Difference between revisions of "BME103:T130 Group 15"

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'''Image Analysis Data Table'''
'''Image Analysis Data Table'''

Revision as of 12:20, 15 November 2012

Owwnotebook icon.png BME 103 Fall 2012 Home
Lab Write-Up 1
Lab Write-Up 2
Lab Write-Up 3
Course Logistics For Instructors
Wiki Editing Help
BME494 Asu logo.png


Name: Malik Alnaim: Research & Development Scientist
Name: Alyssa Alexander
Role: Research & Development Scientist
Name: Sichun Ai
Role: Experimental Protocol Planner
Name: Nehal Jolly
Role: Experimental Protocol Planner
Name: Ben Reising
Role: Open PCR machine Engineer
Name: Mayuri Gupta
Role: Open PCR machine Engineer


Initial Machine Testing

The Original Design
This is the internal view of the PCR machine that was used in the lab experiment.

Experimenting With the Connections
Above is an image of important parts in a PCR machine.

Before we even began our experiment we had to assess and overlook these specific parts so that we could better learn their function and how that contributes to the overall machine. Part 1 displays the Aluminum Plate, which is designed to both contain and give off the heat necessary to perform the experiment once it is put down over Part 2, the Well Plate. The Well Plate is simply designed to hold the test samples and measure the heat around them, in order to ensure the samples are receiving the right temperature.

Part 3 is the PCB (Printed Circuit Board) of the LCD screen, which means it is responsible for the images and readouts that appear on the screen. This part is important because the entire flow of information comes into here, so that the user can gather information on the progress and results of the machine. Part 4 is the heat sink fan, which works to cool the entire machine by blowing the hot air out from the machine, to prevent overheating. Part 5 is the Rsenda ATX (Advanced Technology eXtended), a type of motherboard. A motherboard is essentially a large PCB which holds the CPU (central processing unit) and is in charge of memory. The final part is Part 6, which is the PCB of the open PCR Circuit Board. This is much like the motherboard, but deals with current and continuous flow of information and signals through the wires. No real memory is stored here.

Connection and Process Testing
In order to see the connection between various parts we ran two tests on machine #10 on October 18th, 2012, where we unplugged a single wire in each. When we unplugged PCB of the LCD screen from PCB of open PCR circuit board, the LCD screen did not turn on at all.

When we unplugged the white wire that connects PCB of open PCR circuit board to well plate, strange images began to show up on the screen, as a result of no information flowing from the well plate to the PCB of the open PCR circuit board.


Polymerase Chain Reaction

  1. (Jim Dorsey) Polymerase Chain Reaction. http://www.contexo.info/DNA_Basics/polymerase_chain_reaction.htm. Last accessed 11/01/12.)

Steps to Amplify DNA Samples

  1. Collect three replicate DNA samples from two patients
  2. Create a new program on the Open PCR system
  3. Create three stages
    • Stage 3. 72°C for 3 minutes
  4. Final hold: 4°C

The Components of the GoTaq® Colorless Master Mix

Volumes Used for Mixture

Table 1
Reagent Volume
Template DNA (20 ng) 10.2 μL
10 μM reverse primer 1.0 μL
dH2O 47.8 μL
0 μM forward primer 1.0 μL
GoTaq master mix 50.0 μL
Total Volume 100.0 μL

DNA Samples (8)

Positive Control:
Cancer DNA Template
Tube label A
Replicate 1
Tube Label 1-1
Patient 1 ID: 27762, F, Age: 52
Replicate 2
Tube Label 1-2
Patient 1 ID: 27762, F, Age: 52
Replicate 1
Tube Label 1-3
Patient 3 ID: 27762, F, Age: 52
Negative Control:
No DNA Template
Tube Label B
Replicate 2
Tube Label 2-1
Patient 2 ID: 59484, F, Age: 45
Replicate 2
Tube Label 2-2
Patient 2 ID: 59484, F, Age: 45
Replicate 2
Tube Label 2-3
Patient 2 ID: 59484, F, Age: 45

Flourimeter Measurements
Screen Shot 2012-11-08 at 3.41.08 PM.png
Image above shows the set up of the flourimeter measurments

Flourimeter Assembly Procedure

  4. Using the specific pipette for each component, one drop of buffer was put onto the first and second centered holes of the slide and two water drops were placed on the gathered buffer drops.
  5. The device was then put under the black box provided and the phone was placed into the holder inside the box.
  9. The previous steps were repeated for each sample with the exception of
    • Replacing the water drops with the rest of the samples
    • using a different row on the glass slide each time a sample was used.

Open ImageJ

Research and Development

Specific Cancer Marker Detection - The Underlying Technology

There is a genetic relation to having cancer or not when an individual is over the age of 40. The specific gene, in this case, is located on chromosome 22, r17879961. To test an human's DNA for this cancer gene, we have go through a series of reactions called PCR on the DNA for replication and amplification of the patient's DNA strand.

As previously explained shown in Protocols, primers are needed for the DNA replication, a forward and a reverse. One at the "completing" strand of the double strand, and one as the cancer detecting strand. On chromosome 22, the primer to detect r17879961 defect has to have the changed nucleotide.

The reverse primer used: AACTCTTACA-C-TCGATACAT

The forward primer used: TTGAGAATGT-C-AGCTATGTA

In this particular instance, it is a adenine replaced by the cancerous-related nucleotide cytosine as shown above as the bold C. The primer will only bind to the matching sequence (testing sequence) because of the A-T, and C-G pairings. Since an A has been replaced with a C, the primer can't bind to the DNA strand beyond that specific nucleotide in the sequence. Due to the open double helix, further steps in the reaction will not happen, and no amplification will happen. No amplification means no visible results and the test will run negative.

If the cancer gene is present, then the matching primer will completely bind to the DNA strand. When this happens, the amplification sequence will be able to precede, and this will show up as a positive result.

Image by: Alyssa Alexander

This is all in theory of course, and should work perfectly every time. But there are many factors to consider. For instance, not every one who has cancer has the cancer gene.

Reliability and Accuracy of Specific Cancer Marker Detection

Based on Bayesian reasoning, the probability that someone will test positive that will actually have the disease has approximately 7.8 percent. However, the chance that someone does not test positive, and doesn't have cancer is about 99.8%. These percentages generally mean that the test itself is generally in favor of testing negative, which means there are less chances to have false diagnosis and/or treatments.

These percentages resulted from the following equation:
[math]Positive Predictive Value = (True Positive)/(True Positive + False Positive)[/math]

[math]Negative Predictive Value = (True Negative)/(True Negative + False Negative)[/math]

The values for these above equations are gathered from Bayesian's reasoning. Baye's Theorem:

[math]p(C/T) = (p(T/C)*p(C))/(p(T/C)*p(C)+p(T/nC)*p(nC))[/math]

where p(C/T) is the probability of a person with positive results will have cancer out of the entire patients participating. p(C) is the probability of having cancer present, p(T/C) is the percent of patients who tested positive with have cancer and had it. n = not

The values of the these equations are taken from the general statistics from tests performed on 'x' amount of patients. For instance, the direct results showed that 80% of the people with cancer, tested positive, this value would be used as p(T/C) because that is the test running positive and having cancer. The value p(T/nC) would be the percentage of people who tested positive but do not actually have cancer, which resulted to be 9.6%. Additionally, the general statistics were 90.4% of patients will test negative and will not have cancer, and 20% percent of people with cancer will run negative.

The calculated p(C/T) was used as the "True Positive" in the original two equations (PPV and NPV). The same for the false positive, true negative and false negative can be solved similarly as well.


Image Analysis Data Table BME103 Group15 Data Table.jpg

BME103 Group15 WaterGreen.jpg Water and SYBR Green I Solution

SYBR Green I Solution and DNA Calf Thymus BME103 Group15 DNA Calf Thymus Green.jpg

Sample Integrated Density DNA μg/mL Conclusion
PCR: Negative Control 790480 0.888 Negative
PCR: Positive Control 2614324 3.994 Positive
PCR: Patient 1 ID 27762, rep 1 2597111 3.966 Positive
PCR: Patient 1 ID 27762, rep 2 3654456 5.673 Positive
PCR: Patient 1 ID 27762, rep 3 2620690 4.004 Positive
PCR: Patient 2 ID 59448, rep 1 700607 0.905 Negative
PCR: Patient 2 ID 59448, rep 2 444410 0.491 Negative
PCR: Patient 2 ID 59448, rep 3 331748 0.309 Negative


  • Sample = The samples were different sources of DNA that were analyzed.
  • Integrated Density =
  • DNA μg/mL =
  • Conclusion =