BME103:W930 Group5

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Lab Write-Up 1
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Lab Write-Up 3
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Contents

OUR TEAM

Name: Andrea CarpenterRole(s): Experimental Protocol Planner & DNA Measurement Operator: Smartphone
Name: Andrea Carpenter
Role(s): Experimental Protocol Planner & DNA Measurement Operator: Smartphone
Name:Dana McElwainRole(s): Open PCR Machine Engineer & ImageJ Software Processor
Name:Dana McElwain
Role(s): Open PCR Machine Engineer & ImageJ Software Processor
Name: Michelle NguyenRole(s): R&D Scientist & Protocol Person: Sample Prep & Application
Name: Michelle Nguyen
Role(s): R&D Scientist & Protocol Person: Sample Prep & Application
Name: Nathan HolmanRole(s): Experimental Protocol Planner & Data Compiler & Analyzer
Name: Nathan Holman
Role(s): Experimental Protocol Planner & Data Compiler & Analyzer
Name: MalikMclaurinRoles(s): Open PCR Machine Engineer
Name: MalikMclaurin
Roles(s): Open PCR Machine Engineer
Name: Chris AnastosRoles(s): R&D Scientist & R&D Scientist: Open Wet Ware
Name: Chris Anastos
Roles(s): R&D Scientist & R&D Scientist: Open Wet Ware

LAB 1 WRITE-UP

Initial Machine Testing

. The Original Design
PCR_group_5_labeled_diagram.png
This is a diagram of the inside of the OpenPCR Machine. By connecting this machine through a USB port to a computer, it allows you to amplify DNA to later analyze different markings and base pairs within the sequence.


Experimenting With the Connections

When the LCD screen was unplugged from the Open PCR brains Board, the information for the sample cycle was no longer available on the screen.

When we unplugged the white wire that connects the brain board to the 16 tube PCR block, the machine temperature was unable to be regulated.


Test Run

The initial test date of the Open PCR Machine was October 24, 2012. Our experience with the machine was rather time consuming, with the experiment taking an hour and a half to complete. However, the machine was very straightforward and easy to use.




Protocols

Polymerase Chain Reaction

The Polymerase Chain Reaction is a process controlled by thermal cycling; a system of repeatedly heating and cooling the sample. It is simple and inexpensive, as PCR allows you to make millions of copies of DNA to better analyze data. First you need a small amount of DNA and a small amount of some PCR reaction mix. A program on the machine needs to be created consisting of three stages. During the three stages, the DNA is first heated to separate the DNA double helix, creating two single stranded DNA molecules. The thermal cycler is now cooled and primers match up to the DNA strands before they naturally attempt to pair up. As the cycles progress, DNA is replicated and used as a template to create additional copies. There are several components of a PCR reaction. The template DNA amplifies millions of copies to determine if they are cancerous. The many primers start the binding of complentary strands (specific primers) and they bind to cancer sequences. The taq polymerase is a protein that catalyzes the DNA assembly. There is also a cofactor, which binds to Taq to enable optimal binding speed. Lastly, there are deoxynucleotide tri-phosphates, which builds a new strand of DNA.

Steps to Describe How to Amplify a Patient's DNA Sample:

Step 1: Initiation
During this step the reaction needs to be heated to around 95 degrees Celsius and held there for about three minutes.

Step 2: Heat Denaturation
A DNA molecule carrying a target sequence is denatured by heat (90-95 degrees Celsius) and the two strands are separated.

Step 3: Primer Annealing
As the mixture cools, each strand of DNA molecule becomes annealed with an oligonucleotide primer conplementary to either end of the target sequence.

Step 4: Primer Extension
DNA polymerase is added and complementary strands are synthesized at a temperature of 60-75 degrees Celsius.

Step 5: Termination
During this step, the final hold, the reaction is held at 4 degrees Celsius to stabilize the reaction.


Description of DNA Samples

Patient ID Sex Age
76893 Female 47
92307 Male 59

NOTE: 8 samples total: 3 trials (a, b, & c) of each patient (1 & 2) with a positive control and a cancer DNA template.

Reagent Volume
Template DNA (20ng) 0.2 μL
10 μM forward primer 1.0 μL
10 μM reverse primer 1.0μL
GoTaq Master Mix 50.0 μL
dH2O 47.8 μL
Total Volume 100.0 μL

Fluorimeter Measurements
The following link shows group 5's flourimeter results. The numbers reflect the output of the ImageJ software. http://openwetware.org/images/f/fd/Final_results_Lab_-1_102.txt

Fluorimeter Assembly

1. Empty the given container of the separate parts for the fluorimeter.

2. To create a darker environment for the images, unsnap one side of the container and place the container upside-down so that the unsnapped flap of the container flips up.

3. Place the smartphone to be used for the experiment into the given black stand to provide a stable stand for the camera.

4. Position LED box with the drop in the back of the container with the camera parallel to it so that the camera is viewing light passing through the water droplet.

You are now ready to shoot photos of the experiment. Remove the LED box and camera stand from the container each time to replace the droplet.

Image:Flourimeter.jpg

Drop Preparation:
1. Fill the plastic pipette with liquid from the sample only to the bottom of the black line.
2. Carefully place 2 drops of SYBR green in the middle of the first 2 rows of the slide using the plastic pipette.
3. Add two drops of the sample.
4. Align the drop so that the blue LED light is focused by the drop to the middle of the black fiber optic fitting on the other side of the drop.
5. Repeat for each sample.


ImageJ Procedures and Operation:
Primary function: To take data from image files by using processing and measuring features. Once downloaded onto the computer-

1. Inactivate the designated camera's flash

2. Set ISO to 800+

3. Set white balance to auto

4. Set to max exposure

5. Lower saturation to a minimum

Once Images are saved onto phone:
1. Download photos onto computer with the ImageJ program (this can be done several ways, like using a usb cord to connect the phone to the computer or simply emailing the images to an email address)
2. Open the photos in the program by going under File and selecting Open and choosing the desired images.

Sample Photo:
Image:example1.jpg


NOTE: To ensure there is not contamination between the samples make sure to use a new pipette for each sample when replacing the drops on the LED box.

Research and Development

Specific Cancer Marker Detection - The Underlying Technology

(See above for PCR explanation)

CHEK 2, code rs17879961, is a single nucleotide polymorphism (SNP) that is linked to cancer. It is a symbol for CHK2 Checkpoint Homolog and is located on chromosome 22. Mutations of the CHEK2 gene are related to increased risk of breast cancer. In week three's class, it was concluded that PCR can be used to bind specific DNA primers to the cancerous DNA bases--resulting in cancerous DNA amplification. As a result, the PCR reaction will form normal DNA sequences and the supposed cancerous CHEK2 strain. Possible primers for supposed amplification were used in the experiment and were compared to sample sequences to determine if the DNA in question was amplified.

Extra information: Gene missense, T mutates to cancer associated C for the specific allele. Cancer sequence binding primer: AACTCTTACACTGCATACAT. In this scenario, the risk allele in the highlighted C base.



Image:DNA PCR.jpg


Bayesian Reasoning

P(A|B)=[P(B|A)*P(A)]/P(B)
or
P(C|T)=[P(T|C)*P(C)]/[P(T|C)*P(C)+P(T|~C)*P(~C)]
Where C is the cancer present and T is the positive test. Other values include the following: P(C) is prior probability and P(T|C) and P(T|~C) are conditional probabilities.

Bayesian reasoning is a conditional probability tool used to help understand the limitations of diagnostic testing. The formula for Baye's Rule is given above--it is also given in various forms. Through Baye's reasoning, four important things can be calculated: Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV). Sensitivity is the probability that an individual with the disease will result in a positive, and specificity is the probability that he/she without the disease will result in a negative test. PPV refers to the probability that an individual with a positive test actually has the disease, where as NPV is the probability that an individual with a positive test does not actually have the disease. It is important to utilize Baye's Rule because it will give a better understanding to the reliability of open PCR results based on the probabilities of false positives and false negatives.

Results

Result for water:
Image:water2.jpg

Result for Calf Thymus:
Image:calfthymus2.jpg


Sample Integrated Density DNA μg/mL Conclusion
PCR: Negative Control 9361818 1.03 No signal
PCR: Positive Control 30643859 3.38 Positive
PCR: Patient 1 ID: 76893, 1A 10774328 1.19 Positive
PCR: Patient 1 ID: 76893, 1B 5848412 .64 Positive
PCR: Patient 1 ID: 76893, 1C 1669198 .18 No signal
PCR: Patient 2 ID: 92307, 2A 2265634 .25 Positive
PCR: Patient 2 ID: 92307, 2B 2787876 .31 Positive
PCR: Patient 2 ID: 92307, 2C 2578187 .28 Positive


KEY

  • Sample = The set of DNA contained within one test tube.
  • Integrated Density = The sum of the values of the pixels in the image or selection.
  • DNA μg/mL = This is the concentration of the DNA in the samples. A formula can be used to determine this concentration.
  • Conclusion = The results obtained in the experiment. This determined whether or not the tested gene was positive or negative for cancer.
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