The smartphone was placed in the cradle with the camera app on. The cradle was then moved so that the phone was 12 cm away from from where the drop would be measured. Pieces of bubble wrap were placed in between the phone and the cradle so that the phone was level with the drop.
Distance between the smart phone cradle and drop = 12 cm
Solutions Used for Calibration
Initial Concentration of 2X Calf Thymus DNA Solution (µg/mL)
Volume of 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
Solutions Used for Measuring PCR Products
PCR Product Tube Label
Volume of the Diluted PCR Product solution(μL)
Volume of SYBR Green I Dye Solution (µL)
Dilution 1
Dilution 2
Total Dilution Simplified Fraction
G23 +
80
80
0.166
0.5
0.0833
G23 -
80
80
0.166
0.5
0.0833
G23 1-1
80
80
0.166
0.5
0.0833
G23 1-2
80
80
0.166
0.5
0.0833
G23 1-3
80
80
0.166
0.5
0.0833
G23 2-1
80
80
0.166
0.5
0.0833
G23 2-2
80
80
0.166
0.5
0.0833
G23 2-3
80
80
0.166
0.5
0.0833
Placing Samples onto the Fluorimeter
Line up fluorimeter light with the section on slide where the sample will be placed (middle of two circles)
Put new tip into micropipette and collect 80uL of SYBR green
Place 80 uL of SYBR green into area from step 1, lined with the light
Discard tip, get new tip and collect 80uL of desired sample
Place 80uL of sample on top of SYBR green in the fluorimeter slide
Adjust slide on fluorimeter to allow for the narrowest possible stream of light
Place camera on a timer
Place camera and fluorimeter in a box to block out light, and wait until picture is taken
Data Analysis
Representative Images of Negative and Positive Samples
Sample With No DNA
Sample With DNA
Image J Values for All Calibrator Samples
Final DNA concentration in SYBR Green I solution (µg/mL)
Area
Mean Pixel Value
RAWINTDEN of the Drop
RAWINTDEN of the Background
RAWINTDEN Drop - Background
0#1
117281
62.946
7382324
120175
7262149
0#2
95944
55.867
5360083
82761
5277322
0#3
102480
56.232
5762676
122017
5640659
0.125#1
200967
58.129
11682080
230881
11451199
0.125#2
209397
61.156
12805862
316028
12489834
0.125#3
220281
59.243
13050128
315441
12734687
0.25#1
148276
105.684
15670365
166860
15503505
0.25#2
143862
104.852
15084237
168573
14915664
0.25#3
148848
104.226
15513804
176779
15337025
0.5#1
63004
121.832
7675924
84689
7591235
0.5#2
53899
121.386
6542599
67056
6475543
0.5#3
63250
120.075
7594743
71477
7523266
1#1
101788
159.563
16241620
111100
16130520
1#2
107815
154.656
16674224
121570
16552654
1#3
106365
155.667
16557507
116040
16441467
2.5#1
52043
224.116
11663647
272607
11391040
2.5#2
96387
201.455
19417686
339679
19078007
2.5#3
93996
203.554
19133220
489494
18643726
PCR Product Tube Label
Area
Mean Pixel Value
RAWINTDEN of the Drop
RAWINTDEN of the Background
RAWINTDEN Drop - Background
G23 + #1
81288
211.873
17222739
519304
16703435
G23 + #2
83298
209.743
17471210
477364
16993846
G23 + #3
84916
206.982
17576093
380353
17195740
G23 - #1
63262
47.935
3032495
114182
2918313
G23 - #2
65364
52.551
3434964
126152
3308812
G23 - #3
73048
53.772
3927959
184799
3743160
G23 1-1 #1
63236
217.135
13730756
291772
13438984
G23 1-1 #2
69676
214.228
14926555
274659
14651896
G23 1-1 #3
72935
209.691
15293786
484533
14809253
G23 1-2 #1
68930
223.918
15434635
748054
14686581
G23 1-2 #2
83892
214.71
18012462
542818
17469644
G23 1-2 #3
78844
213.253
16813706
552361
16261345
G23 1-3 #1
79201
218.261
17286505
653016
16633489
G23 1-3 #2
91457
222.257
20326925
1007852
19319073
G23 1-3 #3
77372
225.738
17465838
697357
16768481
G23 2-1 #1
86508
221.876
19194089
849243
18344846
G23 2-1 #2
79512
230.236
18306536
717249
17589287
G23 2-1 #3
84287
228.699
19276328
708500
18567828
G23 2-2 #1
77942
224.541
17501204
264074
17237130
G23 2-2 #2
66272
229.613
15216881
260396
14956485
G23 2-2 #3
69675
229.864
16015757
250409
15765348
G23 2-3 #1
46398
225.599
10467352
271372
10195980
G23 2-3 #2
39267
223.424
8773180
218858
8554322
G23 2-3 #3
54020
228.365
12336293
436545
11899748
Final DNA concentration in SYBR Green I solution (µg/mL)
RAWINTDEN DROP - BACKGROUND
'
'
'
'
1
2
3
Mean
Standard Deviation
0
7262149
5277322
5640659
6060043.333
1056786.151
0.125
11451199
12489834
12734687
12225240
681427.0689
0.25
15503505
14915664
15337025
15252064.67
302990.0229
0.5
7591235
6475543
7523266
7196681.333
625448.0938
1
16130520
16552654
16441467
16374880.33
5044544.086
2.5
11391040
19078007
18643726
16370924.33
4318169.285
PCR Product Tube Label
RAWINTDEN DROP - BACKGROUND
'
'
'
1
2
3
Mean
G23 +
16703435
16993846
17195740
16964340.33
G23 -
2918313
3308812
3743160
3323428.333
G23 1-1
13438984
14651896
14809253
14300044.33
G23 1-2
14686581
17469644
16261345
16139190
G23 1-3
16633489
19319073
16768481
17573681
G23 2-1
18344846
17589287
18567828
18167320.33
G23 2-2
17237130
14956485
15765348
15986321
G23 2-3
10195980
8554322
11899748
10216683.33
Calibration curve
PCR patient label
Mean RAWINTDEN drop-background
PCR product concentration
Initial PCR product concentration
G23 +
16964340.33
2.32 ug/mL
27.84 ug/mL
G23 -
3323428.333
-2.23 ug/mL
-26.76 ug/mL
G23 1-1
14300044.33
1.43 ug/mL
17.16 ug/mL
G23 1-2
16139190
2.05 ug/mL
24.6 ug/mL
G23 1-3
17573681
2.52 ug/mL
30.24 ug/mL
G23 2-1
18167320.33
2.72 ug/mL
32.64 ug/mL
G23 2-2
15986321
1.99 ug/mL
23.88 ug/mL
G23 2-3
10216683.33
.072 ug/mL
0.864 ug/mL
PCR Results Summary
Our positive control PCR result was 27.84 μg/mL
Our negative control PCR result was -26.76 μg/mL
Observed results
Patient 1-33849 : All of the images for patient 1 were bright green. The green was mostly at the bottom, with blue at the top. Patient 1 had an average initial PCR product concentration of 24 μg/mL.
Patient 2-19967 : All of the images for patient 2 were also bright green. The green fluorescence was shown all throughout the drop. Patient 2 had an average initial PCR product concentration of 18.869 μg/mL.
Conclusions
Patient 1-33849 : Patient 1 had an average initial PCR product concentration that was 3.84 μg/mL less than that of the product concentration of the positive sample, and 50.76 μg/mL greater than that the product concentration of the negative sample. Since Patient 1 had a concentration closer to the concentration of the positive sample, Patient 1 is positive. All of Patient 1's initial concentrations were closer to the concentration of the positive sample than the negative sample.
Patient 2-19967 : Patient 2 had an average initial PCR product concentration that was 8.9712 μg/mL less than that of the product concentration of the positive sample, and 45.6288 μg/mL greater than that the product concentration of the negative sample. Since Patient 2 had a concentration closer to the concentration of the positive sample, Patient 2 is positive. All of Patient 2's initial concentrations were closer to the concentration of the positive sample than the negative sample. Positive initial concentrations suggest that the patient is positive, while negative initial concentrations suggest that the patient is negative.
SNP Information & Primer Design
Background: About the Disease SNP
A nucleotide is the basic building block of DNA and other nucleic acids. the four main nucleotides are A,C,T,and G. A polymorphism is a common change in DNA sequences that appears in the phenotypes of different groups of population. The SNP rs16991654 variation is found in homosapiens. This variation is found in the 21:34370656 chromosome. The clinical significance is this SNP is that it is pathogenic. This SNP is associated with the KCNE2 gene. This SNP is linked to the disease of congenital long QT syndromes. KCNE2 stands for potassium voltage-gated channel, Isk-related family, member 2. The molecular function of this gene includes regulating neurotransmitter release, heart rate, insulin secretion, neuronal excitability, epithelial electrolyte transport, smooth muscle contraction, and cell volume. An allele in an alternative to a gene sequence that are done through mutation of a nucleotide base pair. The disease-associated allele contains the sequence CTC. The numerical position of the SNP is 34,370,656. The non-disease forward primer is catggtgatgattggaatgt. The numerical position exactly 200 bases to the right of the disease SNP is 3,430,856. The non-disease reverse primer is cccttatcagggggacattt. The diseases forward primer is catggtgatgattggaatgc. The disease reverse primer is cccttatcagggggacattt.
Primer Design and Testing
When the non-disease forward primer was tested, it was found in the database. This is because a part of the DNA that was selected did not contain the sequence that causes the disease. The disease specific primers did not exist because one nucleotide was changed in the DNA sequence. While this may cause the disease, it affects the entire sequence, it does not exist since it did not change by a group of three nucleotides, or an entire codon.