BME100 s2015:Group6 12pmL5

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OUR TEAM

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

Screenshotnegative.jpg

Positive Control

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) Area Mean Pixel Value RAWINTDEN of Drop RAWINTDEN of Background RAWINTDEN Of Drop-Background
0 30975 17.181 532191 5781 526410
0 31458 20.821 654990 1891 653099
0 32604 17.144 558957 15477 543480
0.125 37978 33.656 1278187 17746 1260441
0.125 38395 32.996 1266896 24457 1242439
0.125 34625 32.829 1136700 21623 1115077
0.25 29562 52.354 1547696 11930 1535766
0.25 26864 49.443 1328240 15886 1312354
0.25 29984 51.175 1534431 13316 1521115
0.5 32864 74.415 2445574 3982 2441592
0.5 34112 73.774 2516595 9849 2506746
0.5 31832 75.691 2409393 10711 2398682
1 50484 87.329 4408713 111899 4296814
1 50484 86.787 4381363 103104 4278259
1 50484 86.227 4353097 101216 4251881
2.5 73954 173.506 12831475 964780 11866695
2.5 73954 173.696 12845494 956051 11889443
2.5 73954 173.442 12826756 1041565 11785191


Calibration Statistics ' ' ' ' '
Final DNA concentration in SYBR Green 1 solution (µg/mL) RAWINTDEN of Drop-Background Mean Standard Deviation
1 2 3
0 526410 653099 543480 574329.6667 68748.10638
0.125 1260441 1242439 1115077 1205985.667 79242.08009
0.25 1535766 1312354 1521115 1456411.667 124972.4827
0.5 2441592 2506746 2398682 2449006.667 54412.22276
1 4296814 4278259 4251881 4275651.333 22579.71582
2.5 11866695 11889443 11785191 11847109.67 54816.13665

Calibration curve

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 Label Volume of DILUTED PCR Product Solution (µL) Volume of SYBR Green I Dye Solution (µL) Dilution 1 Dilution 2 Total Dilution (simplified fraction)
G6 P 80 80 0.167 0.500 1/12
G6 N 80 80 0.167 0.500 1/12
G6 1-1 80 80 0.167 0.500 1/12
G6 1-2 80 80 0.167 0.500 1/12
G6 1-3 80 80 0.167 0.500 1/12
G6 2-1 80 80 0.167 0.500 1/12
G6 2-2 80 80 0.167 0.500 1/12
G6 2-3 80 80 0.167 0.500 1/12
PCR Reaction Image Analysis ' ' ' ' '
PCR Product Tube Label Area Mean Pixel Value RAWINTDEN of Drop RAWINTDEN of Background RAWINTDEN Of Drop-Background
G6 P 39540 120.744 4774226 21528 4752698
G6 P 39948 120.453 4811849 21503 4790346
G6 P 39900 121.476 4846888 23388 4823500
G6 N 42720 25.511 1089830 14272 1075558
G6 N 43552 24.732 1077119 16405 1060714
G6 N 42984 24.245 1042156 17164 1024992
G6 1-1 35540 21.714 771732 9518 762214
G6 1-1 33557 21.219 712030 10974 701056
G6 1-1 28312 18.53 524622 12302 512320
G6 1-2 42539 25.604 1089181 25385 1063796
G6 1-2 42428 24.098 1022441 15859 1006582
G6 1-2 41906 25.339 1061852 18822 1043030
G6 1-3 26488 32.018 848089 8064 840025
G6 1-3 27412 31.644 867428 9569 857859
G6 1-3 24780 29.305 726186 4608 721578
G6 2-1 37262 40.434 1506645 16071 1490574
G6 2-1 37326 39.86 1487802 11371 1476431
G6 2-1 36990 39.389 1457017 13287 1443730
G6 2-2 35341 31.836 1125114 14859 1110255
G6 2-2 36788 29.623 1089761 17610 1072151
G6 2-2 36242 31.976 1158859 15033 1143826
G6 2-3 30728 38.906 1195515 8279 1187236
G6 2-3 30530 39.758 1213806 7825 1205981
G6 2-3 30472 40.324 1228740 4343 1224397


PCR Reaction Statistics ' ' ' ' '
PCR Product Tube Label RAWINTDEN of Drop-Background Mean Standard Deviation
1 2 3
G6 P 4752698 4790346 4823500 4788848 35424.76258
G6 N 1075558 1060714 1024992 1053754.667 25991.428
G6 1-1 762214 701056 512320 658530 130261.6533
G6 1-2 1063796 1006582 1043030 1037802.667 28962.97998
G6 1-3 840025 857859 721578 806487.3333 74072.32313
G6 2-1 1490574 1476431 1443730 1470245 24026.86061
G6 2-2 1110255 1072151 1143826 1108744 35861.38239
G6 2-3 1187236 1205981 1224397 1205871.333 18580.74273
Calculations ' ' '
PCR Product Tube Label Mean RAWINTDENT Drop-Background PCR Product Concentration (μg/mL) Initial PCR Product Concentration (μg/mL)
G6 P 4788848 1.1082 13.2984
G6 N 1053754.667 0.174426667 2.09312
G6 1-1 658530 0.0756205 0.907446
G6 1-2 1037802.667 0.170438667 2.045264
G6 1-3 806487.3333 0.112609833 1.351318
G6 2-1 1470245 0.27854925 3.342591
G6 2-2 1108744 0.188174 2.258088
G6 2-3 1205871.333 0.212455833 2.54947


  • 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

Nondisease6.jpg


Diseased Primer

Disease6.jpg