dna purification

7/28/2019 Dna Purification

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Summary Total Cell DNA

Plasmid DNA

Bacteriophage DNA Read article + assignment


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From Bacteria

1. Bacteria are grown and harvested

2. Cells broken open to release contents3. Cell extract treated to remove all

components except DNA

4. DNA solution is concentrated

DNA Purification > Total Cell


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From Forensic Samples



components except DNA




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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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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, whichare mixtures of unknown chemicals

Example: LB


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Growth can be monitored by optical density



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Growth can be monitored by optical density



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Harvesting Cells

Harvesting

accomplished bylow-speed

centrifugation

Supernatantremoved; pellet

resuspended.



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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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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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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


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Cell Membrane Disruption

Sodium dodecyl sulphate(SDS) is a detergent;

removes lipid molecules



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Centrifuge removes insoluble cell debris



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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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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


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Organic Extraction



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Organic Extraction

Repeated phenol extractions

Not favorable; DNA destruction with mixing

What if there are excessive proteins?

Breakdown with protease before extraction Pronase or proteinase K



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Organic Extraction

Some mRNA removed with phenol

treatment. Remaining RNA can be digested with

ribonuclease (if necessary)



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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.



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Concentration of DNA



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Silica Extraction

Guanidinium thiocyanate added

Denatures non-DNA materialMakes 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 withSilica Particles



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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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Purification of Plant DNA



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Centrifuge


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Ultracentrifuge


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Plasmid DNA(similar to total DNA)



components except plasmid DNA


Plasmid DNA separated from chromosomal DNA

DNA Purification > Plasmid DNA


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Similar to Total DNA

Separation based on differences between

plasmid and bacterial DNA.

1. Size = plasmids much smaller

2. Conformation = plasmids circular; bacterial

DNA linear (broken during prep)



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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 gentlecell disruptiontechniques



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Gently Breaking the Cell

Lysozyme and EDTA treatment in

presence ofsucrose preventsimmediate burst.

Sphaeroplasts formed; cell can be lysed

with addition of non-ionic detergent.



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Separation based on conformationBasis for separation is the supercoiling of

small circular plasmids.



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Separation based on conformation

Narrow pH range where supercoiled DNA

is denatured and regular DNA is not.

Clear lysate is usually used.

1. Base added; regular DNA is denatured

2. Acid added; regular DNA tangled mass

3. Tangled mass pelleted



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Supercoiled DNA also separated from

regular DNA using ethidium bromidecoupled with a caesium chloride (CsCl)

density gradient.

CsCl density gradient formed bycentrifugation vs. diffusion battle.



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Ethidium bromide will bind normal DNA,

but only a limited amount of supercoiledDNA.

This binding causes a shift in band of

normal DNA.



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Phage DNA can be outside the cell; do not have to startfrom cell extract.

Culture centrifuged = bacteria in pellet;

phage is suspension

Difficulty lies in getting a high number ofphages per mL in culture

DNA Purification > Bacteriophage

Bacteriophage DNA


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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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Purifying Phage DNA

Phages may be precipitated with

polyethylene glycol (PEG)


Centrifuged. Supernatant dumped.

Redissolved.

Further purified by CsCl density gradient


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

n/
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