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: Carlos Duarte Research and Design Scientist Name: Ben Alcron Open PCR Machine Engineer Name: Bryce DeSimmone Research and Design Scientist LAB 1 WRITE-UP Initial Machine Testing The Original Design Description of Open PCR Device 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 (Write the date you first tested Open PCR and your experience(s) with the machine) Protocols Polymerase Chain Reaction Reagent Volume Template DNA (20 ng) .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 Fluorimeter Measurements (Add your work from Week 3, Part 2 here) Research and Development Specific Cancer Marker Detection - The Underlying Technology (Add a write-up of the information discussed in Week 3's class) 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 E6 F6 G6 PCR: Positive Control E7 F7 G7 PCR: Patient 1 ID #####, rep 1 E8 F8 G8 PCR: Patient 1 ID #####, rep 2 E9 F9 G9 PCR: Patient 1 ID #####, rep 3 E10 F10 G10 PCR: Patient 2 ID #####, rep 1 E11 F11 G11 PCR: Patient 2 ID #####, rep 2 E12 F12 G12 PCR: Patient 2 ID #####, rep 3 E13 F13 G13 KEY Sample = Integrated Density = DNA μg/mL = Conclusion =