DNA Fingerprinting

One technique used in human DNA fingerprinting is comparing various DNA segments that contain repeated segments (VNTRs: variable number of tandem repeats). One such locus is the D1S80 locus on chromosome 1. In this region, we all have a 16 bp sequence that is repeated anywhere from 16 to 41 times (16x16 to 16x41 bp). Most likely, you have two different "alleles" on your two #1 chromosomes (different number of repeats). In this lab we will PCR amplify the D1S80 region to see which "alleles" you have.

DNA Fingerprinting Protocol

Important Notes:

Wear gloves throughout the procedure.

Wipe down your work table with 70% alcohol before and after the lab exercise.

Collecting DNA from Buccal Cells

Pour 20 ml of sterile 0.9% saline solution from a 50 mL disposable tube into your mouth and vigorously swish for 10 seconds.

Expel saline solution back into the same 50 mL tube.

Carefully pour 15 mL of the saline solution from the 50 mL tube into a 15 mL conical test tube and cap tightly.

Centrifuge at 1000 x g 10 min.

Without disturbing the cell pellet, carefully pour off the supernatant into the 50 mL conical test tube. (Discard the 50 mL tube in the biological waste container.)
Use UltraClean Tissue & Cells DNA Isolation Kit (MoBio) to extract DNA

Shake to mix Solution TD1. To the Dry Bead Tubes provided, add 700 μl of Solution TD1.
Add approximately 200 μL of your cells.
Adhere to a flat vortex then vortex at maximum speed for 10 minutes.
Centrifuge tubes at 10,000 x g for 1 minute at room temperature.
Avoiding the beads, transfer the entire volume of liquid sample to a Spin Filter (provided) and centrifuge at 10,000 x g for 30 seconds.  
Discard the flow through.
Add 400 μL of Solution TD2 and centrifuge at 10,000 x g for 30 seconds.  
Discard the flow through.  
Centrifuge again at 10,000 x g for 1 minute at room temperature to remove residual Solution TD2.  
Carefully place the Spin Filter in a new clean 2 ml Collection Tube (provided).  
Add 50 μL of Solution TD3 to the center of the white filter membrane.  
Centrifuge at 10,000 x g for 30 seconds at room temperature.  
Discard the Spin Filter. DNA in the 2 ml Collection Tube is now ready for PCR.

PCR and Electrophoresis

Set up a PCR reaction with 12.5 μL GoTaq HotStart Colorless Mastermix, 1 μL D1S80-F primer, 1 μL D1S80-R primer, 8.5 μL micropure water, and 2 μL of your DNA sample.

Using the thermal cycler, run a PCR reaction ([94C 30 sec, 68C 30 sec 72C 2 min]x30).

Store at -20C or proceed directly to Electrophoresis.

Run a 4% eGel (agarose gel) using 15-20 μL PCR sample. Use TrackIt as the DNA marker.

Observe, photograph, and analyze.