BME103:T130 Group 6

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: Jocelynn Christensen Role: Experimental Protocol Planner Name: Sam Zimmerman Role: OpenPCR Machine Engineer and Vampire Hunter Extraordinaire Name: Adam Helland R&D Name: Ryan Uchimura Role: Experimental Protocol Planner Name: student Role(s) LAB 1 WRITE-UP Initial Machine Testing The Original Design The images above depict our OpenPCR machine. It performs polymerase chain reactions (PCR), a process in which a particular DNA sequence is amplified. The DNA sequence can then be easily analyzed. The OpenPCR machine accomplishes the amplification of a DNA sequence through a series of heating and cooling sequences. Experimenting With the Connections When we unplugged the LCD screen from the circuit board, the machine stopped displaying information on the LCD screen. When we unplugged the white wire that connects the circuit board to the heating block, the heating block would not heat up. Test Run We first tested our OpenPCR machine (machine #6) on October 25, 2012. We ran the machine through a preprogrammed sample test run. At first we were not getting consistent temperature readings outputting to the LCD screen; however, after we rebooted the OpenPCR machine and ran the test again the machine worked perfectly. Protocols Polymerase Chain Reaction 1. The Polymerase Chain Reaction or PCR works by singling out a single piece of DNA and then multiplying it so there's millions of copies of one strand of DNA. It's a step by step process that first occurs by heating up the DNA to 100°C in order to denature the hydrogen bonds between the two strands of DNA so that both sides of the DNA can be accesible to copy. After the strands are separated, specific primers are added to locate the section of DNA to be amplified. Next, the Taq DNA polymerase is added which actually copies the section of DNA desired and synthesizes the second half of each strand. After this there are only a few copies of the DNA which is why the machine then replicates more strands by repeating the process multiple times until there are millions of copies. 2. 1. Collect DNA from patients 2. Heat Denaturation- the sample is heated to break the bonds between the strands 3. Primer Annealing- the solution cools and the DNA matches up to the primer. 4. Extension- The DNA replicates to produce millions of copies of the one strand of DNA . 3. The GoTaq master mix contains 400µM dATP, 400µM dGTP, 400µM dCTP, 400µM dTTP, and 3mM MgCl2 4. Reagent Volume Template DNA (20ng) 0.2μL 10μM forward primer 1.0μL 10μM reverse primer 1.0μL GoTaq master mix 50.0μL dH2O 47.8μL Total Volume 100.0μL 5. Out of the eight samples we ran the PCR on, one was a positive control with the cancer DNA template, the other was a Negative control with no cancer DNA template. Then we had three samples of a 57 year old male's DNA (patient ID 19185). We also had three samples of a 63 year old female's DNA (patient ID 88142). Flourimeter Measurements (Add your work from Week 3, Part 2 here) Research and Development Specific Cancer Marker Detection - The Underlying Technology Our genes can tell us anything and everything about ourselves. The sooner we can detect cancer, the more effectively it can be prevented or treated. So why not find out about our disposition to cancer with our genes? The science community has identified many DNA sequences that are correlated to incidence of cancer. Through a process known as Polymerase Chain Reaction, (or PCR,) we can make tons of copies of any sequence of DNA from a DNA template. So, let's say we want to find out if someone has a sequence of DNA that may be indicative of a higher cancer risk; how can we do it? r17879961 is a sequence of DNA that has been positively linked with cancer. It is a part of a sequence of DNA that codes for a protein kinase called CHEK2. (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 = Comprised of two drops of DNA solution and two drops of SYBR Green. Integrated Density = The integrated density of the drop minus the integrated density of the background. This calculation account for background noise. DNA μg/mL = The integrated density of sample divided by the integrated density of drop multiplied by two. Conclusion = If the reading is positive then it means that the cancerous mutation is present.If the reading is negative or "no signal," then the mutation is not present.