Relevance of DNA Isolation Isolation of DNA is often the first step before further analysis • DNA profiling • Cloning • Disease diagnosis • DNA sequencing • Genetically modified organisms (GMO) -agriculture, pharmaceutical • Environmental testing, biodefense
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Relevance of DNA Isolation Isolation of DNA is often the first step before further analysis DNA profiling Cloning Disease diagnosis DNA sequencing Genetically.
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Relevance of DNA Isolation Isolation of DNA is often the first
Gently chewing the inside of the mouth combined with a water mouth wash is used to dislodge epithelial cells lining the mouth
Ample cell collection is critical for success.
2. Lysis Buffer
What is Lysis Buffer?
• 50 mM Tris-HCI, pH 8.0• 1% SDS
Tris buffer to maintain the pH of the solution at a level where DNA is stable
1% SDS to break open the cell and nuclear membranes, allowing the DNA to be released into the solution (SDS also denatures and unfolds proteins, making them more susceptible to protease cleavage).
O
S
O
O
O
-
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH3
SDSNa+
3. Why Add Protease?
•Protease is added to destroy nuclear proteins that bind DNA and cytoplasmic enzymes that breakdown and destroy DNA.
•Protease treatment increases the amount of intact DNA that is extracted.
4. Adding Salt
• The protease solution already contains salt
• Na+ ions of NaCI bind to the phosphate groups of DNA molecules, neutralizing the electric charge of the DNA molecules.
• The addition of NaCI allows the DNA molecules to come together instead of repelling each other, thus making it easier for DNA to precipitate out of solution when alcohol is added.
Na+
Na+
Na+
Na+
OCH2
O
P O
O
O Base
CH2
O
P
O
O
O
Base
OH
Sugar
Sugar
O
DNA Structure
5. Adding Ice Cold Alcohol?
•DNA does not dissolve in alcohol.
•The addition of cold alcohol makes the DNA clump together and precipitate out of solution.
•Precipitated DNA molecules appear as long pieces of fluffy, stringy, web-like strands.
•Microscopic oxygen bubbles “aggregate” , or “fuse” together, as the DNA precipitates.
•The larger, visible air bubbles “lift” the DNA out of solution, from the aqueous into the organic phase.
DNA Fingerprinting Real WorldApplications • Crime scene
• Human relatedness
• Paternity
• Animal relatedness
Anthropology studies
Disease-causing organisms
Food identification
Human remains
Monitoring transplants
DNA Restriction Enzymes
• Evolved by bacteria to protect against viral DNA infection
• Endonucleases = cleave within DNA strands
• Over 3,000 known enzymes
Enzyme Site Recognition
• Each enzyme digests (cuts) DNA at a specific sequence = restriction site
• Enzymes recognize 4- or 6- base pair, palindromic sequences (eg GAATTC)
Palindrome
Restriction site
Fragment 1 Fragment 2
5 vs 3 Prime Overhang
• Generates 5 prime overhang
Enzyme cuts
Common Restriction Enzymes
EcoRI– Eschericha coli– 5 prime overhang
Pstl– Providencia stuartii– 3 prime overhang
The DNA DigestionReaction
Restriction Buffer provides optimal conditions
• NaCI provides the correct ionic strength
• Tris-HCI provides the proper pH
• Mg2+ is an enzyme co-factor
DNA DigestionTemperature
Why incubate at 37°C?
• Body temperature is optimal for these and most other enzymes
What happens if the temperature is too hot or cool?
• Too hot = enzyme may be denatured (killed)
• Too cool = enzyme activity lowered, requiring longer digestion time
Restriction Fragment Length PolymorphismRFLP
Allele 1
Allele 2
GAATTCGTTAAC
GAATTCGTTAAC
CTGCAGGAGCTC
CGGCAGGCGCTC
PstI EcoRI
1 2 3
3Fragment 1+2Different Base PairsNo restriction site