BME103 s2013:T900 Group2 L3

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OUR TEAM

 Name: Joe SansoneRole(R&D Scientist) Name: Shang Ruan Open PCR Machine Engineer Name: William ScottRole(R&D Scientist) Name: Andy SonProtocol Planner Mitch Riggs Open PCR Machine Engineer/ Team Leader

LAB 3 WRITE-UP

Original System: PCR Results

PCR Test Results

 Sample Name Ave. INTDEN* Calculated μg/mL Conclusion (pos/neg) Positive Control 518016 0.199 N/A Negative Control 375033 0.032 N/A Tube Label:2 Patient ID: 91562 rep 1 384551 0.043 neg Tube Label:3 Patient ID: 91562 rep 2 312171 -0.042 neg Tube Label:4 Patient ID: 91562 rep 3 371384 -0.031 neg Tube Label:5 Patient ID: 25235 rep 1 467127 0.139 pos Tube Label:6 Patient ID: 25235 rep 2 406664 0.064 neg Tube Label:7 Patient ID: 25235 rep 3 484088 0.154 pos

* Ave. INTDEN (Patient 91562)= 356035.3 *Ave. INTDEN (Patient 25235)= 452626.3

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] = [24/27] = [0.89]
• P(A|B) = [0.931 0r 93.1%]

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

• A = [frequency of "yes" cancer diagnosis] = [6/9] = [0.67]
• B = [frequency of pos test conclusions] = [9/20] = [0.45]
• P (B|A) = [frequency of pos given "yes"] = [0.67]
• P(A|B) = [0.47 or 47%]

New System: Design Strategy

We concluded that a good system Must Have:

• [Must have #1 - Be easy to determine the result. Normally we have bunches of tubes waiting to run the test. Efficiency is important for the test. If it takes sophisticated procedure to determine the result,the test engineer must be highly qualified which is not that common. ]
• [Must have #2 - Fast imaging results. Efficiency is essential when the test we are running is based on tons of samples.]
• [Must have #3& #4 - Simple OpenPCR software to operate and easy sample loading OpenPCR. Basically the same reason as #1. ]
• [Must have #5 - Small sample volume. Operating the whole test is easier with small samples. We don't have to waste time on collecting big amount of sample and then disposing it. ]

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

• [Want #1 - Low cost. Nobody wants to pay more than they have to.]
• [Want #2 - Portable and compact. A small portable machine makes use much simpler and hassle-free for use anywhere.]

We concluded that a good system Must Not Have:

• [Must Not Have #1 - Bad USB connectivity. The whole experiment was dependent on transferring data via USB. If it doesn't work the whole system is compromised.]
• [Must Not Have #2 - Fire hazard. Dangerous equipment is worse than broken equipment. Safety is #1.]

We concluded that a good system Should Avoid:

• [Should Avoid #1 - Inaccurate time readout. Accurately estimating time can increase productivity a lot.]
• [Should Avoid #2 - Slow amplification. Three hours for one batch of DNA is too long to wait in commercial applications.]

[[Image:]]==New System: Machine/ Device Engineering==

SYSTEM DESIGN

KEY FEATURES

We chose to include these new features

• Feature 1 - We thought we would provide the PCR with an additional flourimeter apparatus (designed like a mini-box that a smart phone can be incorporated into) as well as the necessary chemicals.
• Feature 2 - We would provide an app for smart phone that the user buys. The app would in theory do all the necessary calculations in one easy step (pic->result), basically skipping the image j analysis.

INSTRUCTIONS

New System: Protocols

DESIGN

We chose to include these new approaches/ features

• We thought we would provide the PCR with an additional flourimeter apperatus (designed like a mini-box that a smart phone can be incorperated into) as well as the necessary chemicals. The user would have to provide some DNA sample (saliva or blood maybe) and we would provide an app for smart phone that the user buys. The app would in theory do all the necessary calculations in one easy step (pic->result), basically skipping the image j analysis.

MATERIALS
Supplied In Kit
dNTPs
MgCl2
Reaction buffers
Taq DNA Polymerase

Supplied by User
Sample DNA
Forward and Reverse Primers
Fluorescent, cancer-specific probe
SYBR Green dye

PROTOCOLS

• PCR Protocol
• Step 1: The first step was to program the PCR machine and create the Thermal Cycler Program. The set-up used for the program is as follows

Stage one: 1 cycle, 95 degrees Celsius for 3 minutes
Stage two: 35 cycles, 95 degrees Celsius for 30 seconds, 57 degrees Celsius for 30 seconds, 72 degrees Celsius for 30 seconds
Stage three: 72 degrees Celsius for 3 minutes
Final Hold: 4 degrees Celsius

• Step 2: The second step is to add reagents to the tubes. Start by gathering the necessary materials to set-up the DNA samples (pipette, PCR reaction mix, 8 transfer pipettes)
• Step 3: Set the pipette to 50 microliters
• Step 4: Place the transfer pipette onto the pipette to prevent cross contamination (never re-use).
• Step 5: Use the pipette to transfer 50 microliters of each tube in the PCR reaction mix and transfer accordingly to the DNA sample tubes corresponding to the labels.
• Step 6: Place the set of mixed tubes into the Open PCR machine and shut it tightly.
• Step 7: Hook up the machine to a computer and run the Open PCR application with the pre set-up Thermal Cycler program.
• Step 8: Once the application finishes (approximately 1 hour and 30 minutes) the DNA sample has been set up.

• DNA Measurement and Analysis Protocol
• Step 1: The first step was to set up a camera on a smart phone that can take a photo of the reactions mixed with the SYBR Green most accurately. The following is what settings our group had for the camera.

Smart Phone Camera Settings

• The smartphone used for the camera was the Android Google Nexus 4. An app named Camera Self-Timer was installed to create a window in which the camera can shoot a picture in which the fluorimeter could then be covered in darkness for the most accurate results.
• Flash: No flash was used
• ISO setting: Unknown (could not be altered)
• White Balance: White balance was set on auto
• Exposure: Exposure was set on auto
• Saturation: Saturation was set on high
• Contrast: Contrast was set on low
• Step 2: The second step was to dilute the samples in SYBR Green. Each mixture was created by transferring the appropriate solution using a micropipette that was set to 80 μL. The mixtures were created corresponding to the following table...

Solutions Used for Calibration

 Calf Thymus DNA Solution (microg/mL) Volume 2X DNA Solution (uL) Volume SYBR GREEN I Solution (uL) Fina DNA concentration in PicoGreen Assay (ng/mL) 5 80 80 2.5 2 80 80 1 1 80 80 0.5 0.5 80 80 0.25 0.25 80 80 0.125 0 80 80 blank
• Step 3: The solutions above were mixed by combining drops of 80 μL onto the slide on the flourimeter. It was then lined up with the blue LED light that emitted from the flourimeter. After each transfer of a solution onto the slide, a different beaker was used to prevent cross-contamination.
• Step 4: The camera was placed on a cradle that was in approximately equal height of the fluorimeter. If the cradle and camera needed to be taller in order to be of equal height to the fluorimeter, the cradle was then placed on a stacked glass case until the camera was parallel. The distance from the cradle to the fluorimeter was about 7 cm. After creating a solution for calibration the camera was then set on a self-timer of 10 seconds and then the fluorimeter was encased in a box and covered for complete darkness for the most accurate results. After the beep indicating that the picture was taken the cycle was then complete and the process was then repeated for each solution.
• Step 5: The photos are uploaded onto the image j application. Then do the command Split Channels, splitting the pic into 3 new ones. Choose the one labeled green. The measurement tool should be set to measure IntDen (or intergrated density). Using the oval tool, set the oval over majority of the droplet, avoiding reflections as best as possible. Hit the command Measure under Analyze, and a table should come up, giving IntDen value

New System: Research and Development

BACKGROUND

Polymerase Chain Reaction (PCR) is a scientific method that utilizes DNA Polymerase to create a complimentary base strand from a template strand of DNA. Triphosphate nucleotides align with open DNA strands and DNA polymerase works to link the complementary nucleotide bases together growing strands through both condensation and hydroysis reactions. Through these mechanisms it is possible to target specific positions on the template DNA sequence that a scientist intends to amplify(PCR 1). When the PCR process is completed the targeted DNA sequence containing the single-nucleotide polymorphism (SNP) will have manufactured over a billion copies (amplicons). A SNP essentially is a type of gentic variation among organisms which represents a difference in a single nucleotide. For example, a SNP may replace a nucleotide cytosine (C) with a nucleotide thymine (T) in a certain part of an organisms DNA. These SNPs can be utilized as biological markers which in turn can help locate genes that have associative properties that contribute to the formation of harmful diseases.

The targeted SNP for this research was rs17879961. This SNP is found in Humans (Homo sapiens) and represents a variation class SNV, which stands for single nucleotide variation. Furthermore, This SNP is a variant of the CHEK2 gene (Checkpoint kinase 2) which if present in a person's genome may increase their risk of developing breast cancer. This SNV signifies a single base change from a Thymine (T) to a Cytosine (C) located on chromosome 22 and its clinical significance is classified as a pathogenic allele. For example, this mutation would alter the normal alelle ATT and the middle position resulting the cancer associated allele ACT.

DESIGN

Primers for PCR

Cancer allele forward primer: -> TTGAGAATG[TCA]CGTATGTAT
Cancer allele reverse primer: -> AACTCTTAC[AGT]GCATACATA

Disease alleles will yield PCR products because the target amplicon is only associated with the cancer DNA sequences. Thus primer annealing will following base pairing rulese when it binds with the template strand. For example, triphosphate nucleotides align with open DNA strands and DNA polymerase works to link the complementary nucleotide bases together growing strands through both condensation and hydroysis reactions. The presence of a primer is required so that polymerase can proceed with directing the new nucleotides in place. Through these mechanisms it is possible to target specific positions on the template DNA sequence that a scientist intends to amplify. When the PCR process is completed the targeted DNA sequence containing the single-nucleotide polymorphism (SNP) will have manufactured over a billion copies (amplicons).

Our primers address the following design needs

• The PCR method utilized above produces very effective results because the primer annealing follows base pairing rules therefore, by isolating the targeted SNP on the DNA template strand and amplifying them the cancerous genes can be detected easier.