Research and Design Specialist
Open PCR Machine Engineer
Open PCR Machine Engineer
Research and Design Specialist
LAB 3 WRITE-UP
Original System: PCR Results
PCR Test Results
| Sample Name || Ave. INTDEN* || Calculated μg/mL || Conclusion (pos/neg)
| Positive Control || 4967763 || 235.294 || N/A
| Negative Control || 472744 || 28.996 || N/A
| Tube Label: A1 Patient ID: 29013 rep 1 || 382980 || 23.306 || NEG
| Tube Label: A2 Patient ID: 29013 rep 2 || 280396 || 20.694 || NEG
| Tube Label: A3 Patient ID: 29013 rep 3 || 520011 || 28.832 || NEG
| Tube Label: B1 Patient ID: 13146 rep 1 || 5257810 || 250.616 || POS
| Tube Label: B2 Patient ID: 13146 rep 2 || 5240422 || 247.816 || POS
| Tube Label: B3 Patient ID: 13146 rep 3 || 5286556 || 253.391 || POS
* Ave. INTDEN = Average of ImageJ integrated density values from three Fluorimeter images
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 = The frequency of positive cancer DNA sequence conclusions = 9/20 = .45
- B = The frequency of total positive DNA sequences in tests = 26/60 = .433
- P (B|A)= The frequency of total positive DNA sequences in tests given a positive conclusion = 25/26 = .962
- P(A|B) = .999
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 = .55
- B = Frequency of Negative PCR Reactions = 34/60 = .566
- P (B|A) = Frequency of negative PCR given a cancer-negative conclusion = 32/34 = .941
- P(A|B) = .914
Calculation 3: The probability that the patient will develop cancer, given a cancer DNA sequence.
- A = The frequency of a "yes" cancer diagnosis = 7/20 = .35
- B = The frequency of a positive cancer diagnosis = 9/20 = .45
- P (B|A) = The frequency of positive cancer given a "yes" diagnosis = 6/7 = .857
- P(A|B) = .925
Calculation 4: The probability that the patient will not develop cancer, given a non-cancer DNA sequence.
- A = Frequency of patients who did not develop cancer = 13/20 = .65
- B = Frequency of a non-cancer DNA sequence = 11/20 = .55
- P (B|A) = Frequency of non-cancer DNA sequence given a patient did not develop cancer = 10/13 = .769
- P(A|B) = .909
New System: Design Strategy
We concluded that a good system Must Have:
- Results which are easily determined - When doing any kind of diagnostic test, having a clear result is imperative. A system which only distinguishes a positive from a negative result by a narrow margin is not nearly as clear as a system that has a clear distinction between the two. This is even more critical when testing for serious conditions such as cancer because an unclear result may cause a patient to not undergo necessary treatment or to live in fear unnecessarily. Neither option is beneficial.
- Small Sample volume - We need our system to be able to use small volumes of sample material. A system which requires large volumes of sample necessitates large volumes to be collected from a patient. This may result in several issues, the first being the toll on the patient of having it removed. The second problem becomes that of storing and processing a large volume. Smaller samples allow for higher volumes of tests to be done on a single patient's sample as well as multiple patients' samples to be tested in a high throughput method.
We concluded that we would Want a good system to have:
- Fewer steps - Each step allows another opportunity for error. Additional steps also require more hands-on time for processing, reducing the efficiency of the over-all process.
- High volume throughput - In a practical setting, multiple samples are likely to be in line for processing. Even when only doing 3 repetitions of 2 patients, the ability to do more of the process simultaneously would increase the number of readings which could be done per hour or per day. This would allow for more responsive diagnosis and treatment of the patients.
We concluded that a good system Must Not Have:
- Easy to mix samples during imaging - This is a source of error that can very easily be eliminated. Contamination of the samples in the very last step in which they are used is an unnecessary and unwanted source of potential incorrect readings.
- Fire Hazard - Safety issues take precedence. A slightly increased cost to raise the safety quality of an electric item which intentionally produces heat by replacing the wood casing with a less flammable material is a cost worth accepting.
We concluded that a good system Should Avoid:
- Minute adjustments of the phone - Adjustment and careful placement of the phone with each picture is a very time-consuming step in this process. Elimination of this procedure would aid in our other goals of higher volume and more streamlined processing.
- Manual processing of the images - Manual image processing is very time consuming. The processing step alone takes significant amounts of time. Additionally, because the processing is manual, there is more room for error. The area selected is unlikely to be always consistent; the variations in reflections might be improperly included or excluded. An automatic image processing system would eliminate time and error from this procedure.
New System: Machine/ Device Engineering
The Open PCR thermo cycler is a system designed to amplify desired segments of DNA causing an enhanced DNA polymerase chain reaction via multiple cycles of alternating temperatures. The apparatus creates an ideal environment for primers and DNA molecules to interact, and the number of cycles used in the reaction ultimately determines the amount of amplified DNA at the conclusion of the reaction. The open PCR machine is modified with a heating plate that presses against the sample containers to prevent water from condensing on the inside of the capsules from the mixtures.
An important modification that is essential for the open PCR system is to construct it in a way that it does not require a wooden shell. Because the machine has to be plugged into a power source and can be found in labs with other high-tech devices, there is definitely a fire hazard that accompanies the system. This issue can be easily solved by equipping the thermo cycler with a plastic, fire proof shell.
In order to modify the entire system, our team decided not to use a fluorimeter to measure the fluorescence of our PCR samples. The system is inefficient because the steps necessary to preform fluorimetry take too long and produce only fractions of the data neccessary to diagnose patients. Only one drop of sample can be analyzed with a camera, and it is inconvenient to use the imageJ software to digitally measure the data separately.
It would be much more efficient to analyze the PCR samples on a larger scale and in one quick step, so our new design would include a microwell plate and a micro plate spectrophotometer. A microwell plate has multiple wells that can hold up to three hundred samples out of a time, and it is a standard tool commonly used in clinical diagnostic testing laboratories throughout the world. This feature will enhance our ability to analyze large amounts of data, and the micro plate spectrophotometer is the perfect instrument capable of performing this process. Spectrophotometry quantitatively measures the intensity of light through an aqueous solution as a function of wavelength. Because absorbance is proportional to concentration, the amount of light that passes through the solutions will indicate the concentration of desired material, in this case the fluorescent primers involved in the process of diagnosing positive and negative patient samples.
We chose to include these new features
- Microwell plate - using a micro-well plate for the samples will dramatically reduce the chance of samples inadvertently mixing together. It also lends itself to a high through put system, allowing for up to 92 sample runs and controls to be prepared simultaneously.
- Microwell plate Reader - "reading" the samples with the micro-well plate reader will accomplish many of our goals. It allows for high volume processing by measuring multiple samples simultaneously. Using the reader will also eliminate the manual image processing required by use of the fluorimeter/camera/ImageJ system as the reader produces numerical results for each well.
Using the new system for data analysis will require less steps for faster completion. The PCR samples will be loaded into the microwell plate using a multichannel pipette. Once the samples are grouped into containers, the spectrophotometer is plugged in and warmed up. Next the device is connected to a computer via USB to record the data as the samples are analyzed. Finally, a specific wavelength is chosen to detect the fluorescence of the sample, and the well plate is loaded into the device for detection.
New System: Protocols
We chose to include these new approaches/ features
- Non-flamable case on PCR machine - Safety requirements take priority. The replacement of the case will reduce the fire risk of the repeatedly heated equipment.
- Use of microwell plates and a spectrophotometer- The micro well plates will allow hundreds of samples to be prepared simultaneously and read all at once. The spectrophotometer will eliminate the manual analysis of the image results. These aspects meet our goal of a high throughput system with fewer steps and more automated result analysis.
- Cancer detection as a fluorescent probe - This aids the requirement of clear results. Our system uses primers which attach to the DNA around the cancer specific sequence. A probe specific to the cancer marker sequence which releases a fluorescent particle when interacting with DNA polymerase is also added to the solution. When The sample is mixed with SYBR Green dye after amplification, the use of a spectrophotometer will allow the fluorescence of two wavelengths of light to be measured; Response to green light will confirm that the PCR ran successfully and response to orange will indicate that the cancer gene is present.
| Supplied in Kit || Supplied by User
| dNTPs ||Sample DNA
| MgCl2 || Forward and Reverse Primers
| Reaction buffers || Fluorescent, cancer-specific probe
| Taq DNA Polymerase || SYBR Green dye
Thermal Cycler Program
95°C for 3 minutes: Initial DNA strand is separated.
35 cycles of the following steps, each with a duration of 30 seconds:
- 95°C: Double strands of DNA separate.
- 57°C: Primers attach at ends of target DNA segment.
- 72°C: DNA polymerase activates and replicates target segment of DNA.
Final Hold 4°C for 3 minutes: PCR reaction is stopped.
PCR Reaction Mix
- Taq DNA polymerase
Add 25μL of the 2x Master Mix to each reaction tube
DNA Sample/Primer Mix
- 5μL Extracted sample of a particular patient's DNA
- 3μL Forward Primer
- 3μL Reverse Primer
- 4μL Fluorescent probe (cancer specific)
DNA Sample Set-up Procedure
- Prepare the PCR Reaction Mix and DNA/Primer sample solutions as prescribed above, ensuring a positive and negative cancer control are being used for comparison purposes
- Label reaction tubes for each sample or control
- Add 50μL of each DNA sample Mix to the correspondingly labeled reaction tube (using a new pipette tip for each transfer in order to avoid cross-contamination between samples)
- Place the reaction tubes into the thermocycler
- Run the thermocycler program detailed above so that PCR will occur in each reaction tube
Solutions Used for Calibration
| Calf Thymus DNA solution (microg/mL) || Volume of DNA Solution (μL) || Volume of SYBR GREEN I Dye solution (μL) || Final DNA concentration in PicoGreen Assay (ng/mL)
| 0 || 30 || 30 || blank
| .25 || 30 || 30 || .125
| .5 || 30 || 30 || .25
| 1 || 30 || 30 || .5
| 2 || 30 || 30 || 1
| 5 || 30 || 30 || 2.5
- DNA Measurement and Analysis Protocol
- Place 30μL of SYBR green dye in each well of a microwell plate to be used during this reading
- Add 30μL of each PCR product to a corresponding well
- Map locations of each solution so results have meaning
- Include calibration wells as shown above so that a baseline reading for DNA content is available:
- Load microwell plate into spectrophotometer
- Set spectrophotometer to 500nm and read The results produced are for detecting all DNA- All samples should be positive except for the water in the calibration row. If not, something has gone wrong.
- Export data
- Set spectrophotometer to 630nm and read The results produced are for indicating if a sample is cancer-positive or not.
- Export data
- Perform Bayesian analysis on results to determine reliability.
New System: Research and Development
CHEK 2, or Checkpoint Kinase 2, is a gene in the human genome that acts as a suppressor for the growth and division of a cell. When a portion of DNA becomes damaged, this gene is activated which halts the G1 phase of cell division, preventing the cell from entering meiosis. Whenever any damage is done to the DNA, CHEK 2 suppresses growth and also helps repair that damaged DNA. However, an SNP (or single nucleotide polymorphism) can occur in this gene. An SNP causes a single nucleotide to be changed in a way that alters how the sequence is supposed to be, and thus alters its function. The SNP associated with CHEK 2 can actually cause the process of cell death to stop, which means more cell division, indicating a presence of cancer. Specifically, it is most associated with breast cancer. The cells cannot stop growing because they can't die, and thus CHEK 2 does the opposite of what it's supposed to do. The associated sequence for CHEK 2 is ATT, and the cancer-associated sequence is ACT. By knowing this, we can target the allele associated with the cancer SNP of CHEK 2, and thus determine whether a patient may or may not have cancer.
Primers for PCR
Because the cancer gene is a mutation in with the allele "ACT" as opposed to "ATT", a primer can be designed and created that acts as a compliment to the cancer associated strand of DNA. However, rather than creating a fluorescent primer that fluoresces green at the presence of DNA, a dye will be made that fluoresces for the presence of any DNA. What replaces the positive cancer test is a probe that will fluoresce orange when attached to the caner-specific DNA. This way, a negative control is present that tells us there isn't a cancer-segment, whereas the positive control will fluoresce orange if there is a cancer-segment of DNA. Also, we know whether or not the negative control works because it will fluoresce green if negative. If there is no fluorescence, then we know something went wrong with the dye.
Forward Primer: [ A C G T A T G T A T]
Reverse Primer: [ T G C A T A C A T A]
Our primers address the following design needs
- Introduction of a fluorescent, cancer-specific probe - This maintains our goal of having clear results. Reading the samples in the spectrophotometer will allow for clear indication of fluorescence when the probe is activated in cancer-positive samples during replication.
- All sample DNA is amplified by using primers which surround the cancer-specific sequence - Having a signal for all DNA helps to confirm that any negative result for cancer is a real negative result and not simply an indication that PCR (or some other step of the process) went wrong.