DNA purification

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DNA purification

• Dr. Jason Linville
• University of Alabama at Birmingham
• jglinvil_at_uab.edu

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Summary

• Total Cell DNA
• Plasmid DNA
• Bacteriophage DNA
• Read article assignment

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DNA Purification gt Total Cell

• From Bacteria

• Bacteria are grown and harvested
• Cells broken open to release contents
• Cell extract treated to remove all components
  except DNA
• DNA solution is concentrated

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DNA Purification gt Total Cell

• From Forensic Samples

• Bacteria are grown and harvested
• Cells broken open to release contents
• Cell extract treated to remove all components
  except DNA
• DNA solution is concentrated

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DNA Purification gt Total Cell
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DNA Purification gt Total Cell

• Growing and Harvesting Cells

Can grow bacteria in a liquid medium

• Defined Medium
• Components (quantities) are known
• Inorganic elements, vitamins, etc. added
• Example M9

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DNA Purification gt Total Cell

• Growing and Harvesting Cells

Can grow bacteria in a liquid medium

• Undefined Medium (or complex)
• Exact components are not known
• Contains tryptone and yeast extract, which are
  mixtures of unknown chemicals
• Example LB

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DNA Purification gt Total Cell
Growth can be monitored by optical density
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DNA Purification gt Total Cell
Growth can be monitored by optical density
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DNA Purification gt Total Cell

• Harvesting Cells

• Harvesting accomplished by low-speed
  centrifugation

• Supernatant removed pellet resuspended.

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DNA Purification gt Total Cell

• Preparing Cell Extract

Cell must be broken open to release DNA

• Physical Methods
• Mechanical force breaks cell membrane/wall
• Mortar and pestle sonication
• Not usually used for DNA prep

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DNA Purification gt Total Cell

• Preparing Cell Extract

Cell must be broken open to release DNA

• Chemical Methods
• Attack cell wall and cell membrane
• Cell wall lysozyme, EDTA or both
• Cell membrane detergent (SDS)

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DNA Purification gt Total Cell
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DNA Purification gt Total Cell gt Breaking Cell

• Cell Wall Disruption
• Lysozyme digests polysaccharides structural
  components of cell wall
• EDTA (ethylenediamine tetraacetate) binds
  magnesium ions essential for preserving structure
  of cell and inhibits enzymes that could degrade
  DNA

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DNA Purification gt Total Cell gt Breaking Cell
Cell Membrane Disruption Sodium dodecyl sulphate
(SDS) is a detergent removes lipid molecules
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DNA Purification gt Total Cell gt Breaking Cell
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DNA Purification gt Total Cell
Centrifuge removes insoluble cell debris
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DNA Purification gt Total Cell

• Purification of DNA

• In addition to DNA, a large amount of proteins
  and RNA remain.
• These must be removed to avoid interference with
  further analysis

Several way to remove RNA and proteins
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DNA Purification gt Total Cell gt Purification

• Organic Extraction

• Add phenol or phenol/chloroform
• Proteins are precipitated form white layer at
  interface between organic and aqueous layers.
• Aqueous solution removed

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DNA Purification gt Total Cell gt Purification

• Organic Extraction

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DNA Purification gt Total Cell gt Purification

• Organic Extraction

What if there are excessive proteins?

• Repeated phenol extractions
• Not favorable DNA destruction with mixing

• Breakdown with protease before extraction
• Pronase or proteinase K

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DNA Purification gt Total Cell gt Purification

• Organic Extraction

• Some mRNA removed with phenol treatment.
• Remaining RNA can be digested with ribonuclease
  (if necessary)

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DNA Purification gt Total Cell

• Concentration of DNA

• If a small amount of DNA is targeted, the
  resulting solution will be dilute.

• One method is precipitation by ethanol.
  Precipitate centrifuged supernatent removed.

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DNA Purification gt Total Cell

• Concentration of DNA

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DNA Purification gt Total Cell gt Purification

• Silica Extraction

• Guanidinium thiocyanate added
• Denatures non-DNA material
• Makes DNA bind to silica particles
• Silica added directly or sample passed through
  silica column.
• DNA removed by adding water

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DNA Purification gt Total Cell gt Purification

• DNA Purification with Silica Particles

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DNA Purification gt Total Cell

• DNA Extraction from non-bacteria

• Plant cells high carbohydrate content
• Lysozyme has no effect
• Cetyltrimethylammonium (CTAB) added binds
  nucleic acid and precipitates
• Centrifuged supernatant removed

Animal cells no cell wall lyse with SDS
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DNA Purification gt Total Cell

• Purification of Plant DNA

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DNA Purification gt Plasmid DNA

• Plasmid DNA (similar to total DNA)

• Bacteria are grown and harvested
• Cells broken open to release contents
• Cell extract treated to remove all components
  except plasmid DNA
• DNA solution is concentrated

Plasmid DNA separated from chromosomal DNA
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DNA Purification gt Plasmid DNA

• Similar to Total DNA

Separation based on differences between plasmid
and bacterial DNA.

• Size plasmids much smaller
• Conformation plasmids circular bacterial DNA
  linear (broken during prep)

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DNA Purification gt Plasmid DNA

• Separation based on size

• In order to maximize difference, minimal breakage
  of bacterial DNA should occur.

• When breakage is minimal, DNA will pellet with
  cell debris.

• Minimal breakage achieved using gentle cell
  disruption techniques

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DNA Purification gt Plasmid DNA

• Gently Breaking the Cell

• Lysozyme and EDTA treatment in presence of
  sucrose prevents immediate burst.

• Sphaeroplasts formed cell can be lysed with
  addition of non-ionic detergent.

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DNA Purification gt Plasmid DNA
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DNA Purification gt Plasmid DNA

• Separation based on conformation

Basis for separation is the supercoiling of small
circular plasmids.
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DNA Purification gt Plasmid DNA

• Separation based on conformation

• Narrow pH range where supercoiled DNA is
  denatured and regular DNA is not.

• Clear lysate is usually used.

• Base added regular DNA is denatured
• Acid added regular DNA tangled mass
• Tangled mass pelleted

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DNA Purification gt Plasmid DNA
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DNA Purification gt Plasmid DNA

• Separation based on conformation

• Supercoiled DNA also separated from regular DNA
  using ethidium bromide coupled with a caesium
  chloride (CsCl) density gradient.

• CsCl density gradient formed by centrifugation
  vs. diffusion battle.

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DNA Purification gt Plasmid DNA
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DNA Purification gt Plasmid DNA

• Separation based on conformation

• Ethidium bromide will bind normal DNA, but only a
  limited amount of supercoiled DNA.

• This binding causes a shift in band of normal
  DNA.

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DNA Purification gt Plasmid DNA
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DNA Purification gt Plasmid DNA
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DNA Purification gt Bacteriophage
Bacteriophage DNA

• Phage DNA can be outside the cell do not have to
  start from cell extract.

• Culture centrifuged bacteria in pellet phage
  is suspension

• Difficulty lies in getting a high number of
  phages per mL in culture

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DNA Purification gt Bacteriophage

• Obtaining a high titre

• Must ensure phage is in lytic phase

• cI gene keeps phage in lysogenic phase

• cIts (temperature sensitive) mutants will enter
  lytic phase at 42 C

Phages must infect bacteria at just the right
time in the growth cycle.
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DNA Purification gt Bacteriophage

• Purifying Phage DNA

• Phages may be precipitated with polyethylene
  glycol (PEG)

• Centrifuged. Supernatant dumped. Redissolved.

• Further purified by CsCl density gradient