BME100 f2014:Group19 L5

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Contents

OUR TEAM

Name: Andrew Carlson
Name: Andrew Carlson
Name: Stacy Stoddard
Name: Stacy Stoddard
Name: Gareth Palas
Name: Gareth Palas
Name: Josh Hislop
Name: Josh Hislop
Name: Josh Martin
Name: Josh Martin
Name: Jose Elenes
Name: Jose Elenes


LAB 5 WRITE-UP

Procedure

Smart Phone Camera Settings

  • Type of Smartphone: Iphone 4
    • Flash: none
    • ISO setting: Automatically Set
    • White Balance: Automatically Set
    • Exposure: Automatically Set
    • Saturation: Automatically Set
    • Contrast: Automatically Set


Calibration
To set up our camera in front of the fluorimeter we first had to place the lighted platform and glass slide at the back of the box with a tray under the platform to increase the height. Next, we took the phone and placed it into the phone cradle. Then we took the phone cradle and placed it in front of the glass slide, varying the distance until the drop was in focus. Once in position, the phone was not moved.

DNA/Primer testing

  • Distance between the smart phone cradle and drop = 2in


Solutions Used for Calibration

Initial Concentration of DNA solution(µg/mL) Volume of DNA solution(µL) Volume of SYBR Green(µL) Final DNA Solution in SYBR Green(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. Place 80µL of the SYBR green solution as a drop in between the middle two dots on the glass slide and add 80µL of one of the DNA solutions in the first column of the table
  2. Position the drop of solution so that the LED light shines directly through the center of it
  3. Set the timer on the camera and quickly close the light box to prevent any stray light from entering
  4. Take three focused images of the drop
  5. Open the lid of the box and without moving anything inside of it, micropipette the 160µL drop off the slide.
  6. Repeat steps 1-5 for each concentration of DNA solution


Data Analysis

Representative Images of Negative and Positive Samples

Negative Samples

Positive Samples

Image J Values for All Calibrator Samples

Area Mean StDev Mode IntDen RawIntDen (Drop) RawIntDen (Background) Drop - Background
Water T1 28484 29.191 28.401 13 831470 831470 158266 673204
Water T2 28680 31.555 33.786 13 904985 904985 202411 702574
Water T3 27404 33.69 40.547 13 923237 923237 137517 785720
Calf Thymus 0.25 T1 19910 42.983 23.499 41 855792 855792 117585 738207
Calf Thymus 0.25 T2 21006 42.528 23.132 42 893342 893342 119887 773455
Calf Thymus 0.25 T3 22096 44.823 23.529 47 990417 990417 149789 840628
Calf Thymus .5 T1 26576 54.056 33.842 49 1436601 1436601 122697 1313904
Calf Thymus .5 T2 25638 51.179 32.873 49 1312135 1312135 144343 1167792
Calf Thymus .5 T3 26148 52.014 31.669 54 1360057 1360057 161701 1198356
Calf Thymus 1 T1 24056 83.603 37.424 89 2011156 2011156 173370 1837786
Calf Thymus 1 T2 24948 79.871 36.813 95 1992628 1992628 166804 1825824
Calf Thymus 1 T3 24436 80.677 36.227 89 1971426 1971426 155532 1815894
Calf Thymus 2 T1 28154 111.768 41.791 139 3146720 3146720 228364 2918356
Calf Thymus 2 T2 31122 111.973 42.206 143 3484820 3484820 203899 3280921
Calf Thymus 2 T3 29404 105.723 43.869 135 3108670 3108670 210787 2897883
Calf Thymus 5 T1 24263 163.781 55.723 255 3973807 3973807 225008 3748799
Calf Thymus 5 T2 24835 161.367 56.173 255 4007539 4007539 180646 3826893
Calf Thymus 5 T3 25503 164.135 57.517 255 4185938 4185938 159026 4026912


Calibration curve

PCR Results Summary

  • Our positive control PCR result was 28.29804534 μg/mL
  • Our negative control PCR result was -1.136261815 (0) μg/mL

Observed results

  • Patient 58961 : Images glowed bright green, had a high amount of green saturation; Initial PCR Concentration determined to be an average of 31.48969805 μg/mL.
  • Patient 79602 : Images glowed bright green, had a high amount of green saturation; Initial PCR Concentration determined to be an average of 37.50745806 μg/mL.

Conclusions

  • Patient 79602 : Since this patient's sample resembled the positive control and is much closer to the positive control concentration than the negative, it can be concluded that the sample tested positive.
  • Patient 58961 : This patient's results are also fairly close to the concentration of the positive control so it can be concluded that they also tested positive.




SNP Information & Primer Design

Background: About the Disease SNP The disease SNP is found in homo sapiens and is located on the chromosome 21. It is associated with the gene KCNE2 which is a potassium voltage-gated channel that regulates neurotransmitter release, hear rate, insulin, neuronal excitability and other important functions. SNP has a clinical significance because is it pathogenic and linked to the disease long QT. Long QT is a rare inherited heart condition that causes a risk of episodes of torsades de pointes, irregular heartbeat originating from the ventricles. The disease is identified by a change in the allele sequence TTC to CTC.


Primer Design and Testing

From our primer test we were able to locate the forward and reverse primer sequences to take us to the desired SNP gene. To mutate this gene, the last base sequence in the forward primer was changed from TTC to CTC. This single base mutation is the cause of the SNP disease long QT. The test below demonstrates how the non-disease primers lead directly to the desired SNP gene and the diseased ones come up with no results. This lack of results confirms that the disease primers result in a mutation and change on the gene.

DNA/Primer testing

DNA/Primer testing


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