BME100 f2014:Group26 L5

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Lab Write-Up 1 | Lab Write-Up 2 | Lab Write-Up 3
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OUR TEAM

Ambike Bhraguvanshi
Timothy Chen
Andrew Polson
Rachel Ponstein
Rebecca Schiavone
Jiaqi Wu


LAB 5 WRITE-UP

Procedure

Camera Settings

  • Type of Smartphone: iPhone 5S
    • Flash: Inactive
    • ISO Setting: Not adjusted because setting was unavailable on smartphone (N/A)
    • White Balance: N/A
    • Exposure: N/A
    • Saturation: N/A
    • Contrast: N/A


Calibration and Solutions Used

Camera Set-Up

  • Distance between the smart phone cradle and drop = 6.3 cm

The iPhone 5S was placed on the cradle at a distance of 6.3 cm from the fluorimeter and positioned, so that the smartphone formed a 45 degree angle with the bottom of the cradle. In other words, the back of the smartphone rested on the side of the cradle closest to the fluorimeter. This ensured that the camera took focused pictures of the drops from the side.


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


BONUS - Photo of Fluorimeter Set-Up

Fluorimeter set up group 26.jpg

The picture above shows how the fluorimeter was set up. As described above, the iPhone 5S was placed on the cradle at a distance of 6.3 cm from the fluorimeter and positioned, so that the smartphone formed a 45 degree angle with the bottom of the cradle. In other words, the back of the smartphone rested on the side of the cradle closest to the fluorimeter. On the fluorimeter itself, slides holding drops consisting of SYBR Green I dye solution and the PCR product samples were placed to align with the fluorimeter's blue LED light. Bubble wrap and a plastic container were placed below the fluorimeter to raise it to a height at which the edge of the slides would align with the camera of the smartphone.


Placing Samples onto the Fluorimeter

Calibration Samples

  1. Using gloves, place a slide into the fluorimeter with the smooth side down, so that the drops are placed on the rough superhydrophobic side.
  2. Use a micropipette to place an 80 μL drop of SYBR Green I dye solution in the middle of the first two clear circles in the middle column of the slide.
  3. Place an 80 μL drop of one of the calf thymus calibration solutions on top of the SYBR Green I dye solution drop to create a large drop with a total volume of 160 μL.
  4. Align the drop with the blue LED light by adjusting the position of the slide. The blue LED light should illuminate the center of the drop, and the drop should focus the light on the other side.
  5. Cover the fluorimeter setup with the light box, but keep one flap up to access the camera's timer button.
  6. Make sure the camera has a clearly focused view of the drop.
  7. Depress the camera's timer button, and close the flap before the camera takes pictures of the sample. Each drop should have three pictures taken of it.
  8. Remove the light box, making sure not to move the smartphone.
  9. Use a pipette to remove the 160 μL drop from the slide, and move the slide to the next position - the center of the next two circles in the middle column of the slide.
  10. Repeat steps 1-9 for the other concentrations of the calf thymus calibration solutions.


PCR Product Samples

The procedure for placing the PCR product samples onto the fluorimeter is exactly the same as the one used above for the calibration samples, except for steps 3 and 10.

  • Step 3: Place an 80 μL drop of one of the patients' PCR product solutions on top of the SYBR Green I dye solution drop to create a large drop with a total volume of 160 μL.
  • Step 10: Repeat steps 1-9 for the other PCR product solutions.


Data Analysis

Representative Images of Negative and Positive Samples

Negative Control Sample N1ovalg 26.png

Positive Control Sample P1oval g26.png


Data Tables for Calibration Samples

Lab 5 Data Table 1.PNG Lab 5 Data Table 2.PNG


Calibration Curve

Lab 5 Calibration Curve.PNG


Data Tables for PCR Product Samples

Lab 5 Data Table 3.PNG Lab 5 Data Table 4.PNG Lab 5 Data Table 5.PNG Lab 5 Data Table 6.PNG


PCR Results Summary

  • Our positive control PCR result was 9.96 μg/mL
  • Our negative control PCR result was -0.36 μg/mL
    • The negative control PCR result was a negative value rather than a value close to zero because the line of best fit had a large y-intercept, which caused the value to become slightly negative. The value, however, is still considerably close to zero

Observed Results

  • Patient 47894: This patient's PCR product samples fluoresced green (in the images) when placed in the beam of the fluorimeter's blue LED light, which means that the patient has the disease. Although the samples of the third replicate were less fluorescent in the images than the other replicates, this can be attributed to how the initial PCR product concentration for the third replicate was lower at 17.52 μg/mL, in comparison to the first two replicates, which had concentrations of 21.84 μg/mL and 19.44 μg/mL.
  • Patient 77482: This patient's PCR product samples fluoresced green (in the images) when placed in the beam of the fluorimeter's blue LED light as well, which means that the patient has the disease also. Although the samples of the second replicate were less fluorescent in the images than the other replicates, this can be attributed to how the initial PCR product concentration for the second replicate was lower at 15.96 μg/mL, in comparison to the first and third replicates, which had concentrations of 24 μg/mL and 26.88 μg/mL.

Conclusions

  • Patient 47894: This patient had initial PCR product concentrations that were far larger than the positive control initial concentration (9.96 μg/mL). The lowest concentration for patient 47894 was 7.56 μg/mL more than the positive control value, which would mean that this patient had the disease.
  • Patient 77482: Patient 77482 had initial PCR product concentrations that were far larger than the positive control initial concentration (9.96 μg/mL) as well. The lowest concentration for patient 77482 was 6.00 μg/mL more than the positive control value, which would mean that this patient also had the disease.


SNP Information and Primer Design

Background

A nucleotide is an organic building block of DNA and RNA, which is made up of a "backbone" of deoxyribose sugar (in DNA)/ribose sugar (in RNA) and a phosphate group, and one of the nitrogenous bases, adenine (A), thymine (T), cytosine (C), or guanine (G) [in DNA]/adenine (A), uracil (U), cytosine (C), or guanine (G) [in RNA]. A polymorphism is a variation in the DNA sequence that has no adverse effect on the individual carrying the variation. An SNP, which stands for a single-nucleotide polymorphism is a DNA sequence variation occurring regularly within a population, in which just one nucleotide - A, T, C, or G - in the genome is different between members of a specific biological species or set of paired chromosomes. The SNP specifically studied in this lab was the rs16991654 variation, which is found in the species Homo sapiens and located on chromosome 21:34370656. The rs16991654 variation holds clinical significance because it is pathogenic and is associated with the KCNE2 gene (potassium voltage-gated channel, Isk-related family, member 2), which regulates, among other things, the transport of potassium ions out of heart cells, which recharges the cardiac muscle after each heartbeat to maintain a regular rhythm. The sequence of the allele, which is an alternate form of the DNA sequence of a gene that results in different traits and is composed of one or more SNPs, associated with the rs16991654 variation contains a single SNP of TTC -> CTC. Although this SNP only involves one different nucleotide in the disease-associated allele, it is highly consequential, as it is linked to the pathogenesis of congenital Long QT syndrome (LQTS). LQTS is a rare, inherited heart rhythm disorder, which causes fast, chaotic heartbeats. These erratic heartbeats lead to an increased risk of sudden fainting or seizure and may even cause sudden death by prolonged irregularities in heartbeat.


Primer Design and Testing

Non-Disease Primers

  • Non-disease forward primer: C A T G G T G A T G A T T G G A A T G T
  • Non-disease reverse primer: C C C T T A T C A G G G G G A C A T T T
  • Result: chr21:35742936+35743155

Primer SNP disease screenshot.png

The result displays an output of a 220 DNA base pair sequence that contains nucleotides that lie in between the primer pair and includes the forward and reverse primers themselves. The chromosome indicated from the result is the chromosome on which the rs16991654 variation is found. As the primer test utilizes a non-disease human genome, it makes sense for the non-disease primers to work. Capitalization implies that the primer sequence matches the database sequence at that location; everywhere else, the sequence is lowercase. As displayed, the forward primer has a melting temperature of 56.5°C, and the reverse primer has a melting temperature of 60.0°C.


Disease-Specific Primers

  • Disease-specific forward primer: C A T G G T G A T G A T T G G A A T G C
  • Disease-specific reverse primer: C C C T T A T C A G G G G G A C A T T T
  • Result: No matches

Lab 5 Disease-Specific Primers Results.PNG

When the disease-specific forward and reverse primers are inserted into the primer test, the results show that there are no matches. This is because the non-disease human genome used in the primer test does not contain the disease-associated allele (which has the variation of TTC -> CTC), which explains why the disease-specific primers do not work.