BME103:T130 Group 7

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: Emily Thompson Research & Development Scientist Name: Vivian Benjes Experimental Protocol Planner Name: Frances Marrett Experimental Protocol Planner Name: Chris Glass Open PCR Machine Engineer [1] Name: Ryan Frantz Open PCR Machine Engineer [2] Name: Cenric Nigbur Ghost/ TBD LAB 1 WRITE-UP Initial Machine Testing The Original Design This is a PCR (polymerase chain reaction) machine (model: Open PCR 1 Jankowski and Perfetto). This machine is capable of replicating DNA through a series of temperature changes over timed cycles. It uses specialized primers to target specific gene sequences and is capable of amplifying singular stretches of DNA. Experimenting With the Connections When the PCB board was unplugged from the Open PCR circuit board, the machine's LED display ceased to function. When the white wire that connects the Open PCR circuit board to 16 tube PCR block was unplugged, the machine's LED display read -40 Celsius indicating that the temperature could not be read. Test Run October 18,2012; The test program labeled "Simple Test", was performed to ensure proper function of the Open PCR machine. It ran through two cycles at a full range of temperatures, no DNA was present for the test run. The test performed without any flaws. Protocols Polymerase Chain Reaction To amplify samples of DNA, the OpenPCR machine was used to perform a Polymerase Chain Reaction (PCR). This technique worked by cycling a mixture of DNA Template, Primers, Taq Polymerase, Magnesium Chloride, and dNTP's through three specific temperatures to create more copies of the desired sequence. After assembling the PCR mixture, the PCR machine was programmed to perform three stages. In the first stage, the samples went through one cycle at 95⁰C for 3 minutes. The purpose of this stage was to initially denature the DNA and allow the primers to act on the DNA. The second stage put the samples through 35 cycles of 95⁰C, 57⁰C, and 72⁰C each for 30 seconds. The purpose of the first part of the second stage is to break apart the hydrogen bonds between the base pairs, denaturing the DNA sequence into two separate strands. The purpose of the low temperature is to allow primers to bind. The purpose of the middle temperature is to create an environment for Taq Polymerase to assemble a new strand that is the desired product of the entire polymerase chain reaction. The last stage, stage three, puts the samples through one cycle of 72⁰C for 3 minutes. There is a final hold of 4⁰C that preserves the DNA. The samples were then taken out of the PCR machine. The target sequence had been amplified a million times and could now be analyzed with less sensitive equipment! To analyze the sequence, see the Fluorimeter section. Fluorimeter Measurements To analyze the DNA samples after they've been amplified, a smartphone with adjustable settings was used. These settings were used and the provided cradle was implemented to hold the phone in position for photographing: - Exposure to highest setting - ISO to 800+ - White balance to Automatic - Saturation to highest setting - Flash off if necessary - Contrast to lowest setting The Fluorimeter aparatus consists of a hydrophobic Teflon surface on a glass slide. There is an array of 3x10 glass circles on the surface to anchor the drops. First, place two drops of the cybergreen solution on row 2, column 1&2 (Figure 1). With a sterile pipet, place two drops of amplified sample DNA on top of the cybergreen solution. Do not cross contaminate DNA samples! Take a picture using the settings listed above. Prepare for another sample by sucking the liquid off the slide using a pipet and moving the slide back by two columns. Continue down the columns with the cybergreen drops, another DNA sample(with a new, sterile pipet), pictures, and preparations for another sample. This procedure will require using two slides. Interpreting results: If the dot has DNA, the dye with LED will make the drop appear green. Water will glow blue, and a negative result will also not be green Research and Development Specific Cancer Marker Detection - The Underlying Technology Polymerase Chain Reaction, or PCR, is used to ampllify a specific segment of DNA, in this case the segment known to code for cancer. A primer, which is an artifically synthesized piece of complementary DNA, binds to the desired DNA segment, and the enzyme taq polymerase catalyzes the replication of the DNA strand using dNTPs (free bases). This is repeated over and over to produce many copies of the DNA. Once the copies are made, a fluorescent dye that only bonds to DNA double strands is added to the solution. If a solution that shows fluorescence by using a fluorimeter, then that DNA sample contains the cancer-associated sequence. The single nucleotide polymorphism, or SNP, that is linked to cancer is rs17879961. The DNA base sequence associated with cancer is ACT, while the non-cancer sequence is ATT. PCR works to identify ACT from ATT because the primers, which are complementary to DNA strands containing cancer-positive ACT, will not bind to DNA containing ATT, and instead of DNA double strands, the solution will only contain single strands. The fluorescent dye will only bind to double strands and will identify those. Adapted from: http://users.ugent.be/~avierstr/principles/pcr.html Results These Images were taken using a HTC evo4G using the settings described under flourimeter measurements. Water and SYBR green Water and SYBR green under split color conditions for green SYBR green and Calf Thymus 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 = Using the imageJ software, the image was split into its 'red', 'green' and 'blue' color components. This value represents the amount of 'green' light measured from the excitation through the blue LED light shining through the sample and subtracted from the background value, which was 'black' DNA μg/mL = Conclusion =