BME100 s2015:Group6 12pmL5

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Contents

OUR TEAM

Name: Justin Blommer
Name: Justin Blommer
Name: Ethan Mathew
Name: Ethan Mathew
Name: Spencer Cobb
Name: Spencer Cobb
Name: Sarah Jones
Name: Sarah Jones
Name: Abigail Rene
Name: Abigail Rene

LAB 5 WRITE-UP

Procedure

Smart Phone Camera Settings

  • Type of Smartphone: Samsung Galaxy S5
    • Flash: Off
    • ISO setting: N/A
    • White Balance: N/A
    • Exposure: N/A
    • Saturation: Maximum
    • Contrast: Mininum


Calibration

  • The flourimeter was placed on top of the white plastic tray in order to achieve an ideal camera angle.
  • The camera was adjusted to the above settings with a timer delay of 5 seconds. This allowed ample time to place the cover on and for the camera to refocus to the darker lighting.
  • The camera was inserted in the center of the cradle, which was then placed on top of a small book and table to align with the height of the flourimeter. The angle obtained was as close as possible to level with the slide.
  • The camera was placed as close as possible to the tray without sacrificing image quality.
  • Distance between the smart phone cradle and drop = 5.9cm


Solutions Used for Calibration

Initial Concentration of 2X Calf Thymus DNA solution (μg/mL) Volume of the 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



Placing Samples onto the Fluorimeter

  1. Once camera and flourimeter set up is complete, switch the flourimeter light to the "on" position.
  2. Insert the slide, with the rough, hydrophobic side facing up. Adjust its position so that the flourimeter light is focused between the first two rows of dots.
  3. Adjust the micropipettor to 80μL and insert a clean tip.
  4. Draw 80μL of SYBR Green solution and place it between the center two dots on the row where the light is focused. It is very important to dispense the solution slowly and precisely to ensure the drop maintains intact and centered on the slide.
  5. Discard the tip and replace with a clean one.
  6. Draw 80μL of the DNA sample to be tested and dispense it directly onto the SYBR Green solution drop on the slide. Again, dispense slowly and ensure the combined dot remains centered on the slide.
  7. Verify the camera angle and focus is still correct. After pressing the shutter button (to begin the timer countdown), cover the flourimeter and camera immediately to ensure that no outside light is allowed into the picture.
  8. Once the image has been captured, remove the box cover from the flourimeter. Make certain that the image is of good quality and not excessively blurry or dark.
  9. Using the micropipettor, transfer the solution from the slide into a biohazard waste collection container.
  10. Adjust the slide to the next position and repeat Steps 3-9 for the remaining samples.


Data Analysis

Representative Images of Negative and Positive Samples

Negative Control

image:screenshotnegative.jpg

Positive Control

image:screenshotpositive.jpg


  • As per lab TA instructions, the edges of the samples are left out of the selection because they are the saturated points where the light is entering and exiting the sample drop. Therefore, they are not an accurate representation of the fluorescence of the sample.


Image J Values for All Calibrator Samples

Calibration Image Analysis ' ' ' ' '
Final DNA concentration in SYBR Green I solutions (µg/mL)AreaMean Pixel ValueRAWINTDEN of DropRAWINTDEN of BackgroundRAWINTDEN Of Drop-Background
03097517.1815321915781526410
03145820.8216549901891653099
03260417.14455895715477543480
0.1253797833.6561278187177461260441
0.1253839532.9961266896244571242439
0.1253462532.8291136700216231115077
0.252956252.3541547696119301535766
0.252686449.4431328240158861312354
0.252998451.1751534431133161521115
0.53286474.415244557439822441592
0.53411273.774251659598492506746
0.53183275.6912409393107112398682
15048487.32944087131118994296814
15048486.78743813631031044278259
15048486.22743530971012164251881
2.573954173.5061283147596478011866695
2.573954173.6961284549495605111889443
2.573954173.44212826756104156511785191


Calibration Statistics ' ' ' ' '
Final DNA concentration in SYBR Green 1 solution (µg/mL)RAWINTDEN of Drop-BackgroundMeanStandard Deviation
123
0526410653099543480574329.666768748.10638
0.1251260441124243911150771205985.66779242.08009
0.251535766131235415211151456411.667124972.4827
0.52441592250674623986822449006.66754412.22276
14296814427825942518814275651.33322579.71582
2.511866695118894431178519111847109.6754816.13665

Calibration curve

image:trendyline.jpg


Note: Although difficult to see, the error bars are included in the plots. The error bar is visible on the 3rd point on the plot, but very hard to see on any others. They are inserted using the standard deviation as the positive and negative values.

PCR Results Summary

PCR Product Dilutions ' ' ' ' '
PCR Product Tube LabelVolume of DILUTED PCR Product Solution (µL)Volume of SYBR Green I Dye Solution (µL)Dilution 1Dilution 2Total Dilution (simplified fraction)
G6 P80800.1670.500 1/12
G6 N80800.1670.500 1/12
G6 1-180800.1670.500 1/12
G6 1-280800.1670.500 1/12
G6 1-380800.1670.500 1/12
G6 2-180800.1670.500 1/12
G6 2-280800.1670.500 1/12
G6 2-380800.1670.500 1/12
PCR Reaction Image Analysis ' ' ' ' '
PCR Product Tube LabelAreaMean Pixel ValueRAWINTDEN of DropRAWINTDEN of BackgroundRAWINTDEN Of Drop-Background
G6 P39540120.7444774226215284752698
G6 P39948120.4534811849215034790346
G6 P39900121.4764846888233884823500
G6 N4272025.5111089830142721075558
G6 N4355224.7321077119164051060714
G6 N4298424.2451042156171641024992
G6 1-13554021.7147717329518762214
G6 1-13355721.21971203010974701056
G6 1-12831218.5352462212302512320
G6 1-24253925.6041089181253851063796
G6 1-24242824.0981022441158591006582
G6 1-24190625.3391061852188221043030
G6 1-32648832.0188480898064840025
G6 1-32741231.6448674289569857859
G6 1-32478029.3057261864608721578
G6 2-13726240.4341506645160711490574
G6 2-13732639.861487802113711476431
G6 2-13699039.3891457017132871443730
G6 2-23534131.8361125114148591110255
G6 2-23678829.6231089761176101072151
G6 2-23624231.9761158859150331143826
G6 2-33072838.906119551582791187236
G6 2-33053039.758121380678251205981
G6 2-33047240.324122874043431224397


PCR Reaction Statistics ' ' ' ' '
PCR Product Tube LabelRAWINTDEN of Drop-BackgroundMeanStandard Deviation
123
G6 P475269847903464823500478884835424.76258
G6 N1075558106071410249921053754.66725991.428
G6 1-1762214701056512320658530130261.6533
G6 1-21063796100658210430301037802.66728962.97998
G6 1-3840025857859721578806487.333374072.32313
G6 2-1149057414764311443730147024524026.86061
G6 2-2111025510721511143826110874435861.38239
G6 2-31187236120598112243971205871.33318580.74273
Calculations ' ' '
PCR Product Tube LabelMean RAWINTDENT Drop-BackgroundPCR Product Concentration (μg/mL)Initial PCR Product Concentration (μg/mL)
G6 P47888481.108213.2984
G6 N1053754.6670.1744266672.09312
G6 1-16585300.07562050.907446
G6 1-21037802.6670.1704386672.045264
G6 1-3806487.33330.1126098331.351318
G6 2-114702450.278549253.342591
G6 2-211087440.1881742.258088
G6 2-31205871.3330.2124558332.54947


  • Our positive control PCR result was 13.2984 μg/mL
  • Our negative control PCR result was 2.09312 μg/mL

Observed results

  • Patient 11105 (Patient 1):

The samples of Patient 11105's DNA fluoresced very little green in the fluorimeter. The image of the drop was almost completely a dark blue color. There was a lighter color on both edges, but this was only from the fluorimeter light entrance and exit paths through the sample. Once Patient 11105's images were split by color in the ImageJ program, the green channel sample was extremely dark. This indicates that there was a minute amount of green light fluorescing in the sample. Replications 1, 2, and 3 returned Initial PCR Product Concentrations of 0.907446 μg/mL, 2.045264 μg/mL, and 1.351318 μg/mL, respectively.


  • Patient 22923 (Patient 2):

The samples of Patient 222923's DNA also fluoresced very little green in the flourimeter. This patient's samples also was a dark blue color, with lighter blue edges. When Patient 222923's sample images were split using ImageJ, the green channel was slightly brighter than Patient 11105, but still very dark in general. This indicates that there was a very small amount of green light fluorescing in the sample. Replications 1, 2, and 3 returned Initial PCR Product Concentrations of 3.342591 μg/mL, 2.258088 μg/mL, and 2.54947 μg/mL, respectively.


Conclusions

  • Patient 11105 (Patient 1):

Patient 11105's three replicates had an average Initial PCR Product Concentration of 1.434676 μg/mL. This puts it well below the positive control concentration of 13.2984 μg/mL, and much closer to the negative control sample's concentration of 2.09312 μg/mL. Therefore, it can be concluded that Patient 11105 does not carry the disease gene.


  • Patient 22923 (Patient 2):

Patient 22923's three replicates had an average Initial PCR Product Concentration of 2.716716333 μg/mL. This puts it below the positive control concentration of 13.2984 μg/mL, and much closer to the negative control sample's concentration of 2.09312 μg/mL. Therefore, it can be concluded that Patient 22923 does not carry the disease gene.




SNP Information & Primer Design

Background: About the Disease SNP

Nucleotides are organic molecules that serve as the monometers, also known as subunits, of nucleic acids like DNA and RNA. A polymorphism is when there is a variation in a DNA sequence where one nucleotide in the genome differs between paired chromosomes. For example, when an adenine nucleotide is paired with a guanine.

A common SNP (single nucleotide polymorphism) is the rs268. This variation is commonly found in the homo sapien species. It is located on the 8:19956018 chromosome and clinically classified as a pathogenic polymorphism. The rs268 is associated with the LPL gene. It is associated with many major diseases, including metabolic conditions, heart attack, strokes, and high cholesterol. Rs268 is a lipoprotein lipase, known as LPL for short.

An allele is simply a variant form of a gene. In the rs268 SNP, the AAT (non-diseased) gene is modified to the AGT (diseased) variant. The numerical value of the position of the SNP in the human genome is 19956018.


Primer Design and Testing

The diseased primer returned a result of "no match found" when run through the primer test because a healthy human genome does not possess this mutation (AGT). The human genome, does however, produce the healthy non-diseased primer (AAT), which did successfully return a result when run through the primer test.


Non-Diseased Primer

image:nondisease6.jpg


Diseased Primer

image:disease6.jpg

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