BME100 f2014:Group23 L5

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OUR TEAM

Name: Cesar Marin
Name: Brady Dennison
Name: Kassandra Flores
Name: Danielle Beach
Name: Theodore Kyriacou
Name: Joshua Kahn


LAB 5 WRITE-UP

Procedure

Smart Phone Camera Settings

  • Type of Smartphone: Samsung Galaxy Note 3
    • Flash: None
    • ISO setting: 800
    • White Balance: Auto
    • Exposure: Highest Setting
    • Saturation: Highest Setting
    • Contrast: Highest Setting


Calibration

The smartphone was placed in the cradle with the camera app on. The cradle was then moved so that the phone was 12 cm away from from where the drop would be measured. Pieces of bubble wrap were placed in between the phone and the cradle so that the phone was level with the drop.

  • Distance between the smart phone cradle and drop = 12 cm


Solutions Used for Calibration

Initial Concentration of 2X Calf Thymus DNA Solution (µg/mL) Volume of 2X DNA Solution (µL) Volume of 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

Solutions Used for Measuring PCR Products

PCR Product Tube Label Volume of the Diluted PCR Product solution(μL) Volume of SYBR Green I Dye Solution (µL) Dilution 1 Dilution 2 Total Dilution Simplified Fraction
G23 + 80 80 0.166 0.5 0.0833
G23 - 80 80 0.166 0.5 0.0833
G23 1-1 80 80 0.166 0.5 0.0833
G23 1-2 80 80 0.166 0.5 0.0833
G23 1-3 80 80 0.166 0.5 0.0833
G23 2-1 80 80 0.166 0.5 0.0833
G23 2-2 80 80 0.166 0.5 0.0833
G23 2-3 80 80 0.166 0.5 0.0833


Placing Samples onto the Fluorimeter

  1. Line up fluorimeter light with the section on slide where the sample will be placed (middle of two circles)
  2. Put new tip into micropipette and collect 80uL of SYBR green
  3. Place 80 uL of SYBR green into area from step 1, lined with the light
  4. Discard tip, get new tip and collect 80uL of desired sample
  5. Place 80uL of sample on top of SYBR green in the fluorimeter slide
  6. Adjust slide on fluorimeter to allow for the narrowest possible stream of light
  7. Place camera on a timer
  8. Place camera and fluorimeter in a box to block out light, and wait until picture is taken


Data Analysis

Representative Images of Negative and Positive Samples

Sample With No DNA

No DNA.png

Sample With DNA

DNAA.png

Image J Values for All Calibrator Samples


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#1 117281 62.946 7382324 120175 7262149
0#2 95944 55.867 5360083 82761 5277322
0#3 102480 56.232 5762676 122017 5640659
0.125#1 200967 58.129 11682080 230881 11451199
0.125#2 209397 61.156 12805862 316028 12489834
0.125#3 220281 59.243 13050128 315441 12734687
0.25#1 148276 105.684 15670365 166860 15503505
0.25#2 143862 104.852 15084237 168573 14915664
0.25#3 148848 104.226 15513804 176779 15337025
0.5#1 63004 121.832 7675924 84689 7591235
0.5#2 53899 121.386 6542599 67056 6475543
0.5#3 63250 120.075 7594743 71477 7523266
1#1 101788 159.563 16241620 111100 16130520
1#2 107815 154.656 16674224 121570 16552654
1#3 106365 155.667 16557507 116040 16441467
2.5#1 52043 224.116 11663647 272607 11391040
2.5#2 96387 201.455 19417686 339679 19078007
2.5#3 93996 203.554 19133220 489494 18643726
PCR Product Tube Label Area Mean Pixel Value RAWINTDEN of the Drop RAWINTDEN of the Background RAWINTDEN Drop - Background
G23 + #1 81288 211.873 17222739 519304 16703435
G23 + #2 83298 209.743 17471210 477364 16993846
G23 + #3 84916 206.982 17576093 380353 17195740
G23 - #1 63262 47.935 3032495 114182 2918313
G23 - #2 65364 52.551 3434964 126152 3308812
G23 - #3 73048 53.772 3927959 184799 3743160
G23 1-1 #1 63236 217.135 13730756 291772 13438984
G23 1-1 #2 69676 214.228 14926555 274659 14651896
G23 1-1 #3 72935 209.691 15293786 484533 14809253
G23 1-2 #1 68930 223.918 15434635 748054 14686581
G23 1-2 #2 83892 214.71 18012462 542818 17469644
G23 1-2 #3 78844 213.253 16813706 552361 16261345
G23 1-3 #1 79201 218.261 17286505 653016 16633489
G23 1-3 #2 91457 222.257 20326925 1007852 19319073
G23 1-3 #3 77372 225.738 17465838 697357 16768481
G23 2-1 #1 86508 221.876 19194089 849243 18344846
G23 2-1 #2 79512 230.236 18306536 717249 17589287
G23 2-1 #3 84287 228.699 19276328 708500 18567828
G23 2-2 #1 77942 224.541 17501204 264074 17237130
G23 2-2 #2 66272 229.613 15216881 260396 14956485
G23 2-2 #3 69675 229.864 16015757 250409 15765348
G23 2-3 #1 46398 225.599 10467352 271372 10195980
G23 2-3 #2 39267 223.424 8773180 218858 8554322
G23 2-3 #3 54020 228.365 12336293 436545 11899748
Final DNA concentration in SYBR Green I solution (µg/mL) RAWINTDEN DROP - BACKGROUND ' ' ' '
1 2 3 Mean Standard Deviation
0 7262149 5277322 5640659 6060043.333 1056786.151
0.125 11451199 12489834 12734687 12225240 681427.0689
0.25 15503505 14915664 15337025 15252064.67 302990.0229
0.5 7591235 6475543 7523266 7196681.333 625448.0938
1 16130520 16552654 16441467 16374880.33 5044544.086
2.5 11391040 19078007 18643726 16370924.33 4318169.285
PCR Product Tube Label RAWINTDEN DROP - BACKGROUND ' ' '
1 2 3 Mean
G23 + 16703435 16993846 17195740 16964340.33
G23 - 2918313 3308812 3743160 3323428.333
G23 1-1 13438984 14651896 14809253 14300044.33
G23 1-2 14686581 17469644 16261345 16139190
G23 1-3 16633489 19319073 16768481 17573681
G23 2-1 18344846 17589287 18567828 18167320.33
G23 2-2 17237130 14956485 15765348 15986321
G23 2-3 10195980 8554322 11899748 10216683.33

Calibration curve
Final DNA SYBR.png

PCR patient label Mean RAWINTDEN drop-background PCR product concentration Initial PCR product concentration
G23 + 16964340.33 2.32 ug/mL 27.84 ug/mL
G23 - 3323428.333 -2.23 ug/mL -26.76 ug/mL
G23 1-1 14300044.33 1.43 ug/mL 17.16 ug/mL
G23 1-2 16139190 2.05 ug/mL 24.6 ug/mL
G23 1-3 17573681 2.52 ug/mL 30.24 ug/mL
G23 2-1 18167320.33 2.72 ug/mL 32.64 ug/mL
G23 2-2 15986321 1.99 ug/mL 23.88 ug/mL
G23 2-3 10216683.33 .072 ug/mL 0.864 ug/mL

PCR Results Summary

  • Our positive control PCR result was 27.84 μg/mL
  • Our negative control PCR result was -26.76 μg/mL

Observed results

  • Patient 1-33849 : All of the images for patient 1 were bright green. The green was mostly at the bottom, with blue at the top. Patient 1 had an average initial PCR product concentration of 24 μg/mL.
  • Patient 2-19967 : All of the images for patient 2 were also bright green. The green fluorescence was shown all throughout the drop. Patient 2 had an average initial PCR product concentration of 18.869 μg/mL.

Conclusions

  • Patient 1-33849 : Patient 1 had an average initial PCR product concentration that was 3.84 μg/mL less than that of the product concentration of the positive sample, and 50.76 μg/mL greater than that the product concentration of the negative sample. Since Patient 1 had a concentration closer to the concentration of the positive sample, Patient 1 is positive. All of Patient 1's initial concentrations were closer to the concentration of the positive sample than the negative sample.
  • Patient 2-19967 : Patient 2 had an average initial PCR product concentration that was 8.9712 μg/mL less than that of the product concentration of the positive sample, and 45.6288 μg/mL greater than that the product concentration of the negative sample. Since Patient 2 had a concentration closer to the concentration of the positive sample, Patient 2 is positive. All of Patient 2's initial concentrations were closer to the concentration of the positive sample than the negative sample. Positive initial concentrations suggest that the patient is positive, while negative initial concentrations suggest that the patient is negative.




SNP Information & Primer Design

Background: About the Disease SNP

A nucleotide is the basic building block of DNA and other nucleic acids. the four main nucleotides are A,C,T,and G. A polymorphism is a common change in DNA sequences that appears in the phenotypes of different groups of population. The SNP rs16991654 variation is found in homosapiens. This variation is found in the 21:34370656 chromosome. The clinical significance is this SNP is that it is pathogenic. This SNP is associated with the KCNE2 gene. This SNP is linked to the disease of congenital long QT syndromes. KCNE2 stands for potassium voltage-gated channel, Isk-related family, member 2. The molecular function of this gene includes regulating neurotransmitter release, heart rate, insulin secretion, neuronal excitability, epithelial electrolyte transport, smooth muscle contraction, and cell volume. An allele in an alternative to a gene sequence that are done through mutation of a nucleotide base pair. The disease-associated allele contains the sequence CTC. The numerical position of the SNP is 34,370,656. The non-disease forward primer is catggtgatgattggaatgt. The numerical position exactly 200 bases to the right of the disease SNP is 3,430,856. The non-disease reverse primer is cccttatcagggggacattt. The diseases forward primer is catggtgatgattggaatgc. The disease reverse primer is cccttatcagggggacattt.


Primer Design and Testing

When the non-disease forward primer was tested, it was found in the database. This is because a part of the DNA that was selected did not contain the sequence that causes the disease. The disease specific primers did not exist because one nucleotide was changed in the DNA sequence. While this may cause the disease, it affects the entire sequence, it does not exist since it did not change by a group of three nucleotides, or an entire codon.

Non-Disease Forward Primer Results

Part a.png

Disease Specific Primers Results

Part b.png