Download DNA Technology

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?