Name: Amanda Seaney
Role: Protocol Specialist
Name: Carissa Henriksen
Role: Protocol Specialist
Name: Nathan Scheuer
Role: Open PCR Machine Engineer
Name: Samantha Barker
Role: Research and Development Scientist
Role: Open PCR Machine Engineer
LAB 1 WRITE-UP
Initial Machine Testing
The Original Design
The picture to the right shows the design of the open PCR machine used in class (click to enlarge). This PCR machine is compact, inexpensive, and easy to use in comparison to professional grade PCR equipment making it ideal for student and school use. The machine is composed of an LCD screen, heating lid, mother board (circuit board),thermal conductor and a fan.
Experimenting With the Connections
Part 1 of the open PCR machine is the heating lid with a heating plate. This part heats the top part of the test tube as well as prevents heat from escaping. If this part doesn't work heat is likely to escape and the test tubes will not likely heat to the optimal temperature
Part 2 is the test tube rack. This part hold the test tubes in place and acts as the main heating element for the test tubes. If this part does not work the test tubes will not heat to the optimal level
Part 3 is a cable between the circuit board and the LCD display. If this connection fails the LCD will not function.
Part 4 is a fan within the system. This part sucks the hot air out from the inside of the machine and prevents the system from overheating.
Part 6 is a white wire between the circuit board and the rack. This wire is connected to a temperature sensor. Unplugging this wire will disable the temperature reading on the LCD display.
On March 4, 2013 our PCR machine was first tested. We used machine "10" and downloaded the open PCR software on a mac laptop. The software did not download properly on my pc laptop. The system test ran normal without any issues. The time estimation on the software is not 100% accurate.
Thermal Cycler Program
For the reaction to take place, the machine needs to have a certain program set up to run. The program for the Thermal Cycler is as follows. This is also known as the initialization step:
- 1 Cycle at 95°C for 3 minutes
- 35 Cycles at 95°C for 30 seconds, 57°C for 30 seconds, 72°C for 30 seconds
- 72°C for 3 minutes
- Hold the mix at 4°C
Each cycle consists of initialization, denaturation, annealing, and extension. The denaturation step is when the DNA is heated to a point at which the DNA base pair bonds begin to break, which leads to the double-stranded DNA coming apart into two single strands. Then the machine is cooled for the annealing step, which is when the primers attach to their respective sites on the single stranded DNA and begins the DNA formation. The fourth step, extension, takes place at a warmer temperature than annealing, but cooler than denaturation, and consists of the elongation of the DNA strands.
The picture below shows this process demonstrated in the PCR software.
DNA Sample Set-up
|Tube: A, Positive Control: Cancer DNA template
||Tube: B, Patient 1 ID: 43825, Replicate: 1
||Tube: C, Patient 1 ID: 43825, Replicate: 2
||Tube: D, Patient 1 ID: 43825, Replicate: 3
|Tube: E, Negative Control: No DNA template
||Tube: F, Patient 2 ID: 12079, Replicate: 1
||Tube: G, Patient 2 ID: 12079, Replicate: 2
||Tube: H, Patient 2 ID: 12079, Replicate: 3
PCR Reaction Mix
Creating a PCR reaction mix is essential for the DNA amplification to occur properly. In order to do this, groups are presented with 8 tubes, which will be filled with 25μL of the following:
- Taq DNA polymerase
- Magnesium chloride (MgCl2)
- Premium concentrations for the maximum amount of DNA produced by PCR
DNA/ primer mix
Another component for the amplification is creating a DNA/ primer mix. In order to do this, groups are presented with 8 tubes, which will be filled with 25μL of the following:
- Unique DNA template of each patient
- Forward and reverse primers (the same contents in each tube)
DNA Sample Set-up Procedure
- To keep the patient samples separate and to avoid contamination between samples, it is necessary to label the tubes by numbering them one through eight with a permanent marker.
- Take up 25μL of patient DNA into the micropipette. Next, add it to the 25μL of the PCR reaction mix.
- After all 8 tubes are filled with 50μL, place them into the PCR machine, which is connected to the computer.
Research and Development
Specific Cancer Marker Detection - The Underlying Technology
In order for a PCR machine to function in a way that is scientifically efficient, the machine must go through three different phases. It must go from 95ᵒC, to 57ᵒC, to 72ᵒC, and then repeat this cycle thirty times. At each stage, a microbiological change in the DNA occurrs.
When the sample is heated to 95ᵒC, the hydrogen bonds that hold the two strands of DNA together are broken. It is known as unzipping DNA. This allows for the base pairs of the two DNA strands to be exposed.
The sample is then dropped to 57ᵒC to allow for the primer to bind to the DNA template gene. The template DNA is the sample DNA that was extracted from the patient and the primer's function is to bind to the DNA and direct where the template DNA should be amplified. Two primers are needed to ensure the process is efficient: a forward and a reverse primer. The forward primer binds to the forward DNA strand to direct synthesis of the DNA from left to right; while the reverse primer binds to the opposite DNA strand and directs synthesis of the DNA strand from right to left.
As the temperature increases to 72ᵒC, the Taq polymerase is activated. The polymerase is a protein that synthesizes the new DNA strand by using dNTP’s, the extra base pairs in the solution. The Taq polymerase grabs these extra base pairs and synthesizes the DNA strand that matches the template DNA strand.
From here, the solution is reheated to 95ᵒC and all the steps repeat again. Each time this cycle is complete, the amount of replicated DNA strands will have an exponential growth in the amount of synthesized DNA.
For further detail, refer to the diagram below showing each step of this PCR procedure.
The amplification of template DNA gives scientists the ability to analyze what gene segments are present in patients’ DNA. This becomes extremely helpful in detecting DNA sequences that code for cancer. Cancer is caused by a gene mutation of a base pair. With PCR machines, DNA sequences that have been mutated can be found. For example, if a gene sequence is supposed to be synthesized and replicated as ATTCGG, but is mutated to be ATCGG, a PCR machine can detect this. It is done by creating a primer that will bind to the template DNA if and only if it has the mutated gene; the primer will not bind to a sequence that is not identical to it, and there will be no synthesis of DNA, and thus, no cancer mutation. When the mutated gene binds with the primer, it directs the Taq Polymerase to begin synthesis, which results in an exponential growth in synthesized DNA. When there is an exponential growth in DNA it shows that the gene sequence is in fact present in the template, or patient, DNA. The results also validate that the patient does in fact have the DNA sequence that codes for cancer.