BME100 f2014:Group22 L5

From OpenWetWare

Jump to: navigation, search

Contents

Our Team

Name: Aaron Raber
Name: Aaron Raber
Name: Chase Radigan
Name: Chase Radigan
Name: Daniel Sorto
Name: Daniel Sorto
Name: Zach Steidl
Name: Zach Steidl
Name: Lisa Lavergne
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:
Positive Sample Positive 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

Graph 1


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)

Personal tools