DNA Technology Restriction Digests and Gel electrophoresis

DNA Extraction (taking it out) Collect DNA sample (blood, saliva, hair follicle, skin)Collect DNA sample (blood, saliva, hair follicle, skin) Burst open the cells using lysis buffer and free the DNA (like extraction lab)Burst open the cells using lysis buffer and free the DNA (like extraction lab)

Microsatellite regions Almost all DNA between humans is identical (99.9%), except in non-protein coding sites called microsatellite regionsAlmost 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 paternityWhere we look when comparing DNA to solve crimes or for paternity

DNA Amplification PCR=Polymerase chain reactionPCR=Polymerase chain reaction Makes identical copies of the portion of the DNAMakes identical copies of the portion of the DNA

Restriction Digest Now cut up the DNA based on its base pair sequenceNow 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’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?Why would bacteria need enzymes to cut up DNA?

Practice Where would BsuRI cut for the following individuals and how many pieces would result?Where would BsuRI cut for the following individuals and how many pieces would result? Person 1 5’TGGCCATGGCGGCC3’Person 1 5’TGGCCATGGCGGCC3’ 3’ACCGGTACCGCCGG5’ 3’ACCGGTACCGCCGG5’ Person 2 5’TTCCGGCCTCCAGA3’Person 2 5’TTCCGGCCTCCAGA3’ 3’AAGGCCGGAGGTCT5’ 3’AAGGCCGGAGGTCT5’

Volume We will be measuring tiny volumes- microliters written µL.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.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 micropipettesWe use micropipettes Use p20- can measure between 2 and 20 µL accuratelyUse p20- can measure between 2 and 20 µL accurately Top number = tens place, middle= ones place, and bottom=tenths placeTop number = tens place, middle= ones place, and bottom=tenths place

Micropipettes Very expensive-$300 eachVery expensive-$300 each Liquid must never touch the barrel, it must always be in a new tipLiquid must never touch the barrel, it must always be in a new tip Always hold the micropipetAlways hold the micropipetvertically Work at eye levelWork at eye level

Practice volumes How would you dial to 6.5 µL?How would you dial to 6.5 µL? How would you dial to 20 µL?How would you dial to 20 µL? How would you dial to 2 µL?How would you dial to 2 µL?

Diagram Draw pipette diagram on boardDraw 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!A process used to separate our cut DNA by LENGTH! Gel is a meshwork of fibers-agaroseGel is a meshwork of fibers-agarose DNA is NEGATIVEDNA is NEGATIVE What attracts a negative?What attracts a negative? Negative at the top of the gel, positive at the bottomNegative at the top of the gel, positive at the bottom Electric current pulls DNAElectric current pulls DNA through the gel Use a mutagen called ethidiumUse a mutagen called ethidium bromide to stain bromide to stain Which pieces will move faster ifWhich pieces will move faster if it is like the game? it is like the game?

Activity The restriction enzyme you will use will cut at 5’ to 3’ GG CC, between the Gs and CsThe 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 gelDraw where you would cut the sequences and draw a line where they would move to on the gel Which person committed the crime?Which person committed the crime?