Distance between the smart phone cradle and drop = 5 cm
Solutions Used for Calibration
Initial Concentration of 2X Calf Thymus DNA Solution(micrograms/mL)
Volume of the 2x DNA solution (uL)
Volume of the SYBR Green l solution (uL)
Final DNA concentration in SYBR Green Solution (ug/mL)
5
80
80
2.5
2
80
80
1
1
80
80
.5
.5
80
80
.25
.25
80
80
.125
0
80
80
0
Placing Samples onto the Fluorimeter
[Place the slide, smooth side down, in the fluorimeter so that the drops will be placed on the rough side.]
[Place 80 uL drop of SYBR Green I solution using micropipettor onto the rough side of the slide. The drop should be placed on the first two clear circles in the middle of the slide.]
[Place 80 uL drop of sample solution on top of the SYBR Green I solution. The drop should still be on the first two circles in the middle of the slide.]
[The slide should be adjusted so that the light goes straight threw the middle of the drop, and the drop focuses the light on the other side.]
Data Analysis
Representative Images of Negative and Positive Samples
Final DNA concentration in SYBR Green 1 solution (µg/mL)
Calf Thymus DNA Concentration (ug/mL)
Area
Mean Pixel Value
RawIntDen of the Drop
RawIntDen of the Background
(RawIntDen Drop) - (Background)
H2O (0)
0
51372
11.227
576776
493279
83497
0.25
0.125
22608
26.873
607535
114750
492785
0.5
0.25
26840
44.258
1187881
139044
1048837
1
0.5
26048
73.186
1906346
298830
1607516
2
1
28148
99.922
2812606
117583
2695023
5
2.5
31372
127.845
4010749
184072
3826677
Calibration curve
The results of concentration 2.5 was an outlier so we removed it from our data and re-plotted the adjusted data to calculate the equation of our best fit line.
PCR Results Summary
Calculated Results
PCR Product Tube Label
(RawIntDen Drop) - (Background)
PCR Product Concentration (µg/mL)
Initial PCR Product Concentration (µg/mL)
Positive
3557555
1.110617667
13.327412
Negative
518986
0.097761333
1.173136
1-1 Trial
849487
0.207928333
2.49514
1-2 Trial
462711
0.079003
0.948036
1-3 Trial
736243
0.170180333
2.042164
2-1 Trial
468346
0.080881333
0.970576
2-2 Trial
543800
0.106032667
1.272392
2-3 Trial
971808
0.248702
2.984424
Patient 1 ID =
66684
Patient 2 ID =
40117
Our positive control PCR result was 1.110617667 μg/mL
Our negative control PCR result was 0.097761333 μg/mL
Observed results
Patient 66684:
Patient 40117:
Conclusions
Patient 66684:
Patient 40117:
SNP Information & Primer Design
Background: About the Disease SNP
Single nucleotide polymorphisms commonly known as SNPs are genetic variations among people. These genetic variations occur due to a difference in a single nucleotide of DNA. SNPs normally occur throughout a person's DNA, most commonly in the DNA between genes. When an SNP occurs within a gene they play a direct role in certain diseases. One specific SNP (rs16991654) is an example of one of the SNPs that play a more direct role in diseases. This SNP affects Homo sapiens (humans) and is classified as pathogenic or capable of causing disease. The variation is associated with the gene KCNE2 and is located at 21:34370656 or in other words in chromosome 21 at base 34370656. KCNE2’s official name is “potassium voltage-gated channel, Isk-related family, member 2.” This gene has a diverse set of functions and regulates neurotransmitter release, heart rate, insulin secretion, neural excitability, epithelial electrolyte transport, smooth muscle contraction and cell volume. The SNP rs16991654 occurs when the disease allele TTC is instead CTC. This change in the thymine (T) base for a Cytosine (C) base at 21:34370656 is associated with the disease congenital long QT syndromes (LQTSs). LQTS is a rare and clinically heterogeneous inherited disorder characterized by a long QT interval on the electrocardiogram, increased risk of syncope and sudden death caused by arrhythmias.
Primer Design and Testing
The first step of the SNP–specific primer design process is to determine what variation is being analyzed. In this case, the variation is rs16991654 from the human genome. The numerical position of the SNP is 34370656. The non-disease forward primer can be read by identifying the 5’ on the top strand and then recording the 20 bases from left to write ending with the nucleotide at 34370656. However, the PCR reaction needs two primers, therefore, the non-disease reverse primer will be read from the bottom strand, 200 bases to the right and “backwards” because the 5’ is on the opposite side of the strand. This means that it is read from right to left and written from left to right. To design a pair of disease SNP-specific primers only the final base of the non-disease forward primer must be changed to the disease SNP nucleotide (TTC → CTC). The results can be validated using the UCSC In-Silico PCR website. Once inputting the proper settings (Human genome, Feb. 2009 (GRCh37/hg19) for the assembly, a target of the genome assembly, max produce size of 4000, and min perfect and good match of 20), the two non-diseased primers can be tested and shown to match the 220 bp sequence of rs16991654.
BME 100 Fall 2014
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