BME103:W930 Group4

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: Renaad Alawi Experiemental Protocol Planner Name: Lauren Allison Research and Development Name: Jake Krammer Open PCR Machine Name: Jan Simper Open PCR Machine Name: Justus Vangor Research and Development Name: Christian Vargas Experimental Protocol Planner LAB 1 WRITE-UP (Please finish by 11/7/2012) Initial Machine Testing The Original Design The Polymerase Chain Reaction(PCR) machine essentially tests sequences of DNA for variations in nucleotides. This simple device is portable, easy to use, and relatively inexpensive. It is able to test up to 16 samples of DNA at a time and can be connected to a computer for ease of use. The machine heats up DNA samples so the samples disassociate allowing a primer to connect to the sequences of DNA and then the machine cools the DNA samples down with the primer in place. Experimenting With the Connections When we unplugged the mounting plate from the circuit board, the machine the LCD light and the menu on the PCR machine shut off. When we unplugged the white wire that connects the circuit board to PCR block that holds the samples, the temperature on the menu on the PCR machine dropped from room temperature to -40.0 degrees Celsius. The white wire is the temperature sensor wire. Test Run (Write the date you first tested Open PCR and your experience(s) with the machine) Protocols Polymerase Chain Reaction The Polymerase Chain Reaction works by melting the DNA at 95 degrees Celsius which will in turn unzip the DNA to expose its bases and create two one-stranded strips. After primer is added to the solution, the DNA is then cooled down to 57 degrees Celsius so that the said primer can attach to a template sequence to form a forward primer. The DNA is then once more heated to 72 degrees Celsius so that the replication process can be completed. Flourimeter Measurements (Add your work from Week 3, Part 2 here) Research and Development Specific Cancer Marker Detection - The Underlying Technology What is the function of each component of a PCR reaction? Template DNA: A double-stranded segment of DNA that encodes either a cancerous gene or a normal gene Primers: Short segments of DNA that bind to a specific sequence of nucleotides (binds to cancer gene) Taq Polymerase: A protein that serves as the catalyst for the DNA replication; grabs extra nucleotides within the solution and binds them to the "unzipped" strands Magnesium Chloride: A cofactor that binds to the Taq Polymerase and affects the speed of the reaction; positive correlation between amount of magnesium chloride and reaction speed dNTP's: Deoxynucleotide triphosphates; extra nucleotide bases in solution that are able to be grabbed and synthesized by Taq Polymerase to replicate DNA strands beyond the primer sequence What happens during each step of thermal cycling? • At 95° Celsius: DNA melts and "unzips" to create two one-stranded strips, primers are added to the solution • At 57°Celsius: Primers attach to the corresponding template sequence they complement, forming one forward primer and one reverse primer • At 72° Celsius: Taq Polymerase finishes the replication process with the use of dNTP's and magnesium chloride Why does a cancer gene produce a positive result while a normal gene produces a negative? • Because the cancer gene has the specific sequence of nucleotides that the primers can bond to, the process can continue and the DNA can be replicated; however, since the normal gene does not include that specific sequence, the primers can never bond to the strands and the process cannot take place. Relation to Bayes' Rule: In order to achieve accuracy of the amplification process as an actual determinant for cancer, Bayes' Rule must be used. This will compute the probability of true positives in coordination with false positives and false negatives to give a realistic prediction for how reliable the PCR process is in detecting the true cancer patients. Image Credit to OpenPCR.org/use-it/ Results (Your group will add the results of your Fluorimeter measurements from Week 4 here)