Restriction Digests and Gel electrophoresis 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 volumes-microliters 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?

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?