BME103:T130 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: Malik Alnaim: Research & Development Scientist Name: Alyssa Alexander Role: Research & Development Scientist Name: Sichun Ai Role: Experimental Protocol Planner Name: Nehal Jolly Role: Experimental Protocol Planner Name: Ben Reising Role: Open PCR machine Engineer Name: Mayuri Gupta Role: Open PCR machine Engineer LAB 1 WRITE-UP Initial Machine Testing The Original Design Experimenting With the Connections When we unplugged (part 3) from (part 6), the machine ... (did what? fill in your answer) When we unplugged the white wire that connects (part 6) to (part 2), the machine ... (did what? fill in your answer) Test Run (Write the date you first tested Open PCR and your experience(s) with the machine) Protocols Polymerase Chain Reaction Polymerase chain reaction is basically molecular photocopying and the process or technique used to make copies of small segments of DNA because it only targets specific segments of the DNA and that's what makes it useful. PCR works by mixing two DNA fragments, also known as primers which are about 20 bases long. The mixture is then heated and denatured and then the primers bind to their complementary sequences on the separated strands. Then, the polymerase extends primers into new complementary strands and it goes through about 30 cycles. PCR products are useful and can be used in many experiments like DNA fingerprinting and detection of viruses. (Jim Dorsey) Polymerase Chain Reaction. http://www.contexo.info/DNA_Basics/polymerase_chain_reaction.htm. Last accessed 11/01/12.) Steps to Amplify DNA Samples Collect three replicate DNA samples from two patients Create a new program on the Open PCR system Create three stages Stage 1: 1 cycle, 95°C for 3 minutes Stage 2: 30 cycles for 95°C for 30 seconds, 57°C for 30 seconds, 72°C for 30 seconds Stage 3. 72°C for 3 minutes Final hold: 4°C The DNA samples are 50 μL each, get the patient's ID and label the the each tube. PCR reaction mix - Mix contains Taq DNA polymerase, MgCl2, dNTP's, forward primer, and reverse primer. The primers are artificial DNA, designed to match the chain of DNA we want. Taq polymerase is the enzyme that binds to the end of the new chain and recreates the separated DNA. Mgcl2 binds to Taq as a co-factor and helps Taq to function appropriately, and affects the speed of the Taq binding to the loose strands. dNTp's is dioxnucleotidetriphosphate. this is what is used to recreate the second DNA strands. The 8 tubes of mixtures will then go through the cycles in the PCR system. During each step of the thermal cycling, the DNA is unzipped and heated to 95°C to break the H-bonds between the 2 strands. This exposes the part we want in this lab experiment. Then, the primer binds to the trage we want without cancer marker, this primer won't bind. Next, the temperature will be dropped to 57°C in order to bind the primer. Later, it is heat it back up to 72°C with the Taq to reform and duplicate DNA strands. Finally, this thermal cycling is repeated for amplification and add dye that binds specifically to DNA for detection. The Components of the GoTaq® Colorless Master Mix "dNTP's, MgCl2, and reaction buffers at optimal concentration for efficient amplification of DN templates by PCR." Volumes Used for Mixture Table 1 Reagent Volume Template DNA (20 ng) 10.2 μL 10 μM reverse primer 1.0 μL dH2O 47.8 μL 0 μM forward primer 1.0 μL GoTaq master mix 50.0 μL Total Volume 100.0 μL The eight samples we ran in PCR were: Patient ID, Gender, Ages 27762, F, 52 59448, F, 45 PCR steps of amplification Flourimeter Measurements (Add your work from Week 3, Part 2 here aka, steps of assembly to the flourimeter) Research and Development Specific Cancer Marker Detection - The Underlying Technology There is a genetic relation to having cancer or not when an individual is over the age of 40. The specific gene, in this case, is located on chromosome 22, r17879961. To test an human's DNA for this cancer gene, we have go through a series of reactions called PCR on the DNA for replication and amplification of the patient's DNA strand. As previously explained in PCR, a primer is needed for the DNA replication. Two primers that have to be created. One at the "completing" strand of the double strand, and one as the cancer detecting strand. On chromosome 22, the primer to detect r17879961 defect has to have the changed nucleotide. In this particular instance, it is a adenine replaced by the cancerous-related nulceotide cytosine. The primer will only bind to the matching sequence (testing sequence) because of the A-T, and C-G pairings. Since an A has been replaced with a C, the primer can't bind to the DNA strand beyond that specific nucleotide in the sequence. Due to the open double helix, further steps in the reaction will not happen, and no amplification will happen. No amplification means no visible results and the test will run negative. If the cancer gene is present, then the matching primer will completely bind to the DNA strand. When this happens, the amplification sequence will be able to precede, and this will show up as a positive 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 (Your group will add the results of your Fluorimeter measurements from Week 4 here)