DNA TechnologyDNA Technology

Restriction Digests Restriction Digests and Gel and Gel electrophoresiselectrophoresis

DNA ExtractionDNA Extraction

• Collect DNA sample (blood, saliva, Collect DNA sample (blood, saliva, hair follicle, skin)hair follicle, skin)

• open the cells using lysis buffer open the cells using lysis buffer and a heat bath -2 components and a heat bath -2 components detergent to poke holes in detergent to poke holes in membrane and proteinase K to cut membrane and proteinase K to cut apart the histones and free the apart the histones and free the DNA DNA

Microsatellite regionsMicrosatellite regions

• Almost all DNA between humans is Almost all DNA between humans is identical (99.9%), except in identical (99.9%), except in non-non-protein coding protein coding sites called sites called microsatellite regionsmicrosatellite regions

• Where we look when comparing Where we look when comparing DNA to solve crimes or for DNA to solve crimes or for paternitypaternity

DNA AmplificationDNA Amplification

• PCR=Polymerase chain reactionPCR=Polymerase chain reaction• Input: nucleotides, taq polymerase (like our DNA Input: nucleotides, taq polymerase (like our DNA

polymerase but won’t denature with heat), and primers polymerase but won’t denature with heat), and primers for region we want to copy.for region we want to copy.

• Cycles of heat to unzip DNA, temperature for primers to Cycles of heat to unzip DNA, temperature for primers to bond, and temperature for taq polymerase to add bond, and temperature for taq polymerase to add nucleotides-See animationnucleotides-See animation

Restriction DigestRestriction Digest

• Now cut up the DNA based on its base Now cut up the DNA based on its base pair sequencepair sequence

• We use restriction enzymes from We use restriction enzymes from bacteria. They cut at very specific bacteria. They cut at very specific sequences. Example: BsuRI GGCC going sequences. Example: BsuRI GGCC going 5’ to 3’5’ to 3’

• Why would bacteria need enzymes to Why would bacteria need enzymes to cut up DNA?cut up DNA?

PracticePractice

• Where would BsuRI cut for the following Where would BsuRI cut for the following individuals and how many pieces would individuals and how many pieces would result?result?

• Person 1 Person 1 5’TGGCCATGGCGGCC3’5’TGGCCATGGCGGCC3’ 3’ACCGGTACCGCCGG5’3’ACCGGTACCGCCGG5’

• Person 2 Person 2 5’TTCCGGCCTCCAGA3’5’TTCCGGCCTCCAGA3’ 3’AAGGCCGGAGGTCT5’3’AAGGCCGGAGGTCT5’

VolumeVolume

• We will be measuring tiny volumes-microliters We will be measuring tiny volumes-microliters written µL.written µL.

• There are 1000 µL in I mL. There are 1000mL There are 1000 µL in I mL. There are 1000mL in 1 L. So 1 µL is 1,000,000 of a liter=very in 1 L. So 1 µL is 1,000,000 of a liter=very small.small.

• We use micropipettesWe use micropipettes• Use p20- can measure between 2 and 20 µL Use p20- can measure between 2 and 20 µL

accuratelyaccurately• Top number = tens place, middle= ones place, Top number = tens place, middle= ones place,

and bottom=tenths placeand bottom=tenths place

MicropipettesMicropipettes

• Very expensive-$300 eachVery expensive-$300 each• Liquid must never touch the barrel, it Liquid must never touch the barrel, it

must always be in a new tipmust always be in a new tip• Always hold the micropipet Always hold the micropipet

verticallyvertically• Work at eye levelWork at eye level

Practice volumesPractice 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?

DiagramDiagram

• Draw pipette diagram on boardDraw pipette diagram on board

Micropipet UseMicropipet Use

1. Dial to correct volume and lock plunger1. Dial to correct volume and lock plunger2. Put on fresh tip2. Put on fresh tip3. Push plunger down until first 3. Push plunger down until first

stop=resistancestop=resistance4. Insert tip into liquid and release plunger 4. Insert tip into liquid and release plunger

SLOWLYSLOWLY5. Place tip into container to move liquid to5. Place tip into container to move liquid to6. Push down to the 2nd stop. Remove 6. Push down to the 2nd stop. Remove

micropipet BEFORE you release plungermicropipet BEFORE you release plunger

Gel ElectrophoresisGel Electrophoresis• A process used to separate our cut DNA by LENGTH!A process used to separate our cut DNA by LENGTH!• 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 gelthrough the gel

Which pieces will move faster ifWhich pieces will move faster if it is like the game? it is like the game?

ActivityActivity

• The restriction enzyme you will use The restriction enzyme you will use will cut at 5’ to 3’ GG CC, between will cut at 5’ to 3’ GG CC, between the Gs and Csthe Gs and Cs

• Draw where you would cut the Draw where you would cut the sequences and draw a line where sequences and draw a line where they would move to on the gelthey would move to on the gel

• Which person committed the Which person committed the crime?crime?