Cybertory 2017 Paternity test p.

Paternity test by multiplex PCR on STR polymorphic markers

(Practical activity in the Cybertory virtual laboratory)

Fundamentals:
Paternity testing is based on the Mendelian inheritance paradigm together with genetic diversity of
individuals. The first tells us that for every two copies, or alleles, of some region of our genome, one is identical
to one out of the two our mother has, while the other matches one from our father. The second says that some
persons differ from others in the nucleotide sequence present in diverse regions of the genome, what is called
genetic polymorphism.
For paternity tests, as for other identity tests, polymorphic regions in the genome are chosen such that do
not code for any products (either protein or RNA), so they do no represent any functional advantages or
disadvantages and therefore their variants (alleles) are randomly distributed in the population (although
respecting the laws of inheritance)
This assay specifically deals with several polymorphic regions known as STR (short tandem repeats) that
are part of CoDIS, which is one of the groups of international standard markers, well known because of its use
by the FBI. The difference among alleles present in different individuals is in the number of repeating units and,
consequently, in the length of DNA fragments that get amplified by PCR and are analysed by electrophoresis.

Bibliography:
• R.B. Hallick, C. Ryan (2000) Blackett Family DNA Activity 2 [online]. In The Biology Project, Human
Biology, DNA Forensics [visited on 26 June 2018]. Available at
http://www.biology.arizona.edu/human_bio/activities/blackett2/overview.html
• Wikipedia contributors (2018) Combined DNA Index System [online]. Wikipedia, The Free Encyclopedia
[visited on 26 June 2018]. Available at https://en.wikipedia.org/wiki/Combined_DNA_Index_System

How to start:
The virtual laboratory works within a web page, so you should start by opening the web browser in your
computer or tablet. Any modern browser should do, as long as JavaScript is enabled for it (Firefox or Chrome
are recommended).
Navigate to the Cybertory page at http://biomodel.uah.es/en/lab/cybertory/

Assay:
multiplex PCR amplification for paternity determination
Aim:
Analysis of the polymorphic markers to ascertain which of the candidates may be the actual father of a child.
The assay starts with DNA samples from each putative father, from the mother and from the child; after a PCR
amplification for several STR markers, amplified fragments are separated by electrophoresis. By comparing
band patterns, a deduction will be done of which candidate may be the father (or else disregard all of them).
Virtual materials:
Software:
• “Cybertory” virtual molecular biology laboratory, biomodel.uah.es/en/lab/cybertory/
Virtual reagents:
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• Sample set for paternity test (purchased from CygnusLab ), which contains the following DNA samples:
• DNA from the mother (labelled as M).
• DNA from the child (labelled as C).
• Four DNA samples from men whose paternity is to be assessed (labelled F1 to F4).
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• CygnusLab 10x PCR Master Mix containing
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• 250 U/mL CygnusTaq DNA polymerase
• dATP, dGTP, dTTP, cCTP nucleotides, 2 mM each
• 15 mM MgCl2
• 125 mM TAPS buffer, pH=8.5
• PCR primer pairs, specific for CoDIS STR markers D3S1358, D8S1179, D18S51, D21S11, FGA and VWA, as well
as for the sex-specific marker AMEL (amelogenin gen) used as a control
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• CygnusLab Ladder2™ DNA molecular mass marker
• Nuclease-free water
• Yellow pipette tips, usable from 1 to 100 µL
• Acrylamide, bisacrylamide, TEMED, ammonium persulphate, urea (to prepare the polyacrylamide gel)
• 0.5X TBE buffer (Tris-Borate-EDTA)

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• Ethidium bromide
• Gel loading dye (containing bromophenol blue, xylene cyanol and glycerol)
Virtual instrumentation:
• Variable volume micropipette up to 100 µL
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• CygnusLab TempeMatic incubator with thermal regulation
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• CygnusLab GelMatic Plus tank for horizontal agarose gel electrophoresis
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• CygnusLab 1000ZX DC power source (from 10 to 1000 V)
• Ultraviolet transilluminator (attached to the electrophoresis tank)
• Protective screen and goggles against UV light

Procedure:
Start by pressing the “Start Cybertory” button. After a few seconds, a notice will appear prompting you to place
an order with samples and primers; please accept it.
1) Investigate the Online catalog.
2) Press the “Place an order” button.
3) Select “Samples for paternity test” and all the primers you wish to use.
4) At the bottom of the page you will need to provide some data. (This is a simulation of a real-life situation; it is
not important what you type for first name and surname, but it is crucial that the password given is the one assigned
to the institution).
5) Press the “Send order” button and then “Confirm the order”. Wait until the screen is updated
displaying the simulated laboratory (tubes, pipette, etc.).

Using the virtual micropipette

Select the tube in which you want to pipette the
reagents by clicking on it (not on the cap or on the
label); the cap will open up and the pipette will move to
the tube. A click on the top plunger makes it slide, either
expelling or aspirating the tip contents. The volume to
be dispensed is either set by clicking on the adjustment
wheel (left half decreases it, right half increases it) or by
typing it in the volume display slot (please do not press
the Enter key).
The working range of this pipette is 1 to 100 μL, in
1 μL steps.
To avoid contamination, you should change tips
every time you change samples. The tip is expelled by
clicking on the ejector button or on the trash bin.
Get a new tip by clicking on the tip box.

Possible problems:
Should you need, due to any mistake, to start anew with fresh tubes, click on the image of the container to the
left of the tip box.
After clicking on a tube, the pipette, etc., please allow some time for anything to happen. Do not be impatient.
If you click on the pipette plunger and the hand cursor does not disappear, move the pointer away from the
pipette.
You can better see the liquid inside a tube by having the pipette leave the tube; to do so, click again on the
tube.

6) Prepare reaction mixtures in tubes 1 to 6 for the PCR assay. For this, you should add:
• to each tube, 20 µL of one of the 6 DNA samples (M, C, F1, F2, F3, F4);
• to all tubes, 5 µL of the PCR Mix;
• to all tubes, 1 µL of each primer pair for as many markers as you wish to test;
• fill up with water up to a total volume of 50 µL in each tube.
Before you start pipetting, it is convenient that you prepare a table where you plan in advance which reagents and
volumes you will add into each tube.
7) Start the PCR reaction by pressing the “incubate” button (located at the bottom centre, under the timer).
After the required virtual time is up, reactions will be complete. Depending on how the laboratory is
configured, a warning may be displayed informing of what has been added to each tube; please check that it
matches what you intended.

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8) In the main Cybertory menu, select the “Electrophoresis laboratory”

module. Then select in the tank a 5% polyacrylamide gel. Press the
“autoload” button, so the samples prepared in the previous PCR will be
transferred to the wells in the virtual gel; well number 13 will be automatically
loaded with a mixture of size standards (the Ladder2™ DNA ladder).

After loading, the wells are coloured blue due to the loading dyes commonly added to samples
(bromophenol blue and xylene cyanol), which makes it easier to see initially the samples that
are being loaded and, afterwards, the electrophoresis front. The loading solution also includes
some dense compound (like glycerol, sucrose or ficoll) which makes the samples sink to the
bottom of the well. Agarose gels like this one are regularly set horizontally.
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“Autoloading” is a unique feature of the CygnusLab gels; in real life, samples must be
laboriously transferred manually from reaction tubes to gel wells using a micropipette.

9) Adjust the power source to 250 volts and 1.5 virtual hours. Switch the current on to start the
electrophoresis. During the run, you may switch on the UV light (please make sure you have put on your
protective goggles or face mask) and switch off the virtual room lights in order to see the DNA moving
forward and the bands separating.

In any electrophoresis, if the available power source does not indicate current intensity (milliamperes), it is
important that you make sure the current is, indeed, circulating. Observe how bubbles are released from
both electrodes, as a result of electrolysis of the buffer. You will also see that the blue bands of both dyes
move along the gel (as well as do the DNA bands, but you only see these if you switch the UV light on).
If higher voltages are used, samples will progress quicker along the gel, but high voltages produce high
intensity current that will heat up the gel; this may affect the sample integrity (for instance, denaturing DNA)
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and may also melt the agarose. Unlike real life gels, the CygnusLab gels never melt, so the only
inconvenience of using very high voltage is that samples may be lost at the bottom of the gel before you
have time to react.

10) Once the electrophoresis is finished, draw a sketch indicating the position of all bands (you may capture
an image using the digital camera attached to the electrophoresis tank) and interpret your results:
 Can you assert, with a reasonable certainty, that one of the putative fathers is the true parent of
the child?

Angel Herráez doi:10.5281/zenodo.1299838