Name: Sudarshan Iyer
Research and Development Specialist
Name: Ujwala Vaka
Experimental Protocol Planner
Name: Emily Herring
Experimental Protocol Planner
Name: Garrett Repp
Open PCR Machine Engineer
Name: Joseph Del Rosario
Open PCR Machine Engineer
LAB 1 WRITE-UP
The Original Design
PCR Machines allow us to make copies of different DNA samples by heating the DNA to the point of denaturing. Once the two strands of DNA are separated, an enzyme builds complimentary strands using the original strands as a template. The PCR Machine is able to be programmed to running multiple cycles of this heating phase. Each cycle results in doubling the previous amount, so by the end of the 30th cycle, you have just over 1,000,000,000 (one billion) copies of the original DNA. This allows the DNA to be analyzed and tested for any defects.
Experimenting With the Connections
The Open PCR Machine we received was number 13. When we unplugged the PCB Board of the LCD from the Open PCR Circuit Board, the machine's LCD screen turned off.
When we unplugged the white wire that connects the Open PCR Circuit Board to the 16 Tube PCR Block, the machine could not register or measure the temperature. The LCD screen displayed seemingly random numbers including -40 degrees Celsius (which is not possible because the Open PCR Machine was not changing the temperature at that time).
On October 18, 2012, our group first tested the Open PCR Machine number 13. At first, the machine seemed overwhelming in its design. However, after following the instructions and advice from peers and professors, we were able to determine how to properly setup, program, and run a simple test.
Polymerase Chain Reaction
Polymerase Chain Reaction is a technology that amplifies a single piece of DNA. This technology works very similarly to the natural DNA replication cycle. One PCR cycle consists of three basic steps: denaturation, annealing and extension. In the denaturation step, heat (usually about 95 degrees Celsius) is used to separate the two strands of DNA. Then in the annealing step, the temperature is decreased to 50 degrees Celsius and the DNA primer, specific to the target sequence for that organism, anneal to the separated strand of DNA. The primers mark the beginning and the end of the targeted DNA sequence. Finally, the extension step required the temperature to be raised to 72 degrees Celsius so that the DNA polymerase is activated. The DNA polymerase begins synthesis at the DNA primer. This results in two double stranded target DNA sequences. The PCR cycle is repeated many times to amplify the targeted strand.
There are typically many cycles that need to take place in the PCR in order to amplify a patient's DNA.
Cycle 1: the thermal cycler heats up to 95 degrees Celsius, or 203 degrees Fahrenheit, which is almost at boiling point. At this temperature, DNA double helix separates, creating two single-stranded DNA molecules. Gradually the temperature begins to cool to 50 degrees Celsius so the primers will attach. Then the temperature is raised to 72 degrees DNA polymerase is activated and locates primers attached to the single-strand DNA, which will then begin to add complementary nucleotides onto the strand. This process continues until it gets to the end of the strand and falls off.
Cycle 2: the same three steps occurring in cycle happen in cycle 2. The temperature is raised again to separate the DNA strands, the temperature is lowered so that the primers may attach, and the temperature is raised again slightly to stimulate DNA polymerase to copy the strand.
Cycle 3: the two desired fragments begin to appear—two strands that begin with primer one and end with primer two—and these are the DNA copies of the segment of DNA you’ve targeted. These products will increase (become the majority) as the cycle continues.
Cycle 4: at the end of this cycle, you‘ll have 8 fragments that contain only your target sequence (see Table 2).
Cycle 5: at the end of this cycle, you‘ll have 22 fragments that your target sequence and only ten longer length copies.
After 30 cycles there are over a billion fragments that contain only your target sequence and only 60 copies of the longer length molecules. You now have a solution of nearly pure target sequence.
After the DNA has been through the thermal cycler, mix each new DNA sample with the the PCR master mix (Taq DNA polymerase, dNTP's, MgCl2, forward primer, and reverse primer) into 8 different Eppendorf tubes using separate pipettes to reduce contamination (see Table 1).
|Template DNA (20ng)
|10 μM forward primer
|10 μL reverse primer
|GoTaq master mix
|DNA Sample Descriptions (8 Samples)
|Positive Control: Cancer DNA Template
||Patient 1 Replicate 1: 65685
||Patient 1 Replicate 2: 65685
||Patient 1 Replicate 2: 65685
|Negative Control: No DNA Template
||Patient 2 Replicate 1: 58278
||Patient 2 Replicate 2: 58278
||Patient 2 Replicate 3: 58278
||Eppendorf tube Label
|SYBR Green I Solution
||Blue dot at top
|DNA Calf Thymus, 2 microg/mL
||Red dot at top
|Patient 1 Replicate 1
|Patient 1 Replicate 2
|Patient 1 Replicate 3
|Patient 2 Replicate 1
|Patient 2 Replicate 2
|Patient 2 Replicate 3
Fluorimeter Assembly Procedure
1) Turn on the excitation light using the switch for the blue LED.
2) Place a smart phone on the cradle at a right angle from the slide.
3) Adjust your camera settings as follows:turn off the flash, set the ISO to 800 or higher, increase the exposure to the maximum, and turn off autofocus (optional).
4) Move the smartphone in the cradle as close possible to the first two rows of the slide so that you will get a clear image.
5) The pipette should be filled with liquid only to the bottom of the black line. Then use the pipette to place two drops of water (each drop should be between 130-160 microliters) in the middles of the first two rows of the slide.
6) Move the slide so that the blue LED light is focused on the the drops of water to the middle of the black fiber optic fitting on the other side of the drop.
7) Cover the fluorimeter with the light box so that much of the stray light will removed, but make sure you can still access your smartphone to take pictures.
8) While being careful not to move the smartphone, take three picture of the water droplet.
9) When removing the light box, be careful not to move the smartphone because that could make the analysis more complicated.
10) Use a clean plastic pipette to remove the water droplets from the slide.
11) Push the slide in so that you are now in the next set of two holes.
12) Repeat steps 5-10 four more times in 5 different positions.
13) Record the following: type of smartphone used, the distance from the base of the smartphone cradle to the measurement device (in cm), and attach one image for each position of the drop (5 images total).
After assembling the fluorimeter, you can now determine if you've amplified the targeted DNA in your PCR experiment. Using the Fluorimeter, you can calculate the relative amount of DNA through fluorescence, which is generated by excitation and emission wavelengths. In order to detect fluorescence when dsDNA is present, you'll be using SYBR Green I because it's more safer compared to other dyes. With that being said, gloves must be worn when handling any liquid containing SYBR Green I. The fluorimeter itself is a very simple machine because it uses optical caustic, a special type of optics that completely removes the need for lasers, mirrors, or lenses. Also the flourimeter is battery-powered, lightweight and portable; this allows every student to have one of these at their lab table. Following the steps below, you can easily learn how to dye your amplified DNA.
1)On your lab table, you'll find eight samples from the Open PCR, 1 DNA sample(calf thymus standard at 2 micrograms/mL), and water from the scintillation vial (white cap) to analyze.
2)With a permanent marker, label your Eppendorf tubes and number your pipettes (on the bulb part) so that no cross-contamination will occurs. At the end, you should have 10 Eppendorf tubes and 10 pipettes clearly labeled (see Table 3). REMINDER: Use only 1 transfer pipette per sample!!!
3)Transfer each sample separately (using 1 pipette per sample) into an Eppendorf tube containing 400 mL of buffer. Clearly label this tube with the number of the sample and make sure to get all of the sample into the Eppendorf tube. ONLY use the sample number transfer pipette to place a drop onto the fluorescence measuring machine, and then discard it.
4)Take Eppendorf tube labelled SYBR Green I and using the specially labeled pipette, place 2 drops on the first two centered drops.
5)Now take your diluted sample and place 2 drops on top of the SYBR Green I solution drops.
6)Let the smartphone operator take as many pictures as needed.
7)Now you may either rerun the sample again or discard the sample pipette, but keep the SYBR Green I labelled pipette. Also you can only run 5 samples per glass slide. If more are needed ask your lab TA or professor.
8)Before completing the lab, run the water from the scintillation vial as a BLANK using the same procedure.
Transferring the images from your smartphone to the laptop that has ImageJ
1) Connect your smartphone to your laptop using a USB sync cable.
2) Click Start and then click My Computer or Computer where under Portable Devices, you should find your smartphone icon and double-click on this icon.
3) Once you have opened it, double click folder DCIM, and next double-click the folder Camera.
4) From the Camera folder, press down on CTRL and click on the images you want to transfer and right-click and copy these images. NOTE: Do not take your finger off the CTRL key until after you right-click.
5) Under the Libraries folder, click on the Pictures tab and right-click and go to New and select Folder. Name this new folder ImageJ Pictures and double click on this folder. You can now right-click and paste the images taken by your smartphone into this folder.
6) Go to your desktop and double-click on the ImageJ icon and when ImageJ opens, go to the top left of the bar and click on File, next click Open.
7) A folder will appear on your screen, and on the left click the Libraries icon, next double-click the Pictures icon. In the Pictures folder, find the ImageJ Pictures folder you previously created and double-click on that folder.
8) In the ImageJ Picture folder, select an image (you can only select one image at a time) and click Open. In a few seconds, the image will appear on your screen.
Research and Development
Specific Cancer Marker Detection - The Underlying Technology
The reason that DNA with the cancer-associated SNP (single nucleotide polymorphism) rs17879961 will produce a DNA signal while DNA without the SNP will not produce a DNA signal lies in the arrangement of nucleotides at the molecular level. More specifically, the lack of a DNA signal is due to the inability of the reverse primer to bind to the forward strand during the annealing phase of PCR. To detect the cancer-associated sequence of r17879961, the reverse primer is used. This is because the cancer-associated mutation is represented by a single nucleotide in a particular triplet: instead of the normal ATT, the middle T mutates into a C, thus rendering a triplet of ACT (which one can see in the reverse primer shown above). In contrast, the normal sequence with the normal triplet ATT would read . At the protein level, this mutation of 1 nucleotide changes the coded protein from isoleucine to threonine. As a result, the primer will not attach to the normal DNA sequence as it will not have the corresponding nucleotides (AGT) on the forward strand in the particular section of DNA that the mutated sequence would have, but will rather have the triplet AAT.
When attempting to replicate DNA during PCR, two primers are required in order to facilitate extension of both the forward and reverse DNA strands. As mentioned above, the reverse primer for the cancer associated sequence would be , having the mutated C instead of the T. A forward primer is also required in order to facilitate extension of the reverse strand. It is usually around 200 base pairs from the mutated DNA sequence and is also 20 base pairs long. For this strand of DNA, a suitable forward primer would be: . With these two primers in place, Taq polymerase can properly do its job by attaching free dNTP’s to the forward and reverse DNA strands during the extension phase of PCR. An illustration of this concept is shown below:
An important part of any test is its reliability. If a test or diagnostic does not have a level of reliability, it will be ineffective in identifying populations that pass or fail the test. To measure the reliability a diagnostic will have on a certain population, a theorem known as Bayes Rule is utilized. This equation calculates the probability that A will be true given B. For example, A = hc = people who have cancer in a population while B = C = people in the population who have the rs17879961 SNP; therefore Bayes’ Rule would provide the probability that people who have cancer in population will ALSO have the rs17879961 mutation [p(hc|C)]. In this case, the percentage of people who possess the mutation and have cancer is known [p(C|hc)] along with the percentage of people with the mutation in the population of Finland [p(C)]. Therefore, the only percentage that would need to be found would be the percentage of people who have cancer in Finland [p(hc)] for Bayes’ Rule to work. This example of Bayes’ Rule is illustrated below:
The results of Image J analysis of SYBR Green.
|PCR: Negative Control
|PCR: Positive Control
|PCR: Patient 1 ID 65685, rep 1
|PCR: Patient 1 ID 65685, rep 2
|PCR: Patient 1 ID 65685, rep 3
|PCR: Patient 2 ID 58278, rep 1
|PCR: Patient 2 ID 58278, rep 2
|PCR: Patient 2 ID 58278, rep 3
- Sample = The sample refers to the DNA sample from each patient. Patient one and two both had three different samples tested. Negative Control and Positive Control samples were control samples to indicate whether or not the cancer gene was present.
- Integrated Density = The Integrated Density of the sample is the relative "brightness" of the sample fluorescent picture. This number was calculated by adding up the pixels in each picture and calculating the "mean gray value" for each pixel. A measurement is taken of the water droplet and the background then they are subtracted so the noise of the picture is accounted for.
- DNA μg/mL = The DNA Concentration was calculated by the formula: 2*(INTDEN sample with subtracted background)/(INTDEN of Calf Thymus with subtracted background). In which INTDEN refers to the Integrated Density of each picture sample. This formula works because there was a calibration factor (2) which is a known value for the control.
- Conclusion = The conclusion section indicates whether or not the sample tested indicated presence of the cancer gene. The only sample that showed a positive signal for this was the positive control.