/labs/pcr/) To initiate thermal cycling, the template DNA strand must be heated at a temperature of 95°C over 3 minutes. The heat will cause the double helix to straighten and then denature for 30 seconds. When the DNA strands denature, they break apart at the nucleotide bonds, leaving each strand with its own set. Two strands of DNA. (Base picture: http://learn.genetics.utah.edu/content/labs/pcr/ ; Original labels) Next, the temperature is reduced to 57°C for 30 seconds (Anneal Phase) to allow the primers to attach to the target sequence on each DNA strand. (http://learn.genetics.utah.edu/content/labs/pcr/) Once the primers have attached, temperature is raised to 72°C for the Extend Phase and in 30 seconds DNA or Taq polymerase binds to the primers and becomes activated by the Magnesium Chloride. Holding the temperature at 72°C over the next three minutes allows the polymerase to synthesize the entire complementary DNA strand. (http://learn.genetics.utah.edu/content/labs/pcr/) The solution can then be held at 4°C and the final product becomes two complete sections of the target sequence. (http://learn.genetics.utah.edu/content/labs/pcr/) This completes the first cycle of PCR however, more cycles need to be run to isolate the target sequence and amplify it to testable levels. With each complete cycle the target sequence will multiply exponentially and will exist separate from the extra template DNA sequences that you do not wish to study. Schematic of the target sequence (purple) relative to the template DNA (white) after multiple cycles.(http://learn.genetics.utah.edu/content/labs/pcr/)