What are the Steps of DNA Fingerprinting? June 2, 2014 Do Now: What does DNA Stand for? What is the major function of DNA? Why is DNA shaped in a Double Helix?

Agenda VNTR and STR DNA Extraction PCR DNA Fingerprinting Paternity Testing

DNA Fingerprinting VNTR Variable Number of Tandem Repeats 9 to 80 bases long DNA Fingerprinting

DNA Fingerprinting STR Short Tandem Repeats 2 to 5 bases long Easier to use DNA Fingerprinting

Steps in DNA Fingerprinting Extraction PCR Gel Electrophoresis

DNA Extraction Extracting DNA from a sample or evidence Allows us to amplify by PCR and test by DNA Fingerprinting. DNA Extraction

DNA Extraction Remove the green sepals from the strawberries. Place strawberries into a Ziploc bag and seal shut. Squish for a few minutes to completely squash the fruit. Add 10 ml DNA Extraction Buffer (soapy salty water) and squish for a few more minutes. Try not to make a lot of soap bubbles. Filter through a moistened paper towel set in a funnel, and collect the liquid in a clear tube. Do not squeeze the paper towel. Collect about 3 ml liquid.

DNA Extraction Add 2 volumes ice cold isopropyl alcohol to the strawberry liquid in the tube. Pour the isopropyl alcohol carefully down the side of the tube so that it forms a separate layer on top of the strawberry liquid. Watch for about a minute. What do you see? You should see a white fluffy cloud at the interface between the two liquids. That’s DNA! Spin and stir the stirrer in the tangle of DNA, wrapping the DNA around the stirrer. Rinse your funnel. Put the Ziploc bag and paper towel in the garbage.

PCR • Once we have extracted DNA, we may not have enough to complete gel electrophoresis, so we must amplify the DNA by PCR. To complete a PCR, we must use primers, DNA Polymerase, and nucleotides.

PCR During PCR, the DNA is heated, causing the DNA to split into two strands. After heating, the DNA sample is cooled, and primers are attached, which identifies the specific sequence we want to replicate. Primers also allow DNA Polymerase to bind to the single stranded DNA. Once these bind, they generate two strands of DNA by adding nucleotides. After 30 cycles of PCR, we have about 1 billion copies of the desired DNA fragment.

DNA Fingerprinting • Following PCR, we must test our DNA samples by electrophoresis. Gel electrophoresis separates DNA fragments by size, with the smallest moving furthest and the largest moving the shortest distance. The separation process operates by giving DNA fragments a negative charge and having them move toward a positive charge.

DNA Fingerprinting The results from these tests appear as bands on the gel. To view these bands, we must use a combination of UV light and fluorescent probes. By comparing the samples with standards we can estimate the size of our DNA samples. The unique combination of bands creates a “fingerprint” that allows us to identify individuals.

Paternity Testing Paternity tests identify the father of a child. The child gets half of its DNA from mom and half from dad. Paternity Testing

https://www.youtube.com/watch?v=R0joEIFJNEc

Paternity Testing To identify a father, you must find the bands a child has, but the mother does not. After finding these bands, look for the person that could have passed that DNA to the child. That’s the daddy!