U.S. patent application number 14/966269 was filed with the patent office on 2016-04-07 for process for manufacturing a composite sorbent material for chromatographical separation of biopolymers.
The applicant listed for this patent is NExtTec GmbH. Invention is credited to Hamlet Balayan, Gottfried Brem, Robert-Matthias Leiser, Lutz Plobner, Leonti E. Tkachenko.
Application Number | 20160097048 14/966269 |
Document ID | / |
Family ID | 34963789 |
Filed Date | 2016-04-07 |
United States Patent
Application |
20160097048 |
Kind Code |
A1 |
Balayan; Hamlet ; et
al. |
April 7, 2016 |
PROCESS FOR MANUFACTURING A COMPOSITE SORBENT MATERIAL FOR
CHROMATOGRAPHICAL SEPARATION OF BIOPOLYMERS
Abstract
The present invention relates to a sorbent material for
separation and purification of biopolymers, particularly nucleic
acids, having a solid support substantially modified with a
copolymer coating comprising aromatic monomers and crosslinking
compounds and unsaturated esters or ethers preferably attached to
the support via a vinylchlorsilane. The use of these materials for
separation of nucleic acids, particularly a one-step isolation of
DNA from lysates of different biological sources, is an object of
the invention as well as a chromatographic column or cartridge at
least partially filled with the sorbent material of the invention,
a membrane-like device comprising the sorbent material of the
invention, and a kit comprising the sorbent material of the
invention in bulk or packed in chromatographic devices as well as
other devices necessary for performing sample preparations.
Inventors: |
Balayan; Hamlet; (Yerevan,
AM) ; Leiser; Robert-Matthias; (Solingen, DE)
; Plobner; Lutz; (Erkrath, DE) ; Tkachenko; Leonti
E.; (Yerevan, AM) ; Brem; Gottfried;
(Hilgertshausen, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
NExtTec GmbH |
Leverkusen |
|
DE |
|
|
Family ID: |
34963789 |
Appl. No.: |
14/966269 |
Filed: |
December 11, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14033074 |
Sep 20, 2013 |
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14966269 |
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12830676 |
Jul 6, 2010 |
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14033074 |
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11547011 |
Oct 2, 2006 |
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PCT/EP2005/051490 |
Apr 1, 2005 |
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12830676 |
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Current U.S.
Class: |
536/25.4 |
Current CPC
Class: |
C12N 15/101 20130101;
B01J 20/28061 20130101; B01J 20/283 20130101; B01J 20/28059
20130101; C08F 212/08 20130101; C08F 212/14 20130101; B01J 20/28085
20130101; B01J 20/3204 20130101; B01J 20/28088 20130101; B01J
20/289 20130101; B01J 20/28083 20130101; B01J 20/3278 20130101;
B01J 20/261 20130101; C08F 212/14 20130101; C08F 292/00 20130101;
B01J 20/3219 20130101; B01J 20/265 20130101; B01J 20/103 20130101;
C08F 212/08 20130101; C08F 212/36 20130101; C08F 212/14 20130101;
C08F 2/44 20130101; C08F 2/44 20130101 |
International
Class: |
C12N 15/10 20060101
C12N015/10; B01J 20/26 20060101 B01J020/26 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 2, 2004 |
EP |
04008147.3 |
Claims
1-22. (canceled)
23. A process for the purification and/or isolation of nucleic
acids wherein the process comprises lysing cells yielding a sample
containing nucleic acids and proteins, contacting the sample with a
sorbent material binding the proteins, and collecting the eluate
containing the nucleic acids where the sorbent material comprises a
porous inorganic material comprising silica which is at least
partially covered by a polymer coating having the general formula
##STR00006## wherein the mass ratio of copolymer components [A],
[B], and [M] complies with the formula 1:0.03-0.3:0-0.2 and [A] is
derived from a monomer with general formula ##STR00007## R.dbd.H;
2, 3, 4 --OH; --COCH; --NO.sub.2; --Cl; --Br; --F; --OAlk; --OAc;
--NH.sub.2; --NHAc; --N(Alk).sub.2; -Alk (C.sub.1-C.sub.16) [B] is
derived from a monomer of general formula ##STR00008## R.dbd.H; 2,
3, --OH; --COOH; --NO.sub.2; --Cl; --Br, --F; --OAlk; --OAc;
--NH.sub.2; --NHAc; --N(Alk).sub.2; -Alk (C.sub.1-C.sub.15); and
[M] is derived from a monomer of general formula ##STR00009##
R.dbd.H; Ar; Alk (C.sub.1-C.sub.12) R.sub.1.dbd.H; Ar; Alk
(C.sub.1-C.sub.12); --(--CH.sub.2--).sub.n--OH n=2-18 or a monomer
of general formula ##STR00010## n=1-18 R.dbd.H; Ar; Alk,
(C.sub.1-C.sub.12) R.sub.1.dbd.H; Ar; Alk.(C.sub.1-C.sub.12);
CH.sub.2 .sub.mOH m=2 -18
24. The process of claim 23 wherein the support is a porous
inorganic material comprising inorganic metal oxide.
25. The process of claim 24 wherein the porous inorganic metal
oxides show a bidisperse distribution of pore sizes.
26. The process of claim 23 wherein the support has an average pore
size of 2-200 nm.
27. The process of claim 23 wherein the polymer coating has a
thickness of about 10 to 250 Angstrom.
28. The process of claim 24 wherein the inorganic metal oxide is
selected from the group consisting of oxides of aluminum, titanium,
zirconium, silicon oxides, iron oxides, controlled pore glass
(CPG), diatomaceous earth and combinations thereof.
29. The process of claim 25 wherein the porous inorganic metal
oxide shows a bidisperse distribution with mean pore diameters in
the range of 20-100 nm for the larger pore size.
30. The process of claim 29 wherein the porous inorganic metal
oxide has a mean pore diameter in the range of 2-15 nm for the
smaller pore size.
31. The process of claim 29 wherein the ratio of the mean diameter
of the large pore size distribution and the lower pore size
distribution is in the range of 2-15.
32. The process of claim 26, wherein the support has an average
pore size of 2-100 nm.
33. The process of claim 26 wherein the support has a specific
surface area of 20-300 m.sup.2/g.
34. The process of claim 33 wherein the support has a specific
surface area of 20-100 m.sup.2/g.
35. The process of claim 27 wherein the polymer coating has a
thickness of 10 to 100 Angstrom.
36. The process of claim 27 wherein the polymer coating has
micropores of less than 50 Angstrom accessible to water, salts, and
low molecular weight substances.
37. The process of claim 27 wherein the polymer coating is
non-adsorptive towards nucleic acids and adsorptive towards
proteins.
39. The process of claim 23 comprising component [M].
40. The process of claim 38, wherein [M] is selected from the group
consisting of hydroxyethyl methacrylate, methacrylic acid and allyl
alcohol.
41. The process of claim 23, wherein x and y are independent of
each other, x and y are an integer of 1-100 and z is an integer of
0-100.
Description
[0001] The present invention relates to a sorbent material for
separation and purification of biopolymers, particularly nucleic
acids, having a solid support substantially modified with a
copolymer coating comprising aromatic monomers and crosslinking
compounds and unsaturated esters or ethers preferably attached to
the support via a vinylchlorsilane. The use of these materials for
separation of nucleic acids, particularly a one-step isolation of
DNA from lysates of different biological sources, is an object of
the Invention as well as a chromatographic column or cartridge at
least partially filled with the sorbent material of the invention,
a membrane-like device comprising the sorbent material of the
invention, and a kit comprising the sorbent material of the
invention in bulk or packed in chromatographic devices as well as
other devices necessary for performing sample preparations.
[0002] The development of composite sorbent materials for
stationary phases has led to substances with a wide range of
chromatographic properties. These materials with modified surfaces
are widely used in separation processes. Mostly, a hydrophilic
support material, such as silica gel, is modified with hydrophobic
moieties like alkyl chains of different length.
[0003] Many efforts have been made to improve the properties of the
chromatographic material in terms of chemical stability, the field
of applications or selectivity. Modification of the surface
material moderates the properties of the stationary phases and
influences the separation which is based on hydrophilic,
hydrophobic or ion-ion interactions.
[0004] U.S. Pat. No. 4,045,353 describes a chromatographic material
prepared by radiation of polymerisable monomers absorbed on a
microporous inorganic substrate. The sorbent material thus obtained
is suitable for separation of relatively small molecules, but there
is insufficient selectivity for separation of biopolymers like
nucleic acids.
[0005] Efforts have been made to get composite fluorinated
materials, in particular sorbents manufactured on the basis of
solid porous silica gels. This is to combine in the same material
the mechanical strength, determined by the porous nature of the
inorganic matrix and the specific sorption properties of the
fluorinated polymeric modifying compound. EP 1 148 945 discloses a
material having a solid support of controlled pore glass and a
coating of crosslinkable olefinic oligomers. Fluorination of the
oligomer coated support is effected with gaseous xenon difluoride
(XeF.sub.2), optionally under inert gas conditions, or with a
mixture of fluorine and an inert carrier gas. The composite
material thus obtained is suitable for use in the isolation of DNA
out of complex mixtures, where apart of DNA also RNA, proteins, low
molecular substances and salts are present. But, because of the
exclusively hydrophobic nature of these materials, there are
certain difficulties in using them with aqueous solutions of
biopolymers and they are much more used for chromatographic
separations in columns with increased pressure (HPLC) and
operations using hydrophobic interaction chromatography (HIC).
Moreover, preparation of these materials is somewhat complicated,
laborious and ecologically harmful.
[0006] Another suggestion was made to prepare sorbents on the basis
of macroporous silica gels, modified with vinyl monomers like
N-vinyl-pyrrolidone, styrene, ethylene, vinylethyl ether (authors
certificate, USSR, No. 687081, 1979). The sorbent materials are
manufactured in applying said monomers to .gamma.-aminopropylated
or silanised silica gel and subsequent polymerisation. These
sorbents possess good mechanical stability, do not swell in
solvents and may be used for the separation of proteins and other
active biomolecules. No hint is given for using these materials in
the separation of nucleic acids.
[0007] The enormous progress in genetic engineering connected with
PCR-technology leads to a growing need for applications which lead
to the desired products in a fast and economic way, e.g. isolation
and purification of DNA with effective removal of by-products and
impurities providing the capacity for the isolation of biopolymers
without denaturating conditions, and improving the selectivity by
modifying the surface of the coating.
[0008] Therefore, the object of the present invention is to provide
a sorbent material with improved characteristics in separation and
purification capacity and increased selectivity in the specific
action of the sorbent material on biopolymers having an advanced
surface for biotechnological applications, such as isolation and
separation of biopolymers, primarily in aqueous media, with
improved access area of the separation surface in a separation
medium and improved stability of the coating for the construction
of material suitable for chromatographical applications like HPLC
and fast sample preparations via solid phase extraction in compact
cartridges for PCR-applications.
[0009] The object is solved by a polymer obtainable by a process of
polymerizing at least three components [A], [B], and [M] wherein
[0010] [A] is at least one aromatic or aliphatic compound having at
least one polymerisable unsaturated moiety, [0011] [B] is at least
one cross-linkable aromatic or aliphatic compound, and [0012] [M]
is an organic non-saturated polymerisable compound different from
[A] having hetero atoms in the C--C chain or in side chains wherein
the process comprises the steps of [0013] admixing the components
[A], [B], and [M] sequentially or non-sequentially, [0014]
polymerising the resulting mixture composition [0015] removing
unreacted material and [0016] recovering and drying the composite
material.
[0017] In one embodiment the polymer of the invention comprises the
following structure
{[A].sub.x-[B].sub.y-[M].sub.z}.sub.p
[0018] wherein [A], [B], and [M] have the same meaning as in claim
1 and x, y, and z are independent of each other an integer of 1-100
and p is a number between 2 and 5000.
[0019] Subject matter of the invention is also a monomer having the
structure [A].sub.x-[B].sub.y-[M].sub.z wherein [A], [B], and [M]
have the same meaning as in claim 1 and x, y, and z is independent
of each other an integer of 1-100.
[0020] A chromatographic material comprising essentially a polymer
of the invention is also subject matter of the invention as well as
a composite sorbent material having a support which is at least
partially covered by a polymer coating of the invention.
[0021] According to the invention, a sorbent material is provided
having a previously dehydrated solid support having a copolymer
coating comprising substituted or unsubstituted aromatic vinyl
monomers, substituted or unsubstituted aromatic crosslinking
compounds and optionally non-saturated carboxylic acids or esters
or non-saturated alcohols or ethers, respectively. The coating can
be attached to the support via a vinylchlorsilane. Substituents of
the compounds of the copolymer coating modify the
hydrophobic/hydrophilic properties on the surface of the coating
and provide an essentially improved selectivity for selected
applications. For example, the functional groups exhibiting
hydrophilic properties provide an essentially better wetting of the
inner and outer surfaces of the pores of the sorbent material.
[0022] Preferably the support of the sorbent material of the
invention is porous inorganic material selected from the group
comprising inorganic metal oxides, such as oxides of aluminum,
titanium, zirconium, silicon and/or iron. In particular preferred
is silica gel having an average pore size of 100-2000 .ANG.,
preferably 300-1000 .ANG., and a specific surface of 20-300
m.sup.2/g, more preferably 20-200 m.sup.2/g or 20-100
m.sup.2/g.
[0023] Preferably, the support containing inorganic materials is in
particle-like or monolithic membrane-like form and has a porous
structure which shows a bidisperse or oligodisperse distribution of
pore sizes. Such structures build, e. g., the basis for sorbent
materials according to the present invention, which allow
additionally to the separation of biopolymers such as nucleic acids
the improved retention of low molecular weight substances having,
e.g., molecular weights of less than 500 Da, salts and proteins,
whereas nucleic acids are passing without retention. In a preferred
embodiment, the separation of nucleic acids, e.g. DNA, is conducted
in one step. Such bidisperse supports may preferentially be
obtained by means of gelling (gel building) of silica sols,
starting the process with the mixture of two size types of
monodisperse colloidal silica particles. The mass proportion of
these two types of colloidal particles determines the proportion
and distribution of differently sized pores in the final silica
support material.
[0024] Typically, two types of silica sols are prestructured prior
to mixing. Prestructuring occurs, e.g., by temperature treatment or
other methods and partially evaporating water. Preferably, the
ratio of the mean diameter of the large pore size distribution and
the lower pore size distribution is in the range of 2-15 or 3-15
nm, in particular 4-10 nm. Preferably, the mean diameter of the
larger pore size distribution should not be smaller than 25 to 50
nm and should not exceed 200 nm (2000 .ANG.), preferably 100 nm
(1000 .ANG.). Especially preferred mean diameters of the large pore
size distribution are in the range of 20-100, more preferably 25-80
nm. The mass proportion of the smaller to larger pores represents
(90-60, preferably 85-70):(10-40, preferably 15-30) (in %).
[0025] The polymer coating preferably has a thickness of about 10
to 250 Angstrom, preferably 10 to 100 Angstrom and micropores of
less than 50 .ANG. accessable to water, salts, and tow molecular
weight substances being non-adsorptive towards nucleic acids and
adsorptive towards proteins.
[0026] The preparation of the composite sorbent material which is
at least partially covered by a copolymer coating according to the
general formula (with dimethylvinylchlorosilane exemplary chosen as
modifying agent)
##STR00001##
[0027] is characterised by the following process.
[0028] The previously dried support material is modified by
treating it with a boiling solution of a vinylchlorsilane.
Treatment of the surface of the support material is conducted at
boiling temperature of the solvent used. This treatment enables the
following chemosorption of the copolymer and is the basis for
enhanced chemical stability and durability of the final sorbent
material. Washing of the silanised support, e.g. silica gel, for
removal of unreacted silane is done by multiple extraction with
organic solvents (ethanol, acetone, toluene, dioxane).
[0029] This is followed by addition of a) at least one
unsubstituted or substituted styrene or vinylnaphthalene [A] of
general formula
##STR00002##
R.dbd.H; 2, 3, 4 --OH; --COOH; --NO.sub.2; --Cl; --Br; --F; --OAlc;
--OAc; --NH.sub.2; --NHAc; --N(Alk).sub.2; -Alk
(C.sub.1-C.sub.15)
[0030] which may be substituted by at least one hydroxyl-,
carboxy-, nitro-, chloro-, bromo-, fluoro-, alkoxy-, acetoxy-,
amino-, mono- or dialkylamino-, acetamino-,
alkyl(C.sub.1-C.sub.15)- group,
[0031] b) at least one cross-linkable unsubstituted or substituted
(1,4 or 1,2)-divinylbenzene [B] of general formula
##STR00003## [0032] R.dbd.H; 2, 3, --OH; --COOH; --NO.sub.2; --Cl;
--Br; --F; --OAc; --NH.sub.2; --NHAc; --N(Alk).sub.2;
-Alk(C.sub.1-C.sub.15)
[0033] which may be substituted by at least one hydroxyl-,
carboxy-, nitro-, chloro-, bromo-, fluoro-, alkoxy-, acetoxy-,
amino-, mono- or dialkylamino-, acetamino-,
alkyl(C.sub.1-C.sub.15)- group,
[0034] c) optionally a non-substituted carboxylic acid or alkyl,
aryl, or hydroxylalkyl ester thereof [M] of general formula
##STR00004##
R.sub.1.dbd.H; Ar; Alk.(C.sub.1-C.sub.12); CH.sub.2 .sub.nOH n=2-18
[0035] R.dbd.H; Ar; Alk (C.sub.1-C.sub.12)
[0036] respectively a non-saturated alcohol or ether of general
formula
##STR00005##
n=1-18 [0037] R.dbd.H; Ar; Alk.(C.sub.1-C.sub.12) [0038]
R.sub.1.dbd.H; Ar; Alk.(C.sub.1-C.sub.12); CH.sub.2 .sub.mOH
m=2-18
[0039] In a preferred embodiment, the mass ratio of copolymer
components [A], [B], and [M] complies with the formula
[A]:[B]:[M]=1:0.03-0.30:0-0.2.
[0040] Polymerisation of the copolymer composition is performed by
polymerisation in solution of an organic solvent for a period of 2
to 8 h at the boiling temperature of the solvent or in aqueous
solution (emulsion polymerisation) with an emulgator and in the
presence of an initiator for radical polymerisation. Suitable
initiators comprise benzoyl peroxide, potassium peroxodisulfate as
a water soluble initiator, ammonium persulfate, etc. Preferably, a
5-20% solution of copolymer is used. A mass ratio of silica gel
matrix and solution of 1:3-6 is also preferred. Finally unreacted
material is removed and the composite material is recovered and
dried.
[0041] The sorbent material according to the invention is useful in
separation processes, enhancing the ease of handling and the speed
of these processes. Preferably, the substances to be separated are
nucleic acids and/or proteins. A conventionally used
chromatographic column or cartridge can be filled, at least
partially, with the sorbent material of the invention. The sorbent
material of the invention behaves similar to other solid
chromatographic supports so that the methods for filling
chromatographic columns or cartridges can be used in an analogous
manner. The support for carrying out chromatographic separations
can also be provided in the form of a membrane-like item comprising
the sorbent material of the invention, wherein the sorbent material
is embedded in a polymeric matrix such as a nylon membrane. Also
other membrane materials which are used in preparation, isolation
or separation of biomolecules can be used as matrix for embedding a
sorbent material of the present invention.
[0042] In order to ease the use of a chromatographic material of
the invention it is advantageous to provide the sorbent material
according to the invention in bulk or a chromatographic column or
cartridge or membrane-like device together with filter materials,
reagents and/or buffers or other devices or chemicals for
performing fast sample preparations and chromatographic
separations. This item can especially be provided in form of a kit
or a miniaturized device in form of chips or microreactors. The
chromatographic separation is not limited in its scale. It can be
used In any chromatographic operation for separation, isolation,
identification, purification and/or detection of biomolecules, in
particular nucleic acids, in preparative or analytical scale.
[0043] The present invention provides a product with advanced
sorption properties that allows to use this product for
chromatography of biopolymers according to the object of the
invention. The process of the invention leads to a sorbent material
with high binding capacity towards proteins and minimal
non-specific sorption of nucleic acids, which is accomplished by
using copolymers having complementary functional groups. The
inventive process allows manufacturing of a sorbent with adjusted
hydrophilic properties having high mechanical stability and very
good hydrodynamic characteristics.
[0044] The invention is further explained in the following examples
which are understood to be not limiting.
EXAMPLE 1
[0045] 10 g of macroporous silica gel previously dried in vacuo at
200.degree. C. for 3 h having an average pore diameter of 500 .ANG.
are treated with a boiling 5% solution of dimethylvinylchlorosilane
in dry benzene (3.times.25 ml) followed by acetone (3.times.25 ml)
and finally with distilled water (5.times.20 ml), air dried for 1 h
at 20.degree. C. and further for 2 h at 120.degree. C. In vacuo.
The thus obtained hydrophobic silica gel is suspended in 50 ml of
water containing 0.7 g of an emulgator (sodium stearate).
Thereafter 2.6 ml styrene, 0.32 ml divinylbenzene, 0.30 ml
2-hydroxyethyl methacrylate and 0.017 g potassium peroxodisulfate
as polymerisation initiator are added. The polymerisation is
conducted for 4 h at 85.degree. C. The liquid phase is separated by
decanting and the sorbent is washed with dimethylformamide as long
as the refractive index of the washing no longer differs from that
of the solvent (ca. 60 ml). Subsequently washing is continued with
3.times.25 ml of ethanol and distilled water, respectively.
Finally, the sorbent material is dried in vacuo for 3 h at
80.degree. C.
EXAMPLE 2
[0046] The sorbent has been obtained similar to example 1, but with
a polymer composition without component [M]
(2-hydroxyethylmethacrylate).
EXAMPLE 3
[0047] 10 g of macroporous silica gel having an average pore
diameter of 1000 .ANG. is treated with a 3% solution of
dimethylvinylchlorosilane according to example 1. Thereafter the
thus obtained silanized silica gel is transferred into a
three-necked bulb equipped with a stirrer, a thermometer, and a
reflux condenser. The bulb is charged with 30 ml benzene, 2.9 ml
vinylphenol, 0.50 ml divinylbenzene, 0.16 ml methactylic acid, and
0.33 ml benzoyl peroxide. The polymerisation is conducted for 4 h
at 80.degree. C. Further treatment is carried out according to
example 1.
EXAMPLE 4
[0048] The sorbent has been obtained similar to example 3, but with
a polymer composition without component [M] (methacrylic acid).
EXAMPLE 5
[0049] 10 g of macroporous silica gel previously dried in vacuo at
200.degree. C. for 3 h having an average pore diameter of 300 A are
treated with a boiling solution of dimethylvinyichlorosilane
corresponding to example 1. The thus obtained silanized silica will
be transferred into a three bottlenecked flask with a stirrer, a
thermometer and a reflux condenser. To the flask 40 ml toluene, 3
ml chlorovinyl benzene, 0.4 ml divinylbenzene, 0.4 ml allylic
alcohol and 0.30 g benzoil peroxide will be added. The
polymerization is conducted for 6 h at 102.degree. C. The further
treatment will be achieved corresponding to example 1.
EXAMPLE 6
[0050] The sorbent has been obtained similar to example 5, but with
a polymer composition without component [M] (allylic alcohol).
[0051] The results of usage the obtained sorbents according to
examples 1 to 6 for the separation of DNA from proteins and low
molecular weight compounds corresponding to the protocol for lysis
and isolation of genomic DNA from bacteria in example 9 and the
protocols for the estimation of protein retention, retention of
salt and yield estimation for DNA, given in example 9 as well.
Table
Comparison of Separation Efficiencies by Using Sorbent of Examples
1 to 6 Underlining the Importance of Introducing Component [M] into
the Separation of Polymer Coating
TABLE-US-00001 [0052] Protein Salt Chemical retention retention DNA
recovery composition of (% of 750 (% of 1M (% of applied Support
polymeric layers BSA) CuSO.sub.4) E. coli lysate) Silianized 1.
Styrene- 99.5 72.7 45.0 SiO.sub.2 divinylbenzene- hydroxyethyl-
methacrylate (1:0.12:0.13) 2. Styrene- 67.0 59.5 29.2
divinylbenzene (1:0, 0.12) Silianized 3.2-Hydroxyvinyl- 98.9 73.2
43.8 SiO.sub.2 benzene-divinyl- benzene- methacrylic acid
(1:0.16:0.06) 4.2-Hydroxyvinyl- 65.5 51.5 22.2 benzene-divinyl-
benzene (1:0.16) Silianized 5. Chlorovinyl- 99.0 73.0 44.5
SiO.sub.2 benzene-divinyl- benzene- allylalcohol (1:0.10:0.10) 6.
Chlorovinyl- 62.7 49.0 23.8 benzene-divinyl- benzene (1:0.1)
EXAMPLE 7
[0053] For the synthesis of sorbents having a silica gel support
with controlled bldisperse pore structures preparation is conducted
as follows:
[0054] The two starting types of silica sol in water had following
characteristics:
[0055] A: particle diameter: 6 nm; SiO.sub.2 concentration: 22 mass
%; Na.sup.+-stabilised pH: 9.1
[0056] B: particle diameter: 40 nm; SiO.sub.2 concentration: 40
mass %; Na.sup.+-stabilised pH: 9.2
[0057] Water from the two silica sols was evaporated at pH 5.0 in a
water bath at 80.degree. C. by constant stirring until 30 and 60
mass %, respectively. To 100 ml of sol A structured by evaporation
were added 50 ml of structured sol B and the evaporation has been
continued until the formation of a homogeneous gel. The silica
hydrogel obtained after 4 hours sinerethis (partial shrinkage) was
dried, first for 4 hours at 80.degree. C. in a water bath, followed
by 3 hours at 130.degree. C. in a drying hood. Afterwards the
product was treated at 600.degree. C. for 5 hours in a muffel oven.
The ready obtained silica gel was grinded, fractionated and
analysed for pore size distribution both by mercury porometry
(according to DIN 66 133 (1993)) and BET-method (according to ISO
9277). These analyses showed a preferential pore size In two
classes of 5 nm (appr. 85%) and 28 nm (appr. 15%) and a sorption
volume of 0.7 cm.sup.3/g.
EXAMPLE 8
[0058] The two starting types of silica sol in water had following
characteristics:
[0059] A: particle diameter: 10 nm; SiO.sub.2 concentration: 30
mass %; Na.sup.+-stabilised pH: 9.2
[0060] B. particle diameter: 80 nm; SiO.sub.2 concentration: 50
mass %; Na.sup.+-stabilised pH: 9.1
[0061] The silica gel sorbent was prepared as in example 7, with
following variations:
[0062] Water from the two silica sols was evaporated at pH 4.5 in a
water bath at 80.degree. C. by constant stirring until 52 and 60
mass %, respectively. To 100 ml of sol A structured by evaporation
were added 130 ml of structured sol B. Analyses showed a
preferential pore size in two classes of 7 nm (appr. 75%) and 60 nm
(appr. 25%) and a sorption volume of 0.75 cm.sup.3/g.
[0063] The results of usage the obtained sorbents according to
examples 1 to 6 for the separation of DNA from proteins and low
molecular weight compounds corresponding to the protocol for lysis
and isolation of genomic DNA from bacteria In example 9 and the
protocols for the estimation of protein retention, retention of
salt and yield estimation for DNA, given in example 9 as well.
EXAMPLE 9
[0064] Testing of the Sorbents
[0065] Mercury Porometry
[0066] The porogrammes obtained by testing the sorbents based on
the macroporous silica gel show the distribution of the pores in
differential and integral manner and allow to determine the medium
pore size of the sorbent as well as the effective thickness of the
polymeric layer, which is 5-7.5 nm.
[0067] Protocol for Tissue Lysis and Extraction of Genomic DNA from
Tissue Samples in a Kit Comprising the Sorbent of the Invention
[0068] The kit contains all necessary reagents for lysis of cells
or tissue and genomic DNA purification. The resulting DNA is
suitable for most enzymatic reactions (restriction digests, PCR,
sequencing etc.).
[0069] Compared to most other protocols not DNA is retained by the
column resin, but proteins, detergents and low molecular weight
compounds are. DNA flows through the column during a short,
one-step purification procedure.
[0070] Storage Conditions:
[0071] All kit components are stable at room temperature during
shipment. After arrival
[0072] store the kit at +2.degree. C. to +8.degree. C. Columns may
be stored at room temperature.
[0073] Materials:
TABLE-US-00002 Buffer G1 10 vials (blue), each for 5 isolations
Buffer G2 10 vials (blue), each for 5 isolations Nexttec
clean-columns 50 columns
[0074] Materials Not Provided:
[0075] Eppendorf tubes (1.5 ml)
[0076] Tris-HCl, 50 mM, pH 8
[0077] Preparation of Buffers [0078] 1. Immediately before use add
1.6 ml of deionized water to a tube with lyophilised buffer G1.
Dissolve the constituents by vortexing the tube. [0079] 2. Shortly
centrifuge a tube containing buffer G2 to collect the components at
the bottom of the tube. [0080] 3. Transfer the solution of buffer
G1 completely to one aliquot (tube) of buffer G2 [0081] 4. Mix the
buffers to get a homogeneous solution. [0082] 5. The mixture
contains all components necessary for tissue or cell lysis and is
now ready for use. The mixture Is sufficient for 5 isolations. (The
mixture should be used immediately. Therefore, prepare only as much
buffer as needed for the number of samples to be analysed.)
[0083] Cell or Tissue Lysis [0084] 1. transfer cells or a tissue
sample into an Eppendorf tube (<15 mg fresh weight) [0085] 2.
add 300 .mu.l of lysis buffer mixture (see preparation of buffers)
to each cell or tissue sample [0086] 3. Incubate the samples at
60.degree. C. overnight with constant shaking at .about.800 rpm in
an Eppendorf thermomixer. (If fresh tissue is used, shorter
incubation periods may be sufficient for complete lysis). [0087] 4.
clear the lysate by centrifugation for 3 min at 20,000.times.g
[0088] 5. Take 120 .mu.l from the clear supernatant for DNA
purification. The remaining lysate can be stored at -20.degree.
C.
[0089] Purification of DNA [0090] 6. open the spin-columns, add 300
.mu.l Tris-HCl buffer (50 mM, pH 8.0) onto each column. (The buffer
enters the resin.) [0091] 7. centrifuge the columns at 350.times.g
(corresponds to approx. 2,000 rpm in a 24-place Eppendorf rotor of
a microfuge) for 1 min to remove excess of buffer [0092] 8. discard
the collection tubes with the buffer, place the columns into a new
Eppendorf tube and open the columns [0093] 9. transfer 120 .mu.l of
the cleared supernatant from step 4 onto the columns and close the
lids (the lysate enters the resin layer) [0094] 10. incubate the
columns for 3 min at room temperature [0095] 11. spin the tubes
with the columns at 700.times.g (corresponds to approx. 3,000 rpm
in a 24-place Eppendorf rotor of a microfuge) for 1 min [0096] 12.
The flow-through contains the purified DNA. Discard the columns and
use the DNA immediately or store it at -20.degree. C.
[0097] Protocol for Lysis and Isolation of Genomic DNA from
Bacteria using a Kit of the Invention
[0098] The kit contains all necessary reagents for lysis of
bacterial cells and DNA purification. It is approved for many
Gram(-) as well as Gram(+) bacteria. The resulting DNA is suitable
for most enzymatic reactions (restriction digests, PCR, sequencing
etc.).
[0099] Compared to most other protocols not DNA Is retained by the
column resin, but proteins, detergents and low molecular weight
compounds are. DNA flows through the column during a short,
one-step purification procedure.
[0100] Storage Conditions:
[0101] All kit components are stable at room temperature during
shipment. After arrival
[0102] store RNase solution at -20.degree. C. The other kit
components must be stored at +2.degree. C. to +8.degree. C. Nexttec
clean-columns may be stored at room temperature.
[0103] Materials Provided:
TABLE-US-00003 Buffer B1 (basis buffer) 5 vials (white), each for
10 isolations Buffer B2 5 vials (white), each for 10 isolations
Buffer B3 5 vials (white), each for 10 isolations Nexttec
clean-columns 50 columns RNase solution 1 vial (white), for 50
isolations
[0104] Materials Not Provided:
[0105] Lysozyme
[0106] Eppendorf tubes (1.5 ml)
[0107] Tris-HCl, 50 mM, pH 8
[0108] Preparation of Buffers [0109] 6. Prepare a 25 mg/ml lysozyme
solution in pure water (use lyophilized lysozyme for example from
Sigma Kat.-Nr. L-6876 or comparable). The dissolved lysozyme should
be stored frozen at -20.degree. C. [0110] 7. Each vial with buffer
B1 (basis buffer) is sufficient for 10 DNA preparations.
Immediately before use complete the buffer by adding 110 .mu.l
lysozyme stock solution (20 mg/ml) and 220 .mu.l RNase solution.
Mix by vortexing the tube. [0111] 8. Shortly centrifuge a vial
containing buffer B2 to collect the components at the bottom of the
tube, then add 550 .mu.l deionized water and vortex. The prepared
buffer B2 can be stored for 2 days at +4.degree. C. [0112] 9. Add
550 .mu.l deionized water to one vial with buffer B3 and dissolve
the constituents by vortexing. The resuspended buffer should be
used immediately.
[0113] Lysis of Bacterial Cells: [0114] 1. grow an overnight
culture of bacteria in a suitable medium (e.g. LB, CSB) [0115] 2.
transfer 0.5 ml of the culture to 1.5 ml Eppendorf tubes (1.5
OD.sub.600) [0116] 3. pellet the cells by centrifugation at
6,000.times.g for 1 min, remove and discard the supernatant [0117]
4. add 120 .mu.l buffer B1 (containing iysozyme and RNase solution)
to the bacterial cell pellet [0118] 5. gently vortex the tube to
resuspend the cells [0119] 6. incubate the tube for 10 min at
60.degree. C. constantly shaking (1,200 rpm, Eppendorf thermomixer)
[0120] 7. add 50 .mu.l of buffer B2 and incubate for 5 min at
60.degree. C. (1,200 rpm, Eppendorf thermomixer) [0121] 8. then add
50 .mu.l of buffer B3 and continue the incubation at 60.degree. C.
for 25 min (as described in step 7) in the thermomixer [0122] 9. In
most cases the lysate should be clear after the incubation. If it
is not, centrifuge the tube for 3 min at 20,000.times.g to pellet
cell debris.
[0123] Purification of DNA [0124] 10. open the spin-columns, add
300 .mu.l Tris-HCl buffer (50 mM, pH 8.0) onto the column. (The
buffer enters the resin.) [0125] 11. centrifuge the columns at
350.times.g (corresponds to approx. 2,000 rpm in a 24-place
Eppendorf rotor of a microfuge) for 1 min to remove excess of
buffer [0126] 12. discard the collection tubes with the buffer,
place the columns into new Eppendorf tubes and open the columns
[0127] 13. transfer 120 .mu.l of the clear lysate from step 9 onto
the columns and close the lid (the lysate enters the sorbent layer)
[0128] 14. incubate the columns for 3 min at room temperature
[0129] 15. centrifuge the tubes with the columns at 700.times.g
(corresponds to approx. 3,000 rpm in a 24-place Eppendorf rotor of
a microfuge) for 1 min [0130] 16. The flow-through contains the
purified DNA. Discard the columns and use the DNA immediately or
store it at -20.degree. C.
[0131] Protein retention was measured using bovine serum albumin
(BSA) as a model protein. BSA was dissolved in buffer and the
optical density of the resulting solution at a wavelength of 280 nm
was measured before and after passing columns containing different
sorbents. The concentration of BSA in both solutions was then
calculated using a linear regression resulting from a standard
curve of different BSA concentrations In buffer. The applied amount
of BSA was set at 100% and the eluted amount of BSA was
recalculated in % of the applied amount. The difference between
both values gives a protein retention in %.
[0132] Salt retention was measured using copper sulphate
(CuSO.sub.4) as a model salt. A solution of this salt in water
results in a blue coloured liquid. The optical density of the
solution is linearly depending on the CuSO.sub.4 concentration.
CuSO.sub.4 was dissolved in water (c=1M) and the optical density of
the resulting solution at a wavelength of 650 nm was measured
before and after passing columns containing different sorbents. The
concentration of CuSO.sub.4 in both solutions was then calculated
using a linear regression resulting from a standard curve of
different CuSO.sub.4 concentrations In water. The applied amount of
CuSO.sub.4 was set at 100% and the eluted amount of CuSO.sub.4 was
recalculated in % of the applied amount. The difference between
both values gives the salt retention in %.
[0133] To determine the DNA recovery for sorbents during passage of
columns filled with corresponding chromatographic materials a crude
lysate from Escherichia coli cells was obtained. The lysate
contains low molecular weight compounds, proteins (peptides),
nucleic acids and other compounds.
[0134] The concentration of DNA before (crude lysate) and after
(eluate) passing a sorbent containing column was measured using
PicoGreen, a fluorescent dye, which binds only to doublestranded
DNA, according to the manufacturer's instructions (Molecular Probes
c/o Invitrogen). For the preparation of a standard curve a serial
dilution of DNA from the bacteriophage lambda with known
concentration was used. The applied amount of DNA within the lysate
was set at 100% and the eluted amount of DNA was recalculated in %
of the applied amount. The resulting value gives the DNA recovery
in %.
* * * * *