The PCR Machine otherwise known as a Thermocycler or a DNA Amplifyer is use by scientists to create a vast quanity of a specific sequence of DNA. This method is also used widely for biological and medical applicaions. This process relies on the thermal cycles to amplify the DNA to then be able to replicated it. The system is run by means of a software downloaded to either a desktop or laptop. By connecting the PCR machine to a computer it is then possible to run a test with the unique specifications needed; such as tempurature and number of cycles.
PCR diagramExperimenting With The Connections
When taking apart the PCR Machine there are pieces that if not connected properly or were not working apropriatly would cause the test to be inacurate and result in unreliable data. By umpluging the LCD screen from everything this else truned off the screen and we were unable to see the process that the machine was running. Another part of the machine that was important to make sure it was working properly was the battery, motherboard, and tempurature sensor. When the tempurature sensor was unpluged the reading on the LED screen was inaccurate. Once it was reconnected the tempurature retured to the apropriate level and the reading on the screen was reliable. Each of these components are importante and if they were not working properly could cause the test runs to come out negative and false.
Test Run
The first test date was March 5. It began around 10:00am and completed the 4 minute test cycle with no issues.
A preliminary test on March 19 concerned the Machine testers as the PCR Machine was not completing the specifies cycle. The machine was also remaining above 100 degrees and would not cool down to meet the requirements of the test.
After more testing, it was clear to the group that machine 1 was not going to produce reliable results based on the variation in temperature and the inability of the machine to complete the cycle. The group then moved the test samples to the PCR Machine that another group was testing so that the test might be run on a reliable machine. The machine tester of this new group must be relied upon to make sure that the new machine is operational and will produce reliable results.
Protocols
Experimental protocol planner summery
As experimental protocol planner we have taken on the challenge of making sure that the experiment goes as planned as well as the preparing software that was used to evaluate our samples. We have left the technical portions of experiments to our “Open PCR machine testers” and allowed our R&D scientists to educate us on our experiment so that we (Experimental protocol planners) could create a plan and execute it in order for the experiment as a whole to be successful.
Our job as for Experiment Protocol Planner was to :
Prepare the software for analysis
Prepare DNA samples for tests
Analyze the results
We have educated our self via “PCR Virtual Lab” that can be found at http://openpcr.ord/use-it/ then downloaded and test software that was responsible for taking the results from PCR machine and displaying those results on PC, as well as to make commends to the PCR machine. This software also gave us real time update of the PCR machine
Second and most important part of to prepare our sample. Our sample consisted of two rows of four samples. The table representing the samples is shown below. In this part we have also created a total of 8 different dyes also shown below.
The third part was to take the results from the PCR machine and analyze them with the rest of our team, via software. called
Thermal Cycler Program
PCR experiment summery
PCR machine goal is to take a long code of DNA and to amplify or separate a known part of that DNA. This target DNA is to be replicated with the help of PCR machine. We know which part of the DNA needs to be amplified because we know the sequence of bases of that particular part. We use the master mix which already contains the known sequence and will replicate only that known sequence leaving the the rest of the DNA. The PCR machine allows the master sample to perform its job by performing cycles of cooling and heating. The temperature changes allow different enzymes to perform their job in replicating the DNA. When the sample is heating the DNA opens up, the heat rises up to 95 degree Celsius. Then for primers to be allowed to do their job the sample is cooled down to 57 degree Celsius. Next for the primer to add appropriate amine acids in order to build a new DNA strand the sample is heated up to 72 degree Celsius. This sample is repeated multiple times allowing more and more replicates of the targeted DNA to be made, making the amount of the unwanted DNA to be minimal compared to the amount of wanted DNA.
DNA Sample Set-up
ID : 80175(1), 57483(2)
ROW :
Tube 1
Tube 2
Tube 3
Tube 4
Row 1
Contains : rep : Label
+ Control N/A "+"
- Control N/A "-"
Patient 1 1 "1"
Patient 2 1 "2"
Row 2
Contains : rep : Label
Patient 1 2 "+-"
Patient 2 2 "--"
Patient 1 3 "1-"
Patient 2 3 "2-"
"Contains" describes what sample was inserted in the test tube
"rep" is the replicate, we have 3 replicates of each patient
"label" is simply what symbol we have written on the test tube so that we can identify what sample is where
DNA Sample Set-up Procedure
1. Step 1 : Acquire Materials
Materials :
- A set of four dyes (blue, green, yellow, red)
- Sample containing (+,-,P1,P1,P1,P2,P2,P2)
- Pipette and tips
- two rows of empty test tubes
2. Step 2 : Mix dyes
MIX:
blue 50μL
blue 50μL
blue 50μL
yellow 50μL
blue 50μL
green 50μL
yellow 50μL
yellow 50μL
yellow 50μL
green 50μL
red 50μL
red 50μL
red 50μL
green 50μL
green 50μL
green 50μL
3. Step 3 : Prepare the samples, and label the samples (tables is shown above)
4. Step 4 : allow other team members to start PCR machine and then load the samples into the machine
5. Step 5 : Enter following commands to the Thermal Cycler Program so that the PCR machine knows what and how many cycle s to perform
Stage
Cycles
Heat (Celsius)
time
1
1
95
3 minutes
2
35
95 to 52 to 72
each 30 seconds
3
1
72
3 minutes
6. Step 6 : Start the PCR machine using software
PCR Reaction Mix and
DNA/ primer mix
Inside the PCR reaction mix there are six different components. The first is the template, the template is the specific type of DNA or RNA the person wants to replicate. The quality of the template can determine the outcome of the PCR machine. The seconded is the primers, a person can choice many different types of primers, but they all accomplish the same goal they prime the DNA to be ready for replication. The Third piece of the mix is a DNA polymerase; the polymerase greatly affects the outcome of the PCR machine because this is the enzyme that replicates the DNA or RNA. Fourth is the MgCl2 concentration, this concentration is key it soluble complexes with dNTPs to produce the sub straight that the polymerase will recognize. The fifth is Deoxynucleotide triphosphate (dNTP) concentration and lastly is the pH of the solution. The pH is important because there is a sweet spot that produces the maximum results; this is around a pH of 8.3-9.0. Inside the DNA/ primer mix are a few things. The first in the DNA mix is the template strand of DNA that is going to be replicated. Also there is the DNA polymerase that once active will construct the new DNA strand. The primer mix is an enzyme that attaches to the first three nucleotides in a DNA base and thus letting the polymerase attach more nucleotides to the free 3’ end.
Specific Cancer Marker Detection - The Underlying Technology
The goal of this experiment is to understand the DNA amplification process in order to detect cancerous genes when given Template DNA; otherwise known as DNA taken from the patient. DNA amplification will allow the cancerous genes to replicate. The cancerous gene will produce a positive result, while the non-cancer gene will give a negative result, because the primers are designed to amplify cancerous DNA. Therefore, the cancerous mutation cannot bind to normal DNA, ultimately meaning that amplification cannot occur.
Functions of each component of the PCR Chain Reaction: Template DNA: The DNA taken from the patient Primers: Short (20 base pairs) pieces of synthesized DNA Taq Polymerase: is an enzyme that detects the primer-template DNA and snaps the dNtp's back to DNA Magnesium Chloride (MgCl2): serves as a "co-factor" for Taq Polymerase dNTP's: Once the DNA is "unzipped"and recombined the dNTP's are the unincorporated bases
The following “3 steps” are repeated for 30 cycles, each step taking about 30 seconds.
Step 1:Heat up the Template DNA, Primers, Taq Polymerase, and Magnesium Chloride (MgCl2) to 95 degrees Celsius
• What happens?
o The bases need to me detected, in order to do so “melting” or unzipping needs to DNA will expose those base pairs.
o When the base pairs are exposed the primers will create a forward and reverse strand appropriately. The primers are short pieces (about 20 bases pairs long) of DNA that are synthesized DNA that binds to the Template DNA while directing the new strand to be made.
Step 2:The mixture is cooled down to 57 degrees Celsius
• What happens?
o The primers “anneal,” or in other words the bond.
Step 3:Heat the mixture back up to 72 degrees Celsius.
• What happens?
o The Taq Polymerase and Magnesium Chloride will detect where the template DNA and primer and uses the template DNA strand and primer to build a new copy of DNA
BME 103 Spring 2013
