BME103:T130 Group 10 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: Frances Lakers Role: Protocol Planner Name: Tyler Tamasauckas Role: Protocol Planner Name: Jeffery Ramirez Role: R&D Specialist Name: Alexander Baldwin Role: Machine Design 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. System Design Key Features There are two specific features that will be upgraded in the OpenPCR machine - the case and the power source. The case will be changed from a thin wood to a more durable plastic, so that the device can be taken out into the field. The device will also have solar panels, which will provide for the power. This shift allows the device to be used where electricity is not always available, such as rural and undeveloped regions. When combined, these two features open the device up to a larger market. In addition to the Thermal Cycler's modifications, there have been improvements on the process of flourimeter image collection and measurement. Instead on manually adjusting phone settings and using a computer with ImageJ to measure colors, an app has been implemented that automatically adjusts the cameras settings to the correct exposure, as well as having the capability to measure the colors right from the smart phone. Instructions The instructions would remain mostly the same, as the solar panel array would come attached to the plastic on the device's top side. The only changes in procedure would be in the use of an all in one phone application for the flourimeter measurements. Protocols Materials Supplied in Kit Item Quantity PCR Machine 1 unit Camera Phone Stand 1 unit Fluorimeter Stand (LED Light) 1 unit BlackOut Box 1 unit Operator's Manual 1 unit Also included: Access code for smart phone app download. Supplied by User Item Quantity Smart Phone (w/ Camera) 1 unit USB/Phone Cord (if desired) 1 unit Micropipettes (as needed) Hydrophobic Glass Slides (as needed) Eppendorf Tubes Max 16 tubes/trial Sample DNA 0.2μL/tube; 16 tubes/trial GoTAQ Master Mix 50μL/tube; 16 tubes/trial 10 μM Forward Primer 1μL/tube; 16 tubes/trial 10 μM Reverse Primer 1μL/tube; 16 tubes/trial Distilled H2O 47.8μL/tube; 16 tubes/trial SYBR Green 0.1mL/test sample PCR Protocol 1.) Be sure to be in an area with natural lighting for use of the solar battery, or locate an area with a standard power source availible 2.) Connect the PCR machine via USB or Bluetooth to the phone 4.) Label tubes that will contain the subject DNA. Also be sure to include control tubes in this labeling. 5.) After filling the tubes with sample DNA, primers and other reactants. 6.) Load the tubes into the Thermal cycler's tray. 7.) Close and properly tighten the lid. 8.) Adjust the settings for the thermal cycler. 9.) Begin the program. DNA Measurement Protocol 1.) Assemble the flourimeter and place the glass slide in the tray. 2.) Use the pipette to add to drops of water to the slide. These will serve as controls for the measurements. 3.) Adjust the slide to be aligned with the blue LED. The LED should be shining directly on the test drops. 4.) Open the newly introduced all in one flourimeter application on the smartphone. The application will automatically set the phones camera to the correct settings. 5.) Place the smartphone in the holder and use the in app photo timer to take the picture while the black out box is closed. 6.) Analyze the images color measurements using the app. 7.) Save the image properties as the control image for future measurements. 8.) Repeat steps 2 through 6 for each DNA sample. 9.) The app will automatically compare the control images to the sample images and will list the results. Research and Development Background on Disease Markers The disease that we chose was Sickle Cell Anemia. Sickle Cell is a inherited disease that comes from both parents. If only one parent has the Sickle Cell trait, then the child will be a carrier. If both parents have the trait, then the child will have Sickle Cell. This disease causes the red blood cells to change into a crescent shape instead of being disk shape resulting in the loss of efficient oxygen delivery. It can also result in clotting because the blood cells can not make it through small blood vessels very well. The SNP for this disease is 78478128 and the reference SNP is rs78478128. The link to the page with more info on Sickle cell is http://www.ncbi.nlm.nih.gov/pubmedhealth/PMH0001554/ The link to the page with the genetic information and SNP is http://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?rs=78478128#locus Primer Design The forward primer for Sickle Cell is 5'-tcctaagccagtgccagaag-3' The reverse primer for Sickle Cell is 5'-gaattcgtctgtttcccattctaaac-3' The mutation looks like this: TCCTGAGGAG with the bold a being the mutation. A disease allele will give a PCR result and a non-disease allele will not because PCR is designed to amplify the area that corresponds to the mutation on the DNA. If it did not amplify only the target region, there would be too much information and a result would not be possible. Illustration