Name: Alexandra Brunelle Research and Development Specialist
Name: Charul Singh Experimental Protocol Planner
Name: Alex Hoffmann Experimental Protocol Planner
Name: AJ Ciferno Open PCR Machine Engineer
Name: Haley Gjertsen Research and Development Specialist
Name: student Role(s)
LAB 1 WRITE-UP
Initial Machine Testing
The Original Design
Experimenting With the Connections
When we unplugged the LCD screen from the PCR machine, the machine's display screen stopped working, but information continued to transmit to the computer.
When we unplugged the white wire that connects the Thermocouple to the 16-tube PCR block, the machine's temperature measurement was disabled/no longer kept track of heat readings.
Test Run
(October 18th, 20th): PCR machine responded normally with no signs of malfunction
Protocols
Polymerase Chain Reaction
Reagent
Volume
Template DNA (20 ng)
0.2 µL
10 µM forward primer
1.0 µL
10 µM reverse primer
1.0 µL
GoTaq master mix
50.0 µL
dH20
47.8µL
Total Volume
100.0 µL
Sample Descriptions
Sample 1: Positive Control (contains cancer DNA template), Tube label: P
Sample 2: Negative Control (no DNA template), Tube label: N
Set up PCR Proceedures:
1. Label each tube differently and record label.
2. Insert reactants into their respective PCR tube.
3. Connect assembled PCR machine to a computer.
4. Customize settings in program to two stages:
Stage 1: 1 cycle, heat to 95 degrees Celcius for 3 minutes
Stage 2: 35 cycles, heat to 95 degrees Celsius for 30 seconds; cool to 57 degrees Celcius for 30 seconds
Stage 3: heat to 72 degrees Celcius for 30 seconds.
5. Start up the OpenPCR program previously installed on the computer.
6. Place tubes in slots in PCR machine and close lid tightly.
7. Lower the top of the lid until it just touches tops of tubes.
8. Press "Start" to run the PCR machine.
9. Record data.
Using a Flourimeter
Flourimeter Assembly Procedures:
1. Place the slide (glass facing down) onto the device.
2. Using a pipette place two drops of solution onto the center line two dots.
3. Switch on the blue light and adjust slide to allow the light to shine through the solution drops.
4. Now adjust the settings on the smart phone: no flash, contrast on the lowest setting, saturation on the highest setting and exposure on the highest setting
5. Place the phone in the holder facing the device. (should be about two inches away)
6. Cover the device and the phone and holder with the black box to keep out light.
7. Take picture. For best results, set a timer on the phone to be able to close the box and maximize darkness.
ImageJ Set Up Procedures:
1. Download ImageJ onto a laptop using the ImageJ disk.
2. Save desired photos to desk top.
3. Upload photos to ImageJ
4. Ready to work ImageJ
Research and Development
Specific Cancer Marker Detection - The Underlying Technology
PCR stands for Polymerase Chain Reaction. This reaction is used to amplify a specific segment of DNA that codes for cancer.
Some basic components involved in a PCR reaction are: Template DNA: the original strand of DNA that is going to be copied Primers: attach to the sites on the DNA strands that are at either end of the template DNA, they drive the PCR because they are powerful enzymes that drive replication (if they are able to attach the patient is negative for cancer) Taq Polymerase: reads the DNA code and matches new nucleotides with the template strand, it bind the pairs together with hydrogen bonds (example: A-T, C-G) These base pairs make it possible to make several copies of the DNA strands. Magnesium Chloride: a three atom molecule that binds to Taq polymerase as a cofactor to help it function properly dNTP's: deoxinucleotide triphosphate, they are the new nucleotides
A PCR functions by going through a series of thermal cycles:
In the first cycle the PCR machine heats up to 95 degrees Celsius. This unzips the DNA strand creating two separate DNA strand molecules and exposes the nucleotides we wish to look at. In the second cycle the temperature drops to 57 degrees Celsius which allows the primase to bind to the ends of the DNA strands. The third cycle heats up the DNA to 72 degrees Celsius to allow DNA replication by the enzyme Taq polymerase. These three cycles are repeated many more times, allowing for billions of DNA copies to be made. We need multiple copies of the DNA so that we can see if the patient tests positive for cancer.
r17879961
The single nucleotide polymorphism, or SNP, that codes for cancer is AACTCTTACACTCGATACAT; in a normal patient, the C is replaced with T. (AACTCTTACATTCGATACAT). The backwards part to these two sequences are TTGAGAATGTAAGCTATAGTA. The reason the C causes cancer in a patient is because there is an improper base pair, causing the primers not to bind resulting in single strands forming (C does does not code with A). After the PCR's thermal cycles are finished the cancerous patient's DNA will be single stranded and we will be able to see this after fluorescent dye is added and green. The fluorescent dye does not express the double stranded, non-cancerous patients.
Reliability: Bayes Rule:
We saw that the SNP is two hundred base pairs away, meaning every two hundred base pairs the missense mutation is present.
180 people sample size
C=cancer present
T=positive test
Bayes Theorm: p(hclC)=p(Clhc)p(hc)/p(C)
C/T=1.1%, T/T=98.9%
p(Clhc)=7.8% C's found in cancer patients
p(hc)=0.6795%
p(C)=5.3% C's in Finland
Below is a visual representation of what happens during a Polymerase Chain Reaction:
1. DNA Primers and tag polymerase is added to a section of DNA targeted for replication
2. As the temperature is heated to 95 degrees Celsius, the strand denatures
3. Then, the temperature cools, making the strands want to rebind. However, because there are many more primers than strands, the primers are able to attach to the front of each strand.
4. Next, the DNA is heated to 72 degrees to allow DNA replication to occur.
5. In order to produce many copies of this strand for analysis, steps 2-4 are repeated many times.
Results
DNA Concentration Based on INTDEN
Description
INTDEN w/ background subtracted
DNA Concentration, microg/ml
Water Blank
525630
0
DNA Calf Thymus, 2microg/ml
2372315
2
P
2121336
1.788410055
M1
-57048
-.048094793
M2
1436979
1.211457163
M3
2803635
2.363627933
N
76818
.064762057
F1
140996
.118867857
F2
74969
.063203242
F3
2880430
2.4283706
Sample
Integrated Density
DNA μg/mL
Conclusion
PCR: Negative Control
76818
.064762057
Negative
PCR: Positive Control
2121336
1.788410055
Positive
PCR: Patient 1 ID 74013, rep 1
-57048
-.048094793
Negative
PCR: Patient 1 ID 74013, rep 2
1436979
1.211457163
Positive
PCR: Patient 1 ID 74013, rep 3
2803635
2.363627933
Positive
PCR: Patient 2 ID 72825, rep 1
140996
.118867857
Negative
PCR: Patient 2 ID 72825, rep 2
74969
.063203242
Negative
PCR: Patient 2 ID 72825, rep 3
2880430
2.4283706
Positive
KEY
Sample = a set of DNA that is in one plastic tube
Integrated Density = Integrated Density is an extensive quantity. It is the sum of the values of the pixels in the image or in the selected part of the image. This sum is the same as the product of the mean gray value and the area. Integrated density(Background)-Integrated density(sample)=our data
DNA μg/mL =
Conclusion = A positive signal allowed the sample to appear green. No signal resulted in the sample appearing clear.
BME 103 Fall 2012
