Name: Mayuri Gupta Role: Open PCR machine Engineer
LAB 1 WRITE-UP
Initial Machine Testing
The Original Design
This is the internal view of the PCR machine that was used in the lab experiment.
Experimenting With the Connections
Above is an image of important parts in a PCR machine.
Before we even began our experiment we had to assess and overlook these specific parts so that we could better learn their function and how that contributes to the overall machine. Part 1 displays the Aluminum Plate, which is designed to both contain and give off the heat necessary to perform the experiment once it is put down over Part 2, the Well Plate. The Well Plate is simply designed to hold the test samples and measure the heat around them, in order to ensure the samples are receiving the right temperature.
Part 3 is the PCB (Printed Circuit Board) of the LCD screen, which means it is responsible for the images and readouts that appear on the screen. This part is important because the entire flow of information comes into here, so that the user can gather information on the progress and results of the machine. Part 4 is the Heat Sink Fan, which works to cool the entire machine by blowing the hot air out from the machine, to prevent overheating. Part 5 is the Rsenda ATX (Advanced Technology eXtended), a type of motherboard. A motherboard is essentially a large PCB which holds the CPU (central processing unit) and is in charge of memory. The final part is Part 6, which is the PCB of the open PCR Circuit Board. This is much like the motherboard, but deals with current and continuous flow of information and signals through the wires. No real memory is stored here.
Connection and Process Testing
In order to see the connection between various parts we ran two tests on October 18th, 2012, where we unplugged a single wire in each.
When we unplugged Part 3 (PCB of LCD) from Part 6 (PCB of open PCR circuit board), the LCD did not turn on at all.
When we unplugged the white wire that connects Part 6 (PCB of open PCR circuit board) to Part 2 (Well Plate), strange images began to show up on the screen, as a result of no information flowing from Part 2 to Part 6.
Protocols
Polymerase Chain Reaction
Polymerase chain reaction is basically molecular photocopying and the process or technique used to make copies of small segments of DNA because it only targets specific segments of the DNA and that's what makes it useful. PCR works by mixing two DNA fragments, also known as primers which are about 20 bases long. The mixture is then heated and denatured and then the primers bind to their complementary sequences on the separated strands. Then, the polymerase extends primers into new complementary strands and it goes through about 30 cycles. PCR products are useful and can be used in many experiments like DNA fingerprinting and detection of viruses.
Collect three replicate DNA samples from two patients
Create a new program on the Open PCR system
Create three stages
Stage 1: 1 cycle, 95°C for 3 minutes
Stage 2: 30 cycles for 95°C for 30 seconds, 57°C for 30 seconds, 72°C for 30 seconds
Stage 3. 72°C for 3 minutes
Final hold: 4°C
The DNA samples are 50 μL each, get the patient's ID and label the the each tube.
PCR reaction mix - Mix contains Taq DNA polymerase, MgCl2, dNTP's, forward primer, and reverse primer.
The primers are artificial DNA, designed to match the chain of DNA we want.
Taq polymerase is the enzyme that binds to the end of the new chain and recreates the separated DNA.
Mgcl2 binds to Taq as a co-factor and helps Taq to function appropriately, and affects the speed of the Taq binding to the loose strands.
dNTp's is dioxnucleotidetriphosphate. this is what is used to recreate the second DNA strands.
The 8 tubes of mixtures will then go through the cycles in the PCR system.
During each step of the thermal cycling, the DNA is unzipped and heated to 95°C to break the H-bonds between the 2 strands. This exposes the part we want in this lab experiment. Then, the primer binds to the trage we want without cancer marker, this primer won't bind. Next, the temperature will be dropped to 57°C in order to bind the primer. Later, it is heat it back up to 72°C with the Taq to reform and duplicate DNA strands. Finally, this thermal cycling is repeated for amplification and add dye that binds specifically to DNA for detection.
The Components of the GoTaq® Colorless Master Mix
"dNTP's, MgCl2, and reaction buffers at optimal concentration for efficient amplification of DN templates by PCR."
Volumes Used for Mixture
Table 1
Reagent
Volume
Template DNA (20 ng)
10.2 μL
10 μM reverse primer
1.0 μL
dH2O
47.8 μL
0 μM forward primer
1.0 μL
GoTaq master mix
50.0 μL
Total Volume
100.0 μL
DNA Samples (8)
Positive Control: Cancer DNA Template Tube label A
(Add your work from Week 3, Part 2 here aka, steps of assembly to the flourimeter)
Research and Development
Specific Cancer Marker Detection - The Underlying Technology
There is a genetic relation to having cancer or not when an individual is over the age of 40. The specific gene, in this case, is located on chromosome 22, r17879961. To test an human's DNA for this cancer gene, we have go through a series of reactions called PCR on the DNA for replication and amplification of the patient's DNA strand.
As previously explained shown in Protocols, primers are needed for the DNA replication, a forward and a reverse. One at the "completing" strand of the double strand, and one as the cancer detecting strand. On chromosome 22, the primer to detect r17879961 defect has to have the changed nucleotide.
The reverse primer used:
AACTCTTACA-C-TCGATACAT
The forward primer used:
TTGAGAATGT-C-AGCTATGTA
In this particular instance, it is a adenine replaced by the cancerous-related nucleotide cytosine as shown above as the bold C. The primer will only bind to the matching sequence (testing sequence) because of the A-T, and C-G pairings. Since an A has been replaced with a C, the primer can't bind to the DNA strand beyond that specific nucleotide in the sequence. Due to the open double helix, further steps in the reaction will not happen, and no amplification will happen. No amplification means no visible results and the test will run negative.
If the cancer gene is present, then the matching primer will completely bind to the DNA strand. When this happens, the amplification sequence will be able to precede, and this will show up as a positive result.
Image by: Alyssa Alexander
This is all in theory of course, and should work perfectly every time. But there are many factors to consider. For instance, not every one who has cancer has the cancer gene.
Reliability and Accuracy of Specific Cancer Marker Detection
Based on Bayesian reasoning, the probability that someone will test positive that will actually have the disease has approximately 7.8 percent. However, The chance that someone does not test positive, and doesn't have cancer is about 99.8%.
These four percentages generally mean that the test itself is generally in favor of testing negative, which means there are less chances to have false diagnosis and/or treatments.
These percentages resulted from the following equation: [math]\displaystyle{ Positive Predictive Value = (True Positive)/(True Positive + False Positive) }[/math]
where p(C/T) is the probability of a person with positive results will have cancer out of the entire patients participating. p(C) is the probability of having cancer present, p(T/C) is the percent of patients who tested positive with have cancer and had it. n = not
The values of the these equations are taken from the general statistics from tests performed on 'x' amount of patients. For instance, the direct results showed that 80% of the people with cancer, tested positive, this value would be used as p(T/C) because that is the test running positive and having cancer. The value p(T/nC) would be the percentage of people who tested positive but do not actually have cancer, which resulted to be 9.6%. Additionally, the general statistics were 90.4% of patients will test negative and will not have cancer, and 20% percent of people with cancer will run negative.
The calculated p(C/T) was used as the "True Positive" in the original two equations (PPV and NPV). The same for the false positive, true negative and false negative can be solved similarly as well.
BME 103 Fall 2012
