BME103:W930 Group5

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: Andrea Carpenter Role(s): Experimental Protocol Planner Name:Dana McElwain Role(s): Open PCR Machine Engineer Name: Michelle Nguyen Role(s): R&D Scientist Name: Nathan Holman Role(s): Experimental Protocol Planner Name: MalikMclaurin Roles(s): Open PCR Machine Engineer Name: Chris Anastos Roles(s): R&D Scientist LAB 1 WRITE-UP (Please finish by 11/7/2012) Initial Machine Testing . The Original Design http://openwetware.org/images/3/36/PCR_group_5_labeled_diagram.png This is a diagram of the inside of the OpenPCR Machine. By connecting this machine through a USB port to a computer, it allows you to amplify DNA to later analyze different markings and base pairs within the sequence. Experimenting With the Connections When the LCD screen was unplugged from the Open PCR brains Board, the feeder information was no longer available on the screen. When we unplugged the white wire that connects the brain board to the 16 tube PCR block, the machine temperature was unable to be regulated. Test Run (Write the date you first tested Open PCR and your experience(s) with the machine) Protocols Polymerase Chain Reaction The Polymerase Chain Reaction is a process controlled by thermal cycling, a system of repeatedly heating and cooling the sample. It is simple and inexpensive, PCR allows you to make millions of copies of DNA to better analyze data. First you need a small amount of DNA add a small amount of some PCR reaction mix. A program on the machine needs to be created consisting of three stages. During the three stages, the DNA is first heated to separate the DNA double helix, creating two single stranded DNA molecules. The thermal cycler is now cooled and primers match up to the DNA strands before they naturally attempt to pair up. As the cycles progress DNA is replicated and used as a template to create additional copies. There are several components of a PCR reaction. The template DNA amplifies millions of copies to determine if they are cancerous. The many primers start the binding of complentary strands (specific primers) and they bind to cancer sequences. The taq polymerase is a protein that catalyzes the DNA assembly. There is also a cofactor, which binds to Taq to enable optimal binding speed. Lastly, there are deoxynucleotide tri-phosphates, which builds a new strand of DNA. Steps to Describe How to Amplify a Patient's DNA Sample: Step 1: Initiation During this step the reaction needs to be heated to around 95 degrees Celsius and held there for about three minutes. Step 2: Heat Denaturation A DNA molecule carrying a target sequence is denatured by heat (90-95 degrees Celsius) and the two strands are separated. Step 3: Primer Annealing As the mixture cools, each strand of DNA molecule becomes annealed with an oligonucleotide primer conplementary to either end of the target sequence. Step 4: Primer Extension DNA polymerase is added and complementary strands are synthesized at a temperature of 60-75 degrees Celsius. Step 5: Termination During this step, the final hold, the reaction is held at 4 degrees Celsius to stabilize the reaction. Reagent Volume Template DNA (20ng) 0.2 μL 10 μM forward primer 1.0 μL 10 μM reverse primer 1.0μL GoTaq Master Mix 50.0 μL dH2O 47.8 μL Total Volume 100.0 μL Flourimeter Measurements (Add your work from Week 3, Part 2 here) Research and Development Specific Cancer Marker Detection - The Underlying Technology CHEK 2, code rs17879961, is a single nucleotide polymorphism (SNP) that is linked to cancer. It is a symbol for CHK2 Checkpoint Homolog and is located on chromosome 22. Mutations of the CHEK2 gene are related to increased risk of breast cancer. In week three's class, it was concluded that PCR can be used to bind specific DNA primers to the cancerous DNA bases--resulting in cancerous DNA amplification. As a result, the PCR reaction will form normal DNA sequences and the supposed cancerous CHEK2 strain. Possible primers for supposed amplification were used in the experiment and were compared to sample sequences to determine if the DNA in question was amplified. Results (Your group will add the results of your Fluorimeter measurements from Week 4 here)