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DNA Isolation Objectives of this Lecture

DNA Isolation Objectives of this Lecture

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DNA Isolation Objectives of this Lecture

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  1. DNA IsolationObjectives of this Lecture • To understand the basic process of isolation of DNA from various sources eg blood, tissue, bacteria. • To realise that different types of DNA require different methods of isolation. • To realise that the method used is dependent upon the final application. • To understand the basis of gel electrophoresis • To realise that there are different types of gel electrophoresis. MG331/MB331

  2. DNA IsolationWhich Method? • The isolation method of choice is dependent upon • The source of the DNA eg blood, buccal, bacterial, bacteriophage • The final application eg PCR, RE, library construction • The type of DNA eg genomic vs plasmid • To a lesser extent the number of samples to be processed ?robotics/automation. MG331/MB331

  3. Isolation of DNAMethods of Isolating DNA • Tissue • Homogenise, chemically or mechanically • Single cell suspension • Cell wall rupture • Gram -ve lysozyme • Gram +ve lysostaphin • Yeast/fungi zymolase • Cell membrane rupture • Detergents - SDS, sarcosine, triton • Proteinases - Proteinase K, Pronase E • Chelators - EDTA MG331/MB331

  4. Isolation of DNAMethods of Isolating DNA • Cell extraction • Organic - phenol, CHCl3 • high salt • guanidinium HCl • Removal of cell debris • proteins, lipids, polysaccharides • Concentration of DNA • ethanol, isopropranol • DNA absorbing matrix • CTAB, spermidine • Optional steps • Rnase A removal of RNA MG331/MB331

  5. Specific Methods of DNA Isolation • Genomic DNA • SDS/Proteinase K • Qiagen columns • Alkaline method • Automated methods • Plasmid DNA • Alkaline/SDS • Qiagen column methods • Bacteriophage M13 DNA • PEG precipitaton method • Bacteriophage lambda DNA • PEG/Salt precipitation method MG331/MB331

  6. Plasmid Isolation MG331/MB331

  7. Problems • DNA is very delicate • sheared by mechanical action especially if vortexed. This can be overcome in certain applications by embedding the cells in agarose plugs prior to extraction • DNases • released when cells are disrupted and these degrade DNA • Time consuming • automation • kits (eg Qiagen, Wizard) • Dangerous chemicals • Phenol, chloroform, SDS, proteinase K. MG331/MB331

  8. Methods of Separating DNA • Polyacrylamide gel electrophoresis • 20bp - 2000bp • Conventional agarose gel electrophoresis • 300bp - 40,000bp • 100bp-2000bp (special agaroses) • low melting point agaroses • Pulse field/CHEF • 40kbp - 2000kbp MG331/MB331

  9. Gel Electrophoresis(Principal) • -ve charged phosphate groups of the DNA are attracted to the (+) electrode of the electrophoresis tank when a charge (potential) is applied. • DNA has evenly spaced charge, thus it migrates according to size. • The migration is dependent upon the media used. Sometimes we want to resolve small differences - use polyacrylamide whereas other times we may want to resolve larger DNA molecules (agarose). MG331/MB331

  10. Agarose Gel Electrophoresis • Features • size separate • purification of DNA fragments • is relatively simple • is relatively rapid • fragments can be visualised using fluorescent intercalating agents such as ethidium bromide • main type is submersible MG331/MB331

  11. Agarose Gel Electrophoresis MG331/MB331

  12. Polyacrylamide Gel Electrophoresis • Features • made up of two solutions, acrylamide and bis-acrylamide (cross linker) by polymerisation • polymerisation initiated by TEMED and catalysed by ammonium persulfate • highly toxic (neurotoxin) • inhibited by presence of air, hence between glass plates • usually run vertical • separation dependent upon - total concentration (3.5%-20%)and concentration of cross-linker. 3.5%: 100-1000bp, 8%: 60-400bp, 20%: 10-100bp MG331/MB331

  13. Polyacrylamide Gel Electrophoresis MG331/MB331

  14. Polyacrylamide Gel Electrophoresis MG331/MB331

  15. Migration parameters • Molecular size of the DNA • migration inversely proportional to log10Mwt • Matrix concentration • molecular sieve effect. Increase concentration decrease larger molecules separating • Buffers • Tris acetate, Tris borate or Tris phosphate. usually Tris acetate for agarose and Tris borate for polyacrylamide • Conformation of DNA • Applied current • maximum resolution at 5v/cm MG331/MB331

  16. Agarose GelMigration characteristics MG331/MB331

  17. Properties of Agarose • Melting temperature • low melting temp agaroses can be used for isolation of DNA fragments from the gel • Gel strength • affects handling properties • Resolving performance • some agaroses are designed to resolve very small fragments (100-500bp) or very large (50kbp+) DNA fragments MG331/MB331

  18. Pulse Field Gel Electrophoresis • Uses agarose • Main type called CHEF • Contour clamped Homogeneous Electric Field • hexagonal array of electrodes • voltage pulsed from one set of electrodes to another • large DNA molecules tumble through the gel compared to a “slinky spring” • Can resolve complete yeast/bacterial chromosomes MG331/MB331

  19. Questions • Using a flow diagram, describe the method of plasmid extraction called “Alkaline lysis”, highlighting the important steps involved MG331/MB331