The improvement that we are planning is the ability to test for multiple strands of mutations via PCR test. Detecting two or a few different mutations that cause the same type of cancer in order to increase the amount of patients. In theory, many different mutations cause one type of cancer (IE – there are 5 different mutations that cause pancreatic cancer) and therefore if we could include primers that detect for all these different mutations, we determine if the patient has PC with one test, not five different ones.
<!--- A description of the diseases and their associated SNP's (include the database reference number and web link) --->
<!--- A description of the diseases and their associated SNP's (include the database reference number and web link) --->
Our re-design is based upon the Open PCR system originally designed by Josh Perfetto and Tito Jankowski.
System Design
Key Features
Instructions
Protocols
Materials
The following materials are provided in the PCR and Fluorimeter kit:
Supplied in Kit
Amount
Open PCR Machine
1
Fluorimeter
1
Phone Stand
1
Box
1
Superhydrophobic Slides
10
CD Software
1
The following materials will need to be supplied by the user:
Supplied by User
Amount
Eppendorf tubes
10
Pipettes
12
Pipette Tips
8
Power source
1
Smartphone with Camera
1
ImageJ Software*
1
PCR Master Mix
800 μL
SYBR Green Solution
10 mL
DNA Samples
6
DNA Positive Control
1
DNA Negative Control
1
Calf Thymus DNA Sample
1
Water
100 mL
Plastic Tube-holding Grid
1
Note that the PCR machine does not require a computer but ImageJ does. A further improvement to this technology would be to design an app for ImageJ that can be used on smartphones.
PCR Protocol
In order to perform PCR, the samples must first be prepared. This is done by adhering to the following steps:
1. Open up all Eppendorf tubes that will be needed to perform the PCR. Place them in the plastic grid to hold them.
2. Fill each Eppendorf tube with 50 μL of the PCR Master Mix using a pipette.
3. Using a distinct, clean pipette tip per sample (including positive and negative controls), fill each tube with one DNA sample. Be sure to close each tube as you fill it with the DNA sample and label the lid. Throw away each pipette tip and place a new one on each time you are going to use a different sample to avoid contamination.
Now that the samples have been prepared, the PCR machine must then be configured to the specific cycling and temperature needs of your experiment. In this case, the instructions for carrying out PCR in one manner are as follows:
1. Turn on the Open PCR machine and press the forward arrow key to select PCR.
2. Set the lid temperature to 100°C and press the forward arrow key when done.
3. Set the initial temperature to 95°C. Press the forward arrow key and select 3 for the number of minutes to hold the tubes there.
4. Set the number of cycles to 30 and press the forward arrow key.
5. For the first temperature, set it to 95°C, press the forward arrow key, and set it for 30 seconds.
6. Select the second temperature of the cycle to be 57°C, press the forward arrow key and set it to 30 seconds.
7. Select the third temperature of the cycle to be 72°C, press the forward arrow key and set it to 30 seconds.
8. Set the final temperature to 72°C, press the forward arrow key, and select 3 minutes.
9. Set the hold for the tubes to be 4°C.
10. When ready, place the test tubes into the machine, close the lid and twist the top to hold it down, and select the forward arrow key to start the PCR. Note that if a mistake was made you may use the backward arrow key to scroll through the different settings you selected and alter as necessary.
DNA Measurement Protocol
The next part of the process is to measure the amount of replicated DNA and use a fluorimeter to determine whether or not someone has the mutation associated with cancer. The first part of this is to set up the fluorimeter correctly:
1. Pull out all of the materials in the box and open up one side of the box, placing it upside down on the table with the open side facing you.
2. Place a slide on the fluorimeter, glass side down, making sure that at least one row of holes lines up with the light.
3. Place the smartphone in the holder, making sure the camera is ready. This includes increasing the exposure, turning off the flash, and setting it to 800 mp.
4. Download the ImageJ software onto a computer or device that is compatible with the smartphone.
Now collection of the data may begin. This is done first by making sure that the experiment worked using the calf thymus DNA as a standard and then measuring the amount of green light from the added dye there is in order to determine the concentration of DNA:
1. Place a water droplet on the slide in the middle hole.
2. Gently add the SYBR Green dye droplet to the water droplet. Place this in the box with the light on and take a picture. This will be the baseline to determine the concentration of pixels in ImageJ when there is no DNA present.
3. Email this photo to a computer with ImageJ and upload it into the program. Split this image into the three component colors: blue, green, and red. Using only the image that shows green, place a circle around the droplet to determine the amount of green in the drop. Make a similar circle on an area of the image that is pitch black. Subtract the background density from the image density to determine the actual density.
4. Repeat steps 2 and 3 but add a droplet of calf thymus to the water droplet as well. This will ensure that the fluorimeter is working properly. Record the actual density of the pixels. This will become important for calculating the concentration of DNA. In order to calculate this, take the actual density and divide it by the calf thymus density and multiply by 2 (since the calf thymus had 2 μL of DNA).
5. Repeat steps 2 through 4 for each sample, including positive and negative controls, and for the three samples from each patient. If the concentration of DNA is similar to that of the calf thymus, this indicates that the person likely has the mutation associated with colon, rectal and/or pancreatic cancer.
Use of OpenPCR and Fluorimeter for More Cancer Markers
The OpenPCR may also be used to screen for additional types of cancer marker genes in DNA. In order to do so, the same procedure as before with the PCR machine and the fluorimeter must be followed. If the SYBR green shows that there is a cancer marker in the DNA, the specific type may be determined using the PCR machine a second time. In this instance, a sample from the patient who is believed to have a cancer marker will be placed into the PCR machine, but not for the purpose of replicating the DNA. Instead, the PCR machine can be set to various temperatures that are associated with the denaturation of each specific cancer marker since the hydrogen bond arrangement will be different and thus each specific marker will have its own temperature that breaks it down. These known temperatures will be used and the fluorimeter can be used a second time with SYBR green dye solution to determine how much less green light the sample emits. The percentage that it does not emit any more (meaning the difference from the first sample) indicates how much of a presence that specific cancer marker has.
Research and Development
Background on Disease Markers
The improvement that we are planning is the ability to test for multiple strands of mutations via PCR test. Detecting two or a few different mutations that cause the same type of cancer in order to increase the amount of patients. In theory, many different mutations cause one type of cancer (IE – there are 5 different mutations that cause pancreatic cancer) and therefore if we could include primers that detect for all these different mutations, we determine if the patient has PC with one test, not five different ones.
BME 103 Fall 2012
