BME103:T930 Group 16

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Contents

Group 16

Name: Omar Moreno Salinas, and Rachel JuettenOpen PCR, ImageJ Software Processor, Data Compiler, and Analyzers
Name: Omar Moreno Salinas, and Rachel Juetten
Open PCR, ImageJ Software Processor, Data Compiler, and Analyzers
Name: Maggie McClure, and Swetha SwaminathanProtocol Persons: Sample Prep & Application
Name: Maggie McClure, and Swetha Swaminathan
Protocol Persons: Sample Prep & Application
Name: Marianna SinghDNA Measurement Operator
Name: Marianna Singh
DNA Measurement Operator
Name: Muawiya Ali Al-KhalidiR&D Scientist
Name: Muawiya Ali Al-Khalidi
R&D Scientist

LAB 1 WRITE-UP

Initial Machine Testing

The Original Design
Image:group 16.png


Experimenting With the Connections

When the heat sink is unplugged from the circuit board, the LCD screen is turned off. When we unplugged the white wire that connected the circuit board to the heating block the temperature reading on the LCD screen dropped drastically.

Test Run

We first tested open PCR on October 18, 2012. We learned how to take accurate temperatures using the open PCR machine. Using open PCR we were able to make a polymerase chain reaction. In order for this to occur, open PCR had to send the DNA through different sets of temperatures to heat it up to separate the strands and expose the bases, then cool it down for the primers to bind to the sequences, and also heat it back up to attain an extension of the copy of the new DNA. Which was conducted in an hour and thirty minutes.




Protocols

Polymerase Chain Reaction
The open PCR machine makes many copies of a DNA segment. It allows for a large enough sample to be made in order to analyze the DNA. In this lab we made copies of a specific segment of DNA that would allow us to determine whether or not our patients had the gene for cancer.

Sample Procedures and Fluorimeter Picture Procedures

To use the PCR machine we first obtained two patient DNA samples. Then we labeled eight test tubes with the patient number (three test tubes were labeled with patient one and the three others were labeled for patient two's DNA) and the last two were labeled as our positive or negative control. Once the tubes were labeled, we transferred the DNA using pipettes into the corresponding tube that contained solution which would allow the DNA to be copied. This solution was a mixture of Taq DNA polymerase, MgCl2, dNTP's, forward primer and reverse primer. The Taq DNA polymerase is an enzyme that helps to catalyze the matching of the dNTP's (or floating nucleotides) to make copies of the original DNA strand; the MgCl2 helps the Taq be more efficient. After the samples and controls were prepared, we placed the tubes in the open PCR machine and set the correct cycles and temperatures for the DNA to copied. This process took about an hour and a half to complete. Our samples were then collected and incubated until we received them again about two weeks later. Next we began analyzing the samples by creating another solution that would allow positive samples for cancer to glow. To do this we labeled ten transfer pipettes: one with a blue dash for our SYBR green solution, 1 with a red dot for our DNA Calf thymus, a positive control, negative control, 3 for our first patient and the last three for our second patient. The labeling was important so that we would avoid cross-contamination which could heavily skew our results. We first took a picture of our positive control by adding two drops of SYBR green to the slate, then adding two drops of our control to the SYBR green. Next we aligned the light so that it would hit the sample, placed it under the the black box and took the picture using a smart phone. We recorded that it was able to glow. We followed the same procedure using the corresponding pipettes for each sample for all of patient one's samples as well as patient two's samples, our water sample and our sample of DNA Calf Thymus. We recorded its ability to glow based on the pictures to determine which samples were positive for the cancer gene that we amplified (or copied) with the open PCR machine. A picture of the fluorimeter assembly was taken with a smartphone, and then the picture was transferred to a laptop equipped with the ImageJ software via icloud or email. The file was converted to a tiff, and then import in the ImageJ options was selected to obtain the TIFF virtual stack. The image was found in the search box, and then was ready to be analyzed.

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

Here is the patient information:


92136 M 54 Years


62276 F 61 Years

Fluorimeter Measurements

Image:FlouimeterMeasurements.jpg
The Fluorimeter


Fluorimeter with SYBR Green and Water


Fluorimeter with SYBR Green and DNA Calf Thymus


How does the fluorimetry technique work?


The fluorimeter is an instrument that detects fluorescence; the quantity is related to the amount of fluorescent material and is indirectly proportional to the molecule being detected. The slide that enters the fluorimeter is coated with a rough layer of Teflon which allows drops of liquid to form beads on the surface due to the surface tension. Because of this, the light from the Blue LED is focused in the drop which increases the intensity, and the SYBR Green in the liquid causes the presence of DNA to be indicated through a green phosphorescence. In this procedure, two drops of SYBR Green were placed on the slide, and then two drops of the solutions from the samples being tested were placed on top of the SYBR Green. The fluorimeter will give a visual color signal when dsDNA is present, and this can be quantified by taking an image. This is done by placing the smartphone camera (in this case, a Samsung HTC 1) into a diffraction grating and mounting it, and then setting exposure and saturation levels. By the use of a self-timer, the least amount of outside light was allowed in.




Research and Development

Specific Cancer Marker Detection - The Underlying Technology

A Polymerase chain reaction is a machine that amplifies a single or a few strands of DNA to generate millions of copies of that DNA sequence. Using this technology scientists can determine whether a patient has a positive or negative result towards cancer. A method of getting this data is called the PCR detection method, a method that relies on thermal cycling, switching back and forth to melt DNA and then connect primers. This is a method that can be used to detect whether a patient has positive result for cancer, because a sample of DNA can be taken and whether that connects to the primers and creates a chain reaction, scientists can then determine whether this DNA is positive or negative towards cancer. An example of proving this method can be seen using the r17879961 SNP, a cancer-associated sequence, using the PCR detection method we can prove that r17879961 SNP is actually associated with cancer. Because it carries with the Polymerase chain reaction, and to further prove the patient has a positive result for cancer, we use fluorescent dye and if the DNA glows in the solution, then the results are positive for cancer.

(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==

Sample Integrated Density DNA μg/mL Conclusion
PCR: Negative Control 9880697 F6 G6
PCR: Positive Control 4981557 F7 G7
PCR: Patient 1 ID #####, rep 1 14455570 F8 G8
PCR: Patient 1 ID #####, rep 2 8591662 F9 G9
PCR: Patient 1 ID #####, rep 3 13212871 F10 G10
PCR: Patient 2 ID #####, rep 1 12472923 F11 G11
PCR: Patient 2 ID #####, rep 2 22091168 F12 G12
PCR: Patient 2 ID #####, rep 3 11477553 F13 G13


KEY

  • Sample =
  • Integrated Density =
  • DNA μg/mL =
  • Conclusion =


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