BME100 f2014:Group22 L5

Our Team

Name: Aaron Raber

Name: Chase Radigan

Name: Daniel Sorto

Name: Zach Steidl

Name: Lisa Lavergne

Lab B/C Content

Procedure

Note: Due to technical difficulties with the phone camera and the dark box/fluorimeter setup, there was not enough time to capture a full three images per droplet in the calibration. As a result, the final calculated concentrations may not be quite as reliable, though they should still have enough reliability in calculating the concentration of DNA, considering the strong linear correlation between the calibration images' INTDEN values and concentrations.

Camera Settings

The camera used was a Samsung Galaxy S4. Camera settings include:

• ISO: 800
• Photo Size: 4128x3096 pixels
• White Balance: automatic
• Exposure Value: +2.0
• Flash: off
• Photo Timer: 5.0 Seconds

Calibration & Solutions Used

For the calibration solutions, the fluorimeter was set up by placing the fluorimeter device on top of three plastic trays after activating the light. The phone was put in place by setting it inside the phone holder 8.5cm away from and square with the base of the fluorimeter, phone camera facing the fluorimeter. Over both the fluorimeter and the phone holder was a light box, with the back on the fluorimeter side. For each of the different calibration droplets, a fluorimeter slide was placed in the device, and 80 μL of the DNA solution was placed on two adjacent dots in the middle column of the hydrophobic surface. To that, a further 80 μL of CYBR GREEN dye was added to the droplets until the droplets on the two adjacent dots fused into a single, large, oblong droplet, spanning the hydrophobic surface between the two. After the smartphone camera was adjusted to the proper settings (described above), the camera was focused and the camera timer started. While the timer counted down, the flap of the light box was closed over the entire setup. After the picture was taken, the light box was removed and a micropipettor was used to remove the droplet, moving the slide to the next position in preparation for the next droplet after the previous one was removed.

Images Captured of Sample Drops

Positive Sample:	Negative Sample:

Dilutions

PCR Product TUBE LABEL	Volume of the DILUTED PCR Product solution (µL)	Volume of the SYBR GREEN I Dye solution (µL)	Dilution 1	Dilution 2	Total Dilution:
Positive	80 µL	80 µL	1/6	1/2	1/12
G22 1-1	80 µL	80 µL	1/6	1/2	1/12
G22 1-2	80 µL	80 µL	1/6	1/2	1/12
G22 1-3	80 µL	80 µL	1/6	1/2	1/12
G22 2-1	80 µL	80 µL	1/6	1/2	1/12
G22 2-2	80 µL	80 µL	1/6	1/2	1/12
G22 2-3	80 µL	80 µL	1/6	1/2	1/12
Negative	80 µL	80 µL	1/6	1/2	1/12

Procedure

The setup and camera settings for the fluorimeter analysis of the PCR results were unchanged from those of the calibration analyses as described above. The phone and camera settings remained the same, as did the distance between the base of the fluorimeter and the base of the phone holder (8.5cm). Each slide contained ten dots in the middle column, allowing for 5 droplets (taking up 2 dots each) to be analyzed on each slide. With 8 PCR product solutions to be analyzed, 2 slides were used. For each one, the slide was placed in the fluorimeter, carefully moved to the correct position (with the light between two of the dots). Between the two focused dots, 80 μL of DNA solution and 80 μL of CYBR GREEN dye were micropipetted into position, and the picture was taken after putting the light box into place to block outside light. After each picture, the micropipettor was used to thoroughly remove the droplet before carefully moving the slide into position for the next. Between slides, the first slide was carefully removed (avoiding moving the fluorimeter or phone holder), and the next was inserted with equal care. After each picture was taken, the label of the tube from which the sample originated was recorded in order to preserve the proper labeling of each.

Data Analysis

Excel Table 1: Raw Data

Final DNA concentration in SYBR Green I solution (µg/mL)	Area	Mean Pixel Value	RAWINTDEN of the Drop	RAWINTDEN of the Background	RAWINTDEN Drop - Background
0	743	45.51	33813.93	0	33813.93
0.25	762	47.277	36025.074	0	36025.074
0.5	788	50.411	39723.868	0	39723.868
1	788	55.555	43777.34	0	43777.34
2	816	102.832	83910.912	0	83910.912
5	972	164.168	159571.296	0	159571.296

Excel Table 2: Concentrations and Means of background-subtracted Values

Final DNA concentration in SYBR Green I solution (µg/mL)	Rawintden Drop- Background	Mean
0	33813.93	33813.93
0.25	36025.074	36025.074
0.25	39723.868	39723.868
0.5	43777.34	43777.34
1	83910.912	83910.912
2.5	159571.296	159571.296

Graph of Concentration Calibration

Excel Table 3: Raw and background-subtracted data for PCR

PCR Product TUBE LABEL	Area	Mean Pixel Value	Rawintden of the Drop	Rawintden of the Background	Raw Drop- Background
Positive	801	150.125	120250.125	0	120250.125
1-1	764	138.809	106050.076	0	106050.076
1-2	816	163.685	133566.96	0	133566.96
1-3	826	130.857	108087.882	0	108087.882
2-1	767	137.314	105319.838	0	105319.838
2-2	838	132.415	110963.77	0	110963.77
2-3	816	168.19	137243.04	0	137243.04
Negative	810	37.38	30277.8	0	30277.8

Excel Table 4: Means of background-subtracted values for PCR

Final DNA concentration in SYBR Green I solution (µg/mL)	Rawintden Drop- Background	Mean
0	33813.93	33813.93
0.25	36025.074	36025.074
0.25	39723.868	39723.868
0.5	43777.34	43777.34
1	83910.912	83910.912
2.5	159571.296	159571.296

INTDEN = 26185.44*concentration +27959.403 Concentration = (INTDEN-27959.403)/26185.44

PCR Product Concentrations

PCR Product TUBE LABEL	Mean RAWINTDEN Drop - Background	PRC Product Concentration (µg/ml)	Initial PCR Product Concentration (µg/ml)
Positive	120248.5273	1.762222142	21.1466657
1-1	106053.9463	1.491182568	17.89419082
1-2	133565.727	2.016508487	24.19810185
1-3	108094.5933	1.530147867	18.36177441
2-1	105319.0567	1.477150158	17.7258019
2-2	110968.9603	1.5850327	19.0203924
2-3	137245.3857	2.086770027	25.04124032
Negative	30269.64767	0.04411315347	0.5293578416

INTDEN = 52.370.88*concentration + 27959.403 Concentration = (INTDEN-27959.403)/52370.88

Results Summary

Through the process of the calibration of the device, the equation of the standard curve for the different known concentrations was determined to be INTDEN=26185.44*concentration+27959.403 which was then solved for concentration to give the equation, concentration=(INTDEN-27959.403)/26185.44. This equation, along with the measured INTDEN of the unknown samples found using the cell phone fluorimeter device, was used to calculate the concentration of DNA in each of the unknown PCR patient samples. From these calculations it was determined that all the samples taken from both patients 1 and 2, with concentrations raging from 17.7258019 (µg/ml) to 25.04124032 (µg/ml), more closely resembled the positive control sample, with a concentration of 21.1466657 (µg/ml), than the negative control sample which we found to have a concentration of 0.5293578416 (µg/ml). Therefore, since all of the samples were most similar to the positive control sample, which contained DNA, rather than the negative control sample, which was intended to not contain any DNA, all of the samples were determined to be positive for the DNA.

Lab D: SNP Info and Primer Design

Part 1

1. A nucleotide is the monomer of DNA, consisting of a double helix ladder-like structure with a deoxyribose sugar and a phosphate group as the outer "pole" and one of four nitrogen-containing bases as the "rung". These nitrogenous bases include Adenosine (A), Thymine (T), Cytosine (C), and Guanine (G), which form pairs to create a unique sequence that defines a gene.
2. A polymorphism occurs when there are two or more different phenotypes from the same gene, and occurs quite commonly, as is the case with the phenotypes for human hair texture, which can be wavy, curly, or straight.

SNP rs16991654

1. Organism: Homo sapiens sapiens
2. Chromosome: 21
3. Clinical Significance: rs16991654 is a pathogenic allele
4. Genes: the selected SNP is associated with the KCNE2 gene
5. Disease: rs16991654 is associated with Long QT syndrome, which can cause an irregular heartbeat

Part 2

1. KCNE2 is a potassium voltage-gated channel, ISK-related family, member 2.
2. KCNE2 functions to regulate processes such as neurotransmitter release, heart rate, insulin secretion, and smooth muscle contraction.
3. An allele is any variation/mutation on the same gene. For instance, the gene for human hair texture can have two alleles, one for curly hair and one for straight hair; their phenotypes are the physical representations, which include curly, straight, and their heterozygous condition, wavy hair.
4. The disease-associated allele contains the sequence CTC, mutated from TTC, changing the amino acid Phenylalanine to Leucine.
5. The mutation is located in the numerical position 34370656.

Part 3

1. Non-Disease Forward Primer: 5'-CATGGTGATGATTGGAATGT
2. Reverse Primer Position: 34370856
3. Reverse Primer: 5'-CCCTTATCAGGGGGACATTT
4. Disease Forward Primer: 5'-CATGGTGATGATTGGAATGC
5. Disease Reverse Primer: 5'-CCCTTATCAGGGGGACATTT

Results

When testing the disease forward and reverse primers in the UCSC In-Silico PCR Search, the non-disease primers returned a perfect match with genome that was already on record (see Figure 1 below). This is because it was the control, the primers being targeted towards the normal, non mutated human gene, producing a perfect match with the non mutated gene on record.

Figure 1: Non-Disease Primer (Match)

When testing the forward and reverse primers that contained the single mutation associated with Long QT syndrome, the UCSC search returned no matches (see Figure 2 below). This is because the forward primer, with its single nucleotide change, did not meet the 20-nucleotide perfect match minimum of the search to be considered a match against the normal, non mutated human gene.

Figure 2: Disease Primer (Non-Match)