Calibration
To set up our camera in front of the fluorimeter we first had to place the lighted platform and glass slide at the back of the box with a tray under the platform to increase the height. Next, we took the phone and placed it into the phone cradle. Then we took the phone cradle and placed it in front of the glass slide, varying the distance until the drop was in focus. Once in position, the phone was not moved.
Distance between the smart phone cradle and drop = 2in
Solutions Used for Calibration
Initial Concentration of DNA solution(µg/mL)
Volume of DNA solution(µL)
Volume of SYBR Green(µL)
Final DNA Solution in SYBR Green(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
Place 80µL of the SYBR green solution as a drop in between the middle two dots on the glass slide and add 80µL of one of the DNA solutions in the first column of the table
Position the drop of solution so that the LED light shines directly through the center of it
Set the timer on the camera and quickly close the light box to prevent any stray light from entering
Take three focused images of the drop
Open the lid of the box and without moving anything inside of it, micropipette the 160µL drop off the slide.
Repeat steps 1-5 for each concentration of DNA solution
Data Analysis
Representative Images of Negative and Positive Samples
Negative Samples
Positive Samples
Image J Values for All Calibrator Samples
Area
Mean
StDev
Mode
IntDen
RawIntDen (Drop)
RawIntDen (Background)
Drop - Background
Water T1
28484
29.191
28.401
13
831470
831470
158266
673204
Water T2
28680
31.555
33.786
13
904985
904985
202411
702574
Water T3
27404
33.69
40.547
13
923237
923237
137517
785720
Calf Thymus 0.25 T1
19910
42.983
23.499
41
855792
855792
117585
738207
Calf Thymus 0.25 T2
21006
42.528
23.132
42
893342
893342
119887
773455
Calf Thymus 0.25 T3
22096
44.823
23.529
47
990417
990417
149789
840628
Calf Thymus .5 T1
26576
54.056
33.842
49
1436601
1436601
122697
1313904
Calf Thymus .5 T2
25638
51.179
32.873
49
1312135
1312135
144343
1167792
Calf Thymus .5 T3
26148
52.014
31.669
54
1360057
1360057
161701
1198356
Calf Thymus 1 T1
24056
83.603
37.424
89
2011156
2011156
173370
1837786
Calf Thymus 1 T2
24948
79.871
36.813
95
1992628
1992628
166804
1825824
Calf Thymus 1 T3
24436
80.677
36.227
89
1971426
1971426
155532
1815894
Calf Thymus 2 T1
28154
111.768
41.791
139
3146720
3146720
228364
2918356
Calf Thymus 2 T2
31122
111.973
42.206
143
3484820
3484820
203899
3280921
Calf Thymus 2 T3
29404
105.723
43.869
135
3108670
3108670
210787
2897883
Calf Thymus 5 T1
24263
163.781
55.723
255
3973807
3973807
225008
3748799
Calf Thymus 5 T2
24835
161.367
56.173
255
4007539
4007539
180646
3826893
Calf Thymus 5 T3
25503
164.135
57.517
255
4185938
4185938
159026
4026912
Calibration curve
PCR Results Summary
Our positive control PCR result was 28.29804534 μg/mL
Our negative control PCR result was -1.136261815 (0) μg/mL
Observed results
Patient 58961 : Images glowed bright green, had a high amount of green saturation; Initial PCR Concentration determined to be an average of 31.48969805 μg/mL.
Patient 79602 : Images glowed bright green, had a high amount of green saturation; Initial PCR Concentration determined to be an average of 37.50745806 μg/mL.
Conclusions
Patient 79602 : Since this patient's sample resembled the positive control and is much closer to the positive control concentration than the negative, it can be concluded that the sample tested positive.
Patient 58961 : This patient's results are also fairly close to the concentration of the positive control so it can be concluded that they also tested positive.
SNP Information & Primer Design
Background: About the Disease SNP
The disease SNP is found in homo sapiens and is located on the chromosome 21. It is associated with the gene KCNE2 which is a potassium voltage-gated channel that regulates neurotransmitter release, hear rate, insulin, neuronal excitability and other important functions. SNP has a clinical significance because is it pathogenic and linked to the disease long QT. Long QT is a rare inherited heart condition that causes a risk of episodes of torsades de pointes, irregular heartbeat originating from the ventricles. The disease is identified by a change in the allele sequence TTC to CTC.
Primer Design and Testing
From our primer test we were able to locate the forward and reverse primer sequences to take us to the desired SNP gene. To mutate this gene, the last base sequence in the forward primer was changed from TTC to CTC. This single base mutation is the cause of the SNP disease long QT. The test below demonstrates how the non-disease primers lead directly to the desired SNP gene and the diseased ones come up with no results. This lack of results confirms that the disease primers result in a mutation and change on the gene.