BME103:T130 Group 11

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: Timothy Peterson Role:Research and Development Scientist Name: Sharon Gooi Role: Experimental Protocol Planner Name: Derek Biah Role: Data Analyzer Name: Hunter Workman ImageJ Software Processor Name: Steven Casaceli PCR Operator Name: Kory Chisholm Flexible Roles LAB 1 WRITE-UP Initial Machine Testing The Original Design The PCR machine heats up the DNA so that enzymes can "unzip" the two strands of DNA. This process happens in cycles so that the DNA will seperate and duplicate a multitude of times. A certain amount of primer is used to duplicate the DNA specific to the amount of original DNA. By amplifying the amount of DNA, a proper diagnosis of a certain gene can be made. Experimenting With the Connections When we unplugged the LCD screen from the circuit board, the machine's screen shut off. When we unplugged the white wire that connects the circuit board to tube PCR block, the machine stopped reading the temperature. Test Run We first tested the PCR machine on the 18th of October, 2012. We initially ran our sample testing on the 1st of November, 2012 on PCR machine 12. The LCD screen readings matched the reading on the computer PCR program, and the machine worked well and efficiently. Protocols Polymerase Chain Reaction (PCR) The Polymerase Chain Reaction (PCR) is a process that depends on a DNA Polymerase enzyme's ability to synthesize a strand that is complementary to a targeted fragment of DNA in a test tube mixture of all four DNA bases, which are adenine, cytosine, guanine and thymine. Besides that, the test tube mixture also must have two fragments of DNA of about 20 base pairs that are called primers. These primers should have sequences complementary to adjacent areas of each side of the targeted DNA segment. Since these two primers should match exactly with only the targeted DNA sequence, only this area would be defined and copied. For the process of PCR, there are different stages, and they are collectively called the heating-cooling cycle. First of all, the sample to be tested would be added to the GoTAQ PCR master mix. Secondly, the mixture would be heated to separate the sides of the double-stranded DNA. Thirdly, the mixture would be cooled to an optimum temperature for the primers to find and bind to whichever side of the separated DNA strands that they are complementary to. Lastly, the temperature is then raised slightly to reach an optimum temperature for the polymerase, which is included in the test tube mixture, to extend the primers so that new complementary strands are generated. At the end of the first cycle, there will be two copies of the targeted DNA segment. The cycle is repeated multiple times to generate more and more copies of the targeted DNA segments. With the advent of technology in biological sciences and engineering, the entire process can be automated after all the correct components are added into a tube by using a thermocycler or a PCR machine such as the OpenPCR machine. The PCR master mix that was obtained from Promega consists of several different substances. These include nuclease-free water, deoxynucleotide triphosphates (dNTPs), magnesium chloride, and reaction buffers at optimal concentrations for any amplification of DNA. The reaction buffers had a pH of 8.5, and were made of 400μm deoxyadenosine triphosphate (dATP), 400μm deoxyguanosine triphosphate (dGTP), 400μm deoxycytidine triphosphate (dCTP), 400μm deoxythymidine triphosphate (dTTP) as well as 3mM of magnesium chloride. After taking image documents of all the samples on the fluorimeter, the ImageJ processor split the color channels of each image and measured the amount of pixels of the drop selection in the green image. The amount of pixels that were measured was then subtracted from the same background of the same area of selection. This subtraction gave the integrated density of each drop sample. The integrated density was used in the formula to find the DNA concentration in each drop. This table below lists all of the reagents used as well as the volume used during this process. Reagent Volume Template DNA (20ng) 0.2 µL 10 µM forward primer 1.0 µL 10 µM reverse primer 1.0 µL GoTaq master mix 50.0 µL dH2O 47.8 µL Total Volume 100.0 µL During Week 2, there were eight samples that PCR was run on. These samples consisted of a positive control, a negative control, and three samples from Patient 1 and three samples from Patient 2. Below are the details concerning Patient 1 and Patient 2. Patient 1 ID #: 11640 Gender: Female Age: 54 years old Patient 2 ID #: 29292 Gender: Male Age: 63 years old Fluorimeter The eight samples from the Polymerase Chain Reaction experiment were used in this experiment. In addition to that, eight Eppendrof tubes filled with 400ml of buffer to maximize fluorescence, a Eppendorf tube filled with DNA (calf thymus standard at 2 micrograms/ml; the tube was marked with a red dot on the cover), water in a scintillation vial, an Eppendorf tube filled with SYBR GREEN 1 (marked with a blue dot on the cover), several glass slides, a fluorimeter, a black box, a smartphone stand, a smartphone, a marker pen and several pipettes, a pipette with a blue strip, a pipette with a red strip, a pipette with a black strip and a pipette with a blank piece of paper taped to it, as well as a cup were used. The eight Eppendrof tubes were labeled using the marker pen according to the eight samples from the Polymerase Chain Reaction experiment; they were labeled +, -, 1a, 1b, 1c, 2a, 2b and 2c. Similarly, for each Eppendorf tube (ten altogether), a pipette for each tube was given a corresponding label. Using the corresponding pipettes, the eight samples from the Polymerase Chain Reaction experiment were transferred from their PCR tubes to their corresponding Eppendrof tubes. The pipettes were all kept carefully separate from each other. The fluorimeter was set up according to the image shown. The pipette with the blue strip was used to put two large drops of SYBR GREEN I on the first two centered drops on the glass slide in the fluorimeter.(Warning: please do not dispose of any of the pipettes used until the entire process is complete!) Once that was done, The corresponding pipette for the positive control was used to add two drops of the sample of the positive control to the two drops of the SYBR GREEN I that was already on the glass slide. The light in the fluorimeter was aligned to ensure that it was going through the drop. The fluorimeter was covered with the black file box and the smartphone operator was allowed to take as many pictures as needed. After the pictures were taken, the pipette with the black strip was used to dispose of the drop on the glass slide into the cup. The slide was then pushed forward so that the light would be in the general direction of the next two centered holes on the glass slide. The above process was then repeated using the next samples available, which were the negative control sample, sample 1a, 1b, 1c, 2a, 2b and 2c. Once the first five samples were done, shifting the glass slide down two holes after every sample, that glass slide was disposed off, and a new glass slide was used. After these eight samples were run, two drops of SYBR GREEN I were put on the appropriate two centered drops before two drops of the Calf Thymus DNA from the Eppendrof tube with the red dot on top were added using the pipette with the red strip, and the above procedure was repeated. Lastly, after the slide was moved two holes down and two drops of SYBR GREEN I was added to the slide, the pipette with a blank piece of paper taped to it was used to add two drops of water from the scintillation vial on the slides, with the above procedure also being repeated on it. After all of this was done, the slides, pipettes, and all of the tubes containing the samples were disposed of in the correct waste containers. Flourimeter Measurements Tubes Description Eppendorf Tube Label Pipette Label Positive Control + + Negative Control - - Patient 1 Sample 1 1a 1a Patient 1 Sample 2 1b 1b Patient 1 Sample 3 1c 1c Patient 2 Sample 1 2a 2a Patient 2 Sample 2 2b 2b Patient 2 Sample 3 2c 2c DNA Measurement Operator: Smartphone Image Number 2 Drops SYBRGr 2 Drops Comments ✓ 1A Patient 1 Sample 1 Darker blue tone with dark vertical streaks ✓ 1B Patient 1 Sample 2 Lighter Blue tone with very thin, vertical, fluorescent streaks ✓ 1C Patient 1 Sample 3 Lighter Blue tone with very fine, hazy fluorescent streaks ✓ 2A Patient 2 Sample 1 Light blue tone with thick,vertical, hazy, fluorescent streaks ✓ 2B Patient 2 Sample 2 Darker blue tone of drop with a variation of thick and fine, vertical, fluorescent streaks ✓ 2C Patient 2 Sample 3 Darker toned drop with very fine, wispy, fluorescent streaks ✓ Positive Control (+) Positive Control Lighter blue drop with green round shape blobs displaced throughout ✓ Negative Control (-) Negative control Lighter Blue drop with light, vertical streaks. No apparent fluorescence. ✓ Water (H2O) Water Dark blue drop with no fluorescent streaks. ✓ Calf Thymus Calf Thymus Dark blue drop with hazy, green, abstract round shapes distributed around the blob. ImageJ Software Processor Sample or Background ID Area and x, y, w, h, info Mean Pixel Value INTDEN RAWINTDEN INTDEN (IF DIFFERENT) 1a 69668 5.134 357674 same 1a background 69668 4.814 335409 same 1b 82952 14.383 1193134 same 1b background 82952 4.188 347364 same 1c 77078 29.946 2308181 same 1c background 77078 3.655 281721 same 2a 61076 78.280 4781047 same 2a background 61076 3.888 237445 same 2b 52524 50.560 2655589 same 2b background 52524 3.657 192067 same 2c 72068 21.003 1513624 same 2c background 72068 3.894 280632 same + Control 72476 97.093 7036889 same + Control background 72476 4.269 309387 same - Control 48856 51.595 2520721 same - Control background 48856 3.646 178142 same Calf Thymus Standard 64720 76.988 4982678 same Calf Thymus Standard background 64720 4.561 295190 same Data Analyzer Description INTDEN with background subtracted DNA Concentration, micrograms/ml WATER BLANK 0 DNA CALF THYMUS, 2 microg/ml 4687488 2 PCR: Negative Control 2342579 0.999502932 PCR: Positive Control 6727502 2.870408202 PCR: Patient 1 ID 11640, rep 1 22265 0.009499758 PCR: Patient 1 ID 11640, rep 2 845770 0.36086279 PCR: Patient 1 ID 11640, rep 3 2026460 0.864625147 PCR: Patient 2 ID 29292, rep 1 4543602 1.938608483 PCR: Patient 2 ID 29292, rep 2 2463522 1.051105411 PCR: Patient 2 ID 29292, rep 3 1232992 0.526077933 Research and Development Specific Cancer Marker Detection - The Underlying Technology PCR helps to detect certain types of genes. In this case it is used to find out a specific type of cancer. In the process of detecting the cancer, primers are made to compliment a DNA strand that has the cancer gene in it. If a subject has the cancer in their DNA, the primers will bind to strand, whereas a subject without the cancer would not have a primer attach to their DNA strand. The r1787996 SNP is linked to the cancer sequence. The codon ATC is the sequence for cancer where the ATT means there is no cancer. In PCR, the ATC cancer sequence is detected because the primers will only attach to the DNA strands that have the ATC sequence. The ATT, non-cancer, strands will not bind with the primers. Only the combined primer DNA strand will be detected thus alerting for cancer. Bayes' Theorem [math]\displaystyle{ P(A|X) = \frac{P(X|A) P(A)}{P(X|A) P(A) + P(X|-A) P(-A)} }[/math] This rule can be used to determine the association between the probability of testing for cancer and whether or not the patient actually has cancer because this method of testing is not entirely accurate. Results Sample Integrated Density DNA μg/mL Conclusion PCR: Negative Control 2342579 0.999502932 - PCR: Positive Control 6727502 2.870408202 + PCR: Patient 1 ID 11640, rep 1 22265 0.009499758 - PCR: Patient 1 ID 11640, rep 2 845770 0.36086279 - PCR: Patient 1 ID 11640, rep 3 2026460 0.864625147 - PCR: Patient 2 ID 29292, rep 1 4543602 1.938608483 + PCR: Patient 2 ID 29292, rep 2 2463522 1.051105411 + PCR: Patient 2 ID 29292, rep 3 1232992 0.526077933 - KEY Sample = The sample is a portion the DNA that was extracted from each patient. Integrated Density = This is the amount of pixels in an image or a selection of an image. We got these data by subtracting the integrated density of the drop selection and the black background. DNA μg/mL = This was calculated by multiplying the Integrated density of the sample, with the background subtracted, by two and then dividing by the calf thymus integrated density. Conclusion = Positive means that the sample contains a positive test result cancer gene; no signal means the sample contains a negative test result for the cancer gene. The samples that resulted with no signal meant that it produced a concentration that was smaller than that of the negative control. The samples that contained a positive test result produced a higher concentration than the negative control. From the data obtained, it appears that Patient 2 yielded a high level of DNA μg/mL, which resulted in a positive result for cancer in the first and second reps. In conclusion, Patient 2 most likely contains the cancer gene and Patient 1 does not based on the acquired results.