BME100 f2017:Group16 W1030 L6

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Lab Write-Up 1 | Lab Write-Up 2 | Lab Write-Up 3
Lab Write-Up 4 | Lab Write-Up 5 | Lab Write-Up 6
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Name: Alfred Abraham
Name: Hannah Correa
Name: Alexia Kamau
Name: Jaynee Monarrez
Name: Michelle Mungaray



Bayesian Statistics

Overview of the Original Diagnosis System

In 16 groups of 4-6 people, BME students were given two patients samples which had to be tested for a particular disease (SNP). Each patient sample was then divided into 3 smaller tubes using a micropipette into a primer mix that diluted the DNA and allowed it to be tested in a PCR Machine. Along with the given patient samples, students were also given a sample that tested positive (positive control) for the disease and a sample that tested negative (negative control). The negative and positive control were also diluted with the primer mix. Then the 8 samples of DNA (3 from each patient, the negative control, and the positive control) were mixed with buffer and SYBR green in order to test them with the fluorimeter device. Each of the samples was also mixed with Calf Thymus DNA of varying concentrations. The samples were then photographed using a fluriometer device. In order to avoid potential errors, three pictures were taken of each sample. The images were then analyzed on ImageJ, in which the brightness of the samples was analyzed to determine whether or not the patient was negative or positive for the "disease". For the most part, the class was accurate in their final assessments of whether their patients were positive or not, although there were some inconclusive results which could have come from mistakes in calculations or errors in analyzing the images.

What Bayes Statistics Imply about This Diagnostic Approach

Calculation 1 represents the probability that a patient would receive a positive final result given that a positive PCR result was also obtained. The number calculated in this part was close to 1, meaning that the experiment has high sensitivity in predicting if the patient is positive or negative for the disease. Calculation 2 represents the probability of the patient receiving a negative test conclusion given a negative PCR reaction. Since this number is close to 1, this shows a high specificity in predicting whether the patient is positive or negative for the disease. Therefore, since both numbers of sensitivity and specificity are close to 1, it is reasonable to believe that the PCR replicates are good sources when determining whether or not a patient has a certain disease.

Calculation 3 represents the probability that a patient who is given a positive final test result will develop the disease. The number calculated for this part was about 55% which shows a low sensitivity in detecting the disease. Calculation 4 represents the probability that a patient who is given a negative final test result will not develop the disease. The number calculated for this part is close to 1, showing that there is a high specificity in the experiment detecting the disease. While the specificity is high, the sensitivity is significantly low, meaning that the accuracy of the PCR experiment is doubtful due the possibility of giving inaccurate readings (specifically negative results).

One possible source of error during the PCR and detection steps could have been the image analysis on ImageJ. When placing the oval on the droplet, it may have been inconsistent with the area of the droplet or shifted slightly in the process of analyzing. Additionally, the pictures analyzed could have been blurry, therefore making it harder to distinguish the area of the droplet against the background.

Intro to Computer-Aided Design

Our Design

Detachable Flap to Cover Fluorimeter Device
Modified Fluorimeter Device
Modified Fluorimeter Device

The design of the PCR machine was not changed at all. Our design was modified to incorporate an embedded camera within the fluorometer box.The camera would be fixed through a whole of a detachable flap , a component of the box. This design is different from the original fluorometer design because it does not have this altered camera feature. The original fluorimeter design required one to place a smart phone on a phone stand to take pictures of the various DNA droplet concentrations. However, it was observed that when using our smart phone to take the pictures during this previous lab (Week 11:lab D) positioning was the biggest issue. Adjusting the smartphone on the cradle so that it stood close enough across the droplet that lay inside the box and would not produce a blurry image was trickier than anticipated. This design was chosen because it would eliminate this inefficiency and help generate clear , magnified pictures. The camera will be accessible through the hole so that way someone can be able to validate the quality of the pictures being taken. The camera will also have a timer feature. That way, the photos can be taken automatically and the images lens will not be disrupted.

3D Modeling
The software that was used was SolidWorks, an advanced 3-D modeling tool to redesign a more preferable fluorimeter design. This application allowed us to redesign the fluorimeter system by either adding particular components or restyling current aspects of the design. One difficulty that surfaced during the computer-aided design lab was establishing the flap of the box.The first step to redesigning this system was sectioning the different parts of the box and adjusting the dimensions . The flap's dimensions were then modeled after the dimensions of the box in order to ensure a correct fit of the flap. A circular incision was then cut out from the flap measuring to the appropriate circumference of the camera so that the camera fits through the opening of the flap's box. The circular incision was cut directly in the middle to assure proper alignment and distance between the camera's lens and the droplet of DNA.

Feature 1: Consumables

Our packaged kit will include the following consumables -

  • 1.Taq DNA polymerase & Multiplex Buffer
  • 2.PCR mix
  • 3. Primer solution
  • 4. Gloves
  • 5. A pipetter and an abundance of discard tips
  • 6. Pre-labeled plastic tubes
  • 7. SYBR green solution
  • 8. Glass slides for the DNA concentration droplets
  • 9.Plastic cup to discard tips
  • 10. Googles

The labeled tubes would be important for this particular procedure because it reduces the time spent on labeling the positive and negative controls and the patients' ID. Glass slides are an added benefit because they help visualize the fluorescence and of the droplet and its shape better. Gloves and googles help when handling these chemicals and protecting them from entering your eyes.

Feature 2: Hardware - PCR Machine & Fluorimeter

The design of the open PCR machine will be the same as it was for the experiment in the previous lab. The one part of the hardware for this system that is changing is the fluorimeter. The new type of hardware that this part of the system will be utilizing is a built in camera. This camera will be able to capture a clearer image of the contents in the box and record this information in a chip that can then be connected to a computer where the photos can be downloaded.

One of the major weaknesses of the fluorimeter was the way we had to take pictures. The phone stand did not work very well. Multiple phones fell off of it because it would bend backwards and it needed to be properly placed. What we thought would help this fluorimeter is a built in camera and a flap that can be completely taken off and snapped on. Going along with this, the camera will be placed on the flap on the front of this device and it will have sensors that allow it to take the perfect picture. This also takes away a lot of the re-positioning that was done with the phone and the box. Overall, implementing a camera is would be very useful and would allow for a less confusing experimental device.