1. 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.
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2. 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.
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3. 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
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4. 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
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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
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6. Plasmid Isolation
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7. Problems DNA is very delicate DNases Time consuming
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.
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8. Methods of Separating DNA
Polyacrylamide gel electrophoresis
20bp bp
Conventional agarose gel electrophoresis
300bp - 40,000bp
100bp-2000bp (special agaroses)
low melting point agaroses
Pulse field/CHEF
40kbp kbp
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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).
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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
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11. Agarose Gel Electrophoresis
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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%: bp, 8%: bp, 20%: bp
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13. Polyacrylamide Gel Electrophoresis
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14. Polyacrylamide Gel Electrophoresis
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15. Migration parameters Molecular size of the DNA Matrix concentration
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
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16. Agarose Gel Migration characteristics
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17. Properties of Agarose Melting temperature Gel strength
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 ( bp) or very large (50kbp+) DNA fragments
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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
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19. Questions
Using a flow diagram, describe the method of plasmid extraction called “Alkaline lysis”, highlighting the important steps involved
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