BME100 f2014:Group34 L5

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OUR TEAM

Victor Huerta
Xavier Richmond
Ferrin Thomas
Morgan Seburn
Teja Vemulapalli


LAB 5 WRITE-UP

Procedure

Smart Phone Camera Settings

  • Type of Smartphone:Samsung Galaxy S5
    • Flash: Off
    • ISO setting: 800
    • White Balance: Auto
    • Exposure: Auto
    • Saturation: Auto
    • Contrast: Auto


Calibration

1. Place fluorimeter on the table and turn on the blue LED by flipping the switch

2. Place the slide (smooth side down) in the fluorimeter

3. Set timer on camera to 3-5 seconds and insert into cradle a fixed distance from fluorimeter.

4. Adjust height of fluorimeter using plastic trays to get an edge-on view through the camera.

  • Distance between the smart phone cradle and drop = 8cm


Solutions Used for Calibration

Initial Concentration of 2X Calf Thymus DNA Solution (ug/mL) Volume of 2X DNA Solution (uL) Volume of SYBR GREEN I Dye Solution (uL) Final DNA Concentration in SYBR Green I Solution (ug/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 .0125
0 80 80 0



Placing Samples onto the Fluorimeter

  1. Using gloves, find the smooth side of the sample plate
  2. Slide the sample plate, smooth side down, into the fluorimeter and turn blue LED light on
  3. Using proper micropipetting technique, draw and place 80 uL of SYBR Green I on the first 2 circles on the slide
  4. Insert new pipette tip and draw and place 80 uL of sample/calibration solution on top of the SYBR Green I drop
  5. Adjust the slide so light illuminates the center of the drop


Data Analysis

Representative Images of Negative and Positive Samples

Sample with no DNA

Description of image

Sample with DNA

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Image J Values for All Calibrator Samples


Final DNA concentration in SYBR Green I solution (ug/mL) AREA Mean Pixel Value RAWINTDEN OF THE DROP RAWINTDEN OF THE BACKGROUND RAWINTDEN DROP - BACKGROUND
0 28984 11.886 344498 0 344498
0 61956 6.148 380930 0 380930
0 52800 7.303 385613 0 385613
0.25 122221 13.835 1690956 0 1690956
0.25 41840 17.874 747835 111 747724
0.25 38107 17.224 656373 98 656275
0.5 48064 43.756 2103070 1703 2101367
0.5 47681 70.084 3341658 0 3341658
0.5 47017 69.83 3283185 0 3283185
1 47058 56.929 2678965 222 2678743
1 45932 116.4 5346463 4329 5342134
1 51697 123.216 6369878 1362 6368516
2 49277 104.192 5134266 0 5134266
2 47508 174.654 8297468 3489 8293979
2 42232 182.475 7706288 3118 7703170
5 71196 156.683 11155235 10725 11144510
5 45304 215.618 9768366 2898 9765468
5 55848 182.673 10201907 1658 10200249

Calibration curve
Description of image


PCR Results Summary

PCR Product Tube Label MEAN RAWINTDEN DROP-BACKGROUND PCR PRODUCT CONCENTRATION ug/mL INITIAL PCR CONCENTRATION ug/mL
34 P 3206761.667 0.603380833 7.24057
34 N 771576.3333 -0.614211833 -7.370542
34 +1-1 651603 -0.6741985 -8.090382
34 +1-2 1011260 -0.49437 -5.93244
34 +1-3 1319958.667 -0.340020667 -4.080248
34 -1-1 6481576 2.240788 26.889456
34 -1-2 4780935.333 1.390467667 16.685612
34 -1-3 2600978.333 0.300489167 3.60587


  • Our positive control PCR result was 7.24057 μg/mL
  • Our negative control PCR result was -7.370542 μg/mL

Observed results

  • Patient 88697 : All the images for patient 1 were dark. There was no identifiable green fluorescence coming from the drop. Patient 1 had an average initial PCR product concentration of approximately -6.034 ug/ml
  • Patient 48246 : All the images for patient 2 had a clear and indentifiable green fluorescence coming from the drop. Patient 2 had an average initial PCR product concentration of approximately 15.727 ug/mL

Conclusions

  • Patient 88697 : Patient 1 had an average initial PCR product concentration of approximately -6.034 ug/mL. This value differed from the negative control by approximately 1.337 ug/mL and differed from the positive control by approximately 13.275. Since the concentration is closer to the negative control, patient 1 is negative. All 3 of patient 1's initial concentrations were much closer to the negative control.
  • Patient 48246 : Patient 2 had an average initial PCR product concentration of approximately 15.727 ug/mL. This value differed from the negative control by approximately 23.098 ug/mL and differed from the positive control by approximately 8.486 ug/mL. Since the concentration is closer to the positive control, patient 2 is positive. All 3 of patient 2's initial concentrations were much closer to the positive control.




SNP Information & Primer Design

Background: About the Disease SNP

Nucleotides are the building blocks of DNA. They consist of Adenine, Thymine, Guanine, and Cytosine. A polymorphism is a presence of genetic variation within a population. In this section we are focusing on SNPs, or single nucleotide polymorphism. SNP rs16991654 is found in homosapiens and is located in the 21:34370656 chromosome. This SNP has a clinical significance because it is pathogenic with diseases like congenital long QT syndromes being linked to it. KCNE2 stands for potassium voltage-gated channels and its function primarily consists of regulating neurotransmitter release, heart rate, insulin secretion, neuronal excitability, epithelial electrolyte transport, smooth muscle contraction and cell volume.


Primer Design and Testing

To design primer pairs for PCR, the non-disease forward primer contains 20 bases and ends with the disease codon. The reverse non-disease primer also contains 20 bases, and is found 200 base pairs to the right on the bottom strand. To create disease primer pairs, the SNP is applied to the appropriate nucleotide in the forward primer. The reverse primer does not change when diseased. When testing both primers, we found that the search returned the non-diseased sequence since it is part of the healthy human genome. The reason why the diseased variation did not come up in the search is because it is not a part of the healthy human genome, it is a SNP.


Non-disease forward and non-disease reverse primer search

Description of image

Disease-specific primer search

Description of image