Our Team
LAB 1 WRITE-UP
Initial Machine Testing
The Original Design
The Open PCR machine is designed to perform polymerase chain reactions which replicate DNA segments. Enzymes and nucleic acid are added to a sample of DNA. Then, primers are used to select a specific genetic sequences. Due to the thermal cycling that is done by the Open PCR machine the segment of DNA that is selected by the primers is exponentially replicated. Essentially the goal of the Open PCR machine is signal amplification. The signal that is being amplified is the selected DNA segment. This is very useful, because a genetic marker for a disease can be selected and then amplified so that doctors will know whether or not the patient has that specific sequence. This particular Open PCR machine is a relatively cheap and easy to use machine for completing this process.
Experimenting With the Connections
If the Arduino UNO board is disconnected fron the LCD screen, then then LCD screen will not be able to display information; this includes information such as the current cycle of the PCR and the current temperature.
If the Arduino UNO board is disconnected from the 16-tube PCR block, then the LCD wouldn't be able to display any temperatures, this is because temperatures in the 16-tube block are not being monitored.
Test Run
The experiences of first testing the Open PCR machine, on October 24th, 1012, were of mixed results. As for the set up of the Open PCR, things went fairly well. Connecting the Open PCR to a computer was not a problem, and finding a suitable location to let the machine run was very easy. However, the first computer we connected the Open PCR to had problems running the Open PCR software. The experiment design part of the software would not allow for editing of the number of cycle, resting temperature, time length of the cycles and all other variables of the experiment. The software might have been corrupted or the computer may not have been running correctly; whatever the case, the Open PCR had to be moved to another computer in order to solve this problem. The use of the second computer allowed for editing of the experimental variables and the initiation of the experiment. However, further problems arose once the experiment was in progress. From the beginning of the experiment the Open PCR machine being used took a considerable amount of time on the cooling part of the cycle, much longer than the other groups running the experiment. If the machine does not cool down correctly and to the right temperature, then the PCR cannot move onto the next cycle. And eventually, due to this problem, the Open PCR machine became stuck on the cooling part of cycle 5 of 30 and would not move forward in the experiment. After trouble shooting from TA's and the professor the problem could not be reverse and additional amplified DNA samples will have to be created for group 3.
Protocols
Polymerase Chain Reaction
Polymerase Chain Reaction is a biochemical technology that is used in molecular biology to amplify single/multiple copies of a piece of DNA, generating thousands to millions of copies of a targeted DNA sequence. To do so, PCR relies on thermal cycling, which consists of repeated cycles of heating and cooling the samples in order to melt the DNA and have the enzymes replicate the targeted strand if found. Primers, which are short DNA fragments, have complementary sequences to the target strand of DNA, in addition to a DNA polymerase, which allows selective and repeated amplification of the target strand. As the cycles progress, the DNA is used as a template for exponential amplification (or creation of DNA copies).
How to Amplify a DNA Sample with PCR
- The initialization step consists of heating the reaction to a temperature between 94 to 98°C, depending on how thermostable the polymerase is. It is held at this temperature for 1-9 minutes. Note, however, that this step is only required for DNA polymerases that require a "hot start", which reduces non-specific amplification during the set-up stage of the PCR.
- The denaturation step is the first cycle which consists of heating the reaction to 94-98°C for 20-30 seconds. This causes the DNA template to melt by disruption of the hydrogen bonds between complementary bases, which yields single-stranded DNA.
- The annealing step consists of lowering the temperature to 50 to 65°C for 20 to 40 seconds, which allows annealing of the primers to the single-stranded DNA template. Annealing is the process of heating then cooling the DNA strands so as to separate the double-strands into single-strands. Normally, the annealing temperature is about 3 to 5°C below the melting temperature of the primers used. Stable DNA-DNA hydrogen bonds are only formed when the primer sequence very closely matches the template sequence.
- Step 4: Initial Extension
- During the initial extension step the DNA polymerase synthesizes a new DNA strand that is complementary to the DNA template strand by adding dNTPs which are complementary to the template in 5'-3' direction, which condenses the 5'-phosphate group of the dNTPs with the 3'-hydroxyl group at the end of the extending(nascent) DNA strand. The time for the extension to take place depends on the DNA polymerase used and the length of the DNA fragment that is being amplified. A general guideline that can be applied is 1,000 bases a minute, though under optimal conditions the amount of DNA target is doubled at each step, which leads to exponential amplification.
- The final extension step is performed at a temperature of 70-74°C for 5-15 minutes after the last cycle to ensure that any remaining single-stranded DNA is fully extended.
- The final hold step is the step in which the reaction is stored for the short-term at 4-15°C and may be done indefinitely.
Components of GoTaq® Colorless Master Mix
- 2X Colorless GoTaq® Reaction Buffer (pH 8.5)
- 400μM dATP
- 400μM dGTP
- 400μM dCTP
- 400μM dTTP
- 3mM MgCl2
| Reagent
|
Volume
|
| Temple DNA (20 ng)
|
0.2 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
|
8 Samples, 3 each patient, positive and negative control
| Patient ID
|
10840
|
12675
|
| Gender
|
Female
|
Male
|
| Age
|
48
|
59
|
Flourimeter Measurements
Fluorimeter Assembly Procedure
- Step 1) Switch on excitation light for blue LED
- Step 2) Place smartphone on the provided cradle at an angle perpendicular to the slide (make sure to keep cradle in place throughout pictures to ensure consistency).
- Step 3) Adjust camera settings if possible: Turn flash on, set ISO to 800+, increase exposure to maximum and disable auto-focus.
- Step 4) Position cradle as near as possible to the slide so that an clear image will be taken.
- Step 5) The pipette should be used to apply two drops of water (~130-160 microliters per drop) in the center of the first two rows of the slide. (Note that the pipette should only be filled to the bottom of the black line.)
- Step 6) Adjust the slide so that the blue LED light is focused on the drops so that the middle of the black fiber optic is on the other side of the drop.
- Step 7) Create a dark environment by covering the fluorimeter setup with the box (either side (width) can be unbuttoned so that an access slot for the phone to take a photo is available) so as to reduce as much stray light as possible.
- Step 8) Take three separate photos of the water droplet while taking care to move the phone as little as possible to ensure consistency.
- Step 9) Remove the box while again taking care not to change the phone's position, as it is crucial to maintaining a constant.
- Step 10) Dispose of the drops by extracting them with another clean pipette from the slide and disposing of them in the proper hazardous waste receptacle.
- Step 11) Adjust the slide's position so that the following pair of holes are accessible and ready to use.
- Step 12) Reapply steps 5 through 10 in several (at least 5) different positions.
- Step 13) Take note of the following data: Type of smartphone used to take the pictures, distance from the measurement device to the base of the cradle in centimeters, and an attached image of each position of the drops (for a total of at least 5).
Transferal of Images to ImageJ
- Step 1) Connect smartphone that has photos to the laptop with the ImageJ software with a USB cable.
- Step 2) Either press the Windows Key or Start and then browse to My Computer/Computer.
- Step 3) Under the category Portable Devices, the smartphone should be listed (if not check the USB cable connection and try unplugging and replugging it), double-click the icon.
- Step 4) Find the folder labeled DCIM and open it, then the following sub-folder Camera.
- Step 5) If the pictures that you wish to transfer are the only images in the library then drag and select all of the images and right-click and select Copy then Paste them into your preferred destination (Suggested folder: Pictures, under Libraries, alternatively Control + C then Control + V), otherwise hold down Control and left-click each picture that needs to be transferred and repeat the above instructions.
- Step 6) Open the ImageJ software by either double-clicking the icon on the Desktop or by browsing the Start Menu.
- Step 7) When the ImageJ software is open, go to the top left bar and click on File, then select Open.
- Step 8) Browse to the folder you saved the pictures in (For example, Pictures) then select the image.
- Step 9) Repeat steps 7-8 for the rest of the other pictures when necessary.
Research and Development
Specific Cancer Marker Detection - The Underlying Technology
- The key to biomarkers is primer binding. The primer that matches the cancer gene will bind and amplify the genes of interest. Primers are made specifically to bind to the cancer gene by having complementary sequence. If the gene is not present, the primer will not bind, and gene amplification will not occur.
- There are two variances of bases that exist in a DNA sequence. These represent the genes in question and will often identify as the cancer causing gene.
- Include possible primers in the range of the marker (place of amplification) and compare it to the sample sequence to see if the primers match up and amplify. If they do, the person has a 7.8% chance of actually having cancer according to Bayes rule.
Bayes Rule: Predicting the possibility of cancer.
- Bayes Rule is a theorem that aims to show the comparison of the probability of something happening, to the actual occurrence or nonoccurrence of an event.
- According to Bayes rule, out of the sample 10.4% tested positive and only 0.8% of those people actually had cancer. 9.6% of the positive testing group did not have cancer. Out of the large sample, 89.6% tested negative and out of that group 0.2% actually had cancer. The other 89.4% were cancer free.
Bayes Theorem: (.008)/(.008+0.095)=7.8%
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"Polymerase Chain Reaction (PCR)." Contexo.info. N.p., n.d. Web. 07 Nov. 2012. <http://www.contexo.info/DNA_Basics/polymerase_chain_reaction.htm>.
Results
| Sample |
Integrated Density |
DNA μg/mL |
Conclusion
|
| PCR: Negative Control |
2404566 |
0 |
Negative for Gene
|
| PCR: Positive Control |
8620824 |
2 |
Positive for Gene
|
| PCR: Patient 1 ID 10840, rep 1 |
8135995 |
1.667144 |
Positive for Gene
|
| PCR: Patient 1 ID 10840, rep 2 |
2998645 |
0.125993 |
Negative for Gene
|
| PCR: Patient 1 ID 10840, rep 3 |
1047714 |
-0.45928 |
Negative for Gene
|
| PCR: Patient 2 ID 12675, rep 1 |
4610442 |
0.609698 |
Positive for Gene
|
| PCR: Patient 2 ID 12675, rep 2 |
2509549 |
-0.02074 |
Negative for Gene
|
| PCR: Patient 2 ID 12675, rep 3 |
384138 |
-0.65835 |
Negative for Gene
|
KEY
- Sample = The sample is the designated amount of DNA from the target population, which in this case is a cancer test patient.
- Integrated Density = The integrated density is the sum of the gray values of each pixel in a determined area, in other words measuring the brightness of the sample and is found by subtracting the integrated density measurement from the background from the integrated density reading from the drop for all the measurements. For example, in this specific experiment, integrated density was used to measure the brightness of the color green in each sample.
- DNA μg/mL = The DNA microliters per milliliter was calculated by using the negative and positive control samples to create a calibration curve in which each of the integrated density values could be substituted for the x value in the calibration curve equation. For example this curve's equation was y = 3·*10-7·x-0.7736, in which y is the value of the micrograms per milliliter of DNA in each sample and x is the integrated density substituded.
- Conclusion = This states whether or not the test was positive or negative for the cancer gene.
NOTE: It is understood that it does not make sense for the samples of the patients to have a negative value for their concentration of DNA; however, due to the calibration curve equation these are the results gathered and conclusions were still drawn from this data.
|
BME 103 Fall 2012
