DNA/RNA extraction:
An overview of principles and methods
Hugo Moors
Research unit: Microbiology (MIC)
Expert group Molecular and Cellular Biology (MCB)
[email protected]
Copyright © 2011
SCK•CEN
Molecular Biology and Cytometry Course, 5-6 May 2011
  Don’t (always) trust your kits
Economic profit, as a starting point,
is not the ideal companion for
fundamental research
  Don’t believe in miracles
Even in molecular biology all rules
and laws of physics and chemistry
are still valid
Why is DNA/RNA extraction still important?
Forms the basis of a huge number of molecular biology analyses
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Restriction digestion
All kinds of PCR
Micro-array analysis
Nucleotide sequencing
Phylogenetic research
Forensic investigation
Cloning
Mutagenesis
Understanding cellular and metabolic processes
Epigenetic studies
…
DNA/RNA extraction:
Choice of Kit or Protocol?
Kit
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Fast
High DNA purity
Reliable
Reproducible
Black box
Expensive
Low overall yield
Not modular
Not adaptable
Protocol
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Transparant
Modular
Adaptable
Less expensive
Time consuming
Less reliable
Less reproducible
No quality guarantee
Need to know how it works
DNA/RNA extraction:
Why is it difficult with microorganisms?
DNA/RNA extraction: a stepwise overview
1. Cell lysis = making the DNA/RNA accessible
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Chemical lysis
Physical disruption
Enzymatic lysis
2. DNA/RNA purification/conditioning = making DNA/RNA ready for
further analysis
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Chemical purification
Physical cleaning
3. Concentrating/diluting & storing DNA/RNA
•  Chemical conditioning (= stable complexes, nuclease elimination)
•  Physical stabilization (= thermal conditioning, freezing)
Cell lysis
Physical disruption
1.  Freeze/thawing
  Ice dendrites, needles, that can puncture and disrupt the cell
membrane, thereby releasing the content of the cell in the solvent
2.  Grinding in liquid nitrogen
  Samples of cells are frozen in liquid nitrogen and subsequently
crushed (e.g. with the aid of a pestle and a mortar)
3.  Ultra-sonication
  Ultrasonic waves generate friction forces that might disrupt cell
membranes and generate shear in large biological molecules.
4.  Bead milling
  The collision forces generated by shaking beads may disrupt cell
membranes and thereby releasing the content of cells
Cell lysis
Chemical attack
1.  Ionic detergents e.g. sodium dodecyl sulphate (SDS), potassium ethyl
xanthogenate (PEX), cetyl trimethyl ammonium bromide (CTAB)
Ionic detergents are known to:
•  denature proteins
•  inhibit enzymes
•  interact strongly with lipids.
The direct action of detergents is probably cleaning the cell membrane
of its lipids, which ultimately leads to the destruction of the cytoplasmic
membrane causing the complete lysis of the cell
2.  Non-ionic solvents e.g. butanol
Aim is to dissolve certain structural molecules of the cell membrane,
thereby initiating membrane leaks, which may cause cell lysis
3.  Special buffers e.g. Sucrose-method
Drastic change of osmotic environment around cells yield to leaking cell
membranes, which may lead to cell lysis.
Cell lysis
Enzymatic disruption
1.  Lysozyme (= muramidase)
•  Isolated from chicken egg white
•  Hydrolyzes glycosidic bonds (β(1-4) linkages)
2.  Lyticase
•  Mixture of endoglucanase and protease
•  hydrolyzes poly-β(1→3)-glucose
+
3.  Other enzymes e.g. achromopeptidase, Labiase, Lysostaphin
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Isolated from different (bacterial) organisms
DNA/RNA Purification
Chemical purification
and alcohol precipitation
1.  Phenol/Chloroform/Isoamyl alcohol extractions (PCI 25:24:1; pH>7.4)
•  Phenol separates/dissociates proteins from DNA (pH depended!)
•  Trichloromethane (Chloroform) denaturates proteins and lipids
and makes DNA less soluble in the organic/phenolic phase
•  3-methyl-1-butanol (Isoamyl alcohol) acts as anti foaming agent
2.  Alcohol precipitation
•  Salts of nucleic acids and monovalent cations are almost insoluble
in alcohol-water mixtures (precipitate as pellet).
  Used alcohols:
•  ethanol
•  2-propanol
  Suitable +1 cationic salts are:
•  ammonium acetate
•  Lithium chloride
•  sodium chloride
DNA/RNA Purification
Physical cleaning (1):
Isopycnic centrifugation
Density Gradient typically obtained with:
•  CsCl cesiumchloride
•  Sucrose
DNA/RNA Purification
Physical cleaning (2):
Ultra filtration
450 µl
DNA-free
solution
• Centrifugation
• Vacuum
• Pressure
DNA/RNA concentration/dilution & storage
Why do you need DNA/RNA?
Your downstream application determines the
concentration/dilution and storage conditions.
1. Long term = Dried or Lyophylized (+ stabilizing agent)
•  Trehalose
•  Commercially available stabilizers
•  DNAstable® (Biomatrica)
•  QIAsafe™ DNA (Qiagen)
•  GenTegra™ DNA (IntegenX)
2. Short term/dilution needed = in solution
•  Nuclease free water
•  TE-buffer
•  Hind III digested lambda DNA
•  trehalose
3. Temperature: the colder, the more stable
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Room temperature; 4°C; -20°C; -80°C; Liquid nitrogen
DNA/RNA control
Quantity & Quality control?
1. Quantity: Spectrophometric
2. Quality: Electrophoretic mobility
PEX-DNA isolation
Modular and flexible protocol:
Cell lysis
1. Cell lysis = PEX-chemical lysis buffer
•  Potassium Ethyl Xanthogenate (PEX) (62 mM)
•  Disodium-EDTA (100 mM)
•  Sodium Dodecyl Sulphate (35 mM)
•  Ammonium acetate (800 mM)
2. DNA sample + PEX lysis buffer
•  Incubation at 70°C, overnight
Tris-HCl, 100 mM
pH = 8.0
PEX-DNA isolation
Modular and flexible protocol:
Chemical purification
3. Phenol/Chloroform/Isoamylalcohol (25:24:1)
•  Phase lock GelTM
4. Chloroform/Isoamylalcohol (24:1)
•  Phase lock GelTM
PEX-DNA isolation
Modular and flexible protocol:
Alcohol precipitation
5. 2-propanol-Lithiumchloride (800 mM) precipitation
•  “Speed vac” = crude DNA-Pellet
6. Redisolve DNA in nuclease free water
PEX-DNA isolation
Modular and flexible protocol:
Physical cleaning
7. Ultra-filtration/concentration of DNA
n
Add 450 µl
DNase/RNase
free water
500 µl
crude
DNA solution
50 µl retentate
Containing most
Of the DNA
500 µl
DNA solution
Impurities are
10 times diluted
50 µl retentate
All DNA
Impurities 10x
diluted
450 µl
DNA-free
solution
450 µl
DNA-free
solution
+/- 45 µl
Ultra purified
DNA-solution
DNA/RNA Purification
Physical cleaning (3):
Molecular Weight Cut Off guide
MWCO
ds DNA
[Base pairs]
ss DNA/RNA
[Bases]
1K
5 - 16
9 - 32
3K
16 - 32
32 - 65
5K
25 - 50
50 - 95
10K
50 - 145
95 - 285
30K
145 - 285
285 - 570
50K
240 - 475
475 - 950
100K
475 - 1450
950 - 2900
300K
1450 - 2900
2900 - 5700
1000K
4800 - 9500
> 9500
Available from Amicon
PEX-DNA isolation
Quantity & Quality control?
Copyright notice
Copyright © 2011 - SCKCEN
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