BME100 s2015:Group2 12pmL5

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Lab Write-Up 1 | Lab Write-Up 2 | Lab Write-Up 3
Lab Write-Up 4 | Lab Write-Up 5 | Lab Write-Up 6
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OUR TEAM

Name: Levi Riley
Name: Alexandria Clark
Name: Blossom Mendonca
Name: Alina Kilic
Name: Trevor Douglass
Name: Andre Dang


LAB 5 WRITE-UP

Procedure

Smart Phone Camera Settings

  • Type of Smartphone: iPhone 6
    • Flash: Off
    • ISO setting: Auto
    • White Balance: Auto
    • Exposure: Auto
    • Saturation: Auto
    • Contrast: Auto


Calibration

  • Camera should be set in the cradle at least 4 cm away from the drop placed on the slide
  • Camera should be set on a timer of three seconds in order to allow for a proper time of closing the box and no outside light to contaminate the pictures
  • The lens should be level with the drop (all of the drop's spherical surface from top to bottom should be in the picture)
  • Adjust Fluorimeter height if necessary to get it in the proper view of the phone camera
  • Distance between the smart phone cradle and drop = 6.8 cm


Solutions Used for Calibration

Calibration via H2O Calibration Via H2O Calibration via the standard given which was a concentration 0.25 units of DNA per microliter Calibration Via 0.25 mL|
Calibration via the standard given which was a concentration 0.5 units of DNA per microliter Calibration Via 0.5 Calibration via the standard given which was a concentration 1.0 units of DNA per microliter Calibration Via 1.0 |
Calibration via the standard given which was a concentration 2.0 units of DNA per microliter Calibration Via 2.0 mL Calibration via the standard given which was a concentration 5.0 units of DNA per microliter Calibration Via 5.00 mL |


Bonus Picture

Bonus Question Picture

For the set-up, this picture represents what the set up looked like right before the group closed the flap to take the picture. The camera was set in the cradle and the lens was 6.8 cm away from the drop that was on the superhydrophic side of the glass slide. The box covered the Fluorimeter to protect the SyberGreen Dye from exposure to light. After close the flap, three pictures were taken using the timer on the iPhone to delay the taking of the pictures until after the flap was closed.

Placing Samples onto the Fluorimeter

  1. Ensure that superhydrophic side of the slide is facing up when the slide is placed in the Fluorimeter
  2. Turn On Fluorimeter by flipping the switch on the side of the Fluorimeter
  3. Pipette 80 μL of SYBR Green I dye in between the first two clear circles on the glass slide (light from Fluorimeter should be absorbed by drop if the drop is in the proper place)
  4. Replace the pipette tip and then pipette 80 μL of the PCR sample mix or the calibration sample on top of the SYBR Green I dye droplet
  5. Make sure the light shines through the center of the drop and the light is focused through the opposite side of the drop
  6. Ensure the iPhone rests in the cradle at least 4 cm away from the droplet
  7. Set phone timer for 3 seconds and adjust camera as described in above calibration section
  8. Make sure the iPhone camera focuses on the droplet
  9. Cover the Fluorimeter with the box and press the button to take the picture on the iPhone and quickly close the flap to block all outside sources of light
  10. Take at least three pictures of each sample tested to ensure that a good picture is obtained
  11. Remove the used slide and dispose of properly
  12. Repeat the above setup for each sample


Data Analysis

Representative Images of Negative and Positive Samples

Negative Sample

H20 Only

Positive Sample

Green


Image J Values for All Calibrator Samples


Image J table

In the calibration section of the chart listed above, picture 1 correlates to H2O, picture 2 correlates to 0.25 units of DNA per microliter, picture 3 correlates to 0.5 units of DNA per microliter, picture 4 correlates to 1.0 units of DNA per microliter, picture 5 correlates to 2.0 units of DNA per microliter, and picture 6 correlates to 5.0 units of DNA per microliter. Picture 6 served as the positive control while picture 1 served as the negative control when checking the results of the PCR experiment.


Calibration curve
Calibration

Experimental


PCR Results Summary

  • Our positive control PCR result was 0.0833 μg/mL
  • Our negative control PCR result was 0.0833 μg/mL

Observed results

  • Patient 1 ID# 58575 :

For all images of Patient 1, the droplets all did not produce any type of green color when the drop samples were placed on top of the SYBR Green I dye. The drops themselves actually appeared to have a dark center. The concentration of DNA in these samples was the same as the positive and negative controls to which they were compared against. This is because the amount of PCR solution was 100μL which was then pipetted into the 500 μL solution given which resulted in 600 μL. After this solution was created, 80 μL was then pipetted onto the 80 μL of SYBR green dye which produced and amount which was 0.0833 μg/mL. These droplets all did not glow with SYBR Green I dye meaning they looked similar to the negative control which did not glow either.

  • Patient 2 ID# 86157 :

For all the images of Patient 2, the droplets did not produce any green glow. In fact, the droplets appeared to actually have a dark center when the light was concentrated on them. The results of Patient 2 essentially mirrored those of Patient 1. The concentration of DNA in these samples was the same as the positive and negative controls to which they were compared against. This is because the amount of PCR solution was 100μL which was then pipetted into the 500 μL solution given which resulted in 600 μL. After this solution was created, 80 μL was then pipetted onto the 80 μL of SYBR green dye which produced and amount which was 0.0833 μg/mL. These droplets all did not glow with SYBR Green I dye meaning they looked similar to the negative control which did not glow either.


Conclusions

  • Patient 1 ID# 58575 :

The results of Patient 1 were comparable to the results of the negative control. This is due to the fact that neither the negative control nor Patient 1 produced a green glow when paired with SYBR Green I dye on the Fluorimeter. Both samples had the same concentration (0.0833 μg/mL) meaning that their lack of glowing was not due to a discrepancy in the contents, but rather due to the fact that both these samples contained no DNA or negligible amounts of DNA.

Because SYBR green I dye glows green in the presence of DNA, Patient 1's genes were not replicated in PCR. The droplet did not glow green because there was no DNA - or a negligible amount of DNA - to cause it to glow. This means that the PCR did not replicate Patient 1's DNA. The most probable cause of this is due to the presence of the diseased gene. The PCR mix contained the healthy primer which would only bind with healthy, non mutated DNA. The primer cannot bind to DNA that contains a gene mutation because the base pairs do not properly match. This means that Patient 1's lack of glowing in the presence of SYBR green I dye can be accredited to the fact there is a negligible amount of DNA (or no DNA) present. This negligible amount of DNA is due to the fact that Patient 1's DNA could not replicate in the PCR reaction because the genes were diseased and would not match with the primer. All this goes to say that Patient 1 contains the diseased gene.

  • Patient 2 ID# 86157 :

Based upon the lack of glowing green in Patient 2, these results were comparable to the negative control. The negative control was the sample which did not glow. The concentrations of both of these samples was the same as well (0.0833 μg/mL), so the problem was not in a discrepancy of contents.

Because SYBR green I dye glows green in the presence of DNA, Patient 2's genes were not replicated in PCR. The droplet did not glow green because there was no DNA - or a negligible amount of DNA - to cause it to glow. This means that the PCR did not replicate Patient 2's DNA. The most probable cause of this is due to the presence of the diseased gene. The PCR mix contained the healthy primer which would only bind with healthy, non mutated DNA. The primer cannot bind to DNA that contains a gene mutation because the base pairs do not properly match. This means that Patient 2's lack of glowing in the presence of SYBR green I dye can be accredited to the fact there is a negligible amount of DNA (or no DNA) present. This negligible amount of DNA is due to the fact that Patient 2's DNA could not replicate in the PCR reaction because the genes were diseased and would not match with the primer. All this goes to say that Patient 2 contains the diseased gene because it did not glow in the presence of the SYBR Green I dye meaning that the PCR could not replicate the diseased genes.



SNP Information & Primer Design

Background: About the Disease SNP


A nucleotide is composed of a nitrogenous base, a five carbon sugar, and at least one phosphate group. They are organic molecules that serve as monomers or sub units of nucleic acids, like DNA and RNA. A Polymorphism is a variation in the coding and non coding DNA sequence among a population. It is a discontinuous genetic variation resulting in the occurrence of several different forms or types of individuals among the members of a single species. The SNP analyzed is found in the Homo Sapien species and the chromosome in which it is found is 8:19956018. The clinical significance of the SNP is that is a pathogenic mutation which can cause great harm to the recipient of the mutation. In addition, it is associated with the LPL (lipoprotein lipase) gene and linked to coronary heart disease. A few of the functions of LPL are Apolipoprotein Binding, Heparin Binding, Lipoprotein Lipase Activity, Phospolipase activity, and Protein Binding. A polymorphism can result in something known as an allele which is a variant form of a gene. Some genes - which are located on the exact same spot on a chromosome - have a variety of different forms which are all alleles of that gene. The polymorphism which results in this pathogenic associated allele is AGT. The numerical position of AGT is 19956018. The normal chromosome of a healthy individual not containing this diseased allele is AAT. The mutation of an A to a G results in a diseased gene that is linked with coronary heart disease.

Primer Design and Testing

The diseased primer had no matches because the humane genome does not have the mutation of AGT. Therefore, there would be no match in accordance with the natural human genome tested in the website. While polymorphisms, unique alleles and variations are all what helps to make each individual have a slightly unique genome, the AGT mutation is what can lead to a person having coronary heart disease. The diseased primer has no results while the healthy, non-diseased primer produces a result.

Diseased Primer

<a href="http://imgur.com/ruzkEYS"><img src="http://i.imgur.com/ruzkEYS.png" title="source: imgur.com" /></a>

NON-Diseased Primer

<a href="http://imgur.com/TAV9kPT"><img src="http://i.imgur.com/TAV9kPT.png" title="source: imgur.com" /></a>