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To run the PCR test first set up the PCR machine by plugging it into an electrical outlet and downloading the smartphone application. Program the application to fit the needs of your test, set the temperature, and number of cycles needed for your test. The average test will take about two hours to run. The row in the PCR machine has two sample spots, these spots are designated for a positive and negative control. For the positive control sample, add the DNA cancer template sample with the reagent and for the negative, add the negative sample with the reagent. There are six more rows with three sample spots available for six different patient samples. It is useful to have three samples for each patient to get a more reliable result. The final volume of the reaction should be 2 mL. Let the cycles run to completion for results.
Adjusting smartphone settings:
1)First inactivate the flash.
2)Set ISO to 800.
3)Set white balance to auto.
4)Set exposure to highest setting.
5)Set saturation to the highest setting.
6)Set contrast to lowest setting.
DNA Measurement Protocol
THe DNA concentration measurements are to be taken using a fluorimeter. To assemble the fluorimeter, use the following steps:
1) Turn on the excitation light using the switch for the blue LED.
2) Place a smart phone on the cradle at a right angle from the slide.
3) Open the application used to control the PCR machine and go to the DNA measurement page.
4) Move the smartphone in the cradle to the line on the mat designated for the model of your smartphone.
5) The pipette should be filled with liquid only to the bottom of the black line. Then use the pipette to place two drops of water (each drop should be between 130-160 microliters) in the middles of the first two rows of the slide.
6) Move the slide so that the blue LED light is focused on the the drops of water to the middle of the black fiber optic fitting on the other side of the drop.
7) Cover the fluorimeter with the light box so that much of the stray light will removed, but make sure you can still access your smartphone to take pictures.
8) While being careful not to move the smartphone, take three pictures of the water droplet.
9) When removing the light box, be careful not to move the smartphone because that could make the analysis more complicated.
10) Use a clean plastic pipette to remove the water droplets from the slide.
11) Push the slide in so that you are now in the next set of two holes.
12) Repeat steps 5-10 four more times in 5 different positions.
After assembling the fluorimeter, you can now determine if you've amplified the targeted DNA in your PCR experiment. Using the Fluorimeter and the smartphone app, you can calculate the relative amount of DNA through fluorescence, which is generated by excitation and emission wavelengths. In order to detect fluorescence when dsDNA is present, you'll be using SYBR Green I because it's more safer compared to other dyes. With that being said, gloves must be worn when handling any liquid containing SYBR Green I. The fluorimeter itself is a very simple machine because it uses optical caustic, a special type of optics that completely removes the need for lasers, mirrors, or lenses. Also the flourimeter is battery-powered, lightweight and portable; this allows every student to have one of these at their lab table. Following the steps below, you can easily learn how to dye your amplified DNA.
1)On your lab table, you'll find eight samples from the Open PCR, 1 DNA sample(calf thymus standard at 2 micrograms/mL), and water from the scintillation vial (white cap) to analyze.
2)With a permanent marker, label your Eppendorf tubes and number your pipettes (on the bulb part) so that no cross-contamination will occurs. At the end, you should have 10 Eppendorf tubes and 10 pipettes clearly labeled (see Table 3). REMINDER: Use only 1 transfer pipette per sample!!!
3)Transfer each sample separately (using 1 pipette per sample) into an Eppendorf tube containing 400 mL of buffer. Clearly label this tube with the number of the sample and make sure to get all of the sample into the Eppendorf tube. ONLY use the sample number transfer pipette to place a drop onto the fluorescence measuring machine, and then discard it.
4)Take Eppendorf tube labelled SYBR Green I and using the specially labeled pipette, place 2 drops on the first two centered drops.
5)Now take your diluted sample and place 2 drops on top of the SYBR Green I solution drops.
6)Let the smartphone take as many pictures as needed for the smartphone application
7)Now you may either rerun the sample again or discard the sample pipette, but keep the SYBR Green I labelled pipette. Also you can only run 5 samples per glass slide.
8)Before completing the lab, run the water from the scintillation vial as a BLANK using the same procedure.
Uploading Pictures and Analysis Using ImageJ
1)Upload pictures onto your computer.
2)Upload pictures to ImageJ.
3)On the toolbar, click on Analyze, on the drop down bar choose Set Measurements. A new window will open and select Area Integrated Density and Mean Grey Value.
4)Go back to the toolbar,click Image, on the drop down bar choose Color, then select Split Channels in order to make 3 new files.
5)Choose an image that has "green" in it.
6)On the Menu bar, click on the Oval tool.
7)On the image, click and stretch the oval around the "green" or clear drop. Then click Analyze and then click Measure.
8)Now you see some number at the top and those are your measurements. Record these measurements and save them on Excel.
9)Repeat steps 5-8 for all other pictures.
Research and Development
Background on Disease Markers
A particular single nucleotide polymorphism (SNP), rs132630309, is characterized by a mutated C instead of a normal T on the forward strand at position 5448 of the EDA (ectodysplasin A) gene on the X chromosome. The normal DNA sequence around this position is: while the SNP associated DNA sequence is: . At the protein level, this mutation causes a change from arginine (R; associated with the DNA triplet CGG) to leucine (L; associated with the DNA triplet CTG). The condition associated with this SNP is hypohidrotic ectodermal dysplasia; this is one of a group of syndromes known as ectodermal dysplasia which is characterized by "abnormal development of the skin, hair, nails, teeth, or sweat glands" (Taken from the NCBI database). More info on this SNP can be found via this web link: http://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?rs=132630309.
Since this SNP lies on the forward strand, the primer used to facilitate a DNA signal of the mutated DNA will include a forward primer containing the mutation and a reverse primer 200 base pairs to the right of the mutation, with each primer being 20 base pairs long.
Forward Primer (5' to 3'):
Reverse Primer (5' to 3'):
A disease allele will produce a PCR product while a non-disease will not produce a PCR product due to the ability of the forward primer, which contains the mutated T, to bind to the reverse strand. If the reverse strand has a corresponding A at the position opposite the mutated T then the primer will bind. If the reverse strand has a C instead (normal), the forward primer will not bind to the reverse strand. This is illustrated in the figure below.
Bayes' Rule: Test of Reliability
A nucleic acid test is only as useful as its level of reliability. If the test is not reliable and used, people who have a mutated T will be told they have or will have hipohydrotic ectodermal dysplasia when they actually do not. Bayes' Rule provides an equation that can be used to determine the probability that a person who has a mutated T will have hipohydrotic ectodermal dysplasia. The equation with the necessary variables is displayed below.