Name: Tyler Ray
Research and development scientist
Name: Seth Howell
Open PCR machine engineer
Open PCR machine engineer
Data Compiler and Analyzer
Everyone has contributed to this project even though there are only two usernames. Every person used these two users to make edits to the wiki. Dr. Haynes said that this would be sufficient enough to give each member full participation credit for this project
LAB 1 WRITE-UP
Initial Machine Testing
The Original Design
The open PCR is a device that enables the splitting of DNA. It is run through many cycles of heating and cooling that enables the splitting and recombination with polomers. It connects to a computer program to run the cycles. The temperature change is controled by the heating lid and than the information is fed to the LED screen. The heating lid heats tubes that are held in the heat tube holder to the appropriate temperature and then cools them as needed in each cycle.
Experimenting With the Connections
When we unplugged part 3, the LCD, from part 6, the Open PCR Brains Board, the LCD on the machine stopped working and did not show anything on it.
When we unplugged the white wire that connects part 6, the Open PCR Brains Board, to part 2, the heat tube, the machine no longer measures the temperature of the plate and sends it to the LCD.
First test run: 24 Oct. 2012
Experiences with it: It worked very well with minimal problems. The program would not allow us to enter the parameters once they were set. Aside form that, everything functioned with ease.
Polymerase Chain Reaction
Polymerase Chain Reaction uses thermal cycling to artificially replicate DNA in vitro. First, a piece of double stranded DNA is added to a test tube containing a solution of primers, and the enzyme DNA polymerase. This solution is heated to 95 degrees celsius in order to separate (denature) the DNA molecule. Then, the solution is cooled to 57 degrees celsius and the primers bind to the single strands of DNA (provided the base sequences match up), then the solution is heated to 72 degrees celsius and DNA polymerase adds bases to complete the new complimentary strands, doubling the amount of DNA. This is repeated several times.
Procedure for amplifying a person’s DNA
1. Add .2ng’s of a person’s dna to a tube containing the proper primers, water, and the GoTaq master mix. Repeat this step for as many replicates as you need.
2. Create a program on the open PCR machine that will run the proper cycle for replicating DNA.
3. The proper program will include an initial temperature hold of 95 degrees Celsius for three minutes, followed by 35 cycles of 95 to 57 to 72 all for 30 seconds each, then a final hold for 3 minutes at 72 degrees.
4. Place the tubes in the open pcr machine making sure to close the lid.
5. Run the program with the tubes inside the machine.
The Master Mix
GoTaq colorless Master Mix was used in this experiment. It contains taq polymerase, dNTPs(bases), MgCl2, and reaction buffers. Specifically, 2x colorless GoTaq master mix contains 400um of each base, and 3mm MgCl2 and a reaction buffer of pH 8.5. Part of this was then mixed with a few other ingredients as detailed in the table below. (information from www.promega.com)
Contents of PCR vials
| Reagent Table || Volume
| Template DNA (20 ng) || .2 μL
| 10 μM forward primer || 1.0 μL
| 10 μM reverse primer || 1.0 μL
| GoTaq master mix || 50.0 μL
| dH2O || 47.8 μL
| Total Volume || 100.0 μL
The eight samples run in our pcr experiment were a positive control, a negative control, and three replicate samples from each of our patients.
| Patient ID || Gender || Age
| 27150 || Female || 61
| 73439 || Male || 62
1. Place the flourimeter on the table and turn on the blue light.
2. Place provided glass slide on flourimeter track so that the first row of dots is even with the light.
3. Place phone cradle in front of the flourimeter with a smart phone facing perpendicular to the beam of light. (as seen in the picture).
4. Add two drops of green dye on the dots that are even with the light.
5. Place two drops of DNA sample on top of the green dye.
6. Cover the flourimeter and phone by turning the large box over and placing it above both of them.
7. Take a picture of the droplet with the camera.
8. Save the picture and send it to the imageJ operator.
Instructions for opening images in imageJ
1. Take a picture of the fluorimeter assembly with a smartphone.
2. Transfer the picture to a laptop equipped with imageJ via icloud or email.
3. Open imageJ and select file, then open.
4. Find the file on the computer and select it.
5. The image is now open and can be analyzed.
6. The image can be split into three images (blue, green, and red) for better analysis by selecting: image-color-split channels.
Research and Development
What Does PCR Do?
DNA strands across human beings are very similar, but there is a correlation between the discrepancies between DNA sequences and phenotypic conditions such as cancer or heart disease. Because DNA is so small, and has so many components, it is not reasonable to simply look through the entire strand for the sequence of interest. This report used a technique that amplified specific DNA strands which is called Polymerase Chain Reaction or PCR. This technique uses a primer that identifies and replicates a specific Single Nuleotide Polymorphism(SNP), if present. For this to work, the DNA strand and primer need to go through a cycle of temperature shifts that allow the DNA to replicate. The steps are denaturing, annealing and extension. The denaturing process happens when the system is heated to the point where the DNA strands are changed into single strands. After this the annealing step involved cooling the DNA strand and reconstructing of the double stranded DNA. Extension then takes place which is exactly as it sounds, the primer extends or builds a DNA strand that has the gene sequence the primer is looking for, if it is in the DNA. This is where amplification occurs and how we can measure if the DNA at test holds the sequence in question. For measuring purposes, a fluorescent is added, in tandem with the primer.
Looking at rs17879961(CHEK2)
As mentioned above, there are single nucleotide polymorphisms that are consistently found in the population that can indicate phenotypic behaviors in the person. To explain how this method is used, we will consider the sequence, rs17879961. This specific sequence is found on chromosome 22, has been named CHEK2, and is associated with colorectal and breast cancer. Below are the sequences of the "normal" sequence and the cancer associated sequence. Notice that the only difference is marked in brackets and is only a change from a T base to a C base pair. By adding a primer to this strand that attached only to the cancer sequence we knew if the tested DNA has the strand if the sequence was amplified by the primer after a denaturing, annealing, extension cycle. The primer sequence is also shown below in the forward and backward direction.
To interpret the results into something that is meaningful we used Baye's rule which basically gives a number value of the predicative reliability of the test. SO, for instance, if somebody tested positive for a specific allele that was associated with freckles, there will still be a chance that they do not have freckles. THis is true for people that test negative, they may still have freckles. In the bayesian parameters there are four numbers to look at; the positive predictive value(ppc) which is the probability that somebody testing positive will display the associated phenotype, the negative predicative value(npv) which is the probability that somebody testing negative will not display the associated phenotype, false positive which is the probability of somebody testing positive that doesn't display the phenotype, and false negative which is the probility of somebody testing negative that actually has the phenotype. Below is the formula.
To put this in words we can use an example of positive predictive value. It will the number of patients that test positive that have the phenotype divided by the total number patients that tested postive. High PPV and NPV are ideal.
The following are Bayesian values of the CHEK2 SNP and its association with breast cancer as tested by Vahteristo in Finland.
He noted that 2% of people with cancer associated gene had the breast cancer and 1.4% of people without the gene mutation had breast cancer. This means the allele presence has a PPV of 2.0% and a NPV of 98.6%. This doesn't seem very high but when a doctor can look at other associations like breast cancer in family history the PPV grows to as high as 3.1%. It doesn't exactly indicate whether somebody has the cancer but it can let them know they need to be careful and get regular screenings.
| Sample || Integrated Density || DNA μg/mL || Conclusion
| PCR: Negative Control || 815872 || 0 || neg
| PCR: Positive Control || 2469599 || 2 || pos
| PCR: Patient 1 ID 27150, rep 1 || 868816 || .704 || neg
| PCR: Patient 1 ID 27150, rep 2 || 1296676 || 1.05 || pos(borderline)
| PCR: Patient 1 ID 27150, rep 3 || 701619 || .568 || neg
| PCR: Patient 2 ID 73439, rep 1 || 2296110 || 1.860 || pos
| PCR: Patient 2 ID 73439, rep 2 || 1296676 || 1.822 || pos
| PCR: Patient 2 ID 73439, rep 3 || 2795519 || 2.264 || pos
- Sample = A sample was two drops of a flourescent dye that stains DNA green and two drops of DNA solution, either provided as a control, or obtained from open pcr.
- Integrated Density = Integrated density basically measures the amount of brightness there is in a certain area. This is an extensive property (one that depends on how many pixels were in the system) so areas as close in size to each other were used as measurements. The integrated density shown in the tablewas determined by the following means. The integrated density of an area immediately above the droplet was subtracted from the integrated density of the area covering the droplet.
- DNA μg/mL = This value was calculated by dividing the integrated density of the sample by the integrated density of the positive control and multiplying that number by 2 μg/mL (the amount of DNA in the positive control).
- Conclusion = Samples were considered positive for the gene if they contained more than 1 μg/mL of DNA. Samples within .1 μg/mL were arbitrarily determined to be borderline, or tentatively positive.