Calibration
The Samsung Galaxy Note 2 was placed in the phone holder, and positioned in a way that made it almost vertical. The height of the fluorimeter was adjusted to be horizontal to the camera on the smartphone using various sized bases. The phone lens was 10 cm away from the drop being analyzed. The box was placed on top of the system, with one flap left open, which allowed for someone to command the phone to take a photo. Three photos were taken for each test with the aid of a 5 second timer on the phone to allow the flap to be relatively closed upon taking the picture
Distance between the smart phone cradle and drop = 10 cm
Solutions Used for Calibration
Initial Concentration of DNA Solution (µg/mL)
Volume of DNA Solution Added (µl)
Volume of SYBR Green Added (µl)
Final DNA Concentration After Mixing (µl/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
BONUS: Photo of Fluorimeter Set-Up
The picture above shows how the fluorimeter was set up. The Samsung Galaxy Note 2 was placed on the cradle 10cm away from the fluorimeter. The smartphone rested on the cradle and was positioned so that it formed a 45 degree angle with the bottom of the cradle. The slides were placed on the fluorimeter with drops of SYBR Green 1 dye solution and the PCR product samples were aligned with the blue LED light on the fluorimeter. Finally, in order to better position the phone with the fluorimeter bubble wrap/plastic container were placed underneath the fluorimeter to increase its height.
Placing Samples onto the Fluorimeter
Using gloves place the hydrophobic side of the slide face up when sliding the slide into the fluorimeter.
Adjust the smartphone and the fluroimeter, so the lens is horizontal to the slide.
Pipette 80 µl of the SYBR Green I solution using a micropipette into the space between the first two holes.
Pipette 80 µl of the per-determined concentration solution using a micropipette onto the same drop as created in Step 3.
Place the box above the system to block outside light.
Take three photos of the drop using the camera's timer button, making sure that no light enters the box. Remove the drop from the slide with the pipette after setting it to 160 µl and move the slide to the next position.
Repeat steps 3-6 with the various solutions of differing concentration.
Data Analysis
Representative Images of Negative and Positive Samples
Negative SamplePositive Sample
Image J Values for All Calibrator Samples
Calibration curve
PCR Results Summary
Our positive control PCR result was 1.619779 μg/mL
Our negative control PCR result was -0.038469 μg/mL
Observed results
Patient 21611: The three samples were bright green, but varied in brightness. They were around the brightness of the positive control. The calf thymus DNA concentration was 2.0699 μg/mL
Patient 21095: The three samples were green, not quite as bright as Patient 21611, but still nearing the brightness of the control. The samples varied in brightness, but still were not dark. The calf thymus DNA concentration was 1.2062 μg/mL
Conclusions
Patient 21611 : Tested positive as the concentration was higher than the positive control. There was probably some error, but it can certainly assumed the patient tested positive as it had a high concentration than the control.
Patient 21095 : Tested positive as the concentration was closer to the positive benchmark than the negative benchmark. Although it was not on the positive benchmark, the gap can be attributed to error.
SNP Information & Primer Design
Background: About the Disease SNP
DNA is made up of long strains of the four nucleotides: adenine, thymine, cytosine, and guanine. The order of these nucleotides determines what proteins are created and the function of these molecules. A single nucleotide polymorphism is a change in one of these nucleotides that leads to a change in the order of amino acids that make up proteins in the body, therefore altering how a group of genes are physically expressed. In short, a SNP is a small change in genotype that leads to a change in phenotype.
The specific SNP that we have been researching is rs16991654. This is an SNP found in homo sapiens and on the 21:34370656 chromosome. This SNP is pathogenic and is associated with the KCNE2 gene and the congenital long QT syndrome. This gene regulates neurotransmitter release, heart rate, insulin secretion, neuronal escitability, epithellal electolyte transport, smooth muscle contraction, and cell volume.
Primer Design and Testing
Non-disease primers
Non-Disease Forward Primer: CAT GGT GAT GAT TGG AAT GT
The result matches a 220 DNA base pair sequence of nucleotides which contain both the forward and reverse primers. The location where the sequence is capitalized is where the sequence was identified. As a non disease human primer was used, it was able to be matched to a chromosome where the sequence is found. The chromosome which was matched is the chromosome which has rs16991654 variation. The forward primer has a melting point at 56.5 degrees Celsius, and the reverse primer has a melting point at 60.0 degrees Celsius.
Disease specific primers
Non-Disease Forward Primer: CAT GGT GAT GAT TGG AAT GC
There are no results which match the disease-specific forward and reverse primer. This is no match because the non-disease human genome used in the primer test does not contain the disease-associated allele, thus the disease-specific primer will not work.
BME100: PCR LAB D
What is a Nucleotide?
Nucleotides form nucleic acids such as DNA and RNA. They serve as subunits to nucleic acids. There are four types of nucleotides: Adenine, Guanine, Thymine, Cytosine, and Uracil. They have at least one phosphate group and thus are able to move energy within the cell in the form of nucleoside triphosphates.
What is a polymorphism?
Polymorphism is the genetic variation in the DNA within a population. The genetic variation is caused by mutations in the nucleotides. Natural selection relies on polymorphism in order to be able to bring substantial change to a population.
What species is this variation found in? (latin name)
Homo sapiens
What chromosome is this variation found on?
21:34370656
What is listed as the Clinical Significance of this SNP?
Pathogenic
What gene(s) is this SNP associated with?
KCNE2
Click the PubMed link to view summaries of research associated wit the SNP. What disease is linked to this SNP?
Congenital long QT syndrome
What does KCNE2 stand for?
Potassium-voltage-gated channel, lsk-related family, member 2
Click the KCNE2 link. Briefly describe the molecular functions of this gene?
The gene regulates neurotransmitter release, heart rate, insulin secretion, neuronal escitability, epithellal electolyte transport, smooth muscle contraction, and cell volume. The gene encodes a member of the potassium channel, voltage-gated, lsk-related subfamily.
What is an allele?
An alternate form of a gene which arises by genetic variation. It is any of the possible forms in which a gene for a specific trait can occur. Generally one allele is inherited from each parent. If the paired alleles are the same then its called homogenous, and if they are different then they are called heterozygous. Most alternate forms of the gene do not show any observable changes in the phenotype.
The disease-associated allele contains what sequence?
CTC
The numerical position of the SNP is?
Position 34370656
Non-disease forward primer (20 nt)
CAT GGT GAT GAT TGG AAT GT
The numerical position exactly 200 bases to the right of the disease SNP is
BME 100 Fall 2014
