BME103 s2013:T900 Group1 L3: Difference between revisions
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| [[Image:Guitar.jpg|100px|thumb|Raul Monzolo:<br>Open PCR Machine Engineer]] | | [[Image:Guitar.jpg|100px|thumb|Raul Monzolo:<br>Open PCR Machine Engineer]] | ||
| [[Image:BME103 Group1 Johnny.jpg|100px|thumb|Johnny Montez:<br>Open PCR Machine Engineer]] | | [[Image:BME103 Group1 Johnny.jpg|100px|thumb|Johnny Montez:<br>Open PCR Machine Engineer]] | ||
| [[Image: | | [[Image:Sloth.jpg|100px|thumb|Robert Sanchez:<br>Research and Design Specialist]] | ||
| [[Image:BME103_Group1.JPG|300px|thumb|Group 1]] | | [[Image:BME103_Group1.JPG|300px|thumb|Group 1]] | ||
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<!-- If your team decided not to change any of the machinery/ devices, write a short step-by-step list of instructions on how to set up and use each machine. You may want to consider modifying the instructions to improve ease of use. --> | <!-- If your team decided not to change any of the machinery/ devices, write a short step-by-step list of instructions on how to set up and use each machine. You may want to consider modifying the instructions to improve ease of use. --> | ||
Using the new system for data analysis will require less steps for faster completion. The PCR samples | Using the new system for data analysis will require less steps for faster completion. The PCR samples will be loaded into the microwell plate using a multichannel pipette. Once the samples are grouped into containers, the spectrophotometer is plugged in and warmed up. Next the device is connected to a computer via USB to record the data as the samples are analyzed. Finally, a specific wavelength is chosen to detect the fluorescence of the sample, and the well plate is loaded into the device for detection. | ||
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'''Primers for PCR'''<br> | '''Primers for PCR'''<br> | ||
<!-- If your team decided to only amplify cancer-associated DNA, list the "Cancer allele forward primer" sequence and the "Cancer allele reverse primer" sequence. Include a paragraph that explains why a disease allele will give a PCR product and the non-disease allele will not.--> | <!-- If your team decided to only amplify cancer-associated DNA, list the "Cancer allele forward primer" sequence and the "Cancer allele reverse primer" sequence. Include a paragraph that explains why a disease allele will give a PCR product and the non-disease allele will not.--> | ||
Because the cancer gene is a mutation in with the allele "ACT" as opposed to "ATT", a primer can be designed and created that acts as a compliment to the cancer associated strand of DNA. However, rather than creating a fluorescent primer that fluoresces green at the presence of DNA, a dye will be made that fluoresces for the presence of any DNA. What replaces the positive cancer test is a probe that will fluoresce orange when attached to the caner-specific DNA. This way, a negative control is present that tells us there isn't a cancer-segment, whereas the positive control will fluoresce orange if there is a cancer-segment of DNA. Also, we know whether or not the negative control works because it will fluoresce green if negative. If there is no fluorescence, then we know something went wrong with the dye. | |||
Forward Primer: [ A C G T A T G T A T] | |||
Reverse Primer: [ T G C A T A C A T A] | |||
<!-- If your team chose an alternative approach to amplify the DNA, list all relevant primers. Include a paragraph that explains how your system works.--> | <!-- If your team chose an alternative approach to amplify the DNA, list all relevant primers. Include a paragraph that explains how your system works.--> |
Latest revision as of 17:38, 16 April 2013
BME 103 Spring 2013 | Home People Lab Write-Up 1 Lab Write-Up 2 Lab Write-Up 3 Course Logistics For Instructors Photos Wiki Editing Help | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
OUR TEAMLAB 3 WRITE-UPOriginal System: PCR ResultsPCR Test Results
* Ave. INTDEN = Average of ImageJ integrated density values from three Fluorimeter images
Bayes Theorem equation: P(A|B) = P(B|A) * P(A) / P(B)
Calculation 2: The probability that the sample actually has a non-cancer DNA sequence, given a negative diagnostic signal.
Calculation 3: The probability that the patient will develop cancer, given a cancer DNA sequence.
New System: Design StrategyWe concluded that a good system Must Have:
New System: Machine/ Device EngineeringSYSTEM DESIGN The Open PCR thermo cycler is a system designed to amplify desired segments of DNA causing an enhanced DNA polymerase chain reaction via multiple cycles of alternating temperatures. The apparatus creates an ideal environment for primers and DNA molecules to interact, and the number of cycles used in the reaction ultimately determines the amount of amplified DNA at the conclusion of the reaction. The open PCR machine is modified with a heating plate that presses against the sample containers to prevent water from condensing on the inside of the capsules from the mixtures. An important modification that is essential for the open PCR system is to construct it in a way that it does not require a wooden shell. Because the machine has to be plugged into a power source and can be found in labs with other high-tech devices, there is definitely a fire hazard that accompanies the system. This issue can be easily solved by equipping the thermo cycler with a plastic, fire proof shell. In order to modify the entire system, our team decided not to use a fluorimeter to measure the fluorescence of our PCR samples. The system is inefficient because the steps necessary to preform fluorimetry take too long and produce only fractions of the data neccessary to diagnose patients. Only one drop of sample can be analyzed with a camera, and it is inconvenient to use the imageJ software to digitally measure the data separately. It would be much more efficient to analyze the PCR samples on a larger scale and in one quick step, so our new design would include a microwell plate and a micro plate spectrophotometer. A microwell plate has multiple wells that can hold up to three hundred samples out of a time, and it is a standard tool commonly used in clinical diagnostic testing laboratories throughout the world. This feature will enhance our ability to analyze large amounts of data, and the micro plate spectrophotometer is the perfect instrument capable of performing this process. Spectrophotometry quantitatively measures the intensity of light through an aqueous solution as a function of wavelength. Because absorbance is proportional to concentration, the amount of light that passes through the solutions will indicate the concentration of desired material, in this case the fluorescent primers involved in the process of diagnosing positive and negative patient samples.
KEY FEATURES We chose to include these new features
Using the new system for data analysis will require less steps for faster completion. The PCR samples will be loaded into the microwell plate using a multichannel pipette. Once the samples are grouped into containers, the spectrophotometer is plugged in and warmed up. Next the device is connected to a computer via USB to record the data as the samples are analyzed. Finally, a specific wavelength is chosen to detect the fluorescence of the sample, and the well plate is loaded into the device for detection.
New System: ProtocolsDESIGN We chose to include these new approaches/ features
MATERIALS
Thermal Cycler Program Stage 1
Stage 3
Add 25μL of the 2x Master Mix to each reaction tube
New System: Research and DevelopmentBACKGROUND CHEK 2, or Checkpoint Kinase 2, is a gene in the human genome that acts as a suppressor for the growth and division of a cell. When a portion of DNA becomes damaged, this gene is activated which halts the G1 phase of cell division, preventing the cell from entering meiosis. Whenever any damage is done to the DNA, CHEK 2 suppresses growth and also helps repair that damaged DNA. However, an SNP (or single nucleotide polymorphism) can occur in this gene. An SNP causes a single nucleotide to be changed in a way that alters how the sequence is supposed to be, and thus alters its function. The SNP associated with CHEK 2 can actually cause the process of cell death to stop, which means more cell division, indicating a presence of cancer. Specifically, it is most associated with breast cancer. The cells cannot stop growing because they can't die, and thus CHEK 2 does the opposite of what it's supposed to do. The associated sequence for CHEK 2 is ATT, and the cancer-associated sequence is ACT. By knowing this, we can target the allele associated with the cancer SNP of CHEK 2, and thus determine whether a patient may or may not have cancer.
Because the cancer gene is a mutation in with the allele "ACT" as opposed to "ATT", a primer can be designed and created that acts as a compliment to the cancer associated strand of DNA. However, rather than creating a fluorescent primer that fluoresces green at the presence of DNA, a dye will be made that fluoresces for the presence of any DNA. What replaces the positive cancer test is a probe that will fluoresce orange when attached to the caner-specific DNA. This way, a negative control is present that tells us there isn't a cancer-segment, whereas the positive control will fluoresce orange if there is a cancer-segment of DNA. Also, we know whether or not the negative control works because it will fluoresce green if negative. If there is no fluorescence, then we know something went wrong with the dye. Forward Primer: [ A C G T A T G T A T] Reverse Primer: [ T G C A T A C A T A]
Our primers address the following design needs
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