BME100 s2014:T Group6 L5

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Contents

OUR TEAM

Name: Nicole D. Fisk
Name: Nicole D. Fisk
Name: Doan-Nhi T. Tran
Name: Doan-Nhi T. Tran
Name: Mark A. Keppler
Name: Mark A. Keppler
Name: Rayan S. Altayyar
Name: Rayan S. Altayyar
Name: Brittani M. Ogden
Name: Brittani M. Ogden


LAB 5 WRITE-UP

Background Information

Single-Drop Fluorimeter Device
Single-Drop Fluorimeter Device

SYBR Green Dye
SYBR Green Dye is a molecular dye that binds to DNA. The green dye stains double-stranded DNA as well as single-stranded DNA, but with greater preference for the former. The resulting DNA-dye-complex emits a fluorescent green light. SYBR Green Dye is commonly used by scientists due to its adherence to dsDNA.


Single-Drop Fluorimeter
The single-drop fluorimeter is an instrument that detects the amount of fluorescent material. The fluorimeter operates by shining high-intensity light beams at the sample and determining how much light is absorbed and how much is emitted.

How the Fluorescence Technique Works
The fluorescence technique works by exciting the targeted molecules of a sample with a photon of energy in the form of a beam of laser. The light excites the fluorescent dye and causes it to emit a light of lower wavelength. The amount of fluorescence detected by the a fluorimeter reveals the amount of excited SYBR Green dye and consequently the amount of dsDNA in the sample.


Procedure

Smart Phone Set Up
Smart Phone Set Up

Smart Phone Camera Settings

  • Type of Smartphone: iPhone 5S
  • Flash: Inactivated
  • ISO setting: N/A
  • White Balance: N/A
  • Exposure: N/A
  • Saturation: N/A
  • Contrast: N/A


Calibration

  • The smartphone is aligned on the cradle so that the camera is focused on the illuminated drop from the side.
  • Distance between the smart phone cradle and drop = 6.5 cm


Solutions Used for Calibration

Initial 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)
5.0080802.500
2.0080801.000
1.0080800.500
0.5080800.250
0.2580800.125
0.0080800.000


Placing Samples onto the Fluorimeter
1.) First, turn on the excitation light and the camera then place the camera on the cradle with adjusting the height to focus on the drop sideways.

2.) Second, place 80 microliter drop of SYBR Green 1 on the slide then place 80 microliter of the sample over the SYBR Green.

3.) Third, take a photo of the fluorimeter after lowering the lid of the box, so that it removes as much of stray light.

4.) Finally, remove the box without moving the smartphone and record the sample. Repeat for all samples.


Data Analysis

Sample with no DNA
Sample with no DNA
Sample with DNA
Sample with DNA

Representative Images of Samples
[Shown to the right]

Image J Values for All Samples

Final DNA concentration in SYBR Green I solution (µg/mL) AREA Mean Pixel Value Rawintenden of the Drop
05612854.7043070438
0.256018888.2515311680
0.558392107.7476291567
162152135.2458405724
275472124.3739386656
575472201.53515210272


Fitting a Straight Line

Graph 1.

PCR Results Summary

A polymerase chain reaction (PCR) was utilized to amplify the DNA from 2 patients identified here as 46296 and 16946 being screened for point mutations in chromosome 6’s small ubiquitin-like modifier 4 gene (SUMO4). A positive and negative control were amplified alongside the patient’s DNA. PCR results were analyzed by using SYBR Green and a blue light emitting diode (LED) capable of causing the SYBR to fluoresce if DNA was present in the sample.

Before the patient samples could be analyzed, known concentrations of calf thymus DNA had to be used in conjunction with the SYBR to create a curve fit correlating DNA concentration to the intensity of SYBR fluorescence. Since the calf DNA incorporates SYBR in the same manner that human DNA does, the result will be a tool capable of determining the unknown concentration of DNA in the patient PCR tubes.

An iPhone 5 was used to take snap shots of 160 μL droplets, consisting of 80 μL of calf thymus solution that ranged from 0 – 5 μg/mL and 80 μL of SYBR Green. The photos were imported into Image J, a public, java based program available through the National Institute of Health (NIH). Image J has a function that calculates the integrated pixel density of image samples. The integrated density (RAWINTENDEN) of the samples and the known DNA concentrations were incorporated in Graph 1 above.

Each of the 100 μL post PCR human DNA samples were transferred into separate tubes containing 500 μL of buffer solution. The procedure used during calibration was then repeated using the PCR samples in place of calf thymus DNA. The slope intercept equation for the linear trend line established through the calibration procedure was then used to determine the concentrations of human DNA in the sample tubes.

The images for both patients were somewhat dark and opaque with a low intensity of fluorescence. The patient DNA droplet images were both similar to the sample image of the droplet containing no DNA shown above. This qualitative result combined with the relatively low concentration of DNA found by curve fit demonstrates that both patients should be negative for the SUMO4 point mutation.

-Positive control PCR result: 0.186 μg/mL
-Negative control PCR result: 0.065 μg/mL

-Patient 16946: PCR result: 0.029 μg/mL.
-Patient 46296: PCR result: 0.037 μg/mL.

-Patient 16946: Negative
-Patient 46296: Negative




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