Note: Descriptions are shown in the official language in which they were submitted.
WO 00/49031 CA 02362979 2001-08-16 PCT/EP00/01028
r P a ~~ ~sa
_1_
Method for isolating nucleic acids
The invention relates to a method for separating off and selectively
liberating nucleic
acids by use of special bead polymers
So-called genetic diagnosis has become increasingly important recently.
Genetic diagnosis has become involved in the diagnosis of human diseases
(inter alia
detection of pathogens, detection of genome mutations, analysis of circulating
tumor
cells and identification of risk factors for predisposition to a disease).
However,
genetic diagnosis is also now finding applications in veterinary medicine,
environmental analysis and food testing. A further area of application
comprises
investigations in institutes of pathology/cytology or within the framework of
forensic
problems. However, genetic diagnosis is now employed also for the purposes of
quality and process control (for example investigations of blood samples for
freedom
from pathogens), and legislation is planned to extend the number of tests
already
required now by law in the future.
Methods also employed in genetic diagnosis (such as, for example,
hybridization and
amplification techniques such as PCR, bDNA or NASBA, TMA technology) are also
among the routine methods in fundamental scientific studies.
The increasingly wide use of nonradioactive detection methods, which also play
a
part in genetic diagnosis, leads to the expectation that genetic diagnosis
will be used
even more widely in future than at present.
An important step in genetic diagnosis is the obtaining of gene samples from
biological materials such as cells, blood, sputum, CSF, serum or urine.
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The binding of nucleic acids to polyquaternary cationic polymers is disclosed
in
US-A-4 046 750. However, the binding is irreversible, so that the adsorbed
nucleic
acids cannot be liberated again in this method.
S US-A-4 OSS 469 discloses a method for purifying enzymes where nucleic acids
and
unwanted proteins are precipitated with the aid of water-soluble cationic
polymers.
US-A-4 839 231 discloses supports which are coated with vinylpyridine polymer
and
are able to adsorb proteins and nucleic acids. However, the capacity of the
supports is
rather low and the adsorption of the nucleic acids is not quantitative.
WO-A-91/05606 describes a silanized, porous support material with
hydroxyalkylamino groups for chromatographic separation of nucleic acids.
However, this material is less well suited to rapid and maximally quantitative
1 ~ separation of nucleic acids out of biological materials.
DE-A-4 139 664 describes an apparatus and a method for isolating and purifying
nucleic acids with the aid of anion exchangers. The disadvantages of this
method are
that it is possible to desorb the nucleic acids off the anion exchanger only
with buffer
solutions of high ionic strength, and the separation of the salts out of the
buffer
solution requires additional preparation steps.
DE-A-4 333 805 claims the extraction of nucleic acids from a sample with the
aid of
water-soluble carriers such as dextran, acrylamide or carboxymethylcellulose
and
2~ other reagents, with the nucleic acids being precipitated.
EP-A-0 707 077 (corresponds to US-A-5 582 988) describes a method for
isolating
nucleic acids from biological material using soluble, weakly basic polymer. In
this
method, a precipitate is generated from a soluble, weakly basic polymer and
the
nucleic acid in an acidic pH range, the precipitate is separatr~i from the
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unprecipitated constituents of the biological material, and washed, and the
nucleic
acid is liberated again from the precipitate by adjusting a basic pH.
One disadvantage of the methods of DE-A-4 333 805 and EP-A-0 707 077 is that
the
manipulation, in particular the separation and purification of the
precipitate, is
difficult and very time consuming. These methods can moreover be carried out
using
automatic analyzers only under difficult conditions or not at all.
WO-A-96/18731 describes a method for isolating nucleic acids with the aid of a
detergent and of a solid support. Since solid supports without pores and
without
swellability are employed, the binding capacity of the supports is relatively
low.
WO-A-97/08547 describes a method for isolating nucleic acids in which the
nucleic
acids are bound to a solid hydrophilic organic polymer without effective
positive
1 ~ charge, for example to a cellulose. In this method, the binding is
effected by weak
forces such as Van der Waals interactions.
WO-A-97/34909 describes a method for isolating nucleic acids by use of a
specific
particulate polymer with a lower critical solubility temperature (LCST) of 25-
45° C.
A disadvantage of this method is that buffers of high ionic strength must be
used to
liberate the nucleic acids but may interfere with further use of the nucleic
acids.
Because of the small size of the particles used, from 0.05 to 2 Vim, moreover,
the
processing is time-consuming and difficult to automate.
Despite the numerous previously described methods for,isolating nucleic acids,
there
is still a pressing need for a simple method, which can be automated if
possible, for
the effective separation of nucleic acids from biological material, and the
liberation
a~:ain thereof.
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It has now been found, surprisingly, that certain water-insoluble bead
polymers of
polymerized units of amino monomer, crosslinker and vinyl monomer are
outstandingly suitable for isolating nucleic acids.
The invention relates to a method for isolating nucleic acids from a sample,
comprising the following steps
A) mixing the sample with a water-insoluble polymer which is not ionic in the
basic and neutral range, at a pH of 7 or less, with the nucleic acids being
adsorbed,
B) separating off the water-insoluble polymer and
C) mixing the water-insoluble polymer with an aqueous phase with a pH of
1 ~ greater than 7, with the adsorbed nucleic acids being liberated,
characterized in that the water-insoluble polymer is a bead polymer with an
average
particle size of from 3 to 100 pm and consists of polymerized units of
a) 5 to 98% by weight of amino monomer
b) 0.3 to 30% by weight of crosslinker and
c) 0 to 93% by weight of vinyl monomer.
Where appropriate, in a step interpolated after method step A) the biological
material
is lysed.
The present invention preferably relates to a method for isolating nucleic
acids from
a sample, comprising the steps
;0
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A) mixing the sample with a water-insoluble polymer which is not ionic in the
basic and neutral range, at a pH of 7 or less, with the nucleic acids being
adsorbed,
B) separating off the water-insoluble polymer and
1J
C) mixing the water-insoluble polymer with an aqueous phase with a pH of
greater than 7, with the adsorbed nucleic acids being liberated,
characterized in that the water-insoluble polymer is a bead polymer with an
average
particle size of from 3 to 100 ~m and a specific surface area measured by the
BET
method of from 5 to 500 m2/g and consists of polymerized units of
a) 5 to 98% by weight of amino monomer
b) 0.3 to 30% by weight of crosslinker and
c 1 ) 0 to 93% by weight of hydrophobic vinyl monomer
or in that the water-insoluble polymer consists of
bead polymer which is able to swell in water well and has an average particle
size of
from 3 to 100 pm, and which consists of polymerized units of
2~ a) 5 to 79.7% by weight of amino monomer
b) 0.3 to 10% by weight of crosslinker and
c2) 10 to 93% by weight of hydrophilic vinyl monomer.
The present invention particularly preferably relates to a method for
isolating nucleic
acids from a sample, comprising steps A), B) and C) defined above,
characterized in
that the water-insoluble polymer is a macroporous bead polymer with a particle
size
of from 3 to 100 pm and a pore diameter in the range from 10 to 1 000 nm,
which
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has a specific surface area determined by the BET method of 5 to 500 m2/g
(very
particularly preferably 20 to 200 m2/g) and consists of polymerized units of
a) 5 to 98% by weight of amino monomer
b) 2 to 30% by weight of crosslinker and
c 1 ) 0 to 93% by weight of hydrophobic vinyl monomer
or in that the water-insoluble polymer consists of
bead polymer which is able to swell in water well and has an average particle
size of
from 3 to 100 .um, and which consists of polymerized units of
a) 5 to 79.7% by weight of amino monomer
b) 0.3 to 10% by weight of crosslinker and
I ~ c2) 10 to 93% by weight of hydrophilic vinyl monomer.
The method of the invention is suitable for the isolation and/or purification
of nucleic
acids of varying origin, for example from cells, tissue materials, blood or
pathogens.
Before the isolation of the nucleic acids, the material to be investigated is
disrupted
?0 by teclniques known per se, such as, for example, disruption by protease
digestion,
resulting in a sample, a lysate, suitable for subsequent steps A to C. Where
appropriate, the biological material is lysed in a step interpolated after
method step
A). Further suitable disruption methods have been described in DE-A-4 333 805.
The sample is mixed with a water-insoluble polymer at a pH of 7 or less,
preferably
in the range 2 - 6, particularly preferably in the range 2 - 3, at room
temperature. The
water-insoluble polymer is separated off for example by filtration or by
centrifugation. The complex of nucleic acid and polymer which is obtained in
this
wav can then be purified by washing with suitable buffers such as, for
example, TE.
;O
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To liberate the bound nucleic acids from the complex, the pH of the complex is
then
adjusted to pH values above 7, preferably of 8 - 14, particularly preferably
in the
range 12 - 14.
The bead polymers of the invention provide higher rates of adsorption and
liberation
again than the soluble polymers disclosed in EP-A-0 707 077. The isolation can
be
carried out more easily, i.e. with fewer working steps and in shorter times.
The purity
of the isolated nucleic acids is higher and, in particular, they contain fewer
inhibiting
byproducts so that amplification of the nucleic acids, for example by the so-
called
"PCR reaction" and the "RT-PCR", takes place particularly well. The method of
the
invention is also superior to the method described in EP-A-0 707 077 in
relation to
restriction enzyme digestion of the nucleic acids obtained.
The present invention further relates to the macroporous bead polymers,
1 ~ characterized in that they have an average particle size of from 3 to 100
Vim, a p-are
diameter of from 10 to 1000 nm and a specific surface area measured by the BET
method of from 5 to 500 m2/g and consist of polymerized units of
a) 5 to 98% by weight of amino monomer
b) 2 to 30% by weight of crosslinker and
c 1 ) 0 to 93% by weight of hydrophobic vinyl monomer,
and
the bead polymers which are insoluble but swellable in water, characterized in
that
they have an average particle size of from 3 to 100 ~m and consist of
polymerized
units of
a) ~ to 79.7% by weight of amino monomer
b) 0.3 to 10% by weight of crosslinker and
c 1 ) 10 to 93% by weight of hydrophilic vinyl monomer.
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The present invention further relates to a method for preparing water-
insoluble,
macroporous bead polymers with an average particle size of from 3 to 100 pm, a
pore diameter of from 10 to 1000 nm and a specific surface area measured by
the
BET method of from 5 to 500 m2/g, characterized in that a mixture of
a) 5 to 98 parts by weight of amino monomer
b) 2 to 30 parts by weight of crosslinker
c 1 ) 0 to 93 parts by weight of hydrophobic vinyl monomer
d) 10 - 150 parts by weight of porogen and
e) 0.1 - 2.5 parts by weight of free-radical former
is dispersed in an aqueous medium using a protective colloid, subsequently the
resulting dispersion is polymerized by heating to the decomposition
temperature of
1 ~ the free-radical former and, after the polymerization has taken place, the
porogen is
removed by extraction and/or evaporation,
and a method for preparing bead polymers which are insoluble but swellable in
water
and have an average particle size of from 3 to 100 pm, characterized in that a
mixture
of
a) 5 to 79.7% by weight of amino monomer
b) 0.3 to 10% by weight of crosslinker and
c2) 10 to 93% by weight of hydrophilic vinyl monomer
2~ d) 10-1 SO parts by weight of solvent and
e) 0.1 - 2.5 parts by weight of free-radical former
is dispersed in an aqueous medium using a protective colloid, subsequently the
resulting dispersion is polymerized by heating to the decomposition
temperature of
,0 the free-radical former and, after polymerization has taken place, the
solvent is
removed by extraction and/or evaporation.
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Amino monomers (a) for the purposes of the invention are polymerizable,
ethylenically unsaturated compounds with at least one primary, secondary or
tertiary
amino group. The secondary or tertiary amino group may moreover be part of a
cycloaliphatic or aromatic ring. Examples which may be mentioned are
N-vinylimidazole, N-vinylbenzimidazole, 2-vinylpyridine and 4-vinylpyridine.
Very
suitable amino monomers are also the derivatives of acrylic acid and
methacrylic
acid, such as, for example, 2-aminoethyl methacrylate, N,N-dimethylaminoethyl
methacrylate, N,N-dimethylaminopropyl methacrylate, N,N-dimethylaminoethyl
acrylate, N-tert-butylaminopropyl methacrylate, N-(3-
aminopropyl)methacrylamide,
N-(3-imidazoylpropyl)methacrylamide, N-(2-imidazoylethyl)methacrylamide, N-(3-
aminopropyl)acrylamide, N-(3-imidazoylpropyl)acrylamide, N-(2-
imidazoylethyl)acrylamide, N-(l,l-dimethyl-3-imidazoylpropyl)methacrylamide, N-
( 1,1-Dimethyl-3-imidazoylpropyl)acrylamide, N-(3-
benzimidazoylpropyl)methacrylamide and (3-benzimidazoylpropyl)acrylamide.
Very suitable amino monomers are also the products of the reaction of
isocyanatoethyl (meth)acrylate and imidazoylalkylamines, such as, for example,
the
amino monomer of formula (I) which is novel within the framework of the
present
invention
O
O'~/~N~N~/~'~N~N
i
(I)
O
Further suitable amino monomers according to the present invention are the
pyridine
derivatives of the formulae (II) and (III)
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l~
O
~''N~N \
a i
H H
O
O N
(II) (III).
Derivatives of styrene and a-methylstyrene with amino groups are also very
suitable.
Examples which may be mentioned are: 4-N,N-dimethylaminostyrene,
2-N,N-dimethylaminostyrene. 4-N,N-diethylaminostyrene and 4-N,N-
bis(2-hydroethyl)aminostyrene.
Crosslinkers (b) suitable according to the present invention are: ethylene
glycol
dimethacrylate, butanediol dimethacrylate, hexanediol dimethacrylate,
pentaerythritol dimethacrylate, glycerol 1,2-dimethacrylate, glycerol 1,3-
dimethacrylate, triethylene glycol dimethacrylate, tetraethylene glycol
dimethacrylate, trimethylolpropane trimethacrylate, pentaerythritol
trimethacrylate,
pentaerythritol tetramethacrylate, ethylene glycol diacrylate, butanediol
diacrylate,
l~ pentaerythritol diacrylate, glycerol 1,3-diacrylate, triethylene glycol
diacrylate,
trimethylolpropane triacrylate, pentaerythritol triacrylate, pentaerythritol
tetraacrylate, allyl methacrylate, allyl acrylate, methylene-N,N'-
bisacrylamide, p-
divinylbenzene and m-divinylbenzene.
Hydrophobic vinyl monomers (c 1 ) which may be present in the bead polymer of
the
invention and are suitable for the purposes of the present invention are Cl-Cg-
alkyl
acrylates, Cl-Cg-alkyl methacrylates, such as, for example, methyl
methacrylate or
butyl acrylate, acrylonitrile, methacrylonitrile, vinyl chloride, vinylidene
chloride,
vinyl acetate and aromatic vinyl monomers such as, for example, styrene,
vinylnaphthalene, vinyltoluene, ethylstyrene, a-methylstyrene, chlorostyrenes
and
vinvlbenzvl chloride.
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Hydrophilic vinyl monomers (c2) suitable for the purposes of the present
invention
are, for example: 2-hydroxyethyl methacrylate, 2-hydroxypropyl methacrylate,
2-hydroxyethyl acrylate, 2-hydroxypropyl acrylate, triethylene glycol
monomethacrylate, tetraethylene glycol monomethacrylate, acrylamide,
methacrylamide and N,N-dimethylacrylamide.
Porogens are employed for the method of the invention for preparing water-
insoluble
macroporous bead polymers. Suitable for this purpose are liquid water-
immiscible
compounds which dissolve the monomers employed and precipitate the polymer
formed. Examples which may be mentioned are aliphatic hydrocarbons such as
hexane, heptane, octane, isooctane isododecane and alcohols such as octanol.
The
porogen is employed in amounts of from 10 to 150% by weight, preferably from
20
to 100% by weight, based on the total of the monomers and crosslinker
employed.
1 ~ Suitable and preferred free-radical forrners for the purposes of the
present invention
are oil-soluble initiators. Examples which may be mentioned are: peroxy
compounds
such as dibenozoyl peroxide, dilauryl peroxide, bis (p-chlorobenzoyl
peroxide),
dicyclohexyl peroxydicarbonate, tert-butyl peroctoate, 2,5-
bis(2-ethylhexanoylperoxy)-2,5-dimethylhexane and tert-amylperoxy-2-
ethylhexane,
in addition azo compounds such as 2,2'-azobis(isobutyronitrile), 2,2'-
azobis(2,4-dimethylvalereonitrile) and 2,2'-azobis(2-methylisobutyronitrile).
The
initiators are generally used in amounts of from 0.05 to 2.5% by weight,
preferably
0.2 to 1.5% by weight, based on the monomer mixture.
2~ In the preparation of the macroporous gel bead polymers of the invention
there is use,
if appropriate, of protective colloids in the aqueous phase. Suitable
protective
colloids according to the present invention are natural and synthetic water-
soluble
polymers such as, for example, gelatin, starch, cellulose derivatives, in
particular
cellulose esters and cellulose ethers, polyvinyl alcohol,
polyvinylpyrrolidone,
,0 polvacrylic acid, polymethacrylic acid and copolymers of acrylic acid,
methacrylic
acid, methacrylic esters and/or acrylic esters. Copolymers of methacrylic acid
and
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methacrylic ester neutralized with alkali metal hydroxide are particularly
suitable.
The amount of protective colloid employed is generally 0.05 to 2% based on the
aqueous phase, preferably 0.1 to 1 %.
The aqueous phase may additionally contain where appropriate a buffer system.
Preferred buffer systems adjust the pH of the aqueous phase at the start of
the
polymerization to a value between 12 and 5, preferably between 10 and 6. Under
these conditions, dispersants with carboxyl groups are wholly or partly in the
form of
salts. The effect of the protective colloids is beneficially influenced in
this way.
Particularly suitable buffer systems contain phosphate or borate salts.
The amount of the aqueous phase is generally 75 to 1200% by weight, preferably
100
to 500% by weight, based on the total of monomers, crosslinker and porogen.
1 ~ The stirring speed during the polymerization is important for adjusting
the particle
size. In the preparation method of the invention the size of the resulting
bead
polymers decreases with increasing stirrer speed. The exact stirrer speed for
adjusting
a particular predetermined bead size depends in the individual case greatly on
the
size of the reactor, the reactor geometry and the stirrer geometry. It has
proved to be
expedient to determine the necessary stirring speed by experiment. For
laboratory
reactors with a reaction volume of 3 liters and equipped with paddle stirrers,
on use
of copolymers of acrylic acid, methacrylic acid, acrylic esters and/or
methacrylic
esters as dispersant in general bead sizes of from 6 to 30 pm are reached with
speeds
of from 300 to 500 rpm.
The polymerization temperature in the preparation method of the invention
depends
on the decomposition temperature of the initiator employed. It is generally
between
50 and 150°C, preferably between SS and 100°C. Polymerization
takes 0.~ to some
hours. It has proved appropriate to use a temperature program in which the
polymerization is started at low temperature, for example 70°C, and the
reaction
temperature is raised as the polymerization reaction progresses.
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After the polymerization it is possible for the polymer to be isolated by
conventional
methods, for example by filtration or decantation, where appropriate after one
or
more washes, to be dried. The porogen can be removed during the uy iicb. a
~'~:w ,:r~:~e
of low-boiling porogens such as, for example, hexane, it is also possible to
remove
the porogen from the aqueous reaction mixture wholly or partly by distillation
before
isolating the bead polymer.
Preparation of the bead polymers capable of swelling in water takes place in
analogy
to the preparation of the macroporous bead polymers, employing hydrophilic
vinyl
monomers (c2) in place of the hydrophobic vinyl monomers (c I ), and a solvent
in
place of the porogen.
Suitable solids are those which are immiscible with water and dissolve the
monomers
1 ~ and the crosslinker and do not precipitate, but dissolve or swell, the
polymer formed.
Suitable solvents are toluene, xylene, tetrachloromethane, chloroform,
methylene
chloride, dichloroethane and ethyl acetate. The amount of auxiliary solvent is
generally 10 to 200% by weight, preferably 10 to I50% by weight, particularly
preferably 20 to 100% by weight, based on the total of monomers and
crosslinker. If
required, the auxiliary solvent can be removed for example by distillation
after the
polymerization. Toluene can be removed particularly simply by azeotropic
distillation.
The bead polymers capable of swelling in water according to the invention have
swelling indices of from 1.2 to 12, preferably 1.5 to 8, measured at
25°C and pH 7.
The swelling index is defined as the quotient of the volume of the bead
polymer
swollen to saturation in water and the volume of the anhydrous bead polymer.
The present application also relates to compositions for isolating nucleic
acids
,0 comprising water-insoluble macroporous bead polymers with an average
particle size
of from 3 to 100 pm, a pore diameter of from 10 to 1000 nm and a specific
surface
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area measured by the BET method of from 5 to 500 m2/g, consisting of
polymerized
units of
a) 5 to 98% by weight of amino monomer
b) 2 to 30% by weight of crosslinker
c 1 ) 0 to 93% by weight of hydrophobic vinyl monomer
or of bead polymers which are insoluble but swellable in water and have an
average
particle size of from 3 to 100 p.m, consisting of polymerized units of
a) 5 to 79.7% by weight of amino monomer
b) 0.3 to 10% by weight of crosslinker and
c2) 10 to 93% by weight of hydrophilic vinyl monomer.
These compositions are preferably in the form of aqueous dispersions with a
solids
content of 0.1 - 50%, particularly preferably 1 - 10%. The aqueous phase may,
where appropriate, contain buffers which are preferably effective in the range
2 - 6.
Possible areas of use of a composition which is formulated, for example, as a
test kit
are the application examples already mentioned above, for example in the i~c !
:G~:,:~Y ~~u
nucleic acids from cells, tissue materials, blood or pathogens, with all
diagnostic
questions in particular being involved. The described polymers are also
understood
as test kit for routine nucleic acid tests in microtiter plates and/or test
tubes, or other
formats such as, for example, within the framework of chip technology.
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Examples
Example 1
S Preparation of the amino monomer of the formula 1
62.08 g (0.4 mol) of 2-isocyanatoethyl methacrylate were added dropwise over
the
course of 60 min to a stirred solution of 50.07 g (0.4 mol) of
3-aminopropylimidazole, 140 mg of 2,6-di-tert.butyl-4-methylphenol
(stabilizer),
140 mg of dibutyltin dilaurate (catalyst) in 250 ml of chloroform at
20°C with
cooling. The mixture was then heated at 50°C-until (about 5 h) no NCO
band was
detectable in the IR spectrum. 112 g of amino monomer of the formula (I) were
obtained.
1 ~ Examele 2
Preparation of a gel bead polymer
A solution of 5 g of polyvinyl alcohol (Mowiol~ 40-88) and 1.5 g of disodium
hydrogen phosphate in 130 ml of deionized water was introduced into a 250 ml
reaction vessel with paddle stirrer, reflux condenser, thermometer, gas inlet
tube and
gas outlet tube. To this aqueous solution was added, at 20°C while
stirring at
450 rpm, an organic solution of 7.91 g of N,N-dimethylaminoethyl methacrylate,
7 g
of 2-hydroxyethyl methacrylate, 0.178 of triethylene glycol dimethacrylate,
0.225 g
of 2,2'-azobis(2,4-dimethylvalereonitrile) and 37.5 g of chloroform over the
course
of 15 min. The mixture was gently flushed with nitrogen, and the temperature
was
raised to 67°C and kept at this temperature for 20 hours. After
cooling, the bead
polymer which had formed was separated off from the reaction liquor by
decantation
and freed of chloroform in vacuo at SO°C. 13.5 g of bead polymer with
an average
particle size of 20 pm and a swelling index of 5.1 measured at 25° C in
water were
obtained.
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Example 3
Preparation of a macroporous bead polymer
A solution of 42.5 g of polyvinyl alcohol (Moviol 40-88) and 12.75 g of
disodium
hydrogen phosphate in 1240 g of deionized water was introduced into a 2liter
reaction vessel with paddle stirrer, reflux condenser, thermometer, gas inlet
tube and
gas outlet tube. To this aqueous solution was added, at 20° C while
stirring at
280 rpm (revolutions per minute), an organic solution of 23.71 g of
N,N-dimethylaminoethyl methacrylate, 13.04 g of styrene, 10.67 g of
divinylbenzene, 0.71g of 2,2'-azobis(2,4-dimethylvalereonitrile) and 31.62 g
of
hexane over the course of 30 min at 25°C. The mixture was gently
flushed with
nitrogen, and the temperature was raised to 66°C and kept at this
temperature for
1 ~ 20 hours. The hexane was then distilled out at an internal temperature of
70 to 98° C.
After cooling, the resulting bead polymer was separated from the reaction
liquor by
decantation and dried in vacuo at 50° C. 38 g of bead polymer with an
average
particle size of 15 gm and a specific surface area of 62.3 m2/g were obtained.
Example 4
Preparation of a macroporous bead polymer
Example 3 was repeated employing an organic solution of 23.71 g of amino
2~ monomer from Example 1, 13.04 g of styrene, 10.67 g of divinylbenzene,
0.71g of
2,2'-azobis(2,4-dimethylvalereonitrile) and 38 g of hexane. 35 g of
macroporous
bead polymer with an average particle size of 25 ~m and a specific surface
area of
74 mz/g were obtained.
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Biological result
~ 100 ~l of blood were mixed with 105 Caski cells. These cells contain human
papillomavirus of type 16 (HPV).
~ 10 pl of a 2.4% strength particle dispersion from Example 2 were added to
this mixture..
~ After addition of 200 pl of a suitable buffer, for example TE, incubation
took
place at room temperature for 5 min (TE represents 10 mmol of tris HCl and
1 mmol of ethylenediaminetetraacetic acid pH 7.4 in the final concentration).
~ The particles were then sedimented in an Eppendorf centrifuge at 7 000 rpm
for 3 min.
~ Then 200 pl of a suitable lysis buffer, for example 0.5% IGEPAL CA-630
in TE (IGEPAL CA-630~ is a nonionic detergent which can be purchased, for
example, through Sigma, order number I 3021 ), were added to the sediment
1 ~ and incubated anew at room temperature for 5 min.
In place of IGEPAL CA-630~ it is also possible, however, to employ other
lysis buffers. Examples which may be mentioned are classical methods such
as with protease K digestion and subsequent purification using
phenol/chloroform or sodium lauryl sulfate solutions (Sigma order number:
L 6026), for example as 0.5% strength aqueous solution.
~ Renewed centrifugation and sedimentation took place in an Epp~;ndorf
centrifuge at 7000 rpm for 3 min.
~ The particles were subsequently washed 2 x with a suitable buffer (for
example TE). After the second washing step, the supernatant was discarded
and further work was done only with the particles.
~ The liberation of the nucleic acid bound to the particles took place by
adjusting the pH to >12 by adding 1 pl of 0.5 normal NaOH.
~ This was followed by incubation at room temperature for 15 minutes.
~ After centrifugation (3 min, 7000 rpm in an Eppendorf centrifuge), the
concentration of the nucleic acid obtained was determined by a suitable
WO 00/49031 PCT/EP00/01028
CA 02362979 2001-08-16
-18-
method, for example by analysis in a gel system, particularly preferably the
"Submerged Gel Nucleic Acid Electrophoresis System", order No. 170 4406
from BIO-RAD (webpage: www.bio-rad.com).
Compared with beads for nucleic acid extraction known from the method of
EP-A-0 707 077, distinctly better results were surprisingly achieved with the
method of the invention and the bead polymers used therein.
~ I S ul of the supernatant obtained in this way were then employed in an
HPV-specific PCR (polymerase chain reaction), that is to say primer was
employed in a PCR which are specific for human papillomaviruses (HPV).
On analysis of the result in a gel system it was possible to identify the
expected nucleic acid band clearly and distinctly.
