Calibration
Open up the camera on the smartphone and adjust the settings to fit the settings described above. Place the smartphone in the cradle and adjust the height of the fluorimeter in order for the camera to be able to take a picture of the drop sideways. One should be able to see the side of the plate and the drop. Adjust the distance of smartphone in the cradle and the drop in order to be able to have the best possible image that is close to the drop but is not blurry.
Distance between the smart phone cradle and drop = 6 cm
Solutions Used for Calibration
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 (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
Locate the smooth side of the glass slide using gloves.
Turn the fluorimeter on.
Smooth side facing down, place a slide into the fluorimeter.
With the camera app on, put the smartphone on the cradle. Set the camera timer for three seconds.
Make sure the the camera view of the slide is nearly edge-on. If needed, adjust the fluorimeter height.
In the middle of the slide on the first two clear circles, place 80 uL drop of SYBR Green I solution.
On top of the SYBR Green I solution drop, place 80uL of the Sample/calibration solution.
Make sure the light illuminates the center of the drop and the drop is focused on the light of the other side. The slide and be adjusted for this step.
Record the distance between the smartphone and the fluorimeter so the drop is viewed closely, but still in focus.
With the flap up, cover the lightbox.
Double check to make sure the drop is focused.
Take the picture. To do this, lower the flap and depress the dimer on the camera.
Discard of the 160 uL drop from the slide into the liquid waste container.
Move the slide to the center of the next two circles.
Repeat the procedures with the slide for all of the five possible measurement positions.
When the procedures have been carried out, all slides must be placed in the SHARPS container.
Data Analysis
Representative Images of Negative and Positive Samples
Negative PCR Sample
Positive_PCR_Drop.png
Image J Values for All Calibrator Samples
Due to time constraints in the lab, only one trial for each concentration was recorded.
Because only one trial was carried out, a standard deviation is not applicable.
Due to time constraints in the lab, only one trial for each concentration was recorded. The value for this trial is used for the mean.
Calibration curve
PCR Results Summary
Our positive control PCR result was 34.92 μg/mL
Our negative control PCR result was -8.172 μg/mL
Observed results
Patient 50958 : The drops in the images for this patient did not glow. The drop was not florescent because there was no dsDNA present. The patient's concentration results were closer to the negative control. The images for patient 50985 did not show any florescence, indicating that the patient was negative. The DNA concentration for patient 50958 was found to be -0.597 μg/mL.
Patient 95193 : The drops in the images for this patient did not glow.The drop was not florescent because there was no dsDNA present. This patient's concentration results were also much closer to the negative control. The images for patient 95193 did not show any florescence, indicating that the patient was negative.The DNA concentration for patient 95193 was found to be -0.660 μg/mL.
Conclusions
Patient 50958 : This patient's DNA concentration results were much closer to the negative control value and the drop was not florescent because there was no dsDNA present and it did not glow. The patient tested negative.
Patient 95193 : This patient's DNA concentration results were much closer to the negative control value and the drop was not florescent because there was no dsDNA present and it did not glow. This patient also tested negative.
SNP Information & Primer Design
Background: About the Disease SNP
SNP rs16991654 is a variation commonly found in Homo sapiens and is located on the 21:34360656 chromosome and is found in the Homo sapiens species. It is of pathogenic significance due to it being linked to congenital long QT syndrome (LQTS), a heart rhythm disorder that can potentially cause fast, chaotic heartbeats. This SNP is also associated with the KCNE2 gene; KCNE2 is the gene ID that contains SNP rs16991654. KCNE2 stands for potassium voltage-gated channel, the most complex class of voltage-gated ion channels. KCNE2's functions include regulating neurotransmitter release, heart rate, insulin secretion, neuronal excitability, epithelial electrolyte transport, smooth muscle contraction, and cell volume.
Primer Design and Testing
Initially, the group investigated the DNA sequence of the KCNE2 gene, specifically in the region where the rs16991654 polymorphism was present. Based on the nucleotide sequences immediately before the mutation, the forward primers were created taking into account the 20 base pairs preceding the target nucleotide, and ending them with the first nitrogenous base of the non-diseased and the diseased forms of the nucleotide. Then, the same procedure was followed to determine the DNA sequences for the reverse primers. One of the differences in this case was that the second DNA strand was used to create the reverse primer.Since this strand is directed from 3' to 5'(reading frame is from right to left), the sequence had to be reversed when written so that it's direction was shifted to 5' to 3'. For both the forward and the reverse primers of the disease sequence, the thymine at the start of the target nucleotide sequence had to be changed from a thymine to a cytosine. Posteriorly, the primers were evaluated using the UCSC In-Silico PCR program to ensure all the nitrogenous bases of the sequences were in the correct position.
BME 100 Fall 2014
