Distance between the smart phone cradle and drop = 4 cm
Solutions Used for Calibration
Concentration of 2X Calf Thymus DNA solution (micrograms/mL)
Volume of the 2X DNA solution (μL)
Volume of the 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
Set up the fluorimeter so that the phone taking pictures is around 4 cm away and on the same height as the laser.
Insert a new slide rough side up into the fluorimeter.
Adjust the slide so that the small circles are bisected by the laser.
Choose one of the circles intersected by the laser and add 80 μL of SYBR Green I solution.
Choose another circle intersected by the laser that is next to the circle with the SYBR Green I solution just added and add 80 μL of Calibration or Sample.
The drops should meet in between the circles and stay in the beam of the laser.
Take the picture of the sample and make sure the photo is focused.
Data Analysis
Representative Images of Negative and Positive Samples
Positive
Negative
Image J Values for All Calibrator Samples
Image J Values
'
Concentration:Trial
Area
Mean
RawIntDenDrop
RawIntDenBackground
Drop - Background
0:01
30296
6.622
200607
370484
-169877
0:02
33176
5.065
168041
362721
-194680
0:03
42752
16.918
723296
222323
500973
0.25:1
45248
7.407
335160
225188
109972
0.25:2
52264
7.761
406514
235836
170678
0.25:3
53056
8.307
440762
237667
203095
0.5:1
53056
26.256
1393022
303124
1089898
0.5:2
55440
30.559
1694169
351414
1342755
0.5:3
52264
22.941
1198970
293019
905951
1:01
51464
41.373
2129230
383303
1745927
1:02
49484
45.434
2248274
427959
1820315
1:03
53844
46.975
2529329
297738
2231591
2:01
51160
77.946
3987708
540998
3446710
2:02
54956
77.778
4274351
366331
3908020
2:03
53440
80.505
4302196
665487
3636709
5:01
58532
118.406
6930527
473292
6457235
5:02
59608
110.692
6598123
511418
6086705
5:03
56840
115.993
6593021
407957
6185064
1
2
3
Average
Standard Deviation
X-Axis Int
0
-169877
-194680
500973
45472
394670.328189237
0
0.25
109972
170678
203095
161248.333333333
47272.2142736442
0.25
0.5
1089898
1342755
905951
1112868
219306.063023802
0.5
1
1745927
1820315
2231591
1932611
261582.059660062
1
2
3446710
3908020
3636709
3663813
231846.283422875
2
5
6457235
6086705
6185064
6243001.33333333
191939.238224844
5
Calibration curve
PCR Results Summary
Our positive control PCR result was 1.41 μg/mL
Our negative control PCR result was 0.3 μg/mL
Observed results
Patient 54942: Negative
Patient 69456: Positive
Conclusions
Patient 54942 : Not infected with disease
Patient 69456 : Infected with disease
SNP Information & Primer Design
Background: About the Disease SNP
Polymorphism is when there is genetic variation within a population. This can occur through natural selection. Nucleotides are the basic structural units of DNA, when the bases are switched this causes genetic variations or mutations. The SNP rs16991654 is only found in humans (Homo sapiens) and occurs on the 21st chromosome. The clinical significance of this SNP is that it is a pathogenic allele. The gene affected by this SNP is KCNE2, which stands for potassium voltage-gated channel, Isk-related family, member 2. KCNE2 gene has a variety of functions these include: regulating neurotransmitter release, heart rate, insulin secretion, neuronal excitability, epithelial electrolyte transport, smooth muscle contraction, and cell volume. An allele is two or more alternative forms of a gene that are created through mutations and are located on the same place on a chromosome. When this gene contains the disease-associated allele sequence is CTC.
Primer Design and Testing
The primers we created worked as expected. The primers were created by isolating where the disease SNP is located in the genome. With that location, 20 base pairs were matched on either side of the disease coding sequence so that primers could be created at that specific location. In layman's terms, the primers hold the coordinates to the corresponding location on the human genome. The Non-Disease Primers (Forward and Reverse) worked and created the 220bp long sequence where the disease would be. The Disease Primers (Forward and Reverse) contained no matches. This is because the primers are trying to match onto DNA that does not contain the disease. The forward primer cannot attach because of the difference in base pairs between the forward non-disease primer (CATGGTGATGATTGGAATGT) and the forward disease primer (CATGGTGATGATTGGAATGC). The only difference is the T changes to a C in the disease primer. The reverse primers for disease and non-disease are the same. The following pictures are from the test to tell if the primers will work.