BME103:T930 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: Tony Nguyen Machine Tester Name: Ryelle Pattuinan Research and Development Name: Kenze Caulfield ImageJ Technician Name: Jackie Janssen Protocol Name: Ben Hook Protocol Name: Samantha Boccasini Machine Tester LAB 1 WRITE-UP Initial Machine Testing The Original Design This is an Open PCR machine. The purpose of the machines is to fluctuate the temperature of different samples of DNA strands to separate them into a single strand. It allows for a way to amplify DNA sequences by this separation and heating them with primers and DNA polymerase. When raised to a high enough temperature DNA melts, which is where it separates into the two strands, and primers and DNA polymerase are used to fill in the holes and attach to the now single-stranded DNA. The machine is able to amplify a specific sequence of DNA up to 1 billion times. Because of this ability, scientists and investigators can use it to amplify these sequences even if only a small amount of DNA is provided. Single roots in hair or even microscopic splatters of blood left such as at scenes of crime are actually ample amounts of DNA for PCR. Experimenting With the Connections When we unplugged (part 3) from (part 6), the machine's display screen didn't turn on or show up. When we unplugged the white wire that connects (part 6) to (part 2), the machine's temperature reading was altered compared to the accurate values from the computer. Test Run We first tested Open PCR on October 25, 2012. The machine was accurate and precise but the cycling was slow. Protocols Polymerase Chain Reaction The Polymerase Chain Reaction works by attaching MgCl to taq enzyme in the solution and then pulling deoxy nucleotide diphosphates, binding them to the strand that is already present. This gives a base to the DNA that is already there. DNA amplification contains three important steps: heat denauturation,primer annealing, and primer extension. Step One: Heat Denauturation-Heat is applied to the DNA and the two strands separate. Step Two:Primer Annealing-With excess dNTPs, oligonucleotides are added. Step three: Primer Extension-DNA polymerase is added and new strands of DNA are synthesized. The DNA strands combine with the nucleotides to form completed, complimentary strands of DNA. PCR master mix contains Magnesium Chloride, all four Nucleotides needed to create DNA, and reaction buffers. Chart One: Reagents and Their Volumes Reagant Volume Template DNA (20 ng) 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 Charts Two and Three: Vital Patient Statistics Patient 1 Sample ID Sex Age Date/Time + Control 12123 M 61 Thursday 9:30 AM 1-1 12123 M 61 Thursday 9:30 AM 1-2 12123 M 61 Thursday 9:30 AM 1-3 12123 M 61 Thursday 9:30 AM Patient 2 Sample ID Sex Age Date/Time - Control 21312 F 56 Thursday 9:30 AM 2-1 21312 F 56 Thursday 9:30 AM 2-2 21312 F 56 Thursday 9:30 AM 2-3 21312 F 56 Thursday 9:30 AM Flourimeter Measurements The Flourimeter was assembled by taking the black box, flipping it upside down so that the rest of the materials can be placed inside the box with the flap blocking out excess light. The glass slide was then placed into the flourimeter so that the light of the flourimeter was on the first two rows of holes in the glass slide. The flourimeter was then placed inside the overturned black box, and the phone was placed on the stand. The camera on the phone is lined up with the middle row of holes in the glass slide. After the pictures of the droplets of DNA and SYBR green solution were taken, they were sent to a computer through e-mail and downloaded. Left to Right: Flourimeter in action; Apparatus of flourimeter Research and Development Specific Cancer Marker Detection - The Underlying Technology Polymerase Chain Reaction or PCR for short is a method used to view a short segment of DNA and then amplify it by generating thousands of copies of the specific sequence being focused on. This method operates by fluctuating the temperature in order to allow for certain processes to occur within the DNA. 95 degrees Celsius - the initial temperature used to separate the strands of the DNA. 57 degrees Celsius - the temperature is dropped to this point in order to allow the primers to bind to the individual strands of DNA. 72 degrees Celsius - the temperature is again increased to this point to allow the Taq polymerase to bind and replicate the strands of DNA. For the PCR process to work, a template DNA which is the sample that you want to test for, is first needed. Primers that could identify the sequence that you want are also needed. Magnesium Chloride is also used to to help Taq polymerase bind the nucleotides (dNTP's) that are needed to replicate the DNA. PCR can be used for a variety of purposes, in this case however, PCR was used in order to determine whether or not a specific gene codes for cancer or not. If a cancer gene is located, then it will yield a positive result because the primers only bind to a certain sequence in the DNA and if that certain sequence codes for cancer, then it will produce a positive result indicating that it was located in that sequence. The specific sequence that was focused on was r17879961 with the sequence AACTCTTACA[C]TGCATACAT instead of being AACTCTTACA[T]TGCATACAT. The change is from ACT to ATT. The change from the C base into the C base is a missense where the normal T mutates into the cancer C. To identify whether such mutation occurred in the sequence using PCR, primers should first be designed to bind into the specific sequence. The primer needed for the sequence. The forward primer needed for the sequence is TGGTATAAGACATTCCTGT while the reverse primer should be AACTCTTACACTGCATACAT. Using PCR, the primer will attach to this specific sequence, checkpoint kinase 2 in the 22nd chromosome. Once the thermocycling ends, this sequence will be amplified for each samples. The fluorescent green dye is used to tell whether or not the result is positive or negative. Like explained earlier, if the sample carries the mutation, then the sample would test positive. If it does not, then the sample would test negative because the primers would not be able to bind to the DNA because it does not contain the proper sequence. This specific sequence is reported to be susceptible too breast and colorectal cancer. It is also linked to Li-Fraumeni syndrome, Osteosarcoma and even prostate cancer. It was also reported that the non-mutated form of the gene is found in about 98.9% of the population while the mutated form is found in 1.1% of the population. Furthermore, the mutated gene is found in 7.8% of patients with colorectal cancer and 5.3% of the rest of the population without colorectal cancer. Bayes Rule was also used to determine the relative value of one diagnostic test method over another. The equation for Bayes' Rule is P(A|B) P(B) = P(A,B). http://www2.le.ac.uk/departments/emfpu/genetics/explained/images/PCR-process.gif [1] Results Sample Integrated Density DNA μg/mL Conclusion PCR: Negative Control 677571 14.1254862 No Signal PCR: Positive Control 1695980 24.59754216 Positive PCR: Patient 1 ID 12123, rep 1 1469271 9.5987462 Negative PCR: Patient 1 ID 12123, rep 2 983747 16.548578411 Negative PCR: Patient 1 ID 12123, rep 3 748210 9.54789621 Negative PCR: Patient 2 ID 21312, rep 1 424971 11.4587165498 No Signal PCR: Patient 2 ID 21312, rep 2 738858 10.54881694 Positive PCR: Patient 2 ID 21312, rep 3 658764 13.54489251896 No Signal KEY Sample = This describes the sample number and the patient. Integrated Density = This is the integrated density of the drop without that of the background. DNA μg/mL = 2 * IntDen of Sample/IntDen of Calf Thymus (without that of the background) Conclusion = Whether or not the exponential DNA replication occurred.