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 (PCR)
The Polymerase Chain Reaciton (PCR) is a process that depends on a DNA Polymerase enzyme's ability to synthesize a strand that is complementary to a targeted fragment of DNA in a test tube mixture of all four DNA bases, which are adenine, cytosine, guanine and thymine. Besides that, the test tube mixture also must have two fragments of DNA of about 20 base pairs that are called primers. These primers should have sequences complementary to adjacent areas of each side of the targeted DNA segment. Since these two primers should match exactly with only the targeted DNA sequence, only this area would be defined and copied.
For the process of PCR, there are different stages, and they are collectively called the heating-cooling cycle. First of all, the mixture would be heated to separate the sides of the double-stranded DNA. After that, the mixture would be cooled to an optimum temperature for the primers to find and bind to whichever side of the separated DNA strands that they are complementary to. The temperature is then raised slightly to reach an optimum temperature for the polymerase, which is included in the test tube mixture, to extend the primers so that new complementary strands are generated. At the end of these first cycle, there will be two copies of the targeted DNA segment. The cycle is repeated multiple times to generate more and more copies of the targeted DNA segments. With the advent of technology in biological sciences and engineering, the entire process can be automated after all the correct components are added into a tube by using a thermocycler or a PCR machine such as the OpenPCR machine.
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
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; the tube was marked with a red dot on the cover), water in a scintillation vial, an Eppendorf tube filled with SYBR GREEN 1 (marked with a blue dot on the cover), several glass slides, a fluorimeter, a black box, a smartphone stand, a smartphone, a marker pen and several pipettes, a pipette with a blue strip, a pipette with a red strip, a pipette with a black strip and a pipette with a blank piece of paper taped to it, as well as a cup 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.
The pipette with the blue strip was used to put two large drops of SYBR GREEN I on the first two centered drops on the glass slide in the fluorimeter.(Warning: please do not dispose of any of the pipettes used until the entire process is complete!) Once that was done, The corresponding pipette for the positive control was used to add two drops of the sample of the positive control to the two drops of the SYBR GREEN I that was already on the glass slide. The light in the fluorimeter was aligned to ensure that it was going through the drop. The fluorimeter was covered with the black file box and the smartphone operator was allowed to take as many pictures as needed. After the pictures were taken, the pipette with the black strip was used to dispose of the drop on the glass slide into the cup. The slide was then pushed forward so that the light would be in the general direction of the next two centered holes on the glass slide.
The above process was then repeated using the next samples available, which were the negative control sample, sample 1a, 1b, 1c, 2a, 2b and 2c. Once the first five samples were done, shifting the glass slide down two holes after every sample, that glass slide was disposed off, and a new glass slide was used.
After these eight samples were run, two drops of SYBR GREEN I were put on the appropriate two centered drops before two drops of the Calf Thymus DNA from the Eppendrof tube with the red dot on top were added using the pipette with the red strip, and the above procedure was repeated. Lastly, after the slide was moved two holes down and two drops of SYBR GREEN I was added to the slide, the pipette with a blank piece of paper taped to it was used to add two drops of water from the scintillation vial on the slides, with the above procedure also being repeated on it. After all of this was done, the slides, pipettes, and all of the tubes containing the samples were disposed of in the correct waste containers.
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
✓
1A
Patient 1 Sample 1
Darker blue tone with dark vertical streaks
✓
1B
Patient 1 Sample 2
Lighter Blue tone with very thin, vertical, fluorescent streaks
✓
1C
Patient 1 Sample 3
Lighter Blue tone with very fine, hazy fluorescent streaks
✓
2A
Patient 2 Sample 1
Light blue tone with thick,vertical, hazy, fluorescent streaks
✓
2B
Patient 2 Sample 2
Darker blue tone of drop with a variation of thick and fine, vertical, fluorescent streaks
✓
2C
Patient 2 Sample 3
Darker toned drop with very fine, wispy, fluorescent streaks
✓
Positive Control (+)
Positive Control
Lighter blue drop with green round shape blobs displaced throughout
✓
Negative Control (-)
Negative control
Lighter Blue drop with light, vertical streaks. No apparent fluorescence.
✓
Water (H2O)
Water
Dark blue drop with no fluorescent streaks.
✓
Calf Thymus
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)
1a
69668
5.134
357674
same
1a background
69668
4.814
335409
same
1b
82952
14.383
1193134
same
1b background
82952
4.188
347364
same
1c
77078
29.946
2308181
same
1c background
77078
3.655
281721
same
2a
61076
78.280
4781047
same
2a background
61076
3.888
237445
same
2b
52524
50.560
2655589
same
2b background
52524
3.657
192067
same
2c
72068
21.003
1513624
same
2c background
72068
3.894
280632
same
+ Control
72476
97.093
7036889
same
+ Control background
72476
4.269
309387
same
- Control
48856
51.595
2520721
same
- Control background
48856
3.646
178142
same
Calf Thymus Standard
64720
76.988
4982678
same
Calf Thymus Standard background
64720
4.561
295190
same
Data Analyzer
Description
INTDEN with background subtracted
DNA Concentration, micrograms/ml
WATER BLANK
0
DNA CALF THYMUS, 2 microg/ml
2
PCR: Negative Control
0
0
PCR: Positive Control
6727502
2.870408202
PCR: Patient 1 ID #####, rep 1
22265
0.009499758
PCR: Patient 1 ID #####, rep 2
845770
0.36086279
PCR: Patient 1 ID #####, rep 3
-503540
-0.214844283
PCR: Patient 2 ID #####, rep 1
4543602
1.938608483
PCR: Patient 2 ID #####, rep 2
2463522
1.051105411
PCR: Patient 2 ID #####, rep 3
1232992
0.526077933
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
