Name: Christian Vargas Experimental Protocol Planner
LAB 1 WRITE-UP
(Please finish by 11/7/2012)
Initial Machine Testing
The Original Design
The Polymerase Chain Reaction(PCR) machine essentially tests sequences of DNA for variations in nucleotides. This simple device is portable, easy to use, and relatively inexpensive. It is able to test up to 16 samples of DNA at a time and can be connected to a computer for ease of use. The machine heats up DNA samples so the samples disassociate allowing a primer to connect to the sequences of DNA and then the machine cools the DNA samples down with the primer in place.
Experimenting With the Connections
When the mounting plate was unplugged from the circuit board, the machine the LCD light and the menu on the PCR machine shut off.
When the white wire that connects the circuit board to the sample holder was unplugged, the temperature on the menu on the PCR machine dropped from room temperature to -40.0 degrees Celsius. The conclusion is that the white wire was the temperature sensor wire.
Test Run
The date the OpenPCR Machine #4 was first tested was October 24, 2012. The test tubes were put into the machine and the handle was secured over them. There was a little difficulty in determining when to stop turning the handle. The software itself was simple and easy to use and there were no problems in running the OpenPCR software. After the reaction was complete, the tubes were taken out, labeled, and stored in a refrigerated area.
Protocols
Polymerase Chain Reaction
The Polymerase Chain Reaction works by amplifying DNA through the use of a PCR machine and therefore producing a multitude of copies of DNA sequences. These copies can then be further studied to diagnose hereditary and infectious diseases, such as cancer and HIV.
Thermal Cycling Process:
1)The DNA is first heated to 95 degrees Celsius which will in turn unzip the DNA to expose its bases and create two one-stranded strips
2)After primer is added to the solution, the DNA is then cooled down to 57 degrees Celsius so that the said primer can attach to a template sequence to form a forward primer.
3)The DNA is then once more heated to 72 degrees Celsius so that the replication process can be completed.
4)This process is repeated 30 more times to acquire a greater number of DNA samples.
PCR Master Mix Components:
-GoTaq® Colorless Master Mix, 2X 25μl
-upstream primer 10μM
-downstream primer 10μM
-DNA template 1–5μl
-Nuclease-Free Water to 50μl
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
dH2O
47.8 µL
Total Volume
100.0 µL
Patient Sample Descriptions:
Positive Control: Cancer DNA Template
Negative Control: DNA Template
Patient 1, Replicates(1,2,3):
ID: 80175
Gender: Female
Age: 59
Patient 2, Replicates(1,2,3):
ID: 57483
Gender: Male
Age: 56
Specific Cancer Marker Detection - The Underlying Technology
What is the function of each component of a PCR reaction?
Template DNA:
A double-stranded segment of DNA that encodes either a cancerous gene or a normal gene
Primers:
Short segments of DNA that bind to a specific sequence of nucleotides (binds to cancer gene)
Taq Polymerase:
A protein that serves as the catalyst for the DNA replication; grabs extra nucleotides within the solution and binds them to the "unzipped" strands
Magnesium Chloride:
A cofactor that binds to the Taq Polymerase and affects the speed of the reaction; positive correlation between amount of magnesium chloride and reaction speed
dNTP's:
Deoxynucleotide triphosphates; extra nucleotide bases in solution that are able to be grabbed and synthesized by Taq Polymerase to replicate DNA strands beyond the primer sequence
What happens during each step of thermal cycling?
• At 95° Celsius: DNA melts and "unzips" to create two one-stranded strips, primers are added to the solution
• At 57°Celsius: Primers attach to the corresponding template sequence they complement, forming one forward primer and one reverse primer
• At 72° Celsius: Taq Polymerase finishes the replication process with the use of dNTP's and magnesium chloride
Why does a cancer gene produce a positive result while a normal gene produces a negative?
• Because the cancer gene has the specific sequence of nucleotides that the primers can bond to, the process can continue and the DNA can be replicated; however, since the normal gene does not include that specific sequence, the primers can never bond to the strands and the process cannot take place.
Relation to Bayes' Rule:
In order to achieve accuracy of the amplification process as an actual determinant for cancer, Bayes' Rule must be used. This will compute the probability of true positives in coordination with false positives and false negatives to give a realistic prediction for how reliable the PCR process is in detecting the true cancer patients.
Image Credit to OpenPCR.org/use-it/
Results
Sample
Integrated Density
DNA μg/mL
Conclusion
PCR: Negative Control
2773313
0
Negative for gene
PCR: Positive Control
26759481
2
Positive for gene
PCR: Patient 1 ID 80175, rep 1
17085185
1.27694
Positive for gene
PCR: Patient 1 ID 80175, rep 2
12388707
0.92593
Positive for gene
PCR: Patient 1 ID 80175, rep 3
4620549
0.345339
Negative for gene
PCR: Patient 2 ID 57483, rep 1
16031260
1.19817
Positive for gene
PCR: Patient 2 ID 57483, rep 2
11636055
0.869677
Positive for gene
PCR: Patient 2 ID 57483, rep 3
18928511
1.41471
Positive for gene
KEY
Sample = A sample is a set of DNA contained within 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 selection equivalent to the product of the area and mean gray value. We subtracted the integrated density of the background from the integrated density of our sample to obtain our data.
DNA μg/mL = The concentration was obtained by dividing the integrated density of the sample with the background subtracted by the integrated density of the positive control with the background subtracted and multiplying by 2.
Conclusion = Whether or not the sample was positive for the cancer gene.
BME 103 Fall 2012
