The camera was set up in front of the fluorimeter by placing it within a cradle. The cradle was placed 6 cm from the droplet on the plate. Then a box was placed over the camera and drop.
Distance between the smart phone cradle and drop = 6 cm
Solutions Used for Calibration
Initial Concentration of 2X Calf Thymus DNA Solution (mg/mL)
Volume of the 2X DNA Solution (microliters)
Volume of the SYBR Green I Dye Soultion (microliters)
Final DNA Concentration in SYBR Green I Solution (miroliters)
0
80
80
0
0.25
80
80
0.125
0.5
80
80
0.25
1
80
80
0.5
2
80
80
1
5
80
80
2.5
-
Placing Samples onto the Fluorimeter
Step 1, 80 micro-liters of SYBR GREEN I was pipette and placed on the first row in the first column on the Superhydrophobic surface. 80 micro-liters of the Calf Thymus DNA concentration 5 solution was placed in the exact same spot; first row first column. Superhydrophobic surface was moved so the drop of solution was in front of the blue LED light on the Fluorimeter. Camera was focused and three pictures were saved. Pipette tip was discarded and replaced.
Step 2, 80 micro-liters of SYBR GREEN I was pipette and placed on the first row in the second column on the Superhydrophobic surface. 80 micro-liters of the Calf Thymus DNA concentration 2 solution was placed in the exact same spot; first row second column. The drop was aligned in front of the blue LED light and three pictures were saved. Pipette tip was discarded and replaced.
Step 3, The previous steps were repeated for the following solutions in the following location...
80 micro-liters of SYBR GREEN I were pipette and placed on the first row in the third column on the Superhydrophobic surface. 80 micro-liters of the Calf Thymus DNA concentration 1 solution was placed in the exact same spot; first row third column.
80 micro-liters of SYBR GREEN I were pipette and placed on the first row in the fourth column on the Superhydrophobic surface. 80 micro-liters of the Calf Thymus DNA concentration 0.5 solution was placed in the exact same spot; first row fourth column.
80 micro-liters of SYBR GREEN I were pipette and placed on the second row in the first column on the Superhydrophobic surface. 80 micro-liters of the Calf Thymus DNA concentration 0.25 solution was placed in the exact same spot; second row in the first column.
80 micro-liters of SYBR GREEN I were pipette and placed on the second row in the second column on the Superhydrophobic surface. 80 micro-liters of the Calf Thymus DNA concentration 0 solution was placed in the exact same spot; second row in the second column.
Step 4, The patient solutions were placed on the Superhydrophobic surface. 80 micro-liters of DILUTED PCR Product patient number G15 1-1 was pipette and placed on the second row in the third column on the Superhydrophobic surface. 80 micro-liters of SYBR GREEN I was placed in the exact same spot; second row in the third column. Superhydrophobic surface was moved so the drop of solution was in front of the blue LED light on the Fluorimeter. Camera was focused and three pictures were saved. Pipette tip was discarded and replaced.
Step 5, The previous step was completed for the patient solutions in the following locations..
80 micro-liters of SYBR GREEN I was pipette and placed on the second row in the fourth column on the Superhydrophobic surface. 80 micro-liters of DILUTED PCR Product patient number G15 1-2 was placed in the exact same spot; third row in the first column.
80 micro-liters of SYBR GREEN I was pipette and placed on the third row in the first column on the Superhydrophobic surface. 80 micro-liters of DILUTED PCR Product patient number G15 1-3 was placed in the exact same spot; third row in the first column.
80 micro-liters of SYBR GREEN I was pipette and placed on the third row in the second column on the Superhydrophobic surface. 80 micro-liters of DILUTED PCR Product patient number G15 2-1 was placed in the exact same spot; third row in the second column.
80 micro-liters of SYBR GREEN I was pipette and placed on the third row in the fourth column on the Superhydrophobic surface. 80 micro-liters of DILUTED PCR Product patient number G15 2-2 was placed in the exact same spot; third row in the fourth column.
80 micro-liters of SYBR GREEN I was pipette and placed on the third row in the fourth column on the Superhydrophobic surface. 80 micro-liters of DILUTED PCR Product patient number G15 2-3 was placed in the exact same spot; third row in the fourth column.
Step 6, On a new Superhydrophobic surface, 80 micro-liters of the controlled solution labeled positive was placed on first row first column of the slide. Superhydrophobic surface was moved so the drop of solution was in front of the blue LED light on the Fluorimeter. Camera was focused and three pictures were saved. Then, 80 micro-liters of the controlled solution labeled negative was placed on the first row second column of the slide. Superhydrophobic surface was moved so the drop of solution was in front of the blue LED light on the Fluorimeter. Camera was focused and three pictures were saved. Pipette tip was discarded and replaced.
Step 7, slides were discarded, Fluorimeter was put away, work station was cleaned.
Data Analysis
Representative Images of Negative and Positive Samples
Negative Control:
Positive Control:
Image J Values for All Calibrator Samples
Final DNA Concentration of SYBR Green Solution 1 (μg/mL)
Area
Mean Pixel Value
Rawintden of the Drop
Rawintden of the Background
Rawintden of Drop-Background
2.5
1374
170.5
472573
8914
463659
2.5
1535
164.311
4753167
10166
4743001
2.5
1451
166.101
468810
9706
459104
1
1668
171.558
286158
9149
277009
1
1806
167.444
302403
11372
291031
1
1714
168.285
288441
10001
278440
0.5
1195
133.204
174882
7479
167403
0.5
1356
127.15
170501
8731
161770
0.5
1272
128.805
171952
8271
163681
0.25
1336
116.305
154375
9830
144545
0.25
1486
120.649
150508
9627
140881
0.25
1486
121.182
152861
9878
142983
0.125
1356
87.227
118280
11693
106587
0.125
1356
87.388
118498
11373
107125
0.125
1356
89.757
121711
11712
109999
0
1440
63.26
91094
9949
81145
0
1440
61.522
88591
10257
78334
0
1440
58.929
84858
10139
74719
Final DNA Concentration of SYBR Green Solution 1 (μg/mL)
Rawintden of Drop-Background 1
Rawintden of Drop-Background 2
Rawintden of Drop-Background 3
Mean
Standard Deviation
2.5
463659
465151
459104
462638
3150.140156
1
277009
291031
278440
282160
7715.757967
0.5
174882
170501
171952
172445
2231.720637
0.25
167403
161770
163681
164285
2864.608583
0.125
144545
140881
142983
142803
1838.620135
0
81145
78334
74719
78066
3221.371913
Calibration curve
y = 144538x + 111674
PCR Results Summary
PCR Product Tube Label
Area
Mean Pixel Value
RAWINTDEN of the Drop
RAWINTDEN of the Background
RAWINTDEN of the Drop - Background
G15 + Picture 1
1952
183.093
357398
16902
340496
G15 + Picture 2
1952
182.815
356854
16910
339944
G15 + Picture 3
1952
184.06
359286
18573
340713
G15 - Picture 1
1952
103.169
201385
18190
183195
G15 - Picture 2
1952
100.181
195554
18667
176887
G15 - Picture 3
1952
102.21
199514
19908
179606
G15 1-1 Picture 1
1952
183.698
358579
19648
338931
G15 1-1 Picture 2
1952
184.044
359254
19118
340136
G15 1-1 Picture 3
1952
184.44
360027
19380
340647
G15 1-2 Picture 1
1356
129.691
175861
13970
161891
G15 1-2 Picture 2
1356
127.962
173516
13317
160199
G15 1-2 Picture 3
1356
133.532
181069
13344
167725
G15 1-3 Picture 1
1356
134.926
182959
9013
173946
G15 1-3 Picture 2
1356
131.734
178631
9083
169548
G15 1-3 Picture 3
1356
136.993
185763
9013
176750
G15 2-1 Picture 1
1008
205.278
206920
7732
199188
G15 2-1 Picture 2
1008
201.812
203427
7632
195795
G15 2-1 Picture 3
1008
202.999
204623
7482
197141
G15 2-2 Picture 1
1592
157.801
251219
10976
240243
G15 2-2 Picture 2
1592
163.599
260450
10881
249569
G15 2-2 Picture 3
1592
166.413
263265
11931
251334
G15 2-3 Picture 1
1504
190.01
285775
14647
271128
G15 2-3 Picture 2
1504
190.885
287091
16473
270618
G15 2-3 Picture 3
1504
186.031
279790
14347
265443
PCR Product Tube Label
MEAN RAWINTDEN DROP - BACKGROUND
PCR Product Concentration (µg /mL)
Initial PCR Product Concentration (µg /mL)
G15 +
340384.3
1.582
3.164
G15 -
179896
0.472
0.944
G15 1-1
339904.7
1.579
3.158
G15 1-2
163271.7
0.357
0.714
G15 1-3
173414.7
0.427
0.854
G15 2-1
197374.3
0.593
1.186
G15 2-2
247048.7
0.9366
1.8732
G15 2-3
269063
1.089
2.178
Our positive control PCR result was 3.164 μg/mL
Our negative control PCR result was 0.944 μg/mL
Observed results
Patient 50018 : The images appeared to have less green phosphorescence as the trials went on. The first trial was very bright in the color and the second and third appeared less so, nearly colorless. The first trial had a Initial PCR Product Concentration of 3.158 μg/mL, the second 0.714 μg/mL, and the third 0.854 μg/mL.
Patient 69797 : All three trials were green and glowing, the first was more clear than the other two. Numerically, the first trial had 1.186 μg/mL, the second trial 1.8732 μg/mL, and the third trial 2.178 μg/mL.
Conclusions
Patient 50018 : It may be inferred that this patient is negative for the disease as two of the three trials received concentrations under that of the concentration recorded for the negative control.
Patient 69797 : It may be inferred that this patient is positive for the disease as all three trials received concentrations over that of the concentration recorded for the negative control.
SNP Information & Primer Design
Background: About the Disease SNP
What is a Nucleotide?
A nucleotide is the structural component of DNA and RNA. They consist of four base pairs (Adenine, Thymine, Guanine, and Cytosine), a sugar molecule, and a phosphoric acid.
What is a polymorphism ?
A Polymorphism, is a naturally occurring variation in a DNA sequence, gene, or chromosomes. They do not have any adverse effects on and individual occur fairly often in the general population. It involves variants of a DNA sequence, more commonly at a single base pair. Polymorphisms that occur in long stretches of DNA are known as single nucleotide polymorphism, or SNPs.
What species is this variation found in? (latin name)
Homo sapiens.
What chromosome is the variation located on?
The variation is located on the chromosome 8: base pairs 19956018 position 21948.
What is listed as the clinical significance of this SNP?
The clinical significance of the SNP is it is “Pathogenic allele”.
Which gene(s) is this SNP associated with ?
This is associated with the LPL gene.
What disease is linked to this SNP?
This is SNP is linked with Metabolic syndrome.
What does LPL stand for ?
LPL also known as lipoprotein lipase.
What is the function of LPL?
This gene codes for the production of the LPL enzyme which is responsible for breaking down fat in the form of triglycerides. TH enzyme is found on the surface of cells that line the capillaries withing muscles on fatty tissue. This enzyme breaks triglycerides which carry fat to the bloodstream form different organs; when LPL breaks down these triglycerides the fat molecules are used as energy or stored in fatty tissue for later use.
What is an allele?
An allele is two or more versions of a gene. An individual inherits two alleles for each gene, one from each parent. if the alleles are the same they are homozygous and if they are different they are heterozygous. Allele is a term used to describe the variation among genes and non-coding DNA sequences.
The disease associated allele contains the three bases A, G, T while the non-disease allele has base pairs A, A, T.
Primer Design and Testing
The numerical position of the SNP is 19956018
Non-disease forward primer (20 nt): aatctgggctatgagatcaa
The numerical position exactly 200 bases to the right of the disease SNP is: 19956218
Non-disease reverse primer (20 nt): gaaacaccagggctcagggt
Disease forward primer (20 nt): aatctgggctatgagatcag
Disease reverse primer (20 nt): gaaacaccagggctcagggt
BME 100 Spring 2015
