Calibration
First, place a slide into the fluorimeter and turn the camera on. Next, place the camera in the cradle and make sure the camera is aligned with the surface of the slide. You may need to raise or lower the camera or fluorimeter. Once the camera is properly aligned, make sure there is an ample amount of room to slide the box over the camera and fluorimeter so that it will be completely dark when the picture is taken. Finally, measure the distance between the camera and the fluorimeter.
Distance between the smart phone cradle and drop = 5cm
Solutions Used for Calibration
Initial DNA Concentration(µg/mL)
Volume of DNA solution (μL)
Volume of SYBER GREEN (μL)
Final DNA Concentration (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
Mix all solutions in their designated test tubes
Set the micro-pipette to the desired volume
Using and clean tip, push and hold the button to the first stop, place the tip into the solution, and then release the button
Align the pipette in the middle of the first two dots and slowly push the button all the way down, being careful not to splash the solution
Throw the used tip in the waste bucket
Repeat each step for each solution, using a clean tip each time and moving the slide in order to use a clean section of it for each trial
Data Analysis
Representative Images of Negative and Positive Samples
Positive Image
Negative Image
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)
81440
182.157
14834828
349820
14485008
0(2)
47044
216.611
10190270
228061
9962209
0(3)
47504
211.017
10024167
567097
9457070
.125(1)
40392
188.574
4789460
135365
4654095
.125(2)
30552
177.525
3590621
117614
3473007
.125(3)
40392
110.811
4475860
131342
4344518
.25(1)
44228
143.841
6361796
139242
6222554
.25(2)
42304
149.935
6342866
139684
6203182
.25(3)
34376
153.54
5278078
123830
5154248
.5(1)
42752
140.039
5986927
129819
5857108
.5(2)
40392
147.53
5959021
122723
5836298
.5(3)
45240
151.622
6859357
153690
6705667
1(1)
42752
194.347
8308710
131135
8177575
1(2)
45116
195.155
8804618
141223
8663395
1(3)
42304
207.948
8797027
148308
8648719
2.5(1)
40392
219.747
8876005
140522
8735483
2.5(2)
49884
208.879
10419715
173542
10246173
2.5(3)
51572
222.291
11463994
180723
11283271
Final DNA concentration in SYBR Green I solution (µg/mL)
RAWINTDEN DROP -- BACKGROUND
'
'
MEAN
STANDARD DEVIATION
1
2
3
0
14485008
9962209
9457070
11301429
2768604.845
0.125
4654095
4373007
4344518
4457206.667
171104.2595
0.25
6222554
6203182
5154248
5859994.667
611271.2873
.5
5857108
5836298
6705667
6133024.333
496032.2387
1
8177575
8663395
8648719
8496563
276349.153
2.5
8735483
10246173
11283271
10088309
1281209.079
Calibration curve
PCR Results Summary
Our positive control PCR result was 0.18453 μg/mL
Our negative control PCR result was 0.44294 μg/mL
Observed results
Patient 39635 : the image turned out to be a rounded beach ball shape, the coloring seemed to be black and white, but was brighter than the (red) and (blue) filters that were given, 0.18453 μg/mL.
Patient 64133 : the image was similar to Patient 39635, the shape shape was given and the coloring was the same, 0.44294 μg/mL.
Conclusions
Patient 39635 : was positive, the three initial concentrations of 1-1, 1-2, 1-3 were closest to the observed positive control.
Patient 64133 : was inconclusive, the three initial concentrations of 2-1, 2-2, 2-3 were inconclusive because one resulted in a negative initial concentration, one yielded a positive result and one a negative.
SNP Information & Primer Design
Background: About the Disease SNP
A nucleotide is one of the core elements of DNA and RNA, consisting of a base chemical and a molecule of sugar and phosphoric acid. A polymorphism is a variation that exists inside DNA nucleotide where one of the bases has been substituted that may occur when cells replicate. The species that this SNP is found is Homo sapiens, specifically located on the chromosome 21, position on the chromosome is 34370656
The significance of this SNP is that it is likely pathogenic, meaning it probably can cause disease or other harm to an organism. The SNP rs16991654 is associated with KCNE2 gene. The official name for that gene is “potassium voltage-gated channel, Isk-related family, member 2” (genetics home reference). The function based on the name is the control of potassium as it interacts with cells. Channels that are composed with KCNE2 are present in the heart muscle, which is apart of the function that is in charge of recharging that cardiac muscle to continue a regular rhythm. Other functions of the gene include regulating neurotransmitter release, heart rate, insulin secretion, neuronal excitability, epithelial electrolyte transport, smooth muscle contraction, and cell volume (NCBI).
Disease linked to SNP is the congenital long QT syndrome that can cause irregular and spastic heartbeats. An allele is an alternative gene form that may arise through mutation and are located at the same place on a chromosome. in the SNP the disease-associated allele is the CTC sequence rather than what it should contain the sequence TTC. In the Gene the numerical position of the SNP is at 21:34370656
Primer Design and Testing
Primer without Disease
The results below with the two primers is Chr 21:35742936+35743155. This a chromosome where the rs16991654 SNP is found and the primer test is in a human genome without the TQ disease.
Forward Primer:C A T G G T G A T G A T T G G A A T G T
Reverse Primer:C C C T T A T C A G G G G G A C A T T T
Primer with Disease
With these results were made from changing the final base of TTC to CTC on the primer without disease. Results were not found because the primer test does not contain the allele that is associated with the variation of the TTC.
Forward Primer: C A T G G T G A T G A T T G G A A T G C
Reverse Primer: C C C T T A T C A G G G G G A C A T T T
BME 100 Fall 2014
