1. Isolation and purification of nucleic acids
The basic step for further work with
DNA and RNA
Doc. RNDr. Jan Hošek, Ph.D.
hosek@mail.muni.cz
Department of Molecular Pharmacy
FaF MU
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Useful links
o BioTechniques
• The International journal of Life Science Methods
• http://www.biotechniques.com/
o Reseach Gate
• http://www.researchgate.net/
o BitesizeBio
• Brainfood for biolologists
• http://bitesizebio.com/
o Journal of Visualized Experiments (JoVE)
• http://www.jove.com/
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The Aim of NA isolation and purification
Is to obtain the NAs in native state from a natural material in a
sufficient amount and quality for further analysis
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NAs isolation general steps
Lysis of cells
and tissues
Extraction of NAs
Purification of NAs
Characterisation
of the isolated NA
?
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Lysis of cells and tissues
releasing the internal contents of cells
➢ Biochemical lysis
using enzymes, which degrade cell wall
(lysosyme, proteinase K, cellulase …)
➢ Chemical lysis
detergents (e.g. sodium lauryl sulphate),
chelating solutions (EDTA), guanidine salts
➢ Physical lysis
boiling, freezing, grinding, sonication
Mostly combination of
several methods
Lysis of cells
and tissues
Extraction
of NAs
Purification
of NAs
Characterisation
of the isolated NA
Lysis of cells and tissues
releasing internal contents of the cells
Homogenisation
by grinding
Proteinase K
Lysozyme
Detergents
Homogenisation
by grinding
Cellulase
Detergents
Freezing by
liquid nitrogen
Boiling
Sonication
Lysis of cells
and tissues
Extraction
of NAs
Purification
of NAs
Characterisation
of the isolated NA
7
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After the cell lysis
Complex mixture
DNA, RNA, lipids, proteins,
saccharides, carbohydrates
and other low-molecular compounds
Next steps:
separate DNA and/or RNA
from the other components
Lysis of cells
and tissues
Extraction
of NAs
Purification
of NAs
Characterisation
of isolated NA
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Extraction
Extraction is a purification and separation
process, in which one substance passes
from the mixture of compounds in liquid or
solid phase to another liquid phase, i.e.
solvent.
Extraction is suitable for isolation of
temperature sensitive substances because
it can be carried out at room temperature
or under cold conditions.
Lysis of cells
and tissues
Extraction
of NAs
Purification
of NAs
Characterisation
of the isolated NA
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Extraction of the phenol - chloroform mixtures
protein
denaturation at
the interphase
Separation of proteins and NA
based on
light water
heavier organic
phases
Lysis of cells
and tissues
Extraction
of NAs
Purification
of NAs
Characterisation
of the isolated NA
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General steps in phenol extraction
1) Disruption of cell membranes
3) Separation of individual phases by
centrifugation – organic layer
(phenol), interphase (proteins and
rest of the cells), water (NA)
2) Denaturation of proteins and
lipids – phenol, chlorophorm
DNA and RNA
proteins
phenol
Lysis of cells
and tissues
Extraction
of NAs
Purification
of NAs
Characterisation
of the isolated NA
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Principle of phenol extraction
http://bitesizebio.com/
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Phenol extraction modifications
Phenol equilibrated with neutral or alkaline
buffer
DNA and RNA
proteins
phenol
The use of acid phenol
RNA
DNA
phenol and
proteins
http://openwetware.org/
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Application of acid
phenol
P.Zumbo,WEILLCORNELLMEDICALCOLLEGE
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After extraction
Strongly diluted DNA, RNA
Traces of phenol
Traces of chloroform
Next steps:
Concentration and purification
of NAs
Lysis of cells
and tissues
Extraction
of NAs
Purification
of NAs
Characterisation
of the isolated NA
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Purification of NAs by precipitation
• A certain amount of the
precipitating agent (ammonium
sulphate, ethanol, acetone, etc.)
is added to the solution
containing the desired
macromolecule. Macromolecules
are precipitated without
denaturation
• Later they can be dissolved
again and used in their natural,
biologically active state
Lysis of cells
and tissues
Extraction
of NAs
Purification
of NAs
Characterisation
of the isolated NA
Precipitation is one of the basic
methods for isolation and concentration
of biological macromolecules
http://www.vivo.colostate.edu
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General steps in the purification of ethanol
1) Adding ethanol or isopropanol
3) Sample concentration by centrifugation
(sample is cooled down to -70ºC)
2) Adding monovalent ions (K+, Na+…)
4) Sediment (NAs) washing by 70% ethanol
5) NAs solution in water
Lysis of cells
and tissues
Extraction
of NAs
Purification
of NAs
Characterisation
of the isolated NA
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Solubility of DNA in water
DNA IS HYDROPHILIC
Lysis of cells
and tissues
Extraction
of NAs
Purification
of NAs
Characterisation
of the isolated NA
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General steps in the purification of ethanol
adding of NaCl + C2H5OH
NaCl
Na+
Cl-
C2H5OH
Low dielectric constant
Low solubility
Neutralisation of PO3Lowering
the DNA hydrophility
PRECIPITATION OF DNA
in water solution
Lysis of cells
and tissues
Extraction
of NAs
Purification
of NAs
Characterisation
of the isolated NA
Na+
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General steps in precipitation - FINISHING
3) Sample concentration by centrifugation
(sample is cooled down to -70ºC)
4) Washing the sedimented DNA to
eliminate the traces of salts by 70%
ethanol and evaporation of the ethanol
by warming
5) Dilution of NA in water (adding the EDTA
or Tris-HCl)
Native NA concentrated in a small
volume of water solution
Lysis of cells
and tissues
Extraction
of NAs
Purification
of NAs
Characterisation
of the isolated NA
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Purification of NA by chromatography
➢Preparative affinity column
chromatography
➢Column contains a sorbent (matrix) that is
able to specifically bind NAs
➢It is used to prepare greater amounts of
purified molecules
Lysis of cells
and tissues
Extraction
of NAs
Purification
of NAs
Characterisation
of the isolated NA
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Purification of NA by chromatography
Lysis of cells
and tissues
Extraction
of NAs
Purification
of NAs
Characterisation
of the isolated NA
mobile
phase
sample
column
sorbent
sample
elution
gradually collected fractions
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Commercial chromatography columns for NAs
isolation - spin columns
Lysis of cells
and tissues
Extraction
of NAs
Purification
of NAs
Characterisation
of the isolated NA silica columns
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Commercial chromatography columns for NAs
isolation - spin columns
Lysis of cells
and tissues
Extraction
of NAs
Purification
of NAs
Characterisation
of the isolated NA
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How DNA is bound on membrane
en.wikipedia.org
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Commercial chromatography columns for NAs
isolation - example
Lysis of cells
and tissues
Extraction
of NAs
Purification
of NAs
Characterisation
of the isolated NA
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DNA isolation by magnetic beads
o DNA is bound on the surface of magnetic beads
o The surface of beads is coated by:
• Ion-exchange polymer, e.g. Diethylaminoethyl (DEAE)
• Silica
http://www.diagenode.com/
https://nucleusbiotech.com/produkt-kategorie/ngs/magnetic-beads-for-dna-and-rna-purification/
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http://www.fsijournal.org/
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Magnetic beads - protocol
http://blog.labplanet.com/
Low pH
High pH
http://bitesizebio.s3.amazonaws.com/
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Plasmid isolation by alkaline denaturation
➢One of the method for separation of
plasmid molecules from the
chromosomal DNA in bacterial cells
extracts
➢It uses different sensitivity of DNA
strands for denaturation in high
alkaline pH solutions according to
conformation of the strands and their
state
➢For plasmid isolation can also be used
the commercial “spin column“
processes
Lysis of cells
and tissues
Extraction
of NAs
Purification
of NAs
Characterisation
of the isolated NA
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Plasmid isolation by alkaline denaturation
the principle of the method
pH 12.45
Mixture of plasmid
and chromosomal
DNA
Partial denaturation
of plasmid DNA
Complete
denaturation of
chromosomal DNA
Lowering pH
(C2H3KO2)
+ SDS
DNA renaturation
Precipitation
Neutralisation
Centrifugation
SedimentSupernatant
Lysis of cells
and tissues
Extraction
of NAs
Purification
of NAs
Characterisation
of the isolated NA
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Characterisation of isolated DNA
The two of important characteristics of the
isolated NAs are
Concentration
Purity
Lysis of cells
and tissues
Extraction
of NAs
Purification
of NAs
Characterisation
of the isolated NA
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Characterisation of NAs by spectrophotometry
➢ NAs absorb UV light with maximum at 260
nm
Lysis of cells
and tissues
Extraction
of NAs
Purification
of NAs
Characterisation
of the isolated NA
wavelength (nm)
A
b
s
o
r
b
a
n
c
e
maximum
minimum
Characterization of NAs by spectrophotometry
34
By which part the DNA absorbs
the UV light?
Lysis of cells
and tissues
Extraction
of NAs
Purification
of NAs
Characterisation
of the isolated NA
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Characterization of NAs by spectrophotometry
➢ Optical density corresponds to concentration
➢ Absorbation is measured at different
wavelengths (230 – 320 nm)
➢ Ratio of absorbance = purity of sample
Lysis of cells
and tissues
Extraction
of NAs
Purification
of NAs
Characterisation
of the isolated NA
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Optical density corresponds to concentration
A260 = 1.0 (in 1 cm
cuvette)
dsDNA ~ 50 μg/ml
ssDNA ~ 33 μg/ml
ssRNA ~ 40 μg/ml
Dependence of absorption on
concentration is linear in A260 = 0.1 – 0.3
Lysis of cells
and tissues
Extraction
of NAs
Purification
of NAs
Characterisation
of the isolated NA
concentration
absorbance in 260 nm
0.1 0.3
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Low concentration of DNA
Lysis of cells
and tissues
Extraction
of NAs
Purification
of NAs
Characterisation
of the isolated NA
If the absorbance
is LOW (below
0.1), you will read
the concentration
HIGHER than it
really is.
So you
overestimate the
DNA
concentration
concentration
absorbance at 260 nm
0.1 0.3
reading
real value
0.05
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High concentration of DNA
Lysis of cells
and tissues
Extraction
of NAs
Purification
of NAs
Characterisation
of the isolated NA
If the absorbance
is HIGH (over
0.3), you will
read the
concentration
LOWER than it
really is.
So you
underestimate
the DNA
concentration
0.1 0.3
reading
real value
0.4
concentration
Absorbance at 260 nm
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Purity of DNA
Lysis of cells
and tissues
Extraction
of NAs
Purification
of NAs
Characterisation
of the isolated NA
Is determined
according to the
ratio of
absorptions at
different
wavelengths
http://www.biotek.com
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Purity of DNA
A260/A280 = 1.8
< 1.8 = contamination by proteins
> 1.8 = contamination by RNA
A260/A230 > 2.0
< 2.0 = contamination by compounds
included in the commercial kits
Lysis of cells
and tissues
Extraction
of NAs
Purification
of NAs
Characterisation
of the isolated NA
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Congratulation, you have just learnt
one of the most important steps in
the molecular biology
Isolation of nucleic acids