Distance between the smart phone cradle and drop = 6 cm
Solutions Used for Calibration
Initial 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 (μL)
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
Turn on Blue LED Excitation light
Insert a slide into the Fluorimeter
Place camera on the craddle and as close to as possible without getting a blurry picture
Adjust height of fluorimeter so that picture from camera takes a picture of the drop directly from the side
Place an 80 microliter drop of SYBR Green I on the slide so that it looks like a beach ball
Add an 80 microliter drop of the DNA solution to the already placed solution
Align the slide so that the blue LED is focused on the drop
Take the a picture with the timer set and lower the lid of the lightbox
Take a total of 3 pictures then remove the solution from the slide
Move the slide to the next position and repeat the process with the different solutions
Data Analysis
Representative Images of Negative and Positive Samples
Positve:
Negative:
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
2.5
139380
108.933
15183035
37084
15145951
2.5
142636
109.163
15570617
37393
15533224
2.5
147752
107.308
15854933
49037
15805896
1
106708
83.381
8897448
17939
8879509
1
115956
80.25
9305435
21998
9283437
1
113112
90.715
10260974
34066
10226908
0.5
135592
62.432
8465278
64454
8400824
0.5
118410
58.929
6977741
64902
6912839
0.5
115368
58.331
6729582
46971
6682611
0.25
139968
40.608
5683780
59072
5624708
0.25
127236
40.933
5208100
54709
5153391
0.25
133808
42.526
5690375
51402
5638973
0.125
131884
38.479
5074747
49047
5025700
0.125
124424
34.43
4283904
43969
4239935
0.125
132332
37.836
5006942
54115
4952827
0
137400
21.711
2983023
52412
2930611
0
124400
18.681
2323902
45159
2278743
0
114528
19.95
2284888
39148
2245740
Final DNA concentration in SYBR Green I solution (μg/mL)
RawIntDen drop - background
'
'
'
Standard Deviation
1
2
3
mean
2.5
15145951
15533224
15805896
46485071
331626.7478
1
8879509
9283437
10226908
28389854
691469.3802
0.5
8400824
6912839
6682611
21996274
932680.7426
0.25
5624708
5153391
5638973
16417072
276325.015
0.125
5025700
4239935
4952827
14218462
434156.6754
0
2930611
2278743
2245740
7455094
386235.9758
Calibration curve
PCR Results Summary
Our positive control PCR result was .11603 μg/mL
Our negative control PCR result was .0575 μg/mL
Observed results
Patient 47360 : Average initial concentration of .114425, the bubble observed was clearer than the positive control, however much darker than the negative
Patient 31303 : Average initial concentration of .15928, the bubble was much darker the negative control and the patient 1, much closer to the color of the positive control.
Conclusions
Patient POS : 2 out of three of the runs ran closer in similarity to the positive control
Patient POS : All three of the runs were closer to the positive test
SNP Information & Primer Design
Background: About the Disease SNP
An SNP is a single nucleotide polymorphism(SNP). An SNP occurs when a nucleotide pair, the monomers that create DNA, is altered changing the gene it is contained in creating a polymorphism, a genetic variation. These SNPs occur naturally in biological genomes and through natural selection these mutations are controlled. When an SNP occurs it creates a new allele, a variation in the gene, in a population that if not detrimental will be passed on to the next generation. It is usual for there to be only two different alleles for a specific gene which is the case for the SNP being studied. The disease SNP being studied is rs268. This SNP occurs in Homo Sapiens on the eighth chromosome. The clinical significance of this SNP is that it is pathogenic and it is associated with the LPL gene, the lipoprotein lipase. This gene is involved with processes such as apolipoprotein binding, chylomicron remodeling, and anchoring components of the membrane. This disease is responsible for metabolic syndrome that increase blood pressure, cholesterol levels, excess fat, and blood sugar levels which increases a person risk for stroke, diabetes, and heart disease. The normal allele of this gene is AAT but when the disease is present the mutation creates an allele of AGT.
Primer Design and Testing
The primers worked as expected producing one positive test and one negative test. In the positive test the diseased primer was shown to work. In the negative test the non-disease primer was shown to work. The primers worked by bonding to the DNA and allowing replication to occur if they matched properly. By getting replicated DNA it was confirmed that the primers worked because they were attached. The primers were different at two different points in the forward direction. The non-disease primer would bond allowing replication if the allele was negative for the disease because it ended in an 'A' nucleotide which the gene has if it does not have the disease. The disease primer bonded and allowed replication to occur if the diseased allele was because it ended in a 'G' which the gene would have in at that location if the disease was present. The two nucleotide primers can be seen below.