Difference between revisions of "BME103 s2013:T900 Group4 L3"

BME 103 Spring 2013 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: Kinjal Ahir Role:Protocol Name: Zach Young Initial Machine Testing Name: Anna Essex Initial Machine Testing Name: Tuan Phan Research and Design Name: Amelia Lax Research and Design

LAB 3 WRITE-UP

Original System: PCR Results

PCR Test Results

 Sample Name Ave. INTDEN* Calculated (μg/mL) Conclusion (pos/neg) Positive Control 1,450,385 1.60 pos Negative Control 488,789 0.18 neg Tube Label: B2 Patient ID: 17818 rep 1 709,603 0.51 pos Tube Label: C2 Patient ID: 17818 rep 2 642,405 0.41 pos Tube Label: D2 Patient ID: 17818 rep 3 417,721 0.07 neg Tube Label: B1 Patient ID: 85158 rep 1 450,174 0.12 neg Tube Label: C1 Patient ID: 85158 rep 2 387,850 0.03 neg Tube Label: D1 Patient ID: 85158 rep 3 376,360 0.01 neg

* Ave. INTDEN = Average of ImageJ integrated density values from three Fluorimeter images

Bayesian Statistics
These following conditional statistics are based upon all of the DNA detection system results that were obtained in the PCR lab for 20 hypothetical patients who were diagnosed as either having cancer or not having cancer.

Bayes Theorem equation: P(A|B) = P(B|A) * P(A) / P(B)

Calculation 1: The probability that the sample actually has the cancer DNA sequence, given a positive diagnostic signal.

• A = frequency of cancer-positive conclusions = 9 / 20 = 0.45
• B = frequency of positive PCR reactions = 26 / 60 = 0.43
• P (B|A) = frequency of positive PCR given cancer-positive conclusion = 24 / 26 = 0.92
• P(A|B) = 0.96 = 96%

Calculation 2: The probability that the sample actually has a non-cancer DNA sequence, given a negative diagnostic signal.

• A = frequency of cancer-negative conclusions = 11 / 20 = 0.55
• B = frequency of negative PCR reactions = 34 / 60 = 0.57
• P (B|A) = frequency of negative PCR given cancer-negative conclusion = 31 / 34 = 0.91
• P(A|B) = 0.88 = 88%

Calculation 3: The probability that the patient will develop cancer, given a cancer DNA sequence.

• A = frequency of "yes" cancer diagnosis = 9 / 20 = 0.45
• B = frequency of "pos" test conclusion = 26 / 60 = 0.43
• P (B|A) = frequency of pos given yes = 24 / 26 = 0.92
• P(A|B) = 0.96 = 96%

Calculation 4: The probability that the patient will not develop cancer, given a non-cancer DNA sequence.

• A = frequency of "no" cancer diagnosis = 11 / 20 = 0.55
• B = frequency of "neg" test conclusion = 34 / 60 = 0.57
• P (B|A) = frequency of neg given no = 31 / 34 = 0.91
• P(A|B) = 0.88 = 88%

New System: Design Strategy

We concluded that a good system Must Have:

- easily determined results: The easier the results are to read accurately, the less likely a misdiagnosis in either direction. It is undesirable both to give a false negative, where a patient is not treated when care is needed, or to give a false positive, wasting time and resources on those who do not need them. This aspect is central to any diagnostic tool.

- Simple OpenPCR Software: Simplicity increases ease and efficiency in lab experiments and hopefully leads to faster diagnoses. It also makes troubleshooting easier should problems arise. The more straightforward the system, the more quickly users can learn to use the machine.

We concluded that we would Want a good system to have:

- Low cost: Currently an OpenPCR machine costs \$599 and a Fluorimeter costs \$300. An inexpensive material would help reduce cost and increase accessibility, since there is always a limited budget for new equipment. This would not only allow users to increase the amount of tests that can be run at the same time, but also boost sales, which is important for marketing any device.

- integrated camera: phone cameras are easily moveable and vary in size and quality, leading to differing results. Smartphone camera settings can be time consuming or nonexistent. Having a built-in camera increases cost, but it is worth it to increase speed and accuracy. Furthermore, the program is simpler because it does not have to adjust to different cameras and phone sizes and shapes vary enough to make building a cradle to fit them difficult.

We concluded that a good system Must Not Have:

- Troublesome USB Connectivity. USB connectivity should function well in order for OpenPCR machine to work.

- Casing = fire hazard. High temperature with PCR can be dangerous.

We concluded that a good system Should Avoid:

- Avoid slow amplification.

- Hard to adjust phone/ fluorimeter. The phone can be easily moved by accident, which requires readjustment between the phone and the fluorimeter.

New System: Machine/ Device Engineering

SYSTEM DESIGN

Current design of fluorimeter

Rather than drastically change a fairly-efficient PCR machine, we decided that the fluorimeter setup was more in need of modification. The only change to the PCR machine would be improved USB ports, but the fluorimeter would have a built-in camera to remove the complications of positioning a camera phone. The phone would still be used to run the machine, but it wouldn't directly take the pictures. This new camera would take the place of the current cradle and be at a fixed position in respects to the fluorimeter for most efficient photographing. Also, the slots on the board of the fluorimeter would be labeled to avoid confusion in the process of analysis.

KEY FEATURES

Fluorimeter--We chose to include these new features

• Feature 1 - explanation of how this addresses any of the specifications in the "New System: Design Strategy" section
• Feature 2 - explanation of how this addresses any of the specifications in the "New System: Design Strategy" section
• Etc.-->

This new device will hold the camera phone in place. It will be able to connect temporarily to the fluorimeter, allowing the phone to be held in place. The device will also have an adjustable distance from the phone to the fluorimeter, as well as the height for more compability of other different phones, without trying to adding more heights to the phone or the fluorimeter.

INSTRUCTIONS

Step 1: Connect the base to the fluorimeter.
Step 2: Set the camera phone into the base.
Step 3: Set the height and distance.
Step 4: Take Photo.

New System: Protocols

DESIGN

We chose to add an extra device to the fluorimeter. However, protocols should remain the same.

MATERIALS

 Supplied in the Kit Amount Smart phone 1 Reaction mix given more for more reactions Battery Software
 Supplied in the User Amount Filter water SYBR Green Primers DNA sample (negative and positive)

PROTOCOLS

• PCR Protocol
1. Step 1: Reaction mix
2. Step 2: Add 2.5 μL of negative and positive DNA sample
3. Step 3: Fluorometer gave the sample result

• DNA Measurement and Analysis Protocol
1. Step 1: Set up the equipment
2. Step 2: Put the smart phone and fluorometer in the dark box
3. Step 3: Major the distance between the fluorometer and phone.
4. Step 4: Run the samples
5. Step 5: Take a picture of the experiment
6. Step 6: Repeat this trial with different samples
7. Step 7: Use Image J and make a circle around the drop.

New System: Research and Development

BACKGROUND

CHEK2 is a gene located at chromosome 22. It provides instructions for making protein call checkpoint kinase 2. The checkpoint kinase acts as a tumor suppressor. Mutations of CHEK2 gene can lead to breast cancer, Li-Fraumeni syndrome, and other type cancers and diseases.

DESIGN

Primers for PCR

GGAAGTGGGTCCTAAAAACTCTTACA[C/T]TGCATACATAGAAGATCACAGTGGC

Our primers address the following design needs

• Design specification 1 - explanation of how an aspect of the primers addresses any of the specifications in the "New System: Design Strategy" section
• Design specification 2 - explanation of how an aspect of the primers addresses any of the specifications in the "New System: Design Strategy" section
• Etc.

New System: Software

[THIS SECTION IS OPTIONAL. If your team has creative ideas for new software, and new software is a key component included in your new protocols, R&D, or machine design, you may describe it here. You will not receive bonus points, but a solid effort may raise your overall page layout points. If you decide not to propose new software, please delete this entire section, including the ==New System: Software== header.]