Paternity test using Cybertory

(This needs a more detailed development.)

Basic instructions:

Objective:

To analyze the polymorphic markers in order to determine which of the 4 candidates is the father. The assay consists on, starting with the DNA samples of each of the potential fathers, the mother and the child, performing a PCR amplification of the 4 STR markers and separating the amplified fragments by electrophoresis. Comparing the band patterns, one must deduce which of the 4 candidates is the father (or reject all four).

1.

In the order form, you must buy

• The “Paternity Test kit”, that contains the following DNA samples:
  • DNA from a child (boy or girl, C).
  • DNA from his/her mother (M).
  • DNA from four males whose paternity is being ascertained (F1, F2, F3, F4).
• PCR primers for four STR markers (e.g. AMEL, D8S1179, D18S51, D21S11).
• Reagents (“PCR master mix”) to perform multiplex PCR.

Note: each time an order is placed, the DNA samples from parents and child are different.

2.

In the reaction lab, prepare in tubes 1 to 6 reaction mixtures for the PCR assay. You need to put

• In each tube, 20 µL of one out of the 6 DNA samples (M, C, F1, F2, F3, F4).
• In every tube, 5 µL of the PCR mix.
• In every tube, 1 µL each of the primers for the 4 STR markers.
• Complete volume with water up to 50 µL total (that is, 24 µL water).

3.

Start the PCR reaction pressing the “Incubate” button. After the required virtual time has passed, the amplification of DNA of STR markers wil have completed. A message will confirm what has been amplified, just to check if you did something wrong.

4.

In the electrophoresis lab (to reach it, use the main Cybertory menu at the top right corner of the screen).

• Choose the 5% polyacrylamide gel (bottom left, below the gel).
• Press the “Autoload” button, which will transfer the 12 samples prepared in the reaction lab to the fisrt 12 wells in the virtual gel; well number 13 will be automatically loaded with a mixture of molecular size standards (a “DNA ladder”).
• Set the power source to 300 volts and virtual time to 1 hour and 10 minutes. Switch on the power to start electrophoresis. While it runs, you may switch UV light on (make sure first to wear your protective virtual goggles or face shield) and switch virtual room lights off to see how DNA advances and bands separate.

5.

Once electrophoresis has finished, capture the screen or draw a schematic of band position and interpret the results obtained: can you assert, with a certain probability, that one of the 4 candidates is the father?