BME103:T930 Group 4 l2

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: Alex Hoang Role(s): Open PCR Machine Engineer Name: Bobby Ryan Role(s): Open PCR Machine Engineer Name: Sabrina Freeman Role(s): Experimental Protocol Planner Name: Sheanah Conneen Role(s): Experimental Protocol Planner Name: Hayden Hilliard Role(s): Research and Development Scientist LAB 2 WRITE-UP Thermal Cycler Engineering Our re-design is based upon the Open PCR system originally designed by Josh Perfetto and Tito Jankowski. System Design Key Features Instructions Protocols Materials Supplied in the Kit Amount PCR machine 1 Empty sample tubes 30 Pre-mixed reagent 1,000 μL Gloves (latex) 1 pair Open PCR software 1 copy Sample tube carrier 1 Calf thymus 1,000 μL SYBR Green 1,000 μL Supplied by User Amount Micro-pipetter 1 (50 μL volume) Pipette tips As needed Patient samples 1,000 μL Waste container 1 IMAGEJ Application 1 smart phone PCR Protocol Create as many copies of each original DNA sample as needed. Be sure to label each container explicitly. Put the appropriate amount of reagent mixture and patient sample into each container. Place samples in the PCR machine. Start the PCR machine. Create a new program for the PCR machine, set it as follows: Indicate that there will be 15 cycles. Start the samples at 95°C for 30 seconds to unwind the helix. Then lower temperature to 57°C for 30 seconds to activate the primer and strand pairing. Next raise the temperature to 72°C for 1 minute to activate the DNA polymerase for replication. Remove the samples from the PCR machine and store them until it is time to take flourimeter measurements. DNA Measurement Protocol Explicitly label all pipettes and Eppendrof tubes so that it is used for one specific sample. Transferred each sample into and Eppendorf tube containing 400 μL of buffer and label it with the number of the sample. Obtain the Eppendrof tube containing SYBR Green I and place two drops on the first two centered drops of the glass slide inside the flourimeter box. Place two drops of one sample mixed with the buffer on top of the SYBR Green. Align the light so that it is shining through the drop. After opening the IMAGEJ Application on a smartphone place said smartphone in the carriage of the flourimeter box designated for smartphones. Take pictures of each sample using the flourimeter box. To analyze images using the IMAGEJ Application. Using the default settings draw ovals around the drops in the images and press MEASURE. Save the number that is measured and select ANALYZE BACKGROUND. Save all measurements as a single file and either export the file via email or connect the smartphone to a computer to transfer the file. The file will open in Microsoft Excel. Research and Development Background on Disease Markers Much like the cancer-linked genes we observed in our first lab write up, we were able to pinpoint several other diseases that can be linked to genetic variation. The first of these to be discussed will be Alzheimer's disease. Alzheimer's is a degenerative brain disease that results primary in memory loss, as well as loss of other cognitive brain functions. The Reference SNP number for this genetic variation is 63750082, and similar to the mutation that causes cancer because it is a missense mutation. However, rather than a single-nucleotide missense mutation, there are a few different mutations that can occur. Basically, in the sequence GGT, the second G can become an A, C, or T (GAT, GCT, GTT). For more information about the gene alteration, visit here! Another genetic variation we chose to study was muscular dystrophy (MD), which can be identified by the Reference SNP number 5030730. Much like the other two studied, it is the result of a change to the sequence "CGA" to "TGA." Muscular dystrophy is a disease where muscle tissue is weak and often degenerates. Those who suffer from the disease likely do not have normal muscle function, and often need a wheelchair or other mobile device to travel. In some cases, mental retardation may also occur. More information about the genetic variation behind muscular dystrophy can be be found here. Primer Design The forward primers for Alzheimer's and muscular dystrophy are 5' TTTGCTGTGG 3' AND 5' ATGTCGCATC 3' respectively. Similarly, the reverse primers are the complements of each sequence, because when DNA is paired in strands, each C (cytosine) is paired with a G (guanine), and each A (adenine)is paired with a T (thymine). Therefore, the reverse primers for Alzheimer's and MD respectively are 3' AAACGACACC 5' AND 3' TACAGCGTAG 5'.The way PCR (polymerase chain reactions) are designed is so that they replicate DNA when they come across a certain sequence. Therefore, it is very beneficial to those trying to discover if someone caries the mutated gene sequence for a disease like Alzheimer's or MD. For instance, if the gene were discovered in a person, they could likely expect an early onset of Alzheimer's and prepare for such. Similarly, if a child was found to have the mutated gene for MD, they could also prepare to keep the atrophy at bay for as long as possible. As far as detection goes, PCR primers are specialized so that they have the mutated gene on them. By having the mutated gene as the primer, it will only bind to DNA that contains the mutated gene. Therefore, only those who have the gene for the disease will have DNA replication occur during PCR operation. Illustration