What is a PCR?
A PCR machine stands for Polymerase Chain Reaction machine. When you get the PCR machine, you have to connect it to a computer via USB cable and have the OpenPCR software downloaded. This lets you in control of programming the PCR in a way you want it to work. The main function of this machine is that it uses a segment of DNA to generate billions of copies of that specific segment. DNA replication occurs naturally in the body where an enzyme known as Polymerase uses only one strand of DNA to make a complimentary strand which results in two double identical strands. A PCR however does this process repeatedly and within hours, it creates billions of copies in 35 cycles. The way it works is that the PCR goes through multiple heating cycles to produce duplicates of the segment of DNA. The very first step a PCR goes through is the process of heating up for three minutes. This is where the temperature rises up to 95 degrees C. After that, the denaturing step follows for 30 seconds which melts the DNA strand and splits the two strands apart connected by the sugar backbones. Second step is annealing for 30 seconds. This is where the temperature cools down to about 57 degrees C which then lets primers attach to the opposite ends of each strands of DNA (3' end). Because of the primers attached to the specific region of DNA, an enzyme called TAQ polymerase knows where to direct the synthesis of strands. This is known as the extending process which also lasts 30 seconds. TAQ polymerase gathers free nucleotides and binds them on each strand of DNA. The first cycle then comes to an end. The second cycle starts right away and instead of ending up with two strands, we end up with 4 strands. The next cycle we end up with 8 strands. The process keeps going on like this and we get our specific DNA segment to grow exponetially in hours.
PCR Design
(using third image above). There are several parts to the PCR design. Every part is dependent on each other. First there is the power source (heater) where it controls the temperature of the heating lid. The heating lid encloses the sample holder which holds the DNA segments you want to amplify. The fan is on the right side of the PCR which makes sure that the PCR doesn't overheat. The temperature rises up to 95 degrees C during the initial heating period and to cool it down, the fan turns on so overheating doesnt cause the whole machine to be shut down. Right underneath that is the circuit board which is the main component because it relays information to the rest of the PCR machine. When we open the PCR software on computer, we can choose what temperatures to keep the PCR in, time for each process, and the number of cycles and the circuit board relays this information to the rest of the parts. Finally, there is the LCD screen (blue) which displays information on what cycle the PCR is on and the temperatures.
Experimenting With the Connections
When we unplugged the LCD display (part 3) from the circuit board (part 6), the LCD display on the machine turned off. Part 6 was the circuit board and part 3 was the LCD screen display. The circuit board is the motherboard of the PCR machine. If the wire from that is disconnected from the LCD display sceen, then the circuit board wouldn't be able to relay information to the LCD screen on what needs to be displayed.
When we unplugged the white wire that connects the circuit board (part 6) to the heating plate (part 2), the temperature dropped down.
Test Run
We started the test run of our PCR number 17 on Wednesday October 23rd at 12:55 p.m. After the test run, we concluded that the test run was a success and it ran exactly the way we programmed it on the software. Overall, we completed 25 of the 35 cycles which was enough to let us know if the device was working properly.
Protocols
Thermal Cycler Program
DNA Sample Set-up
DNA Sample Set-up Procedure
Gather the required materials.
Label each tube, and fill it with 50 μL of the corresponding DNA/primer mix (based on the table above) using a pipette. (only one disposable tip pipette is to be used for each sample to avoid cross-contamination.)
Add 50 μL of reaction mix into each of the PCR reaction tubes.
Place the tubes in the PCR machine and press "Start" to run the DNA thermocycling process.
PCR Reaction Mix
What is in the PCR reaction mix?
DNA/ primer mix
What is in the DNA/ primer mix?
Research and Development
PCR - The Underlying Technology
PCR Functions
The template DNA provides the segment of the DNA that we want to be replicated in the PCR. The primers attach to sites on both DNA strands at the opposite ends. They match the segment of DNA you want to copy. They also tell the DNA polymers where to start. The Taq Polyamerase is a protein whose function is to copy a cell's DNA before it divides in two. It finds the primers and starts adding nucleotides to the DNA strand. The magnesium Chloride is a co-factor to the DNA polyamerase. It's role is similar to that of a catalyst and the PCR can't start without it. DNA polyamerase adds dNTP's to the complimentary bases of DNA. These are what DNA is made of.
Steps of Thermocycling
During the initial step, the machine is set to 95 degrees for 3 minutes. During this time the DNA polyamerase is broken down. During the denature stage the machine heats up the sample to 95 degrees for an additional 30 seconds. During this period the double helix is separated into two single strands of DNA. The anneal stage is when the sample is cooled down to 57 degrees for 30 seconds. It is then that the single-strand DNA molecules attempt to naturally pair up. The primers crowd in and lock onto targets before strands rejoin. During the extend phase, the sample is heated back up to 72 degrees for 30 seconds and the DNA polyamerase is activated. The final step includes maintaining the 72 degree temperature for three more minutes. This is when the DNA polyamerase locates a primer attached to a single DNA strand. I begins to add complimentary nucleotides to the strand.
DNA Molecules
DNA is made up of four types of molecules called nucleotides. Base-pairing allows DNA strands to stick together, and allows primers to stick to the template strand. Adenine(A) pairs with Thymine(T), Thymine(T) with Adenine(A), Cytosine(C) with Guanine(G), and Guanine(G) with Cytosine(C).
BME 100 Fall 2013
