BME103:T130 Group 16

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 Brayden Gallimore Research & Design Specialist/Data Compiler and Analyzer Name: Adam M White Experimental Protocol Planner/DNA Measurement Operator:Smartphone Name: Heather Borgard Open PCR Machine Tester 1/ImageJ Software Processor Name: Ashley Guerrero Open PCR Machine Tester 2000 and Open Wet Ware Research & Design Scientist Name: Zonash Zainab Protocol Person: Sample Prep and Application/Research and Design Specialist LAB 1 WRITE-UP The Original Design () Experimenting With the Connections When the wire connecting the LCD screen to the circuit board is unplugged, the LCD will not show, but the machine will still function. The screen will not present any premature values or any details about the thermal cycler. When the white wire connecting the circuit board to the temperature sensor is unplugged, the temperature will not be changed correctly. This would cause problems in the process of DNA amplification and the primers would most likely be unable to attach to the active sites. Test Run We first tested the PCR machine on 10/25/12 The Polymerase Chain Reaction Machine we used is a quick and inexpensive way amplify small sections of DNA. The main function of the PCR machine is to act as a DNA Thermal Cycler to isolate a specific section of DNA from a strand that has an excessive amount of genetic information. A target area on a strand of DNA is amplified and identical copies are generated using changes in temperature. The machine quickly and precisely heats and cools the DNA segments at different times over the span a a little over an hour. In cylce one, the thermal cycler heats to 95 degrees Celsius. The DNA begins to separate into two single strands. The thermal cylcer then cools to 50 degrees Celsius. The primers that were added in the tubes before the experiment then bind the target sites before the single strands pair up again. When the thermal cycle heats again to 72 degrees Celsius, DNA polymerase finds a primer to add complementary nucleotide to the strand.In cycle two the temperature is raised again to separate the DNA strands. When the temperature is lowered, the primers attach again. and the same process is repeated. In cycle three, two strands that begin with primer one and end with primer two appear. At then end of cylce four, there are even more copies of this fragment. As the cylces continue more and more copies of the target sequence will be generated. The this is especially useful to genetic mapping and detecting bio-markers. The PCR Machine is composed of a heated lid that presses against the tubes of DNA to heat during the cycles and thermal block where the tubes of DNA are placed, a heat sink, and a fan to absorb heat and cool during the different cycles, and an lcd screen to read the temperature and cylces that the thermal cycler is going though. Protocols Polymerase Chain Reaction (Add your work from Week 3, Part 1 here) Flourimeter Measurements (Add your work from Week 3, Part 2 here) Research and Development Specific Cancer Marker Detection - The Underlying Technology Using: DIY Fluorescence Measurement to Detect DNA after PCR We took the DNA samples from the open PCR and put it into an Eppendorf tube containing 400mL of buffer. Patient 1: ID 99949 Samples: + P1R1 P1R2 P1R3 Patient 2: ID 61909 Samples: - P2R1 P2R2 P2R3 Recapitulation: Template DNA from the patient (non-human) was extracted, then put into a primer (synthetic sections of DNA that, in this case, bind with the template DNA if it is cancerous). Using a PCR, the DNA was thermally cycled. First, the PCR heated the the solution to 95 degrees (C), in which the DNA was spit into 2 helicase. Then it was cooled to 57 degrees (C), where the primers attach to the specific sequence. Then it is heated to 72 degrees, where the polymerase extends DNA strand by attaching the nucleotides in order (polymerization). This process was repeated for 34 cycles, then refrigerated. There were 2 Eppendorf tubes containing: SYBR Green I (used with blue pipette) DNA calf thymus (2 microg/mL, used with a red pipette) The DNA calf thymus is green when viewed on a Smartphone when a certain dsDNA is present. First, we placed a glass slide on the fluorimeter, then put 2 drops of SYBR Green I on 2 vertically consecutive dots, connecting them. Then we placed 2 drops of + on top of the SYBR Green I, took a picture, then used a pipette to throw away the liquid waste. This procedure was repeated with P1R1, P1R2, P1R3, -, P2R1, P2R2, P2R3, and DNA calf thymus in said order. A positive result will be dyed green (because SYBR Green I dye only binds to double stranded DNA), while clear (blue on a Smartphone) indicates a negative result (non-cancerous). A negative result occurs because the primers do not bind with the DNA sequence. Primer Binding There are 4 nitrogenous bases associated with DNA: Adenine (A), Thymine (T), Cytosine (C), and Guanine (G). In a double stranded DNA sequence, guanine is paired with cytosine (GC) while adenine is paired with thymine (AT). Structure of double-stranded DNA: (Source: http://hshgp.genome.washington.edu/teacher_resources/modules-view.htm) PCR Process: 1) At 95 degrees Celsius, the double stranded DNA is denatured. 2) The primers anneal to the complementary bases at 57 degrees Celsius. 3) Polymerization: The Taq polymerases amplify the DNA by extending the single-stranded DNA, making a new double-stranded DNA. 4) This was repeated 34 times. Patients with no cancer have the ATT (nitrogenous bases adenine thymine thymine) sequence, while the ACT (nitrogenous bases adenine cytosine thymine) sequence replaces the ATT sequence in cancer patients. (BONUS points: Use a program like Powerpoint, Word, Illustrator, Microsoft Paint, etc. to illustrate how primers bind to the cancer DNA template, and how Taq polymerases amplify the DNA. Screen-captures from the OpenPCR tutorial might be useful. Be sure to credit the source if you borrow images.) Results The following chart shows the results from the imagej software processing. Each image that was taken with the smart phone was analyzed through the imageJ program to establish the amount of fluorescence each "drop" produced. The images below the table show the substances that were known. Calf Thymus DNA was expected to have fluorescence, as the Water was expected to have no fluorescence. This showed that the detection method was working correctly. Water: tested negative for cancer Calf Thymus DNA: tested positive for cancer 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 = DNA μg/mL = Conclusion =