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Relevance of DNA Isolation

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

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  1. 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

  2. Cell Bio 101: What are the Structures of the Cell?

  3. Cell Structures

  4. Protocol Highlights: Genomic DNA Extraction • • Use a simple water mouthwash to collect cheek cells • • Add Lysis buffer to cells to break open cell and nuclear membranes and release nuclear contents • • Digest sample with protease to degrade proteins • • Precipitate DNA with cold alcohol in high salt

  5. Genes in a Bottle Kit • Why Perform Each Step?

  6. Cell CollectionGently 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.

  7. CH3 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 O O O S - O SDS 2. Lysis BufferWhat is Lysis Buffer?• 50 mM Tris-HCI, pH 8.0• 1% SDSTris buffer to maintain the pH of the solution at a level where DNA is stable1% 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). Na+

  8. 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.

  9. 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.

  10. O Na+ O P O Base Na+ O O CH2 Sugar O Na+ O O P Base Na+ O O CH2 Sugar OH DNA Structure

  11. 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.

  12. DNA Fingerprinting Real WorldApplications • • Crime scene • • Human relatedness • • Paternity • • Animal relatedness • Anthropology studies • Disease-causing organisms • Food identification • Human remains • Monitoring transplants

  13. Model of DNADNA is Comprised of Four Base Pairs

  14. Deoxyribonucleic Acid (DNA)

  15. DNASchematic O Phosphate O P O Base O O CH2 Sugar O O O P Phosphate Base O O CH2 Sugar OH

  16. DNA Restriction Enzymes • Evolved by bacteria to protect against viral DNA infection • Endonucleases = cleave within DNA strands • Over 3,000 known enzymes

  17. Enzyme Site Recognition Restriction site Palindrome • Each enzyme digests (cuts) DNA at a specific sequence = restriction site • Enzymes recognize 4- or 6- base pair, palindromic sequences (eg GAATTC) Fragment 2 Fragment 1

  18. 5 vs 3 Prime Overhang Enzyme cuts • Generates 5 prime overhang

  19. Common Restriction Enzymes EcoRI – Eschericha coli – 5 prime overhang Pstl – Providencia stuartii – 3 prime overhang

  20. 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

  21. 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

  22. PstI EcoRI Restriction Fragment Length PolymorphismRFLP GAATTC GTTAAC CTGCAG GAGCTC Allele 1 1 2 3 CGGCAG GCGCTC GAATTC GTTAAC Allele 2 3 Fragment 1+2 Different Base Pairs No restriction site M A-1 A-2 Electrophoresis of restriction fragments M: Marker A-1: Allele 1 Fragments A-2: Allele 2 Fragments +

  23. AgaroseElectrophoresisLoading • • Electrical current carries negatively-charged DNA through gel towards positive (red) electrode Buffer Dyes Agarose gel Power Supply

  24. AgaroseElectrophoresisRunning • • Agarose gel sieves DNA fragments according to size • – Small fragments move farther than large fragments Gel running Power Supply

  25. Analysis of Stained Gel • Determine • restriction fragment • sizes • • Create standard curve using DNA marker • • Measure distance traveled by restriction fragments • • Determine size of DNA fragments • Identify the related • samples

  26. Molecular Weight Determination Fingerprinting Standard Curve: Semi-log • Size (bp) Distance (mm) • 23,000 11.0 • 9,400 13.0 • 6,500 15.0 • 4,400 18.0 • 2,300 23.0 • 2,000 24.0

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