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DNA Technology Restriction Digests and Gel electrophoresis DNA Extraction • Collect DNA sample (blood, saliva, hair follicle, skin) • open the cells using lysis buffer and a heat bath -2 components detergent to poke holes in membrane and proteinase K to cut apart the histones and free the DNA Microsatellite regions • Almost all DNA between humans is identical (99.9%), except in non-protein coding sites called microsatellite regions • Where we look when comparing DNA to solve crimes or for paternity DNA Amplification • PCR=Polymerase chain reaction • Input: nucleotides, taq polymerase (like our DNA polymerase but won’t denature with heat), and primers for region we want to copy. • Cycles of heat to unzip DNA, temperature for primers to bond, and temperature for taq polymerase to add nucleotides-See animation Restriction Digest • Now cut up the DNA based on its base pair sequence • We use restriction enzymes from bacteria. They cut at very specific sequences. Example: BsuRI GGCC going 5’ to 3’ • Why would bacteria need enzymes to cut up DNA? Practice • Where would BsuRI cut for the following individuals and how many pieces would result? • Person 1 5’TGGCCATGGCGGCC3’ 3’ACCGGTACCGCCGG5’ • Person 2 5’TTCCGGCCTCCAGA3’ 3’AAGGCCGGAGGTCT5’ Volume • We will be measuring tiny volumesmicroliters written µL. • There are 1000 µL in I mL. There are 1000mL in 1 L. So 1 µL is 1,000,000 of a liter=very small. • We use micropipettes • Use p20- can measure between 2 and 20 µL accurately • Top number = tens place, middle= ones place, and bottom=tenths place Micropipettes • Very expensive-$300 each • Liquid must never touch the barrel, it must always be in a new tip • Always hold the micropipet vertically • Work at eye level Practice volumes • How would you dial to 6.5 µL? • How would you dial to 20 µL? • How would you dial to 2 µL? Diagram • Draw pipette diagram on board Micropipet Use 1. Dial to correct volume and lock plunger 2. Put on fresh tip 3. Push plunger down until first stop=resistance 4. Insert tip into liquid and release plunger SLOWLY 5. Place tip into container to move liquid to 6. Push down to the 2nd stop. Remove micropipet BEFORE you release plunger Gel Electrophoresis • A process used to separate our cut DNA by LENGTH! • DNA is NEGATIVE • What attracts a negative? • Negative at the top of the gel, positive at the bottom • Electric current pulls DNA through the gel Which pieces will move faster if it is like the game? Activity • The restriction enzyme you will use will cut at 5’ to 3’ GG CC, between the Gs and Cs • Draw where you would cut the sequences and draw a line where they would move to on the gel • Which person committed the crime?