LAB 5 WRITE-UP
Smart Phone Camera Settings
- Type of Smartphone: Verizon iPhone 5s
- Flash: OFF
- ISO setting: N/A
- White Balance: N/A
- Exposure: N/A
- Saturation: N/A
- Contrast: N/A
- Distance between the smart phone cradle and drop = 5 cm
Solutions Used for Calibration
|Initial Concentration of 2X DNA solution (micrograms / mL)||Volume of 2X DNA solution (microliters)||Volume of SYBR Green I Dye (microliters)||Final DNA concentration in green dye (micrograms / mL)
| 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
Solutions Used for PCR Analysis
|PCR Product Tube Label||Volume of Diluted PCR Product solution (microliters)||Volume of SYBR Green I Dye (microliters)||Dilution 1 || Dilution 2 || Total Dilution
| G12 (+) || 80 || 80 || 0.167 || 0.5 || 0.0835
| G12 (-) || 80 || 80 || 0.167 || 0.5 || 0.0835
| G12 1-1 || 80 || 80 || 0.167 || 0.5 || 0.0835
| G12 1-2 || 80 || 80 || 0.167 || 0.5 || 0.0835
| G12 1-3 || 80 || 80 || 0.167 || 0.5 || 0.0835
| G12 2-1 || 80 || 80 || 0.167 || 0.5 || 0.0835
| G12 2-2 || 80 || 80 || 0.167 || 0.5 || 0.0835
| G12 2-3 || 80 || 80 || 0.167 || 0.5 || 0.0835
This image shows the fluorimeter device itself. The LED light is switched on with the slide put into place.
The cradle which held the smartphone in place is shown here. A certain distance was measured between the cradle
and the fluorimeter and was maintained for the rest of the experiment.
This image shows the entire setup which was used during the experiment.
Placing Samples onto the Fluorimeter
- Assuming the safety and fluorimeter setup protocols are in place, begin by placing an 80 microliter sample of the SYBR green dye on the superhydrophobic side of the slide between the middle of the first two rows on the slide.
- Once done, dispose of the micropipette tip used for the green dye sample and replace it.
- With a new micropipette tip, place an 80 microliter water blank sample on top of the green dye sample.'
- The drop must be aligned with the blue LED light so that the light will be focused on the fibre optic fitting on the other side of the drop. Do so.
- Ensure that the timer on the smartphone used is set. If so, focus the camera on the drop to improve image quality. Place the light box on the fluorimeter and start the timer once done. Close the lid to reduce the amount of light.
- Once the images are taken, remove the light box.
- With the micropipette, remove the 160 microliter sample into the waste disposal cup provided. Move the slide to its new position.
- Repeat steps for each sample until completion.
Representative Images of Negative and Positive Samples
Image J Values for All Calibrator Samples
Image J Values for All PCR Samples
For the tables provided, the research team was unable to acquire the necessary number of images for the experiment. The use of ImageJ was necessary to complete the task required. Each sample was analyzed using the green, red and blue split color channels on ImageJ. This provided the amount of data that was sufficient for the study. However, the accuracy of the data may be affected by using this process, as only the green color channel would have been used if the team had enough images.
PCR Results Summary
- Our positive control PCR result was 0.015135182 μg/mL
- Our negative control PCR result was 0.015143014 μg/mL
- Patient 83526 : The sample appears to have a noticeable amount of green pigmentation enveloped by the drop. The value of each sample coinciding with the patient's ID is positive.
- Patient 31092 : The sample seems to lack any sort of green dye coloring within the drop. Each sample corresponding with the patient's ID appear to be negative in value.
- Patient 83526 : Each value seems to compare well with the positive control, as there does not seem to be a radical difference or shift in nature. Since the values seem to be closely related to the positive control, the conclusion of positive can be made.
- Patient 31092 : The negative control value is positive, but the patient samples are negative in value. This may seem to be a part of miscalculation during the study. It is due to this that the conclusion of inconclusive can be made, due to a lack of accuracy.
SNP Information & Primer Design
Background: About the Disease SNP
SNP, known as single nucleotide polymorphism, is a process in which a single nucleotide (A,T,C,G) in a DNA fragment differs from another DNA fragment's nucleotide between members of a species. It is believed that SNP is caused by errors when copying DNA. SNP's modification of bases within DNA gives way to genetic mutations, affecting the impact of a disease or ailment. Despite this, SNP has value in the pursuit of science and medicine. Its nature allows studies such as predicting how a certain drug reacts within the human body to be more accurate and effective.
Nucleotide: a building block of DNA that corresponds to the letters A,T,C,G.
Polymorphism: Two or more different phenotypes exist in the same population of a species.
What species is this variation found in? (latin name)
What chromosome is the variation located on?
What is listed as the Clinical significance of this SNP?
Which gene(s) is this SNP associated with?
What disease is linked to this SNP?
What does LPL stand for?
What is the function of LPL?
- Apolipoprotein binding, heparin binding, lipoprotein lipase activity
What is an allele?
- An allele is a gene that has a form which can vary in different ways.
Primer Design and Testing
Two human subjects were used for studying the DNA sequence between them. This was to determine how at the biological level, diseases were able to affect the DNA of each subject. One patient had a particular disease while the other was healthy and did not have any medical issues. During the experiment, it was shown that DNA copying, or amplification, is able to occur for smaller fragments of DNA by primers binding to specific areas on the DNA sequence. DNA amplification takes place at two ends of DNA, known as the 5' (five prime) end and the 3' (three prime) end. The following information shown below describes what was discovered when testing each disease and non-disease primer.
The disease-associated allele
contains what sequence?
The numerical position of the SNP is:
Non-disease forward primer (20 nt):
AAT CTG GGC TAT GAG ATC AA
The numerical position exactly 200 bases
to the right of the disease SNP is:
Non-disease reverse primer (20 nt):
GAA ACA CCA GGG CTC AGG GT
Disease forward primer (20 nt):
AAT CTG GGC TAT GAG ATC AG
Disease reverse primer (20 nt):
GAA ACA CCA GGG CTC AGG GT
The results for comparing non-disease primers are shown here.
The results for comparing disease primers are shown here.