The PCR machine heats up the DNA so that enzymes can "unzip" the two strands of DNA. This process happens in cycles so that the DNA will seperate and duplicate a multitude of times. A certain amount of primer is used to duplicate the DNA specific to the amount of original DNA. By amplifying the amount of DNA, a proper diagnosis of a certain gene can be made.
Experimenting With the Connections
When we unplugged the LCD screen from the circuit board, the machine's screen shut off.
When we unplugged the white wire that connects the circuit board to tube PCR block, the machine stopped reading the temperature.
Test Run
We first tested the PCR machine on the 18th of October, 2012. The LCD screen readings matched the reading on the computer PCR program, and the machine worked well and efficiently.
Protocols
Polymerase Chain Reaction
Reagent
Volume
Template DNA (20ng)
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
During Week 2, there were eight samples that PCR was run on. These samples consisted of a positive control, a negative control, three samples from Patient 1 and three samples from Patient 2.
Patient 1
ID #: 11640
Gender: Female
Age: 54 years old
Patient 2
ID #: 29292
Gender: Male
Age: 63 years old
Fluorimeter Protocol
The eight samples from the Polymerase Chain Reaction experiment were used in this experiment. In addition to that, eight Eppendrof tubes filled with 400ml of buffer to maximise fluorescence, a Eppendorf tube filled with DNA (calf thymus standard at 2 micrograms/ml), water in a scintillation vial, an Eppendorf tube filled with SYBR GREEN 1, several glass slides, a fluorimeter, a black box, a smartphone stand, a smartphone, a marker pen and several pipettes were used.
The eight Eppendrof tubes were labeled using the marker pen according to the eight samples from the Polymerase Chain Reaction experiment; they were labeled +, -, 1a, 1b, 1c, 2a, 2b and 2c. Similarly, for each Eppendorf tube (ten altogether), a pipette for each tube was given a corresponding label. Using the corresponding pipettes, the eight samples from the Polymerase Chain Reaction experiment were transferred from their PCR tubes to their corresponding Eppendrof tubes. The pipettes were all kept carefully separate from each other.
The fluorimeter was set up according to the image shown.
Flourimeter Measurements
Tubes
Description
Eppendorf Tube Label
Pipette Label
Positive Control
+
+
Negative Control
-
-
Patient 1 Sample 1
1a
1a
Patient 1 Sample 2
1b
1b
Patient 1 Sample 3
1c
1c
Patient 2 Sample 1
2a
2a
Patient 2 Sample 2
2b
2b
Patient 2 Sample 3
2c
2c
DNA Measurement Operator: Smartphone
Image Number
2 Drops SYBRGr
2 Drops
Comments
✓
(insert info)
Patient 1 Sample 1
Darker blue tone with dark vertical streaks
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Patient 1 Sample 2
Lighter Blue tone with very thin, vertical, fluorescent streaks
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Patient 1 Sample 3
Lighter Blue tone with very fine, hazy fluorescent streaks
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Patient 2 Sample 1 |
style = "width: 25px;" | Light blue tone with thick,vertical, hazy, fluorescent streaks
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Patient 2 Sample 2
Darker blue tone of drop with a variation of thick and fine, vertical, fluorescent streaks
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Patient 2 Sample 3
Darker toned drop with very fine, wispy, fluorescent streaks
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Positive Control
Lighter blue drop with green round shape blobs displaced throughout
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Negative control
Lighter Blue drop with light, vertical streaks. No apparent fluorescence.
(insert data here)
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Water
Dark blue drop with no fluorescent streaks.
(insert data here)
(insert data here)
Calf thymus
Dark blue drop with hazy, green, abstract round shapes distributed around the blob.
ImageJ Software Processor
Sample or Background ID
Area and x, y, w, h, info
Mean Pixel Value INTDEN
RAWINTDEN
INTDEN (IF DIFFERENT)
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Data Analyzer
Description
INTDEN with background subtracted
DNA Concentration, micrograms/ml
WATER BLANK
0
DNA CALF THYMUS, 2 microg/ml
2
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Research and Development
Specific Cancer Marker Detection - The Underlying Technology
PCR helps to detected certain types of genes. In this case it is used to find out a specific type of cancer. In the process of detecting the cancer, primers are made to compliment a DNA strand that has the cancer gene in it. If a subject has the cancer in their DNA, the primers will bind to strand, whereas a subject without the cancer would not have a primer attach to their DNA strand.
The r1787996 SNP is linked to the cancer sequence. The codon ATC is the sequence for cancer where the ATT means there is no cancer. In PCR, the ATC cancer sequence is detected because the primers will only attach to the DNA strands that have the ATC sequence. The ATT, non-cancer, strands will not bind with the primers. Only the combined primer DNA strand will be detected thus alerting for cancer.
(BONUS points: Use a program like Powerpoint, Word, Illustrator, Microsoft Paint, etc. to illustrate how primers bind to the cancer DNA template, and how Taq polymerases amplify the DNA. Screen-captures from the OpenPCR tutorial might be useful. Be sure to credit the source if you borrow images.)
BME 103 Fall 2012
