BME103 s2013:T900 Group1 L3: Difference between revisions
Line 168: | Line 168: | ||
'''We chose to include these new approaches/ features''' | '''We chose to include these new approaches/ features''' | ||
* Non-flamable case on PCR machine - Safety requirements take priority. The replacement of the case will reduce the fire risk of the repeatedly heated equipment. | * Non-flamable case on PCR machine - Safety requirements take priority. The replacement of the case will reduce the fire risk of the repeatedly heated equipment. | ||
* Use of microwell plates and a spectrophotometer | * Use of microwell plates and a spectrophotometer- The micro well plates will allow hundreds of samples to be prepared simultaneously and read all at once. The spectrophotometer will eliminate the manual analysis of the image results. These aspects meet our goal of a high throughput system with fewer steps and more automated result analysis. | ||
* Cancer detection as a fluorescent probe - This aids the requirement of clear results. Our system uses primers which attach to the DNA around the cancer specific sequence. A probe specific to the cancer marker sequence which releases a fluorescent particle when interacting with DNA polymerase is also added to the solution. When The sample is mixed with SYBR Green dye after amplification, the use of a | * Cancer detection as a fluorescent probe - This aids the requirement of clear results. Our system uses primers which attach to the DNA around the cancer specific sequence. A probe specific to the cancer marker sequence which releases a fluorescent particle when interacting with DNA polymerase is also added to the solution. When The sample is mixed with SYBR Green dye after amplification, the use of a spectrophotometer will allow the fluorescence of two wavelengths of light to be measured; Response to green light will confirm that the PCR ran successfully and response to orange will indicate that the cancer gene is present. <br> | ||
'''MATERIALS''' | '''MATERIALS''' | ||
Line 220: | Line 220: | ||
'''DNA Sample Set-up Procedure''' | '''DNA Sample Set-up Procedure''' | ||
# Prepare the PCR Reaction Mix and DNA/Primer sample solutions as prescribed above | # Prepare the PCR Reaction Mix and DNA/Primer sample solutions as prescribed above, ensuring a positive and negative cancer control are being used for comparison purposes | ||
# Label reaction tubes for each sample or control | # Label reaction tubes for each sample or control | ||
# Add 50μL of each DNA sample Mix to the correspondingly labeled reaction tube (using a new pipette tip for each transfer in order to avoid cross-contamination between samples) | # Add 50μL of each DNA sample Mix to the correspondingly labeled reaction tube (using a new pipette tip for each transfer in order to avoid cross-contamination between samples) |
Revision as of 23:43, 15 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
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.
Our primers address the following design needs
New System: Software[THIS SECTION IS OPTIONAL. If your team has creative ideas for new software, and new software is a key component included in your new protocols, R&D, or machine design, you may describe it here. You will not receive bonus points, but a solid effort may raise your overall page layout points. If you decide not to propose new software, please delete this entire section, including the ==New System: Software== header.]
|