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 This is the internal view of the PCR machine that was used in the lab experiment. Experimenting With the Connections Above is an image of important parts in a PCR machine. Before we even began our experiment we had to assess and overlook these specific parts so that we could better learn their function and how that contributes to the overall machine. Part 1 displays the Aluminum Plate, which is designed to both contain and give off the heat necessary to perform the experiment once it is put down over Part 2, the Well Plate. The Well Plate is simply designed to hold the test samples and measure the heat around them, in order to ensure the samples are receiving the right temperature. Part 3 is the PCB (Printed Circuit Board) of the LCD screen, which means it is responsible for the images and readouts that appear on the screen. This part is important because the entire flow of information comes into here, so that the user can gather information on the progress and results of the machine. Part 4 is the heat sink fan, which works to cool the entire machine by blowing the hot air out from the machine, to prevent overheating. Part 5 is the Rsenda ATX (Advanced Technology eXtended), a type of motherboard. A motherboard is essentially a large PCB which holds the CPU (central processing unit) and is in charge of memory. The final part is Part 6, which is the PCB of the open PCR Circuit Board. This is much like the motherboard, but deals with current and continuous flow of information and signals through the wires. No real memory is stored here. Connection and Process Testing In order to see the connection between various parts we ran two tests on machine #10 on October 18th, 2012, where we unplugged a single wire in each. When we unplugged PCB of the LCD screen from PCB of open PCR circuit board, the LCD screen did not turn on at all. When we unplugged the white wire that connects PCB of open PCR circuit board to well plate, strange images began to show up on the screen, as a result of no information flowing from the well plate to the PCB of the open PCR circuit board. 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 DNA Samples (8) Positive Control: Cancer DNA Template Tube label A Replicate 1 Tube Label 1-1 Patient 1 ID: 27762, F, Age: 52 Replicate 2 Tube Label 1-2 Patient 1 ID: 27762, F, Age: 52 Replicate 1 Tube Label 1-3 Patient 3 ID: 27762, F, Age: 52 Negative Control: No DNA Template Tube Label B Replicate 2 Tube Label 2-1 Patient 2 ID: 59484, F, Age: 45 Replicate 2 Tube Label 2-2 Patient 2 ID: 59484, F, Age: 45 Replicate 2 Tube Label 2-3 Patient 2 ID: 59484, F, Age: 45 Flourimeter Measurements Image above shows the set up of the flourimeter measurments Flourimeter Assembly Procedure First, the glass side of the slide was placed faced down onto the device. A different pipette was used for transferring each content from the small tubes to the bigger ones. After labeling the tubes and pipettes,gloves were worn to ensure a contamination free procedure. Using the specific pipette for each component, one drop of buffer was put onto the first and second centered holes of the slide and two water drops were placed on the gathered buffer drops. The device was then put under the black box provided and the phone was placed into the holder inside the box. After customizing the photo settings in the phone according to the instruction sheet, a shot of the drop sample was taken and saved. The number of the photo was recorded in a table to keep track of the photos. The photo was sent to the e-mail of the group member who was responsible for analyzing the photo. The previous steps were repeated for each sample with the exception of Replacing the water drops with the rest of the samples using a different row on the glass slide each time a sample was used. Sample Flourimeter Image - Patient 1, Rep 2 Sample Flourimeter Image - Patient 2, Rep 1 Open ImageJ 1. By using a USB cable, connect the camera phone to the desired computer that has already ImageJ installed 2. Under my computer, choose portable devices where you could find the smartphone listed; double-click on it 3. After localizing the DCIM folder and opening it, you should select camera 4. The desired photos can then be transferred by simply putting them into the created folder 5. Open ImageJ and go to file; click on it and choose open 6. Select browse then pick the desired picture from the same folder created earlier 7. To continue opening different pictures, you should only repeat steps 5 and 6 Sample ImageJ Green - Patient 1, Rep 2 Sample ImageJ Green - Patient 2, Rep 1 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 shown in Protocols, primers are needed for the DNA replication, a forward and a reverse. 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. The reverse primer used: AACTCTTACA-C-TCGATACAT The forward primer used: TTGAGAATGT-C-AGCTATGTA In this particular instance, it is a adenine replaced by the cancerous-related nucleotide cytosine as shown above as the bold C. 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. Image by: Alyssa Alexander This is all in theory of course, and should work perfectly every time. But there are many factors to consider. For instance, not every one who has cancer has the cancer gene. Reliability and Accuracy of Specific Cancer Marker Detection Based on Bayesian reasoning, the probability that someone will test positive that will actually have the disease has approximately 7.8 percent. However, the chance that someone does not test positive, and doesn't have cancer is about 99.8%. These percentages generally mean that the test itself is generally in favor of testing negative, which means there are less chances to have false diagnosis and/or treatments. These percentages resulted from the following equation: [math]\displaystyle{ Positive Predictive Value = (True Positive)/(True Positive + False Positive) }[/math] [math]\displaystyle{ Negative Predictive Value = (True Negative)/(True Negative + False Negative) }[/math] The values for these above equations are gathered from Bayesian's reasoning. Baye's Theorem: [math]\displaystyle{ p(C/T) = (p(T/C)*p(C))/(p(T/C)*p(C)+p(T/nC)*p(nC)) }[/math] where p(C/T) is the probability of a person with positive results will have cancer out of the entire patients participating. p(C) is the probability of having cancer present, p(T/C) is the percent of patients who tested positive with have cancer and had it. n = not The values of the these equations are taken from the general statistics from tests performed on 'x' amount of patients. For instance, the direct results showed that 80% of the people with cancer, tested positive, this value would be used as p(T/C) because that is the test running positive and having cancer. The value p(T/nC) would be the percentage of people who tested positive but do not actually have cancer, which resulted to be 9.6%. Additionally, the general statistics were 90.4% of patients will test negative and will not have cancer, and 20% percent of people with cancer will run negative. The calculated p(C/T) was used as the "True Positive" in the original two equations (PPV and NPV). The same for the false positive, true negative and false negative can be solved similarly as well. Results Image Analysis Data Table Water and SYBR Green I Solution SYBR Green I Solution and DNA Calf Thymus Sample Integrated Density DNA μg/mL Conclusion PCR: Negative Control 790480 0.888 Negative PCR: Positive Control 2614324 3.994 Positive PCR: Patient 1 ID 27762, rep 1 2597111 3.966 Positive PCR: Patient 1 ID 27762, rep 2 3654456 5.673 Positive PCR: Patient 1 ID 27762, rep 3 2620690 4.004 Positive PCR: Patient 2 ID 59448, rep 1 700607 0.905 Negative PCR: Patient 2 ID 59448, rep 2 444410 0.491 Negative PCR: Patient 2 ID 59448, rep 3 331748 0.309 Negative KEY Sample = The samples were different sources of DNA that were analyzed. Integrated Density = The integrated density was calculated by subtracting the measured integrated density of the drop from the measured integrated density analyzed from the background. This calculation takes into account the background noise in the sample. DNA μg/mL = This was calculated by taking the integrated density of the sample divided by the integrated density of the drop and multiplying that by two. Conclusion = If the sample is considered "positive" then it will glow green and the cancerous mutation is said to be present. However; if the sample is considered to have "no signal" then it will just be clear and the cancerous mutation is said to be not present.