Initial Concentration of 2X Calf Thymus DNA solution (µg/mL)
Volume of the 2X DNA solution (µL)
Volume of the 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
Calibration
Distance between the smart phone cradle and drop = 5.5cm or 0.055m
Placing Samples onto the Fluorimeter
Turn on the excitation light using the switch and turn on your camera and check the settings. The fluorimeter will be setup in the light box.
Place the camera on the cradle and adjust the height of the fluorimeter so that the camera takes a picture of the drop from the side.
Adjust the distance between the camera and the first two rows of the slide to make the drop as big as possible without making it blurry and record the distance.
Insert the slide so the smooth side is on the bottom and the hydrophobic side is on top.
Place a 80 microliter drop of SYBR Green I on the middle dots of the first two rows of the slide and then add 80 microliters of the calf thymus solution.
Adjust the slide so that the LED light is focused in the middle of the drop and fiber optic that is on the other side of the light.
Use a timer on the camera in order to take a picture in the dark after covering the fluorimeter with the light box. The box should remove as much light as possible. Take three pictures.
After taking the pictures, remove the box and the 160 microliter drop, and then move the slide to the next position. Do not move the camera.
Repeat steps 5-8 with the other concentrations of the calf thymus solutions.
Data Analysis
Representative Images of Negative and Positive Samples
Negative Sample
Positive Sample
Image J Values for All Calibrator Samples
Calibration curve
PCR Product Tube Label
Volume of the DILUTED PCR Product solution (µL)
Volume of the SYBR GREEN I Dye solution (µL)
Dilution 1
Dilution 2
Total Dilution
5
80
80
1/2
1/6
1/12
2
80
80
1/2
1/6
1/12
1
80
80
1/2
1/6
1/12
0.5
80
80
1/2
1/6
1/12
0.25
80
80
1/2
1/6
1/12
0
80
80
1/2
1/6
1/12
PCR Results Summary
Our positive control PCR result was 0.73 μg/mL
Our negative control PCR result was 0.14 μg/mL
Observed results
Patient 17567: The drop glowed green, just like the positive sample. The mean calculated initial concentration was 0.602 μg/mL.
Patient 57701: The drop glowed green, just like the positive sample. The mean calculated initial concentration was 0.617 μg/mL.
Conclusions
Patient 17567: This patient had characteristics very similar to the positive sample. The concentration and glow were very close to the positive sample. Therefore this patient has the disease because the targeted genetic sequence is very similar to the positive sample when amplified.
Patient 57701: This patient had characteristics very similar to the positive sample. The concentration and glow were very close to the positive sample. Therefore this patient has the disease because the targeted genetic sequence is very similar to the positive sample when amplified.
SNP Information & Primer Design
Background: About the Disease SNP
SNP is when a single nucleotide on a strand of DNA is changed. This can result in a different phenotype being expressed, but usually these changes in the DNA happen in regions that are noncoding. Occasionally they could also create beneficial mutations. SNPs can be detrimental by creating mutations that lead to the wrong production of amino acids creating broken proteins. The SNP (rs16991654) that we are studying in this lab resulted in a change in the 21st chromosome that led to a protein KCNE2 being malformed. This would lead to irregularities in the heartbeats as the protein channel would make it harder for potassium and other nutrients to pass through heart cells.
Primer Design and Testing
Non-Disease Primer Test
This shows that these primers exist, meaning it is possible to test using these primers.
Disease-Specific Primer Test
This shows that these primers do not exist, meaning that it is not possible to test using these primers.
Part D
3) What is a nucleotide?
They are organic molecules that serve as the monomers or building blocks of nucleic acids. Nucleotides are composed of a nitrogenous base, a five-carbon sugar and at least one phosphate group. An example of this is ATP
What is a polymorphism?
A polymorphism is when there is a single change of a nucleotide on a strand of DNA. This can occasionally cause a change in the phenotype being expressed.
4) What species is this variation found in?
Homo sapiens
What chromosome is the variation located on?
21
What is listed as the Clinical significance of this SNP?
Pathogenic
Which gene(s) is this SNP associated with?
KCNE2
What disease is linked to this SNP?
Congenital long QT syndrome
Part 2
6) What does KCNE2 stand for?
Potassium voltage-gated channel, Isk-related family, member 2
Briefly describe the molecular function of this gene.
The function is regulating neurotransmitter release, heart rate, insulin secretion, neuronal excitability, epithelial electrolyte transport, smooth muscle contraction, and cell volume.
7) What is an allele?
One of two or more alternative forms of a gene that arise by mutation and are found at the same place on a chromosome.
The disease-associated allele contains what sequence?
CTC
8) The numerical position of the SNP is:
34370656
11) To design a non-disease forward primer,write down the sequence that is 20 bases long and ends with the nucleotide at the position you wrote in step 8.
5'-CATGGTGATGATTGGAATGT
12) Every PCR reaction needs two primers to amplify DNA. So, you will need to also design a non-disease reverse primer. In the sequence window, scroll 200 bases to the right of position you recorded in step 8.
34370856
13) Write down the sequence that starts with the base at the position you wrote in step 12, plus 19 bases toward the left.
5'-CCCTTATCAGGGGGACATTT
14) Next, design a pair of disease SNP-specific primers. Simply change the final base of the non-disease forward primer to the disease SNP nucleotide, and leave the reverse primer the same.
BME 100 Fall 2014
