BME103:T130 Group 5

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: Wade Patrick Machine Engineer Name: Liann Klein Machine Engineer Name: Haylee Poncy Protocol Planner Name: Kyle Labban Protocol Planner Name: Alexandria Lam R&D Scientist LAB 1 WRITE-UP Initial Machine Testing The Original Design The Polymerase Chain Reaction (PCR) machine (shown above) is used to replicate a large quantity of a specific strand of DNA. The PCR Machine performs this task by first splitting up the DNA. Since DNA is double stranded, and melts to become two seperate strands at a certain temperature, the PCR Machine heats the DNA to the specific temperature so that the DNA becomes to seperate strands of DNA. Then the PCR Machine uses primers, which are strands of DNA that contain a certain number of nucleotides, to adhere to the two seperate strips of DNA. Then the polymerase, which is an enzyme used to fill in the holes of DNA, completes the strand, resulting in two seperate, double strands of DNA. Experimenting With the Connections When the wire from the board for the LCD screen was unplugged from the main board, the LCD screen on the machine turned off and went blank. When the white wire that connects the main board of the machine and the temperature system was unplugged, the temperature reading decreased and was not accurate. Test Run We administered a test run on October 25 and followed the protocol provided. Everything ran smoothly as the numbers on the Open PCR display screen matched the numbers shown on the computer. (Write the date you first tested Open PCR and your experience(s) with the machine) Protocols Polymerase Chain Reaction Polymerase Chain Reaction (PCR) is a process that uses DNA polymerase to synthesize a large number of copies of a target DNA sequence. PCR is dependent on short DNA fragments called primers. After the DNA has been denatured by heating and then cooled to a temperature suitable for the primers to bind to their complementary sequences, the primers bind to areas adjacent to each side of the targeted DNA sequence. Once the primers are in place, the polymerase extends them into large complimentary strands. The DNA is then denatured once again, then cooled, the primers bind to the complimentary sequence and then the polymerase extends them. Repeating this process results in an exponential amplification of the target DNA sequence. Amplifying a patient's DNA sample using PCR can be done as follows: Collect biological samples from patients or target group. Combine samples with reagents primers to the sample. These primers will enable the DNA to "unzip" and duplicate the target region using the extra base pairs mixed into the solution. Place the DNA sample and reagents into a PCR machine, and program the machine to carry out the desired sequences. Allow the machine to cycle. Once complete, collect the amplified DNA and test. In our experiment, a PCR master mix from Promega containing bacterially derived Taq DNA polymerase, dNTPs, MgCl2 and reaction buffers was used. Reagent Volume Template DNA (20 ng) 0.2μL 10μM forward primer 1.0μL 10μM reverse primer 1.0μL GoTaq master mix 50μL dH2O 47.8μL Total Volume 100.0μL Sample Description (8 samples) Positive control: cancer DNA template Patient 1: 68754, F, 57y Patient 1: 68754, F, 57y Patient 1: 68754, F, 57y Negative control: no DNA template Patient 2: 66913, M, 66y Patient 2: 66913, M, 66y Patient 2: 66913, M, 66y Flourimeter Measurements (Add your work from Week 3, Part 2 here) Research and Development Specific Cancer Marker Detection - The Underlying Technology       The sequence r17879961 represents a specific sequence where a Thymine is replaced by Cytosine due to a missense mutation on chromosome 22. It affects gene CHK2 that is linked to colorectal cancer. A primer binds to a specific sequence on the template DNA and tells Taq polymerase where to begin reading and adding nucleotides to synthesize a new strand of DNA. Primers are very specific in that they can only bind to a certain sequence. A backwards primer consists of 20 nucleotides that specifically are ACT TCT TAC ATT CGA TAC AT. The forward primer is TGT GAT CTT CTA TGT ATG CA. These primers will only bind to that specific sequence of r17879961 where the Cytosine is present and not the Thymine.       If the sequence is present, the primers will bind to both leading and lagging strands of the template DNA. Taq polymerase can then bind and begin synthesizing the strand. The test will come out positive because the DNA will synthesize to create double stranded DNA that the SYBR Green I dye will then bind to. This will cause the DNA to fluoresce and yield a positive result.       If the r1789961 SNP is not present, the primer will not bind to the DNA template. Because the primers tell Taq polymerase where to replicate, Taq polymerase will not have anywhere to bind. Replication will not occur with the product of a double-stranded DNA, but linearly. The SYBR Green I dye only binds to double-stranded DNA, so it will not show because it is single-stranded. There would not be enough double-stranded DNA to fluoresce if the cancer gene is not present. Therefore, the test will display a negative result. (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 =