BME103 s2013:T900 Group2

BME 103 Spring 2013	Home People Lab Write-Up 1 Lab Write-Up 2 Lab Write-Up 3 Course Logistics For Instructors Photos Wiki Editing Help
OUR TEAM Andy Son Protocol Planner William Scott R&D Scientist Joe Sasnone R&D Scientist Mitch Riggs Open PCR Machine Engineer Name: student Role(s) Name: student Role(s) LAB 1 WRITE-UP Initial Machine Testing The Original Design The Open PCR machine is a small wooden box used to copy DNA. It achieves this by going through several cycles of heating and cooling. The length, temperature, and number of cycles can be changed with a computer hooked up to the device via USB. Experimenting With the Connections When we unplugged (part 3) from (part 6), the screen turned off. When we unplugged the white wire that connects (part 6) to (part 2), the screen showed the wrong temperature. Test Run We first ran the machine on Tuesday, March 5. We used unit 3. We performed a simple test with the computer. The temperatures shown on the computer corresponded with the temperatures shown on the device indicating a successful test. Protocols Thermal Cycler Program The set-up used for the program is as follows Stage one: 1 cycle, 95 degrees Celsius for 3 minutes Stage two: 35 cycles, 95 degrees Celsius for 30 seconds, 57 degrees Celsius for 30 seconds, 72 degrees Celsius for 30 seconds Stage three: 72 degrees Celsius for 3 minutes Final Hold: 4 degrees Celsius DNA Sample Set-up Positive control: cancer DNA template Tube label: 1 Patient 1 ID: 91862 Replicate 1 Tube label: 2 Patient 1 ID: 91862 Replicate 2 Tube label: 3 Patient 1 ID: 91862 Replicate 3 Tube label: 4 Negative Control: no DNA template Tube label: 5 Patient 2 ID: 28235 Replicate 1 Tube label:6 Patient 2 ID: 28235 Replicate 2 Tube label: 7 Patient 2 ID: 28235 Replicate 3 Tube label: =) DNA Sample Set-up Procedure Step 1: First gather the necessary materials to set-up the DNA samples (pipette, PCR reaction mix, 8 transfer pipettes) Step 2: Set the pipette to 50 microliters Step 3: Place the transfer pipette onto the pipette to prevent cross contamination (never re-use). Step 4: Use the pipette to transfer 50 microliters of each tube in the PCR reaction mix and transfer accordingly to the DNA sample tubes corresponding to the labels. PCR Reaction Mix The PCR reaction mix contains 8 tubes of 50 microliter samples that contain a mix of Taq DNA polymerase, MgCl2, dNTP's, forward primers, and reverse primers. DNA/ primer mix The DNA/primer mix consists of samples of DNA of various patients. Research and Development Specific Cancer Marker Detection - The Underlying Technology Polymerase Chain Reaction (PCR) is a scientific method that utilizes DNA Polymerase to create a complimentary base strand from a template strand of DNA. Triphosphate nucleotides align with open DNA strands and DNA polymerase works to link the complementary nucleotide bases together growing strands through both condensation and hydroysis reactions. One major issue with DNA polymerase is that DNA strands are anti parallel, and polymerase is only able to add nucleotides to the free 3'OH group hence it can only build new strands in the 5'-3' direction (Sadava 279). Therefore, to correct for this issue the presence of a primer is required so that polymerase can proceed with directing the new nucleotides in place. Through these mechanisms it is possible to target specific positions on the template DNA sequence that a scientist intends to amplify(PCR 1). When the PCR process is completed the targeted DNA sequence containing the single-nucleotide polymorphism (SNP) will have manufactured over a billion copies (amplicons). The SNP included in the amplicon for this experiment was denoted as rs17879961. This polymorphism is a variant of the CHEK2 gene which if present in a person's genome may increase the risk of developing breast cancer (Brennan et al 1795). This SNP is signified by a single base change from a Thymine (T) to a Cytosine (C) located on chromosome 22. With the PCR system, forward and reverse primers can be designed to target the cancer gene abnormality and amplify/multiply it. The normal DNA strands may multiply, but not as exponentially as the abnormal cancer DNA strands. Sources: Brennan, Paul et al. Uncommon CHEK2 Mis-sense Variant and Reduced Risk of Tobacco-related Cancers: Case-control Study. Rep. 15th ed. Vol. 16. Oxford: Advance Access, 2007. Human Molecular Genetics. Oxford University Press, 21 May 2007. Web. 25 Mar. 2013. PDF. <http://hmg.oxfordjournals.org/content/16/15/1794.full.pdf>. "PCR Introduction." NCBI. U.S. National Library of Medicine, n.d. Web. 25 Mar. 2013. <http://www.ncbi.nlm.nih.gov/projects/genome/probe/doc/TechPCR.shtml>. Sadava, David E. Life: The Science of Biology. Sunderland, MA: Sinauer Associates, 2011. Print.