LAB 5 WRITE-UP
Smart Phone Camera Settings
- Type of Smartphone: Samsung Galaxy Note 2
- Flash: Deactivated
- ISO setting: 800
- White Balance: Auto
- Exposure: +2.0
- Timer: 5.0 Seconds
- Photo Size: 4128x3096 pixels
- Distance between the smart phone cradle and drop = 7 cm
Solutions Used for Calibration
The fluorimeter was set up initially by placing it on top of several trays. The phone was adjusted properly by placing it into a phone holder 7 cm away from the base of the fluorimeter, with the camera facing the fluorimeter. To shade the fluorimeter from light, a light box was placed over both the fluorimeter and phone holder. The light box's flap was closest to the phone holder. For each calibration droplet, a fluorimeter slide was placed in the fluorimeter and 80 μL of the DNA solution was placed between the two dots closest to the phone camera on the hydrophobic surface of the slide. Then, 80 μL of the SYBR GREEN dye was added to the DNA solution until the two droplets combined to form a larger, ellipsoid shaped droplet touching the two front dots of the hydrophobic fluorimeter slide. The phone settings were adjusted accordingly (see above), the camera was focused and the timer was activated. During the 5 second timer, the flap of the light box was moved down to remove the light exposure of the droplet from the lights in the room. Once the picture was taken, the light box was removed and a micropipettor was used to remove the ellipsoid shaped droplet. The slide was then moved into the next position for the next droplet to be tested.
Table: Solutions Used for Calibration
Placing Samples onto the Fluorimeter
- Line up the light with the site where the drops are being placed on the slide
- Pipet 80 microliters of SYBR Green Solution on the slide with the hydrophobic side up
- Pipet 80 microliters of PCR dilution onto the SYBR Green Solution already on the slide
- Make sure light is lined up with the drop, set the timer for the camera and close Fluorimeter
Representative Images of Negative and Positive Samples
Image J Values for All Calibrator Samples
Table 1: Calibration Samples- Raw & Background-Subtracted Data
Table 2: Calibration Samples - Concentrations, Means, Standard Deviation of Background Subtracted Values
Below is a graph of the calibration curve, which will be used to calculate the final concentration for each PCR sample.
Data Tables for PCR Product Samples
PCR Results Summary
- Our positive control PCR result was 12.76 μg/mL
- Our negative control PCR result was 2.87 μg/mL
- Patient 56859: An average of 9.6 μg/mL (10.7, 9.2, 8.8). This patient has a significant amount of the disease DNA, because the solution fluoresced green indicating presence of that DNA.
- Patient 50591: An average of 10.3 μg/mL (12.8, 9.6, 8.6). This patient has even more of the disease DNA. The solution fluoresced even brighter, indicating presence of the DNA in question.
- Patient 56859: This patient's values are two-thirds closer to the positive results than the negative. There is a very high probability that this patient has the disease.
- Patient 50591: This patient's results are very similar to the positive results. This patient almost certainly has the disease in question.
SNP Information & Primer Design
Background: About the Disease SNP
The disease SNP is found in homo-sapiens on the 21st chromosome. This SNP is a pathogenic disease that is linked to Congenital Long QT Syndromes (LQTSs). The SNP is associated with the KCNE2 gene, which stands for “Potassium Voltage-gated Channel, Isk-related Family, Member 2” and controls the release of neurotransmitters and the secretion of insulin. LQTS is a rare heart condition that causes irregular heartbeats. The change in the allele sequence from TTC to CTC indicates that an individual has the disease.
SNP stands for Single-Nucleotide Polymorphism which occurs when a single nucleotide differs from either another biological species or a paired chromosome. In other words, a single nucleotide in the sequence differs from a comparable sequence making a difference in the species where it is present.
Primer Design and Testing
After completing the primer test, it was concluded that the non-disease specific primers matched a sequence of DNA in healthy humans. However, the disease-specific primers resulted in "no matches". Using this information, it is evident that this change in nucleotide sequence does not allow the normal primers to function.