Phenol as a cleaning agent for DNA

Based on the discovery that phenol is suitable to extract proteins from aqueous
solutions (Grassmann & Deffner, 1953), Kirby reported phenol as an agent for nuc
leic acid purification (Kirby, 1956). Using a phenol-water mixture he extracted
a tissue homogenate and showed that RNA was separated in the aqueous phase, whil
e proteins associated with DNA were transferred to the interphase. Shortly after
, Kirby found, that in the presence of certain anionic salts (e.g. p-aminosalicy
late and sodium benzoate) both nucleic acid species, DNA and RNA, are enriched i
n the aqueous phase (Kirby, 1957). Today, Kirbys method is still up-to-date, apar
t from a few adaptions (anionic detergents like SDS replaced the anionic salts,
addition of chloroform and isoamyl alcohol, and equilibration of the pH).

Polar, hydrophilic compounds like DNA, RNA and proteins commonly dissolve best i
n polar solvents (with water as the solvent offering maximum polarity). But in c
ontrast to nucleic acids, proteins provide a number of non-polar structures as w
ell. The non-polar side chains of phenylalanine, leucine, isoleucine, valine, pr
oline, methionine and alanine enable the protein to stay in solution when expose
d to a less polar or even non-polar solvent. The proteins rearrange exposing the
non-polar side chains to the surface, while the charged and polar residues beco
me buried inside the protein complex. These features enable the extraction of pr
oteins out of an aqueous phase by a less or even non-polar solvent. Phenol is cl
early less polar than water despite its electronegative oxygen atom; because the
phenyl ring renders the electron density spread all over the molecule but not c
oncentrated on the oxygen atom.
For DNA isolation, the phenol has to be pH-equilibrated with tris to a final pH
of >7.8 to ensure that the DNA is negatively charged and therefore insoluble in
the organic phase. Starting with the cell lysate, an equal volume of tris-buffer
ed phenol-chloroform (1:1), or tris-buffered phenol-chloroform-isoamyl alcohol (
25:24:1) is added and the solution is mixed by vortexing (small DNA molecules of
<10 kb), gently shaking (10-30 kb) or slowly inverting or rotating (>30 kb). Ch
loroform efficiently denatures proteins, avoids the retention of water in the or
ganic phase and improves the phase separation by increasing the density of the o
rganic phase. The addition of a small volume of isoamyl alcohol reduces foaming
during the extraction process, and, aiming at RNA isolation, guarantees the deac
tivation of RNases (Green & Sambrook, 2012).
Centrifugation accelerates the separation of the two phases, resulting in the aq
ueous phase with the lower specific gravity on top. But caution: A high salt con
tent or high amounts of sucrose in the aqueous solution can result in an inversi
on of the two phases! So it is highly recommended to make sure the right solutio
n is further processed. Since equilibrated phenol commonly contains 8-hydroxyqui
noline as a stabilizer, the organic phase can be identified by its yellow color.
After organic extraction, DNA (and RNA, if no RNase A has been added during lys
is) is still in the aqueous phase while denatured proteins have moved into the i
nterphase and lipids have been transferred to the organic phase.
After repeated extractions with the phenol-containing solvent, the DNA-containin
g aqueous phase is extracted a few times with chloroform (or chloroform/isoamyl
alcohol), to remove residual phenol. Finally, the pure DNA is precipitated from
solution using ethanol or isopropanol.
AppliChem s products for Phenol-Chloroform Extraction of DNA
Prod. No.
Description
Comment
A1153 Phenol equilibrated, stabilized
Pure phenol is a colorless and crystalline substance. Liquefied phenol is suscep

tible to oxidation (especially when the pH is equilibrated with Tris) and the ph
enolic oxidation products introduce strand breaks into nucleic acid molecules an
d promote cross-linking. Phenol solutions should be clear and colorless, pink or
brownish solutions should be discarded. To prevent oxidation, 8-hydroxyquinolin
e is frequently added to liquefied phenol. The shelf life of equilibrated and 8hydroxyquinoline-stabilized phenol is approximately 9 months. As a positive side
effect, 8-hydroxyquinoline partially inhibits ribonucleases.
A0971 Phenol equilibrated, non stabilized
A1624 Phenol water-saturated, stabilized
A1578 Phenol water-saturated, non-stabilized
A1594 Phenol crystalline
A0889 Phenol equilibrated, stabilized : Chloroform : Isoamyl alcohol 25:24:1

A3691 Chloroform
A2610 Isoamyl alcohol
A2107 1-Bromo-3-chloropropane
Bromochloropropane is less toxic than ch
loroform and forms a tighter interphase (Chomczynski & Mackey, 1995a).
back

The all in one solution: Acidic guanidinium thiocyanate-phenol extraction

Admittedly, this technique does not really aim at DNA isolation in the first pla
ce, but nevertheless it is possible to use it on this purpose. And since this te
chnique offers interesting possibilities it should be mentioned in this article.
Invented by Chomczynski & Sacchi in 1987 for isolation of RNA, acidic guanidiniu
m thiocyanate-phenol extraction allows cell lysis and successive separation of R
NA, DNA and proteins using one reagent. The technique combines the effect of cha
otropic salts on structure and solubility of macromolecules with the extraction
properties of acidic phenol. Commercialized under names like Trizol, TRI REAGENT a
nd TRItidy G, Chomczynskis reagent conquered the laboratories and still belongs to
the number one choices for RNA isolation.

The birth of monophasic reagents

The history of acidic guanidinium thiocyanate - phenol extraction (or the singlestep method) invented by Chomczynski & Sacchi is in first place the history of RN
A isolation. In 1951, the usage of guanidinium chloride in RNA isolation was fir
stly described (Volkin & Carter, 1951). After Kirbys introduction of phenol as a
deproteinization reagent, guanidinium salts temporarily seem to lose their impor
tance, but guanidinium chloride in the isolation of nucleic acids was focused agai
n in the 1960s (Cox 1968). In 1979, Chirgwin et al. published a method to isolat
e undegraded RNA from ribonuclease-rich tissues employing guanidinium thiocyanat
e instead of guanidinium chloride. In 1987, Chomczynski & Sacchi initially used
a combination of guanidinium thiocyanate and phenol-chloroform extraction for RN
A isolation. Based on the same principle, the method was expanded for simultaneo
us isolation of RNA, DNA and proteins (Chomczynski, 1993). Two years later, furt
her modifications were published employing bromochloropropane (Chomczynski & Mac

key, 1995a) which is less toxic than chloroform and forms a tighter interphase,
and isopropanol (Chomczynski & Mackey, 1995b) for improved isolation of RNA from
polysaccharide- and proteoglycan-rich sources. The development finally also foc
used on the isolation of genomic DNA (Chomczynski et al., 1997).
The monophasic solution contains water, phenol, guanidinium thiocyanate (also of
ten referred to as guanidine thiocyanate), -mercaptoethanol and a detergent. The
chaotropic salt guanidinium thiocyanate lyses the cells, denatures the released
macromolecules and inactivates RNases and other enzymes. As a detergent, lauroyl
sarcosine (sarkosyl) is a good choice, since, in contrast to SDS, lauroylsarcosine
shows high solu
ility in chaotropic high salt
uffers. Addition of sarkosyl to
cell or tissue homogenates improves the purity of the RNA isolated
y guanidiniu
m salts and reduces foaming during homogenisation (MacDonald et al., 1987).
After lysis of the cell in the reagent (e.g.
y repetitive pipetting), chlorofor
m (or alternatively
romochloropropane) is added, leading to the generation of a
second phase. While DNA and proteins enrich in the newly formed organic phase a
nd the interphase, RNA is selectively retained in the aqueous phase. RNA, DNA an
d proteins are isolated
y alcohol precipitation.
Why is hydrophilic DNA transferred into the organic phase? And how is it possi
l
e that RNA, in contrast, is not extracted
y the phenol-chloroform mixture? DNA
and RNA seem to
e very similar at first sight and, under neutral conditions (pH
7-8)
oth molecules remain in the aqueous phase as expected. However, usage of
non-equili
rated, and therefore acidic phenol solution, ena
le the enrichment of
DNA molecules in the non-polar organic phase. So why do these two types of nucl
eic acids
ehave differently under acidic conditions? The answer mainly lies in
the structural differences
etween DNA and RNA: at the prevailing pH of 4-5, the
phosphate groups of the dou
led stranded (and less acidic) DNA are protonated a
nd the affinity of the now non-charged (
ut still dou
le stranded) DNA molecules
to the organic solvent strongly increases. The phosphate
ack
one of the single
stranded RNA is largely protonated as well,
ut due to the exposure of the puri
ne and pyrimidine
ases, the RNA is a
le to form hydrogen
onds with the surroun
ding water molecules (Zum
o, 2011). As a result, RNA does not lose its hydrophil
ic properties and still prefers the aqueous phase.
AppliChem's monophasic reagents: acidic guanidinium thiocyanate-phenol extractio
n
Prod. No.
Description
Comment
A2867 RNAtidy G
Ready-to-use solution for the isolation of small and lar
ge RNA species (0.1 - 15 k
) from
iological material; provides high purity RNA
(DNA and protein-free); suited for small and large samples.
A4051 TRItidy G
Ready-to-use solution for sequential isolation of RNA, D
NA and proteins.
A3418 DNA - Isolation reagent for genomic DNA
Phenol-free ready-to-use
reagent for the isolation of genomic DNA from human, animal (incl. mouse tail),
plant, yeast,
acterial and viral origin.

Fast desalting: Ethanol precipitation

Today, ethanol precipitation is often used as the final step in DNA isolation
y
organic extraction or anion-exchange chromatography. The possi
ility to precipi
tate DNA out of solution ena
les not only removal of alcohol solu
le salts, resi
dual organic solvents and detergents; it also offers an opportunity to concentra

te DNA.
In neutral aqueous solutions, the negatively charged phosphate
ack
one of DNA m
olecules is saturated with the highly polar water molecules that prevent interac
tion with cationic molecules. Increasing concentrations of ethanol lead to a dis
ruption of the hydrate shell and allow the formation of ionic
onds
etween the
phosphate groups and positively charged ions. As a consequence, in solutions con
taining at least 65% ethanol and a sufficient amount of cations, the previously
negatively charged DNA molecules are neutralized. The loss of charge minimizes t
he repulsive forces
etween the molecules and finally makes DNA to precipitate.
The accurate salt concentration is crucial to ensure that on the one hand, all D
NA is recovered from solution, and on the other hand, the anionic salts do not c
o-precipitate. To improve the efficiency, the target nucleic acids often are coprecipitated with nuclease-free inert carriers such as glycogen or linear polyacry
lamide.
The most common salts used in ethanol precipitations are ammonium acetate, sodiu
m acetate and sodium chloride.
A sodium acetate solution at acidic pH of 5.2 is the standard reagent for nuclei
c acid precipitation. Sodium chloride is the first choice if SDS is included in
the sample ensuring its solu
ility in the presence of alcohol and ena
ling the p
recipitation of detergent-free DNA. If the sample is contaminated
y dNTPs or ol
igosaccharides (which includes DNA samples o
tained from agarose gels
y agarase
digestion), ammonium acetate is most suita
le since the ammonium cations preven
t co-precipitation of these two species (source: Green & Sam
rook, 2012).
Isopropanol can
e used alternatively for DNA precipitation; it is mostly used f
or large volumes since only half of the quantity is required to dehydrate the DN
A molecules. Unfortunately, the solu
ility of salts in solutions with 35% isopro
panol is reduced compared to 65% ethanol, increasing the risk of salt co-precipi
tations. Furthermore, it is less volatile than ethanol and therefore harder to r
emove, increasing the risk of alcohol carry-over into the final sample.
AppliChem's products for alcohol precipitation of nucleic acids
Prod. No.
Description
A3678 Ethanol a
solute
A3928 2-Propanol
A4555 Sodium acetate anhydrous
A2936 Ammonium acetate
A6587
DNA-PrecipitAid
.
Synthetic produced polyacrylamide carrier for precipitating picogram amounts of
nucleic acids. DNA-PrecipitAid ena
les to completely recover DNA fragments large
r than 20
ase pairs. DNA-PrecipitAid can also
e used to precipitate RNA with e
thanol. Coprecipitant of choice for PCR/RT-PCR, since no contamination
y nuclei
c acids is detecta
le.

ack

The convenient method: Commercial kits employing pure silica and anion exchange

columns
Silica-
ased spin kits:
The a
ility of DNA to
ind rapidly and selectively to silicates at high salt con
centrations under alkaline conditions was first descri
ed in 1979 (Vogelstein &
Gillespie, 1979): DNA was removed from agarose gels and
ound to glass in the pr
esence of sodium iodide.
Positively charged ions shield the acidic silica surface promoting adsorption of
the DNA molecules through the negatively charged DNA
ack
one. The type of
rid
ging cation determines the strength of
inding (Romanowski et al., 1991). Washin
g with chaotropic salt solutions removes residual protein impurities without aff
ecting the immo
ilized DNA. Not only salts, also addition of alcohol influences
the interaction
etween matrix and DNA. The silica-
ound DNA withstands washing
procedures employing 70% ethanol solutions that displace the excess of salts, me
ta
olites, RNA, car
ohydrates, and other alcohol solu
le
iomolecules. Only elut
ion with pure water or low salt solutions (e.g. TE
uffer) releases the DNA. Thi
s is
ecause the large excess of water molecules replaces the ionic
onds and re
hydrates DNA as well as the surface of the silica particles.
The silica-
ased spin kits are very popular for isolation of genomic DNA as well
as for isolation of plasmids. The purity of the eluted DNA is high, and due to
the low salt elution conditions, further desalting of the DNA is not necessary.
The method is fast, easy to perform, and provides reproduci
le quantities and qu
ality. Unfortunately, the columns are not suita
le for very small DNA fragments.
The smaller the DNA molecules, the tighter the
inding
etween silica matrix an
d DNA. As a consequence, small DNAs cannot effectively
e recovered from the col
umn (Green & Sam
rook, 2012). A further draw
ack is that the
inding capacity of
silica is only moderate. Therefore, these mini columns are only suita
le for smal
l quantities of DNA, typically up to 20 g.

The meaning of chaotropic salts in nucleic acid isolation processes.

Chaotropic agents like guanidinium salts, urea or lithium perchlorate show a num

er of positive effects on the isolation process: 1. lysis of the cell mem
rane,
2. denaturation of proteins, DNA and other macromolecules, 3. inactivation of n
ucleases, 4. promotion of nucleic acid
inding to pure silica material. All thes
e features are
ased on the same mode of action. The chaotropic su
stances inter
fere with intracellular interactions
ased on hydrogen
onds, hydropho
ic effect
s and van der Waals forces resulting in denaturation of proteins (as a consequen
ce protein activity is significantly reduced) and DNA,
ut also in reorganizatio
n and finally collapse of the mem
rane. The accumulation of chaotropic salts in
the hydropho
ic region of the lipid
ilayer strongly compromises mem
rane integr
ity. By replacing the existing hydrogen
onds with adjacent water molecules
y s
alt
ridges, the chaotropic salts destroy the hydration shell that is surroundin
g the nucleic acids, decrease their solu
ility, mask charges and mediate adsorpt
ion to silica surfaces.
Anion exchange-
ased spin kits
The principle of anion exchange totally differs from pure silica-
ased DNA purif
ication. In contrast to the negatively charged silicate surface, the matrix mate
rial possesses a high density of positive charges. The hydrophilic anion exchang
e resin consists of large-pored silica
eads coated with cationic groups. Maximu
m
inding of DNA takes place under slightly acidic low-salt conditions that ena

le a direct and undistur
ed ionic interaction of the DNAs phosphate groups with t
he cationic surface of the resin. Impurities are removed by washing with medium
salt buffers (~ 0.8 - 1.0 M NaCl, depending on the pH). The DNA is eluted with h
igh salt solutions at a pH around 8. The excess of anions replaces the bound DNA
molecules and saturates the surface of the anion exchange matrix. Anion exchang

e materials guarantee a very pure DNA and, compared to pure silica, offer a stro
ngly increased binding capacity due to its high charge density. Therefore, anion
exchange matrices are preferentially used for large scale DNA isolations. Disad
vantage are the elution conditions, namely the requirement of very high salt con
centrations to release the DNA from the column. Subsequent desalting is often es
sential for downstream processes.
Columns of commercial spin kits are commonly discarded after a single use, since
5-10 % of the DNA remains in the matrix. Of course, this is a waste of resource
s and money. And it is not mandatory because the matrix itself is still working
perfectly (Green & Sambrook, 2012). AppliChem offers a product for regeneration
of pure silica and anion exchange columns that even improves the binding capacit
y. The column regeneration kits maxXbond are based on the ExitusPlus technology (s
ee chapter about Decontamination), bio-degradable and non-hazardous.
back
.
AppliChem s column-based DNA and RNA Purification Spin-Kits
[AX: Anion Exchanger; SI: Pure Silica]
Prod. No.
Description
Comment
Isolation of genomic DNA from various samples
A5185 Geno/mini DNA Isolation Spin-Kit
Standard Isolation Kit [SI] for
genomic DNA
A5179 Geno/miracle DNA Isolation Spin-Kit
AX-based
Isolation of plasmid DNA
A5172 Plas/mini Isolation Spin-Kit
Standard Miniprep Kit [SI] for plasmid i
solation.
A5182 Plas/midi Isolation Spin-Kit
AX-based
Purification of DNA and DNA fragments
A5266 DNA Enzyme-free Isolation Spin-Kit
Standard DNA Purification Kit [S
I] for PCR products and enzymatic digested DNA fragments.
A5193 DNA Isolation Spin-Kit Agarose Standard Gel Extraction Kit SI] for Isol
ation of DNA from agarose gels
Isolation of RNA from various samples
A5189 Total RNA Mini SI Isolation Spin-Kit
Standard Kit [SI] for the total
RNA isolation from different types of samples
Decontamination & Regeneration of spin-columns
MB007 maxXbond
Silica matrices are a key technology for the purification of DNA
. Today the rapid isolation of pure DNA samples is essential for a variety of mo
lecular biology protocols in research and commercial applications. Products with
silica matrices are of high quality and high value. Their major disadvantage is
that they can only be used once because after elution substantial amounts of DN
A remain attached to the silica matrix and the binding capacity is reduced. To s
olve this problem AppliChem GmbH developed the first regeneration system for sil
ica matrices: maxXbond for pure silica and maxXbondAX for anion exchange columns.
Two innovative solutions remove all nucleic acids and extraneous material from t
he matrix and restore the original binding capacity.
MB1AX maxXbond AX
MB008 maxXmore Mini Refill Buffer Sets for pure silica DNA binding columns
MB009 maxXmore AX Midi
Refill Buffer Sets for silica-based anion exchange DNA b
inding columns

back

Ultrapure DNA: Size exclusion chromatography

Salts are frequent impurities of nucleic acid isolates and, unfortunately, high
salt concentrations are not tolerated by many subsequent applications like seque
ncing, PCR or transformation. Further purification steps are therefore required
to obtain an appropriate DNA quality. Superior to ethanol precipitation, size ex
clusion chromatography (SEC, also called gel filtration) is suitable for desalti
ng, buffer exchange or removal of other low-molecular impurities (e.g. dyes, bio
tin).
The principle of SEC is simple. Molecules are separated according to their size,
but in contrast to the sieve effect, the large molecules are passing first. Thi
s is due to the longer path the smaller molecules must travel. The SEC matrix is
made up of small beads of defined diameter with a defined pore size. Small mole
cules (<5-10 kDa) enter the pores and therefore have a longer path compared to t
he larger macromolecules that pass the matrix unhindered.
AppliChem offers ready-to-use gravity and spin columns for size-exclusion chroma
tography of nucleic acids and other macromolecules. The matrix is a beaded compo
site material composed partially of polymerized dextran. It exhibits high chemic
al stability, ensuring minimal effects of buffer and pH on the high resolution l
evel. Therefore, buffer conditions, pH value and temperature during the filtrati
on process may be adapted according to the needs of the molecules but not the co
lumn material.
The size exclusion cut-off for AppliChems DextraSEC gravity columns is 10 bp for
nucleic acids, which means that DNA fragments >10 bp quickly elute, while smalle
r oligonucleotides, dNTPs, as well as other small molecules like salts or dyes a
re decelerated.