BME100 s2015:Group5 12pmL5

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OUR TEAM

Name: Brandon Favre
Name: Daniella Orlando
Name: Martisha Clyde
Name: David Shumate
Name: Nick Valverde
Name: Fernanda Diva


LAB 5 WRITE-UP

Procedure

Smart Phone Camera Settings

  • Type of Smartphone: Apple iPhone 5s
    • Flash: Not used
    • ISO setting: Default
    • White Balance: Default
    • Exposure: Default
    • Saturation: Default
    • Contrast: Default


Sometimes the flash went off with the timer, so eventually we just stopped using the timer on the phone.

Calibration

  1. First, turn on Blue LED excitation light.
  2. Next turn on the smartphone camera and adjust the camera setting as those listed in the lab manual or as close as you can get.
  3. Place the phone on the cradle as close to a 90 degree angle as possible. This is done so that you can take a picture of the SYBR GREEN/Calf Thymus DNA solution drop sideways at a profile view. You may need to raise the fluorimeter to the drop directly.
  4. Adjust the distance between the smartphone the first two rows of the slide so that it is as close as possible without causing a blurry image. This distance is should be at least 4 cm away from the drop and should be measured.
  5. Record this distance and don't to move the camera, cradle, or fluorimeter. This can change the image due to the difference in light collect between the different lengths.
  • Distance between the smart phone cradle and drop = 6 cm


Solutions Used for Calibration

Initial Concentration of 2X Calf Thymus
DNA Solution
(micrograms/mL)
Volume of the 2X
DNA Solution (µL)
Volume of the SYBER GREEN I
Dye Solution (µL)
Final DNA concentration in
SYBR Green I solution (µL/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. Using the micropipette and a new tip from the tip box add an 80 uL drop of SYBR GREEN I in middle of the first rows of the glass slide.
  2. Next, add an 80 uLs drop of the 2X DNA solution directly on top of the drop of the SYBER GREEN I, again with a new tip.
  3. Position drop in front of the blue LED light so that the light shines through the middle of the drop.
  4. Set timer on your smartphone camera for three seconds to take a picture of the drop so that you can close the light box.
  5. Take 3 images of the drop. Be sure to keep the camera focused and not to reposition of the phone.
  6. Remove the box without moving the phone. If needed, adjust any accidental movement of the cradle, the phone, or the fluorimeter setup at any time, by using a ruler to measure the initial position of said object. Mark with masking tape and place back exactly where it was. This is not recommended.
  7. Use the pipette to remove the 160 uL drop from the surface. Dispose of in the red cup provided for bioharzardous materials.
  8. Move the slide to the next clean position on the slide as to not contaminate the next drop.
  9. Repeat steps 1-8 for all the other concentrations of Calf Thymus. Use more than one slide if necessary.


Data Analysis

Representative Images of Negative and Positive Samples


Negative
11121662 1082344501782382 651454040 n.jpg
Positive
11117870 1082343908449108 1016009617 n.jpg


Image J Values for All Calibrator Samples

PCR product tube label area mean pixel value rawintden of the drop rawintden of the background rawintden drop- background
1-1 G5 89868 65.598 5895183 80956591 -75061408
1-1 G5 84532 71.437 6038702 84155325 -78116623
1-1 G5 86020 74.773 6431957 87447401 -81015444
1-2 G5 111928 104.169 11659465 64345394 -52685929
1-2 G5 107684 101.261 10904137 65346844 -54442707
1-2 G5 124856 102.537 12802394 68212014 -55409620
1-3 G5 106820 142.652 15238081 88542773 -73304692
1-3 G5 126612 142.364 18025006 94615657 -76590651
1-3 G5 122760 152.241 18689129 97200456 -78511327
2-1 G5 121400 92.921 11280639 68908576 -57627937
2-1 G5 124760 95.389 11900696 72250724 -60350028
2-1 G5 128936 96.023 12380877 74411536 -62030659
2-2 G5 132348 73.744 9759910 65310188 -55550278
2-2 G5 120176 79.379 9539438 71430629 -61891191
2-2 G5 126672 76.843 9733891 73537271 -63803380
2-3 G5 117912 90.119 10626081 70436005 -59809924
2-3 G5 109756 94.938 10420014 74829901 -64409887
2-3 G5 112440 100.434 11292792 76400948 -65108156
Y G5 144276 140.511 20272431 83746409 -63473978
Y G5 139736 148.135 20699815 80264456 -59564641
Y G5 140168 153.394 21500889 86871311 -65370422
N G5 115324 78.876 9096277 98065923 -88969646
N G5 126488 80.795 10219628 98733669 -88514041
N G5 123060 82.457 10147171 99776702 -89629531


Calibration curve

11130573 1082343575115808 1415387870 n.jpg

PCR Results Summary

  • Our positive control PCR result was -65370422 μg/mL
  • Our negative control PCR result was-88969646μg/mL

Observed results

  • Patient 1: 75239 :
  • Patient 2: 75444 :

Conclusions

  • Patient 1: 75239 : The final conclusion that can be made for Patient 75239 is that they are positive since they're values were very close to that of the positive control.
  • Patient 2: 75444 : The final conclusion that can be made for Patient 75444 is that the patient is also positive. The values of that were produced from analyzing the pictures with ImageJ showed that they were very close to the positive control.




SNP Information & Primer Design

Background: About the Disease SNP

The language of DNA is similar to the language in which people write in. Much like written languages, errors can occur as a result of the misspelling of a word. The language of DNA is not written in letters or numbers, but instead in molecules called, "nucleotide bases," which are composed of a nitrogenous base, a pentose (a five-carbon sugar), and at least one phosphate group, and are found in DNA as one of four bases: Adenine, Thymine, Guanine, and Cytosine (A,T,C, and G). The equivalent of typos in genetics are found in SNPs. SNPs, or Single Nucleotide Polymorphisms, are variations in a genome at the base-pair level, where during replication, a single base pair can be copied incorrectly during DNA replication. This leads to the base-pair being substituted, added to, or replaced in a way that is not a correct copy of the original genome, which can lead to millions of outcomes. In fact, it is believed that the 10 million SNPs in the human genome are what account for the differences between humans. Among other things, SNPs can lead to differences in appearance (e.g. hair color or skin tone), responses to medical treatments (e.g. allergies or resistances), and even susceptibility to disease (e.g. hereditary predispositions to diseases like cancer). The specific consequence of an SNP in the genome that is being examined in this experiment is the "disease SNP."

The "disease SNP" is actually a missense SNP (where the correct nucleotide is replaced by an incorrect allele) in the species Homo Sapien, or humans, with pathogenic significance. Alleles are alternative forms of a gene that are created via mutations and can be found in the same place in a chromosome. The disease SNP is found in one such allele, which normally contains a sequence of the bases Adenine- Adenine--Thymine (AAT). The disease-causing allele, however, features an SNP in the second adenine base, changing the sequence to Adenine-Guanine-Thymine (AGT), and is found at position 19956018. A direct consequence of this SNP is the condition of type I hyperlipoproteinemia (a form of Hyperlipidemia), which stems from an overabundance of cholesterol in the body, and is hereditary. The link between the missense SNP in question and type I hyperlipoproteinemia is that the allele that the "disease SNP" affects is responsible for the production of lipoprotein lipase (LPL). LPL finds its role in the body as a homodimer (a quaternary structural protein) in the heart, muscle, and adipose tissue, but more importantly, it is responsible for the breaking down of triglycerides (triglyceride hydrolase) and as a ligand/bridging factor for the complex process of lipoprotein uptake. Because of this integral role that LPL plays, any mutation (or SNP) that affects the production of LPL is cause for extreme medical concern and immediate research.


Primer Design and Testing

Every PCR reaction requires two primers. The first primer is a twenty-base long sequence, ending in the correct nucleotide of the affected allele (A). This sequence is 5'AATCTGGGCTATGAGATCAA. The second primer, the reverse primer, is found in 200 bases forward from the affected "A" SNP, and is 5'GAAACACCAGGGCTCAGGGTT. Upon sequencing these two primers in an online sequencing service, it was determined that the sequence was 220 base-pairs (bp) long, meaning that the two primers are valid, as the first primer is set 20bp before the affected allele, and the second is set 200bp after. Because the two match up, and allow for a successful sequencing, the two primers are valid and correct, and ready to be used. The results of the sequencing of the two primers is shown below:

Primer Readout.jpg

Naturally, because there must be an experimental group, the primers must also be prepared for the diseased allele. All that must be done for this is the modification of the final "A" in the forward sequence, as that base is the affected base. The primer sequence, thus, is changed to 5'AATCTGGGCTATGAGATCAT. The reverse sequence 200bp from the allele is unaffected, as a switching of the base doesn't add or subtract length from the entire genome, meaning that the reverse sequence remains 5'GAAACACCAGGGCTCAGGGTT. Upon attempting to sequence these two primers, an error is encountered, as the first sequence is not a valid sequence in the human genome when matched with the second sequence, as the SNP is a mutation. The result of the attempted sequence is shown below:

Primer Readout Failed.jpg