BME103:T130 Group 12 l2

BME 103 Fall 2012	Home People Lab Write-Up 1 Lab Write-Up 2 Lab Write-Up 3 Course Logistics For Instructors Photos Wiki Editing Help
OUR TEAM Name: James Open PCR machine engineer Name: Heidi Hall Open PCR machine engineer Name: Justin Landstrom Student Protocol Planner Name: Chaio Protocol Planner Name: Jakob Wells R&D scientist LAB 2 WRITE-UP Thermal Cycler Engineering Our re-design is based upon the Open PCR system originally designed by Josh Perfetto and Tito Jankowski. New System Design The two parts below that will be removed are the the knob and the bolt attached to it. These parts are present on the lid currently as a tightening mechanism but they will be removed to improve the design. On the side of the picture are the two heating plates present in the lid. To compensate for removing the tightening mechanism, the plates will be lowered to the point it makes contact with the samples in the heating block at the correct point in which the lid snaps into place. Below is the top part of the general body of the PCR machine. It will be altered so buttons can be placed next to the LED screen. Key Features The design changes are predominately focused on the lid and the top body part next to the LED screen. Regarding the lid, the tightening mechanism was deemed ineffective due to not knowing how much to tighten the knob. To compensate for removing the mechanism, the heating plates will be lowered onto a specified height that it will make contact with the samples when the lid snaps into its natural place. Since sample containers are universally similar, one specified height will relinquish the need of having to set the heating plates themselves. Design changes on the top body part will include changing the layout so that it may fit input buttons that will be connected and programmed internally later. These buttons will allow an individual to set up cycling details and will remove the need of an external computer to run the device. Instructions For the lid design 1. Remove the knob on the lid 2. Once the knob is removed, the bolt will be able to be removed. 3. Lower the bottom heating plate to the desired height 4. Lower the higher heating plate so the difference in space between the two plates was similar as before For the top body part 1.Attach the part as you would normally 2.Place the buttons in their appropriate spots in the new areas on the top board. 3.Connect the input buttons to the circuit board. Protocols Materials Supplied in the Kit Amount PCR Machine (assembled) 1 Power Cord 1 Flourimeter Box 1 Flourimeter 1 Hydrophobic Slides 5 Supplied by User Amount Camera Phone 1 DNA Samples & Target Mix As many as desired Bulb pipettes As many as desired SYBR Green As much as desired Computer w/ ImageJ Software 1 PCR Protocol A polymerase chain reaction (PCR) is based on the enzyme DNA Polymerase's ability to synthesize complementary DNA strands. Through a series of steps involving polymerase breaking apart a DNA strand and then synthesizing a specified complementary piece, a PCR machine is able to isolate and amplify a desired strand of DNA. Steps to Amplify a Patient's DNA Sample 1. PCR is a process that uses controlled temperature changes to make copies of DNA. Heat (about 95°C) separates double-stranded DNA into two single strands; this process is called denaturation. 2. "Primers", or short DNA strands, binds to the very end of the complimentary sequence that is being replicated. This step is called annealing, which takes place between 40°C and 65°C. The temperature that we use for this is 57°C. 3. Once the annealing process is done, the temperature is raised to about 72°C and DNA polymerase then extends from the primers copying the DNA. 4. PCR amplifies a segment of a DNA sequence from the original DNA strand. In the end, there will be two new DNA strands identical to the original strand. Components of PCR Master Mix • A modified form of the enzyme Taq DNA polymerase that lacks 5´→3´ exonuclease activity. • dNTPs • MgCl2 • Colorless Reaction Buffer (pH 8.5) Reagent Volume Template DNA (20 ng) 0.2μL 10μM forward primer 1.0μL 10μM reverse primer 1.0μL GoTaq master mix 50.0μL dH2O 47.8μL Total Volume 100μL DNA Measurement Protocol 1. With a permanent marker, the transfer pipettes were numbered at the bulbs so that one pipette was used per sample. With the permanent marker, the Eppendorf tubes were numbered at the top. 2. Each sample was transferred separately (using one pipette per sample) into an Eppendorf tube containing 400 mL of buffer. This tube was labeled with the number of the sample. The entire sample was transferred into this Eppendorf tube. The pipette with the corresponding sample number was used to place the sample onto the fluorescent measuring device. 3. The specially labeled Eppendorf tube containing SYBR Green I was transferred using the specifically labeled pipette. Only two drops were placed on the first two centered drops as seen on the video. 4. The diluted sample was then taken and two drops were placed on top of the Syber Green I solution drops. 5. The light going through the drop was aligned, as seen in the video. 6. After setting up the Flourimeter and the samples, the Smartphone’s photo settings was setting to the ones listed: Inactivate the flash Set ISO To 800 (or higher if possible) Set White Balance to Auto Set Exposure to Highest Setting Set Saturation to the Highest Setting Set Contrast to Lowest Setting 7. The flourimeter was placed into the light box. 8. The smartphone operator had taken as many pictures using the light box as he/she wants. Their goal is to take pictures clear enough so ImageJ can take data from the images. 9. Once they have taken enough photos of that sample, the flourimeter was taken back to the sample preparer to prepare the next sample. 10. Now the sample was either rerun again or that sample and its pipette was discarded. The SYBR Green I labeled pipette was kept. 11. This procedure was then repeated for all the samples. 5 samples can be run per glass slide. 12. As the last sample was run, the water from the scintillation vial as a blank was run using the same procedure as with the other samples. 13. ImageJ was used to analyze the images taken by the smartphone. To upload the image onto ImageJ, the ANALYZE tab was clicked and SET MEASUREMENTS was chosen. AREA INTEGRATED DENSITY and MEAN GREY VALUE were selected from the menu. 14. The MENU tab was selected and the COLOR tab was chosen, the function SPLIT CHANNELS was used; three separate files were then created. SYBR GREEN fluoresces green, sothe image name with "green" next to it was used. 15. The oval selection was used to draw an oval around the green drop. Then, the MEASURE tab was selected from the ANALYZE tab, and the sample number and the numbers measured from the image were recorded. 16. To get the readings from the background of the image, another oval of approximately the same size was drawn in the background green image and the MEASURE tab was selected from ANALYZE tab. The sample number and the numbers measured from the image were recorded , this data was labeled as "background". 17. The measurements were saved in an excel file by clicking SAVED AS from the FILE tab. 18. The INTDEN was collected for the positive and negative controls and the patient samples. 19. The DNA μg/mL was calculated with this equation: 2*INTDEN of sample/INTDEN of DNA Calf Thymus. Research and Development Background on Disease Markers Human Immunodeficiency Virus, or HIV is a horrible disease and is running rampant worldwide. HIV is especially prevalent in underdeveloped countries. This virus isn't limited to undeveloped countries however, as there are also many people with HIV in developed countries such as the United States. This disease affects chromosome 17 and the genome build 36.3. After researching the SNP for HIV, which is rs1024611, the mutation in the sequence was discovered to be ATA. So then a primer to be used along side a PCR was developed. http://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?rs=1024611 Primer Design The primer that can be used to detect HIV is CGTCTGTCGATAGTGAAAGG and its reverse GCAGACAGCTATCACTTTCC. A diseased allele will only give results because of the way the PCR works and how the primers will attach. If the sample has the disease then the primer will attach to the DNA and as the PCR goes through its cycles there will be a multitude of the same DNA strand with the SNP rs1024611. If the sample does not have the disease allele then there will be no interaction with the DNA and the primer, therefore there will be no change from the original and the end results. Illustration The primer will only bond to the diseases allele because of the specific genetic pattern. For each cycle the amount of binded primers will double creating an immense amount of the strips of DNA. Bayesian Stats The following equation can be used to find the Bayesian statistics: source: http://www-rohan.sdsu.edu/~ling354/n-grams.html