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DNA Isolation Objectives of this Lecture

DNA Isolation Objectives 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.

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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

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