BME103:T930 Group 15

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: Suhail Hiermandi Role(s): Research and Development, Experiment Protocol Name: Andrew Trujillo Role(s): Research and Development Name: Elias Mloukhiyeh Role(s): Open PCR Machine Tester Name: Emma Goddery Experiment Protocol Planner LAB 1 WRITE-UP Initial Machine Testing The Original Design An Open PCR Machine is a device that is used to learn if a sample of DNA reacts with a select gene. It does this by fluctuating the temperature of the samples. With the temperature fluctuating, strands of DNA break apart and the chemical reacts withe the single strand of DNA by completing the strand. Experimenting With the Connections When we unplugged the LCD display from the circuit board, the machine LED display powered down and did not display anything. When we unplugged the white wire that connects the circuit board to the heat sink, the temperature reading on the LED display screen showed the incorrect temperature. Test Run During the "Test Run" portion of the lab on the 25th of October 2012, the OpenPCR machine did as it was supposed to. It fluctuated the heat of the test samples and it also had the same readings on the computer program as well as the LED display screen of the OpernPCR machine. Protocols Polymerase Chain Reaction The Open PCR machine works by heating a sample of DNA to 95 degrees Celsius to separate the sample into single strands. Then, the machine cools to 57 degrees Celsius so that primers can attach to the strand to serve as a probe to a matching sequence. The primer that was used in our experiment was r17879961, a cancer-associated sequence, that will detect if the DNA sample tested positive for cancer. Then, the machine heats up to 72 degrees Celsius where MgCl12 attaches to Taq enzyme, so that Taq can take the free floating dioxynucleotide triphosphates and attach them to the stand to form two new separate and similar DNA strands. Now if the process produce new strands, then the DNA tested positive. If none were present, the DNA tested negative. The reason behind this is that r17879961 only can bind to a sequence that matches its own on the single stranded DNA. This sequence is 5’-AAACTCTTACACTCCATACAT-3’. The cancer mutation site is located at the 12 base pair, 5’-ACT-3’. If the base pair is a C, it is positive for cancer mutation. The base pair should truly be a T, making the sequence 5’-ATT-3’. The particular cancer that is associated with the r17879961 primer is colon rectal cancer. The information regarding this particular cancer sequence can be found on the National Center for Biotechnology Information, in the file “.0002 LI- Faurmen Syndrome”. A study done in the file tested the C mutation r17879961. The results that were found were that 7.8% of C mutations were found in cancer patients, and that 5.3% of C mutations were in Finland. The results then can show that 7.8% of the cancer patients had a positive test, and of that 7.8%, 5.3% are in Finland. How to amplify a patient's DNA sample: 1. Put extracted DNA into a PCR tube. 2. Add primers to the PCR tube. 3. Add nucleotides to the PCR tube. 4. Add DNA Taq Polymerase to the PCR tube. 5. Place this DNA tube into DNA Thermal Cycler. 6. Thermal cycler heats to 95 degrees Celsius for 30 seconds. 7. Thermal cycler cools to 57 degrees Celsius for 30 seconds. 8. Thermal cycler heats to 72 degrees Celsius for 30 seconds. 9. These cycles happen 30 times. Reagent Volume Template DNA (20ng) .2 uL 10 uM forward primer 1.0 uL 10 uM reverse primer 1.0 uL GoTaq master mix 50.0 uL dH2O 47.8 uL Total Volume 100.0 uL Samples we tested: Positive control: cancer DNA template (+) Negative Control: no DNA template (-) Patient 1: ID 12329(*) Replicate: 1 Label: P1R1 Patient 1: ID 12329 Replicate: 2 Label: P1R2 Patient 1: ID 12329 Replicate: 3 Label: P1R3 Patient 2: ID 61058(**) Replicate: 1 Label: P2R1 Patient 2: ID 61058 Replicate: 2 Label: P2R2 Patient 2: ID 61058 Replicate: 3 Label: P2R3 *Patient 1: Female, 61 years of age **Patient 2: Male, 57 years of age. Flourimeter Measurements Flourimeter Assembly: Research and Development Specific Cancer Marker Detection - The Underlying Technology (Add a write-up of the information discussed in Week 3's class) (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 =