BME103:T930 Group 14

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: Jake Lindquist Protocol Planner Name: Breanna Pratt Protocol Planner Name: Kirsten Jefferys Open PCR Machine Engineer Name: Ben Alcron Open PCR Machine Engineer Name: Carlos Duarte Research and Design Scientist Name: Bryce DeSimmone Research and Design Scientist LAB 1 WRITE-UP Initial Machine Testing The Original Design Description of Open PCR Device The Open PCR device is thermo-cycler that helps to create strands of DNA. PCR stands for polymerase chain reaction. The Open PCR consists of a heating lid that clamps down on mini test tubes that are held 16-tube PCR main heating block. It also has a cooling fan to help bring down the temperature when it is necessary in the running experiment. The Open PCR alternates between temperatures in order to properly separate deoxyribose nucleic acid (DNA) strands, let primers bind to these strands, and then finish replication of DNA. Then the process repeats for several cycles. The LED screen displays the temperature, the cycle number over how many cycles total, and the ETA time until the test in completed. It heats up to 110 degrees Celsius by default, while its cool temperature sits at 25 degrees Celsius. Experimenting With the Connections When we unplugged the LED from the Open PCR circuit board, the machine stop displaying information on the LED screen. When we unplugged the white wire that connects the Open PCR circuit board to main heat sink, the machine incorrectly displays the temperature as negative 40 degrees Celsius. Test Run First test was an experimental run to be sure the machine was running properly. This test was conducted on October 25th, 2012 and the test was successful. Protocols Polymerase Chain Reaction The Polymerase Chain Reaction is a process that relies on thermal cycling allowing the DNA to be broken apart and replicated. This biochemical process works by separating a strand of DNA allowing a primer to target and begin replicating a segment of DNA. This separation, replication, and amplification of a specific DNA strand occurs within the machine during the thermal cycling. Procedure: Obtain DNA samples and clearly lab each tube with the sample name. 50 μL of the PCR reaction mixture was added to each 50 μL sample of patient DNA. Three samples of each patient were prepared along with one sample each of a positive control and a negative control. The eight prepared samples were placed into the PCR Machine, the lid must be screwed down until it rests on the test tubes. The PCR was run with a program of 30 cycles of the following steps: 90ºC for 30 seconds 57ºC for 30 seconds 72ºC for 30 seconds. PCR Master Mix Components GoTaq® Colorless Master Mix, 2X Upstream primer, 10μM Downstream primer, 10μM DNA template Nuclease-Free Water Note: GoTaq® DNA Polymerase is supplied in 2X Colorless GoTaq® Reaction Buffer (pH 8.5), 400μM dATP, 400μM dGTP, 400μM dCTP, 400μM dTTP and 3mM MgCl2. Reagent Volume Template 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 The Samples Patient ID Sex Age Fluorimeter Setup Flourimeter setup Sample Drop Procedure: The hydrophobic slide was placed onto the LED box and the first two rows of nodes were centered with the LED. For each sample two drops of the Sybr Green Dye was added to the center nodes of the slide. One drop for each node, or until the two drops coalesced. Two drops of the sample patient (or positive or negative control as appropriate) solution were added to the dye. The LED was turned on and the phone camera was centered onto the drop, held up by the stand. The dark box was placed over the whole setup and closed as completely as possible. A picture was taken and sent to the ImageJ program to be analyzed. The drop was removed and disposed of and the slide was re centered on the next two nodes. The whole procedure was repeated for each sample with the phone in the same place in relation to the drop throughout the entire procedure. ImageJ Procedure Attach the image to an E-mail to be sent from the smart phone to the ImageJ Software Operator Save the image to a computer with ImageJ software, opening the image to be analyzed. In the ImageJ software open the analyze toolbar and mark the following boxes under Set Measurements: Area, Mean Grey Area Value, Integrated Density Once the image is opened in ImageJ use the top drop down menu to select "Image" and then "Color" and then "Split Screen" Only use the green channel, closing out the red and blue channel Use the oval tool to select only the drop of liquid, getting as little background as possible while not cutting any of the drop out. After selecting only the drop press Ctrl+M Move the circle to the background image without changing the size by clicking and dragging the circle Finally, save the results as an excel spreadsheet (set by default) Fluorimeter Measurements Image Number Drop 1 Drop 2 1 Sybr Green Calibration 2 Sybr Green Positive Control 3 Sybr Green Patient 1 Sample A 4 Sybr Green Patient 1 Sample B 5 Sybr Green Patient 1 Sample C 6 Sybr Green Negative Control 7 Sybr Green Patient 2 Sample A 8 Sybr Green Patient 2 Sample B 9 Sybr Green Patient 2 Sample C 10 Sybr Green Water Blank (Add your work from Week 3, Part 2 here) Research and Development Specific Cancer Marker Detection - The Underlying Technology To understand how a Polymerase Chain Reaction machine works, first an understanding of how DNA replicates in the human body must be explained. DNA must replicated to form new identical cells so that the organism the cells are supporting can grow and adapt to conditions it faces. The replication process is very complicated and involves a series of enzymes. In a nutshell, DNA begins with a double helix structure. Once unwound, by the enzyme helicase, the base pairs (Adenine, Thymine, Guanine, and Cytosine) that were held together by hydrogen bonds are now exposed and able to bind with free floating dNTPs with the help of DNA Polymerase III after a primer has been laid down. The step of adding a primer is extremely important. Without an initial strand of RNA to allow DNA Polymerase III to bind, replication would not occur. This is extremely important for our experiment involving the PCR machine. We are testing to see if a cancer gene is present in patients, and without the initial DNA prier being able to bind, we can tell if the cancerous genes are present in that person because the DNA would not be amplified. More on that later. When using an Open PCR (Polymerase Chain Reaction) machine for the first time, you must first make sure that you have a computer capable of running the software and download it. After the software is loaded, plug in the Open PCR machine and prepare your DNA samples that were submitted to be tested for the cancer marker. First, you must prepare the samples for amplification by adding the sample DNA, Taq Polymerase (for replication), MgCl2, and the dNTPs. The DNA primer being used for the detection of this cancer sequence is going to be ATATTCTGTAAGGACAGGAC. The way to know if the sample DNA has a positive result for the cancer gene is if amplification occurs. The primer will bind to the DNA if the cancer sequence is present and the strands will replicate exponentially, whereas if the cancer marker is not present, the primer will not bind to the DNA and it will not replicate because Taq Polymerase will not be able to bind. After the samples are prepared for amplification, the PCR program must be set up. To mimic the replication process of DNA in human cells, we manipulate the system by adding Taq Polymerase instead of DNA Polymerase III because the Taq Polymerase is heat resistant, and heat is going to be used to take place of the enzymes that originally initiate and carry out the DNA replication process. The temperatures that need to be programmed into the PCR are 95˚C (separates the DNA strands to prepare for replication), 57˚C (causes the primers to bind to the DNA), and 72˚C (causes Taq Polymerase to be initiated). There should be 30 cycles entered into this program to make sure ample amplification has occurred. After the 30 cycles are complete, the user can take out the sample and freeze them for later use. When the reaction is finished, the samples can be tested to see if the cancerous gene is present. As discussed before, the primer has to bind with a very specific section of the DNA to initiate replication (Adenine can only bind to Thymine and Guanine can only bind to Cytosine). If the DNA has been replicated exponentially and a large quantity of the target DNA now lies within your sample, you know that the specific primer matched with the DNA strand and the cancerous gene is present. If no replication has occurred and a small amount of DNA is still in the sample, however, you can conclude that the DNA is free of the cancer gene because the primer could not match up with the strand. That would make Taq Polymerase unable to bind and add the dNTPs so the DNA could be copied. (BONUS points: Use a program like Powerpoint, Word, Illustrator, Microsoft Paint, etc. to illustrate how primers bind to the cancer DNA template, and how Taq polymerases amplify the DNA. Screen-captures from the OpenPCR tutorial might be useful. Be sure to credit the source if you borrow images. Results Sample Integrated Density DNA μg/mL Conclusion PCR: Negative Control 2980332 0.5449091318 G6 PCR: Positive Control 12138637 2.219368 G7 PCR: Patient 1 ID #####, rep 1 5320075 0.912696 G8 PCR: Patient 1 ID #####, rep 2 3769062 0.6891166 G9 PCR: Patient 1 ID #####, rep 3 2996412 0.547849 G10 PCR: Patient 2 ID #####, rep 1 4731227 0.865034096 G11 PCR: Patient 2 ID #####, rep 2 6332582 1.1578179076 G12 PCR: Patient 2 ID #####, rep 3 5511982 1.0077834706 G13 KEY Sample = Integrated Density = DNA μg/mL = Conclusion =