DNA Isolation

Doc. Ruslan Kalendar

Visit Program to Egypt:

FCRI-Institute of Field Crops Research, ARC, Egypt; 30.03 - 04.04.2014
 DNA extraction
To understand the basic process of isolation of DNA from various
sources: blood, tissue, bacteria etc.;

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.

DNA structure
 DNA isolation which method?
The isolation method of choice is dependent upon:

The source of the DNA: blood, tissue, bacterial, virus etc.;

The final application: PCR, restriction, sequencing, fingerprinting,

library construction etc.;

The type of DNA: genomic vs plasmid;

To a lesser extent the number of samples to be processed

robotics/automation.
 Isolation of DNA
Methods of Isolating DNA
Tissue
Homogenise, chemically or mechanically

Single cell suspension

Cell wall rupture

Bacteria (Gram-) - lysozyme
Yeast/fungi - zymolase

Cell membrane rupture

Detergents - SDS, sarcosine, triton X-100, CTAB
Proteinases - Proteinase K, Pronase E
Chelators - EDTA
Guanidine thiocyanate/chloride, urea
 Isolation of DNA
Methods of Isolating DNA
• Cell extraction
Organic: phenol, CHCl3 (chloroform)
high salt of guanidinium thiocyanate/chloride (GuTC, GuCl)

• Removal of cell debris

proteins, lipids, polysaccharides

• Concentration of DNA
ethanol, isopropanol with salts: Na+, Li+, NH4+
DNA absorbing matrix (silica - silicon dioxide)
CTAB, PEG, spermidine

• Optional steps
RNAse A removal of RNA
 DNA extraction methods
Several common methods:
• Organic extraction
• Advantage: Yields high quality DNA
• Disadvantages: Toxic and time-consuming

• Chelex extraction
• Advantage: Very fast, Not toxic
• Disadvantage: Product impure
• Not be suitable for DNA sequencing, restriction etc.

• Spin column extraction

• Advantage: Fast, Yields high quality DNA, to be processed
robotics/automation
• Disadvantages: Still toxic (GuTC) and technology consuming
Specific Methods of DNA Isolation
• Genomic DNA
SDS/Proteinase K
Silica columns (Guanidine thiocyanate, CTAB)
Alkaline method
Automated methods

• Plasmid DNA
Alkaline/SDS
Silica column methods

• Bacteriophage DNA
PEG/Salt precipitation method
 Organic method
• Takes advantage of highly negative charge on nucleic acids

• Lyse cells with SDS/PK /(DTT)

• SDS = detergent (solubilizes cell membrane)
• PK = proteinase K (degrades proteins
• DTT = reducing agent; breaks disulfide bonds in strong proteins like
protamines

• Add equal volume of phenol

• Protein fragments and lipids attracted to hydrophobic phenol
• Nucleic acids attracted to water
 Organic method
• Vortex and centrifuge
• Remove aqueous layer
• Add more phenol
• Repeat procedure
• Concentrate DNA
• Ethanol precipitation
• Size exclusion column

Centrifugation separates layers into

phenol (organic) and aqueous phases
 DNA storage
Solvent:

• DNA, RNA and oligonucleotide are storage in 1xTE solution (1 mM EDTA,

Tris-HCl, pH 7.0-8.0);

Temperature:
• Everyday use: +4°C;
• Storage for long time: -20°C or -80°C;

• Under 70% ethanol as a pellet, DNA/RNA can be stored at +4°C almost

indefinitely;
 DNA precipitation
Stock solution Final concentration

5М NaCl 0.2 M
10М Acetate ammonium (CH3COONH4) 2.0 - 2.5 M
10M LiCl 0.8 M
3M Acetate sodium (CH3COONa) 0.3 M
40% PEG 6000-8000 10 %
CTAB (Cetrimonium bromide) 0.5% - 1% w/v and 0.4 М NaCl
Spermine or spermidine 1-10 мМ
LiClO4 and acetone (for oligonucleotides only) 10 volumes of acetone with 2% LiClO4
 DNA precipitation
The precipitation of DNA and RNA in the presence of salt:

using 2 - 3 volumes of 96% ethanol (60% - 80% final concentration of ethanol),

or ½ - 2 volumes isopropanol (35% - 65% final concentration of isopropanol);

Precipitation of DNA by polyethylene glycol (PEG 6000-8000) is carried out in the

presence of 0.4 - 0.6 M NaCl or presence of 10 mM MgCl2, final concentrations of
PEG 6000 from 5% to 15% solution;

The mixture was stirred vigorously and incubated at -20°C for several hours or
overnight .

DNA or RNA is precipitated by centrifugation and the pellet wash with 70 % ethanol
to remove any residual salt or PEG.

The final concentration of 5M LiCl in solution precipitates only RNA without addition
of ethanol.
Measurement of nucleic acid using
spectrophotometry
• The bases in nucleic acids have max. absorption at 260 nm;
• Proteins have a max. absorption at 280 nm;
• Polyphenols/Polysaccharides have a max. absorption at 230 nm;

• A solution which has 50 g/ml of dsDNA has an absorption of 1 at 260 nm;

• A solution which has 40 g/ml of ssDNA has an absorption of 1 at 260 nm;

• OD260/OD280 = 1.8 (protein-free DNA);

• >1.8 - probably contaminated with RNA;

• OD260/OD230 > 2.0 (polysaccharide compounds - free DNA );

• <1.8 - contaminated polysaccharide, poor quality DNA;
Maximum wavelength absorbance
for DNA
 Separation of nucleic acids
• DNA molecules of different sizes can be separated by gel electrophoresis.
• Larger molecules migrate more slowly than smaller ones through the matrix
of the gel.

• Gel electrophoresis includes:

 Agarose
 Polyacrylamide
 Pulse field

• Agarose is polysaccharide which is extracted from seaweed. It is used to

separate DNA fragments of 300-10,000 bp at 0.5-2%;

• The agarose form a solid matrix which allows DNA to migrate through an
electric field based on size;
 Visualization of DNA

• The DNA can be visualized by

staining with ethidim bromide
(EtBr).
• EtBr is an intercalating agent which
will insert itself within the bases of
the DNA and will exhibit
florescence under UV light.
• ssDNA and RNA will also bind EtBr
but to a lesser extent.
 Polyacrylamide gels
• Polyacrylamide gel:
• Have smaller pores than agarose.
• Can separate DNA fragments which range in size from 10-500
bp;
• DNA fragments which differ in size by one nucleotide can be
separated from each other.
• Polyacrylamide gel electrophoresis is also used to separate
protein molecules.
 Agarose gel DNA verification
• Agarose gel electrophoresis
can be used to separate DNA
fragments of different sizes; DNA

• Different forms of a DNA

molecule of the same size
can also be separated by rRNA 18S, 23S, 28S
agarose gel electrophoresis;

• Because of its compact size

supercoiled DNA will move
faster than relaxed or nicked
circular forms;

tRNA, 5S rRNA
RNA degradation
 Agarose gel DNA verification

Genomic DNA

Degraded DNA