Summary

Total Cell DNA

Plasmid DNA Bacteriophage DNA

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DNA Purification > Total Cell

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

DNA Purification > Total Cell

From Forensic Samples

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

DNA Purification > Total Cell

DNA Purification > 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

DNA Purification > 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

DNA Purification > Total Cell

Growth can be monitored by optical density

DNA Purification > Total Cell

Growth can be monitored by optical density

DNA Purification > Total Cell

Harvesting Cells

Harvesting accomplished by low-speed centrifugation Supernatant removed; pellet resuspended.

DNA Purification > 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

DNA Purification > 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)

DNA Purification > Total Cell

DNA Purification > Total Cell > 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

DNA Purification > Total Cell > Breaking Cell

Cell Membrane Disruption

Sodium dodecyl sulphate (SDS) is a detergent; removes lipid molecules

DNA Purification > Total Cell > Breaking Cell

DNA Purification > Total Cell

Centrifuge removes insoluble cell debris

DNA Purification > 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

DNA Purification > Total Cell > Purification

Organic Extraction

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

DNA Purification > Total Cell > Purification

Organic Extraction

DNA Purification > Total Cell > 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

DNA Purification > Total Cell > Purification

Organic Extraction

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

DNA Purification > 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; supernatant removed.

DNA Purification > Total Cell

Concentration of DNA

DNA Purification > Total Cell > 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

DNA Purification > Total Cell > Purification

DNA Purification with Silica Particles

DNA Purification > 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

DNA Purification > Total Cell

Purification of Plant DNA

Centrifuge

Ultracentrifuge

DNA Purification > Plasmid DNA

Plasmid DNA (similar to total DNA)

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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

DNA Purification > Plasmid DNA

Similar to Total DNA

Separation based on differences between plasmid and bacterial DNA.
Size = plasmids much smaller 2. Conformation = plasmids circular; bacterial DNA linear (broken during prep)
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DNA Purification > 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

DNA Purification > 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.

DNA Purification > Plasmid DNA

DNA Purification > Plasmid DNA

Separation based on conformation

Basis for separation is the supercoiling of small circular plasmids.

DNA Purification > Plasmid DNA

Separation based on conformation

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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 > Plasmid DNA

DNA Purification > 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.

DNA Purification > Plasmid DNA

DNA Purification > 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.

DNA Purification > Plasmid DNA

DNA Purification > Plasmid DNA

DNA Purification > 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

DNA Purification > 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.

DNA Purification > Bacteriophage

Purifying Phage DNA

Phages may be precipitated with polyethylene glycol (PEG)

Centrifuged. Supernatant dumped. Redissolved. Further purified by CsCl density gradient

Links
http://learn.genetics.utah.edu/content/labs/extractio

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