Docstoc

DNA Isolation Objectives of this Lecture

Document Sample
DNA Isolation Objectives of this Lecture Powered By Docstoc
					          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.
• To understand the basis of gel
  electrophoresis
• To realise that there are different
  types of gel electrophoresis.


               MG331/MB331
         DNA Isolation
         Which 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
        Isolation of DNA
   Methods 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
         Isolation of DNA
    Methods 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
  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
Plasmid Isolation




     MG331/MB331
               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
 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
      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
 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
Agarose Gel Electrophoresis




          MG331/MB331
      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
Polyacrylamide Gel
  Electrophoresis




     MG331/MB331
Polyacrylamide Gel
  Electrophoresis




     MG331/MB331
       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
     Agarose Gel
Migration characteristics




        MG331/MB331
     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
 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
          Questions

• Using a flow diagram, describe
  the method of plasmid
  extraction called “Alkaline
  lysis”, highlighting the
  important steps involved




             MG331/MB331

				
DOCUMENT INFO
Shared By:
Categories:
Tags:
Stats:
views:19
posted:7/20/2011
language:English
pages:19