Name: Nathaniel Bennett
Role(s): PCR Technician/Research and Development Specialist
Name: Brian Hedden
Roles: ImageJ Software Processor/Data Compiler & Analyzer
Name: Hanna Rahman
Name: Hope Haddad
Role(s): Protocol/Research and Development Specialist
LAB 1 WRITE-UP
Initial Machine Testing
The Original Design
The Open PCR machine is fairly easy to use. Simply plug it in to the USB port on your computer and turn it on. From your computer, you can adjust the temperature, add or remove steps to the process, and adjust the number of cycles.
Experimenting With the Connections
When we unplugged the LCD from the Circuit Board, the LCD turned off.
When we unplugged the white wire that connects the circuit board to heat block, the LCD was unable to accurately read the temperature.
The very first time that our group used the Open PCR machine was October 28, 2012. The process went fairly smoothly, which was unexpected. All we did was put the samples into the machine, set the peak temperature and number of cycles, then let it run for an hour and forty-nine minutes.
Polymerase Chain Reaction
The polymerase chain reaction (PCR) is a biochemical machine used in biological chemistry to produce numerous copies of a particular piece of DNA, generating multiple duplicates of DNA sequences. The PCR machine works similar the cycle of DNA replication at the cellular level. The machine consists of four individual steps, initiation, denaturation, annealing, and extension. The initiation step is solely to prepare the DNA samples to be put through the thermal cycler program. During the denaturation step, the DNA strand is split into two separate strands. After, the annealing step is where the DNA primer attaches to the targeted DNA sequence. The primer only attached to a specific site on the strand, not necessarily the entire strand. The purpose of the primer is to mark the beginning and the end of the targeted DNA sequence. Lastly, in the extension step, the DNA polymerase is first activated, which begins to synthesize the DNA primer. This results in two double stranded target DNA sequences. The PCR machine cycles numerous times to amplify the specific sequence. In order to complete the reaction several components are required such as:
- A PCR reaction mix that contains: Taq DNA polymerase, MgCl2, dNTP’s, and a forward and reverse primer
Most PCR methods use thermal cycling, alternating heating and cooling steps. These thermal cycling steps are necessary to separate the two strands in a DNA double helix at a high temperature in a process called DNA melting. At the lower temperature, the DNA polymerase to amplify a particular target DNA uses each DNA strand as a template in DNA synthesis. The primers aid discrimination of the DNA to target the specific region for amplification under specific thermal conditions. The stages of the PCR machine are listed below:
- Acquire the DNA samples that have been submitted for testing
- Run the DNA samples through the thermal cycler program (see Stage 1)
- Stage 1 (Initiation): 1 cycle at 95°C for 3 minutes, to separate the DNA strand.
- Stage 2 (Denaturation): 35 cycles: first at 95°C for 30 seconds, and then gradually decrease the temperature to approximately 57°C for 30 seconds, and then raise the temperature to approximately 72°C for 30 seconds, so the DNA polymerase can be activated. This is also an example of heat shock, and is effective to initiate the addition of complementary nucleotides onto the DNA strand, which the DNA polymerase does.
- Stage 3 (Elongation) : At this step, hold the DNA at 72°C for 3 minutes.The temperature is held here so that the DNA polymerase can copy the strand. Also, this is where 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 began with.
- Stage 4: Final hold until the DNA stabilizes at 4°C. At the end of this cycle you will have 8 fragments of the DNA (see Table 2)
- After the DNA has been through the numerous cycles, you will have over thousands of fragments of the same DNA sequences. After the DNA has been through the thermal cycle, mix each DNA sample with the PCR reaction mix (Taq DNA polymerase, MgCl2, dNTP’s, and a forward and reverse primer), using a separate pipette each time to reduce cross-contamination into 8 separate tubes (see Table 2).
| Template DNA ( 20 nanograms)
|| 0.2 microliters
| 10 micrometers forward primer
|| 1.0 microliters
| 10 micrometers reverse primer
| GoTaq Master Mix
|| 50.0 microliters
| Distilled Water
|| 47.8 microliters
| Sample Descriptions (8 Samples)
| Positive Control: Cancer DNA Template
|| Patient 1 Replicate 1: 44231
|| Patient 1 Replicate 2: 44231
|| Patient 1 Replicate 3: 44231
| Negative Control: No DNA Template
|| Patient 2 Replicate 1: 57447
||Patient 2 Replicate 2: 57447
||Patient 2 Replicate 3: 57447
|| Eppendorf tube Label
|| Pipette 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:
- Turn on the excitation light using the switch for the Blue LED.
- Place the smartphone on the cradle at a right angle from the slide.
- Turn on the camera setting on the smartphone. Reminders:
- Turn off the flash.
- Set the ISO to 800 or higher
- Increase the exposure to maximum
- If possible, turn off autofocus, to make sure you can take an image where the drop on the slide is.
- Adjust the distance between the smartphone on its cradle and the first two rows of the slide so that it is as close as you can get without having a blurry image.
- The pipette should be filled with liguid only to the bottom of the black. Then, carefully place two drop of water in the middle of the first two rows of the slide using the plastic pipette. Then add 2 more drops. The drop should then be pinned and look like a beach ball. It should be between 130-160 microliters.
- Align the drop by moving the slide so that the blue LED light is focused by the drop to the middle of the black fiver optic fitting on the other sisde of the drop.
- Cover the fluorimeter with the light box, but make sure you can access your smartphone to take the image. The light box should be used to remove as muhc stray light as possible.
- Take three images of the drop of the water. Make sure not to move your smartphone.
- Remove the box (make sure not to move your smartphone). If you need to adjust for any movement, use the ruler provided to measure the distance so that you can return to that location. You can also use ImageJ to compensate for moving the camera, but it makes the analysis more complicated.
- Use a clean plastic pipette to remove the water drop from the surface.
- Push the slide in so that you are now in the next set of two holes.
- Repeat steps 5-10 four more times so that you have now imaged all 5 positions on the slide.
Type of smartphone used: Iphone 4s
Distance-base of smartphone cradle to measurement device:
In this lab, we used SYBR Green I, which is used to detect a particular DNA strand. In this lab, our fluorimeter was created using optical caustic because it eliminated the need for lasers, mirrors, and lenses. A fluorimeter is a device used to measure parameters of fluorescence: it's intensity and and wavelength distribution. The quality is related to the amount of florescent material and indirectly proportional to the molecule being detected. The objective of this part is to determine if you have actually amplified the target DNA in your PCR experiment. Through this you will be able to visually observe the fragments and calculate the relative amount of DNA. During the first procedure, you amplified your DNA, so now that you are going dye it with the following procedure:
- You will have 8 sample from the OpenPCR instrument and 1 DNA(calf thymus standard at 2 micrograms/mL) sample and water from the scintillation vial to analyze.
- With a permanent marker, number the transfer pipettes at the bulb so that you limit cross-contamination and only use ONE per solution, and number your Eppendrof tubes at the top. At the end, you should have 10 Eppendorf tubes and 10 pipettes clearly labeled. (See Table 3)
- Transfer each sample separately ( using one pipette per sample) into an Eppendorf tube containing 400 mL of buffer. Label this tube with the number of your sample. Make sure to transfer all of the sample into the Eppendorf tube. Use the same pipette to place ONLY this sample's drop onto the fluorescent measuring device.
- Take the specially labeled Eppendorf tube containing the SYBR GREEN I and using it's assigned pipette, place two drops on the first two centered drops.
- Now take the diluted sample and place two drops on top of the SYBR GREEN I solution drops.
- Align the light going through the drop.
- Take pictures with the smartphone, as many as desired.
- If you are not satisfied with that sample, you may rerun that sample again or move on to the next sample.
- Be sure to only run 5 samples per slide.
- Before completing the lab, run the water from the scintillation vial as a BLANK using the same procedure listed above.
This will allow you to observe whether or not you amplified your DNA.
Transferring images from your smartphone to the laptop that has ImageJ
- Connect your smartphone to your laptop using a USB cable.
- Click Start and then click My Computer. Under Portable Devices, you should find your smartphone icon, double-click this icon.
- Once it is open, double click the DCIM folder, and then double-click the Camera folder.
- Now hold down CTRL and select the images you wish to transfer over to your laptop. Right-click and copy them.
- Make and label a new Folder - ImageJ. Paste your pictures into this folder.
- Open up your ImageJ program, go to File and click Open. Open up the ImageJ folder that has all your pictures.
- Now, selecting one picture at a time, open them in the ImageJ program.
Research and Development
Specific Cancer Marker Detection - The Underlying Technology
Polymerase Chain Reaction
In order to replicate DNA to work on, we must copy strands of DNA through a process called DNA replication. Polymerase Chain Reaction(PCR) is used to determine a specific point on DNA that is to be copied. In DNA replication, a bubble of DNA primers and fluorescent tags will form around a specific piece of DNA and divide, or unzip (annealing) DNA. Any unneeded reagents in the solution will simply not bind to a base pair. In order for this to occur, the solution is heated to 95 degrees Celsius for about 3 minutes. Here, when the molecule is split into two, polymerase will add corresponding base pairs creating a first copy. For this to occur, the temperature needs to be brought down to about 57 degrees Celsius. At the same moment, polymerase is creating base pairs for the other half of the original DNA strand. The thermal cycler heats and cools the solution to complete the process. This process will cycle a few times in order to create duplicate DNA that is able to be tested for different reasons such as cancer.
(Image taken from biology.kenyon.edu)
The dye we added to each sample will fluoresce under UV light when DNA is present. If our eyesight was good enough, we would see that all the samples fluoresce slightly, however, because that is not the case, we do not. Instead, what we see fluoresce are samples that have a large amount of DNA present. We know from the experiment we created that we replicated cancerous DNA, specifically the r17879961 cancer associated sequence, meaning there would be excess amounts of the DNA in the sample if the patient has this cancerous sequence. Therefore, if a sample fluoresces bright enough for the naked eye to see, an excess amount of DNA is present and the patient does have cancer.
The Bayes Theorem allows a scientist to interpret new data based on their expectations. The calculations show to false positives and inconsistencies in data. This is important in experiments such as the probability of a patient having cancer through a PCR testing such as this.
Pr (A/B): Probability of a positive outcome
P (B/A): Probability of a true positive
P (A): Chance of having the cancer
|| Integrated Density
|| DNA μg/mL
| PCR: Negative Control
| PCR: Positive Control
| PCR: Patient 1 ID 43417, rep 1
| PCR: Patient 1 ID 43417, rep 2
| PCR: Patient 1 ID 43417, rep 3
| PCR: Patient 2 ID 11260 , rep 1
| PCR: Patient 2 ID 11260, rep 2
| PCR: Patient 2 ID 11260, rep 3
- Sample = Sample number and patient
- Integrated Density = The integrated density of the drop of water minus that of the background
- DNA μg/mL = 2 * IntDen of Sample/IntDen of Calf Thymus
- Conclusion = Whether the DNA replicated or not