BME100 s2014:T Group10 L5

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OUR TEAM

Sean-Christopher Bradbury
Thursday Group 10 Member
Suyen Go
Thursday Group 10 Member
Nathaniel Kirkpatrick
Thursday Group 10 Member
Maya Robinson
Thursday Group 10 Member
Lissette Valle
Thursday Group 10 Member

LAB 5 WRITE-UP

Background Information

SYBR Green Dye

SYBR Green Dye is a dye that stains nucleic acids by binding to DNA or RNA. The dye is used to visualize DNA through staining agarose gels, biochip applications, fluorescence imaging techniques, real-time PCR, and more. SYBR dye fluoresces when combined with dsDNA (Zipper Hubert Brunner 1). However, unbound SYBR Green is unreactive to light. This feature allows scientists to determine the concentration of a target DNA sample by measuring the intensity of the fluorescent light being emitted by the sample. The higher the intensity of the light being emitted, the more concentrated the sample is with the target DNA. SYBR Green, as its name implies, fluoresces with a green color. Accordingly, it is important to use a different wavelength of light to excite the sample in order to prevent distorting the results. There are several types of fluorescent dyes similar to the SYBR Green used in this lab, but SYBR Green was chosen for its environmental friendliness and high level of light emission intensity to allow for easier detection of binding to the target DNA.


Single-Drop Fluorimeter

A fluorimeter is a device used to measure and identify fluorescence in a medium. Fluorimeters measure certain wavelengths of light and have two light detectors, one of which detects absorbency while the other detects fluorescence emission (So Dong 1). Single-Drop Fluorimeters do this with a single drop of a sample that is placed on a hydrophobic slide in the path of a beam of single wavelength light. For example, in our setup we used blue LED light to allow for light absorption and prevent contaminated intensity data that would occur if we used a green LED since the SYBR Green Dye fluoresces in the green range. A light detector or camera placed at a uniform distance from this sample is then used to capture the intensity of the fluorescence being emitted from the sample. Cameras are acceptable for this laboratory set-up even though they are unable to measure light intensity because it is assumed that light intensity is proportional to brightness; the brighter an object is in a photo, the higher the light intensity.


Single-Drop Fluorimeter setup without covering.
(Figure: Our Single-Drop Fluorimeter setup. A sample is placed in the path of the LED light on the white hydrophobic slide and its light intensity is captured by taking a picture with the iPhone. Image by Nathan Kirkpatrick)

Single-Drop Fluorimeter setup with covering.
(Figure: Our Single-Drop Fluorimeter covered by a box to prevent ambient light interference. A timer is used to take the picture of the sample once the box is closed. Image by Nathan Kirkpatrick)


How the Fluorescence Technique Works
In this experiment, the concentration of DNA was determined by measuring the amount of fluorescence in a medium that had been dyed with SYBR Green Dye. Since light intensity is assumed to be proportional to brightness, images taken with a camera using the Single-Drop Fluorimeter setup described above can be used to determine the intensity of the SYBR Green Dye and, accordingly, the concentration of target DNA in the sample. After a single-drop fluorimeter was used to measure the fluorescence and an image was taken, ImageJ software was used to determine the concentration of DNA. We use the ImageJ software to split the color channels and work with the green channel, thereby eliminating intensity contamination from the blue LED. Then we draw an oval around the drop and use the software to calculate the RawIntDen of the drop that is the intensity reading of the part of the image within the oval (the drop). Since the pictures were taken at a uniform distance from the drop and the total volume of each drop is the same, we can assume that the area of the drop is constant and are thusly able to compare the intensity values directly without any conversion. For the calibration, we also took a background sample oval of the same area in ImageJ to get a baseline intensity for that image that was subtracted from the intensity value of the drop to get more accurate data. The intensity of the drops of patient samples are then compared to positive and negative controls to determine if they contain the target DNA sequences. For the PCR Data, we conducted a quick calibration of three SYBR Green I and DNA sample concentrations then plotted their intensities on a graph. The line of best fit for this graph can then be used to determine the concentration of target DNA sequences in for our PCR samples with unknown amounts of the target DNA sequences.



Procedure

Smart Phone Camera Settings

  • Type of Smartphone: Apple iPhone5S
    • Flash: no flash
    • ISO setting: 2500
    • White Balance: Auto
    • Exposure: Highest Setting
    • Saturation: N/A
    • Contrast: N/A


Calibration

  • Distance between the smart phone cradle and drop = 8cm
  1. Place a slide with a hydrophobic side in the fluorimeter tray with the hydrophobic side up.
  2. Turn on the blue LED light and make sure it is aligned with the sample drop.
  3. Prepare the camera by ensuring the drop is in focus, centered in the frame, the flash is off, and the ISO is locked at 2500 (we were unable to control the other settings listed in the laboratory workbook.
  4. Verify that the distance from the drop to the camera is 8 cm.
  5. Place 80 μL drop in between two holes in the hydrophobic slide to prevent it from moving.
  6. Add 80 μL of the calf thymus solution or water blank on top of the SYBR Green drop.
  7. Make sure it is lined up with the blue LED light.
  8. Double check that the iPhone camera is 8 cm from the drop.
  9. Set the timer of the iPhone camera to 5 seconds.
  10. Place the black-out box over the whole set-up carefully.
  11. Close the cover.
  12. Check to make sure picture is in focus and valid.
  13. Check the iPhone-Drop distance again.
  14. Take two more pictures.
  15. Remove the drop with a pipette.
  16. Repeat these step for all calibration concentrations using different locations on the hydrophobic slide for each new concentration.

Aerial view of Single-Drop Fluorimeter Set-up.  the distance from the iPhone's camera to the drop is 8 cm.
(Figure: Aerial view of Single-Drop Fluorimeter Set-up. the distance from the iPhone's camera to the drop is 8 cm.)


Solutions Used for Calibration

Initial Concentration of 2X Calf Thymus DNA Solution (μg/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 80 80 2.5
2 80 80 1
1 80 80 0.5
0.5 80 80 0.25
0.25 80 80 0.125
0 80 80 0


Placing Samples onto the Fluorimeter

  1. Make sure you are wearing proper personal protective equipment.
  2. Make sure the hydrophobic slide has the hydrophobic side face up.
  3. Make sure slide is lined up with the LED.
  4. Make sure micropipette is set to 80 μL.
  5. Use good micropipette practices to add 80 μL of SYBR Green I to in between two holes in the middle column of the hydrophobic slide.
  6. Use good micropipette practices to add 80 μL of the calibration solution on top of this drop.
  7. Follow steps listed above to get pictures.
  8. Use pipette to remove the dot.
  9. Put used solution mix into liquid biohazard waste container.
  10. Move slide to the next set of holes.
  11. Repeat for each calibration concentration.
  12. When finished place the slide in the biohazard sharps container.
  13. Dispose of pipette, micropipette tips, gloves and other used materials in the biohazard container.


Data Analysis

Representative Images of Samples

ImageJ Green-Channel isolated Negative Control image with circle around droplet.
(Figure: ImageJ Green-Channel isolated Negative Control image with circle around droplet)


ImageJ Green-Channel isolated Positive Control image with circle around droplet.
(Figure: ImageJ Green-Channel isolated Positive Control image with circle around droplet)


Image J Values for All Samples

Calibration Data

Final DNA Concentration in SYBR Green I Solution (μg/mL) Area Mean Pixel Value RAWINTDEN of the Drop RAWINTDEN of the Background Subtracted RAWINTDEN
0 35438 29.469 1044333 5349 1038984
0 35508 28.895 1026007 4082 1021925
0 38040 27.429 1043189 2237 1040952
0 33727 28.897 974620 8357 966263
0.25 34864 56.034 1953555 10309 1943246
0.25 36644 47.857 1753686 11015 1742671
0.25 32122 45.717 1468518 6105 1462413
0.25 31920 46.667 1489608 6226 1483382
0.5 36018 66.312 2388411 6733 2381678
0.5 35838 66.157 2370925 7255 2363670
0.5 34096 70.996 4240685 10984 2409701
0.5 33313 64.808 2158943 7057 2151886
1 32710 100.82 3297817 7473 3290344
1 32380 101.547 3288080 7660 3280420
1 36709 87.955 3228755 7600 3221155
1 37464 87.787 3288829 8455 3280374
2 36244 112.346 4071880 11409 4060471
2 33123 105.502 3494534 30121 3464413
2 30036 105.369 3164875 16870 3148005
2 27950 115.15 3246380 5312 3241078
5 31172 129.814 4046570 11325 4035245
5 30780 130.063 4003335 11554 3991781
5 33575 128.746 4322651 4541 4318110
5 33727 129.1 4356861 373 4356132


Calibration Data Plot
(Figure: Plot of the calibration data with Calf Thymus DNA concentration on the x-axis and INTDENS Readings after background correction on the y-axis with trend line.)


PCR Product Sample Measurements

PCR Product Tube Label Area (pixel^2) Mean Pixel Value RawIntDen of the Drop
PC 1 30528 123.781 3778777
PC 2 28794 124.966 3598262
PC 3 28349 126.204 3577769
NC 1 26035 49.289 1283252
NC 2 25986 38.364 1002239
NC 3 24081 34.513 831118
1R1 1 26780 40.389 1081609
1R1 2 28678 38.826 1113459
1R1 3 27348 39.13 1070129
1R2 1 27328 43.119 1178362
1R2 2 29178 45.662 1332320
1R2 3 26307 47.495 1249444
1R3 1 22214 44.459 987619
1R3 2 26264 32.101 843096
1R3 3 25368 33.248 843428
2R1 1 22296 126.872 2828741
2R1 2 25085 130.523 3274161
2R1 3 24628 130.723 3219456
2R2 1 26100 129.338 3375712
2R2 2 26272 128.394 3373160
2R2 3 23711 129.676 3074751
2R3 1 23900 128.988 3082802
2R3 2 23824 131.695 3137491
2R3 3 27428 131.43 3604871


The table below shows the concentrations of the PCR Products. We used the line of best fit from our quick calibration to determine these values. The PCR Product Concentrations was determined by subtracting the Average INTDENS value of the drop by the intercept of the best fit line (2,000,000 INTDENS) and dividing by the slope of the best fit line (1,000,000 INTDENS/concentration). The concentrations then need to be adjusted for the total dilution that took place in the experiment. When 100 μL of the PCR reaction was added to 500 μL of buffer, the PRC reaction wad diluted by 1/6. Additionally, when 80 μL of the diluted PCR reaction was added to 80 μL of SYBR Green I solution, the PCR reaction was further diluted by 1/2. In total, the PCR reaction was diluted by 1/12. Accordingly, the PCR product concentrations need to be adjusted by multiplying by the denominator of the total dilution (12). These values are reflected in the fourth column of this table.

PCR Product Tube Label Average INTDENS Value of the Drop Average PCR Product Concentration μg/mL Corrected Average PCR Product Concentration, μg/mL
PC 3651602.667 1.651602667 19.819232
NC 1038869.667 -0.961130333 -11.533564
1R1 1088399 -0.911601 -10.939212
1R2 1253375.333 -0.746624667 -8.959496
1R3 891381 -1.108619 -13.303428
2R1 3107452.667 1.107452667 13.289432
2R2 3274541 1.274541 15.294492
2R3 3275054.667 1.275054667 15.300656


For the sake of clarity, the samples are listed below with their corresponding Patient ID Number and corrected PCR Product Concentration. The Positive and Negative controls are also included.

Sample Corrected PCR Product Concentration (μg/ml)
Postive Control 19.819232
Negative Control -11.533564
Patent: 21489 Replicate 1 -10.939212
Patent: 21489 Replicate 2 -8.959496
Patent: 21489 Replicate 3 -13.303428
Patent: 90434 Replicate 1 13.289432
Patent: 90434 Replicate 2 15.294492
Patent: 90434 Replicate 3 15.300656


Fitting a Straight Line


PCR Data Plot
(Figure: PCR Product Sample Quick Calibration measurements with a line of best fit.)


PCR Results Summary
Instructor's summary: You completed 8 PCR reactions in a previous lab. You used the SYBR Green I staining and imaging technique to measure the amount of amplified DNA in each PCR reaction. You used a standard curve (based on known concentrations of calf thymus DNA) to convert INTDEN values into DNA concentration. Your positive control and negative control samples should be used as threshold values for determining whether an unknown (patient) sample is truly positive or negative.

  • Your positive control PCR result was 19.819232 μg/mL
  • Your negative control PCR result was -11.533564 μg/mL


Write-in each patient ID and give both a qualitative (what the images looked like) and a quantitative description (μg/mL) of what you observed

  • Patient 21489 : The samples containing this patient's PCR-duplicated DNA with SYBR Green I did not appear to exhibit SYBR Green I-affiliated fluorescence. In other words, the droplet did not glow at all. The Average Corrected PCR Product Concentration of -11.06737867 μg/mL for all three replicates of this patient supports this visual observation.
  • Patient 90434 :The samples containing this patient's PCR-duplicated DNA with SYBR Green I did appear to exhibit SYBR Green I-affiliated fluorescence. In other words, the droplet was visually glowing. The Average Corrected PCR Product Concentration of 14.62819333 μg/mL for all three replicates of this patient supports this visual observation.

Compare each patient's results to the positive control value and the negative control value. Draw a final conclusion for each patient (positive or negative) and explain why you made that conclusion.

  • Patient 21489 : The Average Corrected PCR Product Concentration of -11.06737867 μg/mL for all three replicates of this patient is very close to the negative control threshold value of -11.533564 μg/mL. From this, we can conclude that Patient 21489 does not contain the DNA sequence in question. We came to this conclusion because the SYBR Green I fluoresced as much in this patient's samples as it did in the negative control where no target DNA sequences were present.
  • Patient 90434 : The Average Corrected PCR Product Concentration of 14.62819333 μg/mL for all three replicates of this patient is very close to the positive control threshold value of 19.819232 μg/mL. From this, we can conclude that Patient 21489 does contain the DNA sequence in question. We came to this conclusion because the SYBR Green I fluoresced as much in this patient's samples as it did in the positive control where a significant amount of target DNA sequences were present. Unfortunately, Patient 90434 has the condition associated with this allele.

Works Cited

Zipper, Hubert, Herwig Brunner, Jurgen Bernhagen, and Frank Vitzthum. "Investigations on DNA Intercalation and Surface Binding by SYBR Green I, Its Structure Determination and Methodological Implications." Ncbi.nih.gov. US National Library of Medicine, 12 July 2004. Web. 3 Apr. 2014.

So, Peter TC, and Chen Y. Dong. "Fluorescence Spectrophotometry." Scribd. Macmillan Publishers Ltd, Nature Publishing Group, 16 Jan. 2009. Web. 03 Apr. 2014.