The flourimeter was placed on top of the white plastic tray in order to achieve an ideal camera angle.
The camera was adjusted to the above settings with a timer delay of 5 seconds. This allowed ample time to place the cover on and for the camera to refocus to the darker lighting.
The camera was inserted in the center of the cradle, which was then placed on top of a small book and table to align with the height of the flourimeter. The angle obtained was as close as possible to level with the slide.
The camera was placed as close as possible to the tray without sacrificing image quality.
Distance between the smart phone cradle and drop = 5.9cm
Solutions Used for Calibration
Initial Concentration of 2X Calf Thymus DNA solution (μg/mL)
Volume of the 2x DNA solution (μL)
Volume of SYBR Green I Dye solution (μL)
Final DNA concentration in SYBR Green I solution (μg/mL)
5
80
80
2.5
2
80
80
1
1
80
80
0.5
0.5
80
80
0.25
0.25
80
80
0.125
0
80
80
0
Placing Samples onto the Fluorimeter
Once camera and flourimeter set up is complete, switch the flourimeter light to the "on" position.
Insert the slide, with the rough, hydrophobic side facing up. Adjust its position so that the flourimeter light is focused between the first two rows of dots.
Adjust the micropipettor to 80μL and insert a clean tip.
Draw 80μL of SYBR Green solution and place it between the center two dots on the row where the light is focused. It is very important to dispense the solution slowly and precisely to ensure the drop maintains intact and centered on the slide.
Discard the tip and replace with a clean one.
Draw 80μL of the DNA sample to be tested and dispense it directly onto the SYBR Green solution drop on the slide. Again, dispense slowly and ensure the combined dot remains centered on the slide.
Verify the camera angle and focus is still correct. After pressing the shutter button (to begin the timer countdown), cover the flourimeter and camera immediately to ensure that no outside light is allowed into the picture.
Once the image has been captured, remove the box cover from the flourimeter. Make certain that the image is of good quality and not excessively blurry or dark.
Using the micropipettor, transfer the solution from the slide into a biohazard waste collection container.
Adjust the slide to the next position and repeat Steps 3-9 for the remaining samples.
Data Analysis
Representative Images of Negative and Positive Samples
Negative Control
Positive Control
As per lab TA instructions, the edges of the samples are left out of the selection because they are the saturated points where the light is entering and exiting the sample drop. Therefore, they are not an accurate representation of the fluorescence of the sample.
Image J Values for All Calibrator Samples
Calibration Image Analysis
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Final DNA concentration in SYBR Green I solutions (µg/mL)
Area
Mean Pixel Value
RAWINTDEN of Drop
RAWINTDEN of Background
RAWINTDEN Of Drop-Background
0
30975
17.181
532191
5781
526410
0
31458
20.821
654990
1891
653099
0
32604
17.144
558957
15477
543480
0.125
37978
33.656
1278187
17746
1260441
0.125
38395
32.996
1266896
24457
1242439
0.125
34625
32.829
1136700
21623
1115077
0.25
29562
52.354
1547696
11930
1535766
0.25
26864
49.443
1328240
15886
1312354
0.25
29984
51.175
1534431
13316
1521115
0.5
32864
74.415
2445574
3982
2441592
0.5
34112
73.774
2516595
9849
2506746
0.5
31832
75.691
2409393
10711
2398682
1
50484
87.329
4408713
111899
4296814
1
50484
86.787
4381363
103104
4278259
1
50484
86.227
4353097
101216
4251881
2.5
73954
173.506
12831475
964780
11866695
2.5
73954
173.696
12845494
956051
11889443
2.5
73954
173.442
12826756
1041565
11785191
Calibration Statistics
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Final DNA concentration in SYBR Green 1 solution (µg/mL)
RAWINTDEN of Drop-Background
Mean
Standard Deviation
1
2
3
0
526410
653099
543480
574329.6667
68748.10638
0.125
1260441
1242439
1115077
1205985.667
79242.08009
0.25
1535766
1312354
1521115
1456411.667
124972.4827
0.5
2441592
2506746
2398682
2449006.667
54412.22276
1
4296814
4278259
4251881
4275651.333
22579.71582
2.5
11866695
11889443
11785191
11847109.67
54816.13665
Calibration curve
Note: Although difficult to see, the error bars are included in the plots. The error bar is visible on the 3rd point on the plot, but very hard to see on any others. They are inserted using the standard deviation as the positive and negative values.
PCR Results Summary
PCR Product Dilutions
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PCR Product Tube Label
Volume of DILUTED PCR Product Solution (µL)
Volume of SYBR Green I Dye Solution (µL)
Dilution 1
Dilution 2
Total Dilution (simplified fraction)
G6 P
80
80
0.167
0.500
1/12
G6 N
80
80
0.167
0.500
1/12
G6 1-1
80
80
0.167
0.500
1/12
G6 1-2
80
80
0.167
0.500
1/12
G6 1-3
80
80
0.167
0.500
1/12
G6 2-1
80
80
0.167
0.500
1/12
G6 2-2
80
80
0.167
0.500
1/12
G6 2-3
80
80
0.167
0.500
1/12
PCR Reaction Image Analysis
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PCR Product Tube Label
Area
Mean Pixel Value
RAWINTDEN of Drop
RAWINTDEN of Background
RAWINTDEN Of Drop-Background
G6 P
39540
120.744
4774226
21528
4752698
G6 P
39948
120.453
4811849
21503
4790346
G6 P
39900
121.476
4846888
23388
4823500
G6 N
42720
25.511
1089830
14272
1075558
G6 N
43552
24.732
1077119
16405
1060714
G6 N
42984
24.245
1042156
17164
1024992
G6 1-1
35540
21.714
771732
9518
762214
G6 1-1
33557
21.219
712030
10974
701056
G6 1-1
28312
18.53
524622
12302
512320
G6 1-2
42539
25.604
1089181
25385
1063796
G6 1-2
42428
24.098
1022441
15859
1006582
G6 1-2
41906
25.339
1061852
18822
1043030
G6 1-3
26488
32.018
848089
8064
840025
G6 1-3
27412
31.644
867428
9569
857859
G6 1-3
24780
29.305
726186
4608
721578
G6 2-1
37262
40.434
1506645
16071
1490574
G6 2-1
37326
39.86
1487802
11371
1476431
G6 2-1
36990
39.389
1457017
13287
1443730
G6 2-2
35341
31.836
1125114
14859
1110255
G6 2-2
36788
29.623
1089761
17610
1072151
G6 2-2
36242
31.976
1158859
15033
1143826
G6 2-3
30728
38.906
1195515
8279
1187236
G6 2-3
30530
39.758
1213806
7825
1205981
G6 2-3
30472
40.324
1228740
4343
1224397
PCR Reaction Statistics
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PCR Product Tube Label
RAWINTDEN of Drop-Background
Mean
Standard Deviation
1
2
3
G6 P
4752698
4790346
4823500
4788848
35424.76258
G6 N
1075558
1060714
1024992
1053754.667
25991.428
G6 1-1
762214
701056
512320
658530
130261.6533
G6 1-2
1063796
1006582
1043030
1037802.667
28962.97998
G6 1-3
840025
857859
721578
806487.3333
74072.32313
G6 2-1
1490574
1476431
1443730
1470245
24026.86061
G6 2-2
1110255
1072151
1143826
1108744
35861.38239
G6 2-3
1187236
1205981
1224397
1205871.333
18580.74273
Calculations
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PCR Product Tube Label
Mean RAWINTDENT Drop-Background
PCR Product Concentration (μg/mL)
Initial PCR Product Concentration (μg/mL)
G6 P
4788848
1.1082
13.2984
G6 N
1053754.667
0.174426667
2.09312
G6 1-1
658530
0.0756205
0.907446
G6 1-2
1037802.667
0.170438667
2.045264
G6 1-3
806487.3333
0.112609833
1.351318
G6 2-1
1470245
0.27854925
3.342591
G6 2-2
1108744
0.188174
2.258088
G6 2-3
1205871.333
0.212455833
2.54947
Our positive control PCR result was 13.2984 μg/mL
Our negative control PCR result was 2.09312 μg/mL
Observed results
Patient 11105 (Patient 1):
The samples of Patient 11105's DNA fluoresced very little green in the fluorimeter. The image of the drop was almost completely a dark blue color. There was a lighter color on both edges, but this was only from the fluorimeter light entrance and exit paths through the sample. Once Patient 11105's images were split by color in the ImageJ program, the green channel sample was extremely dark. This indicates that there was a minute amount of green light fluorescing in the sample. Replications 1, 2, and 3 returned Initial PCR Product Concentrations of 0.907446 μg/mL, 2.045264 μg/mL, and 1.351318 μg/mL, respectively.
Patient 22923 (Patient 2):
The samples of Patient 222923's DNA also fluoresced very little green in the flourimeter. This patient's samples also was a dark blue color, with lighter blue edges. When Patient 222923's sample images were split using ImageJ, the green channel was slightly brighter than Patient 11105, but still very dark in general. This indicates that there was a very small amount of green light fluorescing in the sample. Replications 1, 2, and 3 returned Initial PCR Product Concentrations of 3.342591 μg/mL, 2.258088 μg/mL, and 2.54947 μg/mL, respectively.
Conclusions
Patient 11105 (Patient 1):
Patient 11105's three replicates had an average Initial PCR Product Concentration of 1.434676 μg/mL. This puts it well below the positive control concentration of 13.2984 μg/mL, and much closer to the negative control sample's concentration of 2.09312 μg/mL. Therefore, it can be concluded that Patient 11105 does not carry the disease gene.
Patient 22923 (Patient 2):
Patient 22923's three replicates had an average Initial PCR Product Concentration of 2.716716333 μg/mL. This puts it below the positive control concentration of 13.2984 μg/mL, and much closer to the negative control sample's concentration of 2.09312 μg/mL. Therefore, it can be concluded that Patient 22923 does not carry the disease gene.
SNP Information & Primer Design
Background: About the Disease SNP
Nucleotides are organic molecules that serve as the monometers, also known as subunits, of nucleic acids like DNA and RNA. A polymorphism is when there is a variation in a DNA sequence where one nucleotide in the genome differs between paired chromosomes. For example, when an adenine nucleotide is paired with a guanine.
A common SNP (single nucleotide polymorphism) is the rs268. This variation is commonly found in the homo sapien species. It is located on the 8:19956018 chromosome and clinically classified as a pathogenic polymorphism. The rs268 is associated with the LPL gene. It is associated with many major diseases, including metabolic conditions, heart attack, strokes, and high cholesterol. Rs268 is a lipoprotein lipase, known as LPL for short.
An allele is simply a variant form of a gene. In the rs268 SNP, the AAT (non-diseased) gene is modified to the AGT (diseased) variant. The numerical value of the position of the SNP in the human genome is 19956018.
Primer Design and Testing
The diseased primer returned a result of "no match found" when run through the primer test because a healthy human genome does not possess this mutation (AGT). The human genome, does however, produce the healthy non-diseased primer (AAT), which did successfully return a result when run through the primer test.