U.S. patent application number 13/182691 was filed with the patent office on 2011-11-03 for use of tde for isolation of nucleic acids.
Invention is credited to Frank Bergmann, Horst Donner, Nina Lassonczyk, Marcus Schmid, Manfred Watzele.
Application Number | 20110266172 13/182691 |
Document ID | / |
Family ID | 38109608 |
Filed Date | 2011-11-03 |
United States Patent
Application |
20110266172 |
Kind Code |
A1 |
Donner; Horst ; et
al. |
November 3, 2011 |
USE OF TDE FOR ISOLATION OF NUCLEIC ACIDS
Abstract
The invention provides the use of tetraethylene glycol dimethyl
ether for adsorbing nucleic acids to solid phases such as those
with silica surfaces. To this end, the invention also provides
compositions comprising TDE. Methods are disclosed and claimed to
purify nucleic acids from samples, as well as kits useful for
performing these methods. Particularly, the invention encompasses
methods for the purification of nucleic acids with low molecular
weight. The nucleic acids purified by a method of the invention are
suited for assays aiming at the detection of a target nucleic
acid.
Inventors: |
Donner; Horst; (Penzberg,
DE) ; Bergmann; Frank; (Iffeldorf, DE) ;
Lassonczyk; Nina; (Penzberg, DE) ; Watzele;
Manfred; (Weilheim, DE) ; Schmid; Marcus;
(Wessobrunn, DE) |
Family ID: |
38109608 |
Appl. No.: |
13/182691 |
Filed: |
July 14, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12481807 |
Jun 10, 2009 |
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13182691 |
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PCT/EP2007/010793 |
Dec 11, 2007 |
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12481807 |
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Current U.S.
Class: |
206/223 ;
252/364; 252/62.51R; 435/6.12; 536/25.41 |
Current CPC
Class: |
C12N 15/1006
20130101 |
Class at
Publication: |
206/223 ;
536/25.41; 435/6.12; 252/364; 252/62.51R |
International
Class: |
B65D 71/00 20060101
B65D071/00; C12Q 1/68 20060101 C12Q001/68; C07H 1/06 20060101
C07H001/06 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 13, 2006 |
EP |
06025779.7 |
Claims
1-24. (canceled)
25. A composition effective for isolating nucleic acids, the
composition comprising between about 10% and 75% tetraethylene
glycol dimethyl ether (TDE) by volume, an aqueous buffer, and a
chaotropic agent, with the proviso that acetate at a pH of from 1
to 6 and a concentration of acetate from 5 mM to 200 mM are
excluded.
26. The composition according to claim 25, wherein the nucleic
acids comprise 150 or more nucleotides and the composition
comprises between about 10% and 40% TDE by volume.
27. The composition according to claim 25, wherein the nucleic
acids comprise less than 150 nucleotides and the composition
comprises between about 40% and 75% TDE by volume.
28. The composition of claim 26, wherein the concentration of TDE
in the composition is about 20% by volume.
29. The composition of claim 25, further comprising a compound
selected from the group consisting of 1,3-dioxolan, 1,3-dioxan,
5-hydroxy-1,3-dioxane, and 4-hydroxymethyl-1,3-dioxolane.
30. The composition of claim 25, further comprising a
detergent.
31. The composition of claim 30, wherein the detergent is selected
from the group consisting of sodium dodecyl sulfate, lithium
dodecyl sulfate, cetyltrimethylammoniumbromide, deoxycholic acid,
sodium lauroyl sarcosine, TRITON-X100, TWEEN 20, octyl
beta-D-glucoside, Nonidet P40, BRIJ 35P, and Sulfobetaine 14.
32. The composition of claim 25, wherein the pH is between about 4
and 7.5.
33. The composition of claim 25, wherein the chaotropic agent is
selected from the group consisting of guanidine hydrochloride,
guanidine thiocyanate, guanidine isothiocyanate, urea, sodium
acetate, an alkali perchlorate, and an alkali halogenide.
34. The composition of claim 25, wherein the chaotropic agent has a
concentration between about 0.5 M and 10 M.
35. A method for adsorbing a nucleic acid onto a solid phase, the
method comprising the steps of: providing a sample comprising the
nucleic acid, dissolving the sample in a liquid composition
comprising between about 10% and 75% tetraethylene glycol dimethyl
ether (TDE) by volume, an aqueous buffer, and a chaotropic agent,
contacting the liquid composition containing the dissolved sample
with a solid phase whereby the nucleic acid is adsorbed onto the
solid phase.
36. The method according to claim 35, wherein the nucleic acid
comprises 150 or more nucleotides and the composition comprises
between about 10% and 40% TDE by volume.
37. The method according to claim 35, wherein the nucleic acid
comprises less than 150 nucleotides and the composition comprises
between about 40% and 75% TDE by volume.
38. The method of claim 35, wherein the solid phase comprises a
porous or non-porous silica substrate.
39. The method of claim 35, wherein the solid phase comprises a
substrate selected from the group consisting of glass fibers and
quartz fibers.
40. The method of claim 35, wherein the solid phase comprises
magnetic particles with a silica surface.
41. The method of claim 35, further comprising the steps of:
separating the solid phase with the adsorbed nucleic acid from the
liquid composition, contacting the solid phase with the adsorbed
nucleic acid with a desorption solution, thereby desorbing the
nucleic acid from the solid phase and dissolving the nucleic acid
in the solution, separating the solution with the nucleic acid from
the solid phase, thereby purifying the nucleic acid, and
optionally, precipitating the nucleic acid from the solution and
isolating the precipitated nucleic acid, thereby further purifying
the nucleic acid.
42. The method of claim 41, further comprising the step of, after
separating the solid phase with the adsorbed nucleic acid from the
liquid composition, washing the solid phase with a washing solution
comprising water and TDE whereby the nucleic acid remains bound to
the solid phase.
43. The method of claim 42, wherein the washing solution
additionally comprises a salt.
44. A composition comprising tetraethylene glycol dimethyl ether
(TDE) and magnetic particles with a silica surface.
45. A method for purifying a nucleic acid with low molecular weight
comprising the steps of: providing a sample comprising the nucleic
acid with low molecular weight, dissolving the sample in a liquid
composition comprising an aqueous buffer, tetraethylene glycol
dimethyl ether (TDE) at a concentration between 10% and 75% by
volume, and a chaotropic agent, contacting the liquid composition
containing the dissolved sample with a solid phase whereby the
nucleic acid with low molecular weight is adsorbed onto the solid
phase, separating the solid phase with the adsorbed nucleic acid
with low molecular weight from the liquid composition, washing the
solid phase with a washing solution comprising an organic solvent
at a concentration between 40% and 100%, contacting the solid phase
with the adsorbed nucleic acid with low molecular weight with an
aqueous desorption solution containing solutes in a lower
concentration compared to the liquid composition, thereby desorbing
the nucleic acid with low molecular weight from the solid phase and
dissolving the nucleic acid with low molecular weight in the
desorption solution, separating the solution with the nucleic acid
with low molecular weight from the solid phase, thereby purifying
the nucleic acid with low molecular weight; and optionally
precipitating the nucleic acid with low molecular weight from the
solution and isolating the precipitated nucleic acid with low
molecular weight, thereby further purifying the nucleic acid with
low molecular weight, wherein the nucleic acid with low molecular
weight is selected from the group consisting of single-stranded
nucleic acids having a size between 10 and 150 bases and
double-stranded nucleic acids having a size between 5 and 75
bases.
46. The method of claim 45 wherein the concentration of TDE in the
composition is about 40% by volume.
47. A method for determining the presence of a nucleic acid in a
sample comprising the steps of: dissolving a sample containing the
nucleic acid in a liquid composition comprising, an aqueous buffer,
a chaotropic agent, and about 10% to about 75% tetraethylene glycol
dimethyl ether (TDE) by volume, whereby the sample is dissolved in
the liquid composition, contacting the liquid composition
containing the dissolved sample with a solid phase, whereby the
nucleic acid is adsorbed onto the solid phase, separating the solid
phase with the adsorbed nucleic acid from the liquid composition,
contacting the solid phase with the adsorbed nucleic acid with an
aqueous desorption solution containing solutes in a lower
concentration than the liquid composition, thereby desorbing the
nucleic acid from the solid phase and dissolving the nucleic acid
in the desorption solution, separating the solution with the
nucleic acid from the solid phase, thereby purifying the nucleic
acid, and detecting in the solution the presence of the nucleic
acid, thereby determining the presence of the nucleic acid.
48. The method of claim 47 wherein the nucleic acid is RNA or
DNA.
49. The method of claim 47 wherein the nucleic acid is RNA and the
detection step comprises: reverse transcribing the RNA to form a
cDNA, amplifying, by means of a polymerase chain reaction, the
cDNA, and detecting the presence of the cDNA, thereby determining
the presence of the nucleic acid.
50. A kit of parts comprising an instruction manual, packaging
material, containers, tetraethylene glycol dimethyl ether (TDE), a
concentrated stock solution of a buffer salt and a chaotropic agent
selected from the group consisting of guanidine hydrochloride,
guanidine thiocyanate, guanidine isothiocyanate, an alkali
perchlorate, and an alkali iodide, and chromatographic and
filtering material comprising a material with a surface capable of
interacting with the phosphate residues in the backbone of nucleic
acids.
51. A kit of parts comprising an instruction manual, packaging
material, containers, a suspension of silica-coated magnetic
particles in tetraethylene glycol dimethyl ether (TDE), and a
concentrated stock solution of a buffer salt and a chaotropic agent
selected from the group consisting of guanidine hydrochloride,
guanidine thiocyanate, guanidine isothiocyanate, an alkali
perchlorate, and an alkali iodide.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Ser. No.
12/481,807 filed Jun. 10, 2009, which is a continuation of
PCT/EP2007/010793 filed Dec. 11, 2007 and claims priority to EP
06025779.7 filed Dec. 13, 2006.
FIELD OF THE INVENTION
[0002] The present invention is directed to the purification of a
nucleic acid. Particularly, the invention is directed to methods of
adsorbing a nucleic acid present in an aqueous adsorption solution
to a solid substrate.
BACKGROUND OF THE INVENTION
[0003] Since the structure of DNA was deciphered by Watson &
Crick in 1953 (Watson, J. D. and Crick, F. H. C., Nature 171 (1953)
737-738 investigation and handling of nucleic acids becomes an
integral part of biochemistry molecular biology. Despite the
availability of a number of isolation methods an commercial kits
for performing such methods, new developments for fast and easy
isolation or purification of nucleic acids with high yield and
purity are still of major importance.
[0004] Nucleic acids are highly susceptible to enzymatic
degradation. In 1968 Cox described the chaotropic agent guanidine
HCl as an inhibitor of enzymatic nuclease activity (Cox, R. A.,
Methods Enzymol. 12B (1968) 120-129). Besides a strong denaturing
effect on proteins high concentrations of chaotropic agents also
mediate cell lysis. Therefore chaotropic agents, particularly
guanidine isothiocyanate, are widely in use for nucleic acid
isolation.
[0005] A first principle of nucleic acid isolation from a
biological sample uses an organic solvent, particularly phenol, for
the separation of nucleic acids from the remaining organic sample
components. The phenol extraction is followed by a salt
precipitation of the nucleic acid from an aqueous phase (Stallcup,
M. R. and Washington, L. D., J. Biol. Chem. 258 (1983) 2802-2807,
and Schmitt, M. E. et al., Nucl Acid Res 18 (1990) 3091-3092).
Although this method results in nucleic acids with high yield and
purity the major drawbacks are the use of poisonous reagents, the
time consuming and labor intensive workflow. Due to these
disadvantages automation of this isolation principle is not
amenable to automation, or only to a very limited extent.
[0006] Another principle of nucleic acid isolation makes use of
solid inorganic material, particularly silica, to which nucleic
acids are adsorbed from an aqueous liquid phase such as a lysate of
a biological sample. In 1979 Vogelstein and Gillespie described a
method for isolating nucleic acid from agarose gel slices by
binding nucleic acids to silica particles in presence of highly
concentrated sodium iodide (Vogelstein, B. and Gillespie, D., Proc.
Natl. Acad. Sci. USA 76 (1979) 615-619).
[0007] In addition it was found that the binding of nucleic acids
to the solid phase was increased by the addition of anionic or
cationic or neutral detergents, in particular TRITON-X100 (Union
Carbide Chemicals & Plastics Technology Corporation), sodium
dodecyl sulfate, NP40, and TWEEN 20 (ICI Americas Inc.).
[0008] Adsorption of a nucleic acid to the solid phase is usually
performed in the presence of a potent denaturant such as a
chaotropic agent (Boom, R., et al., J. Clin. Microbiol. 28 (1990)
495-503; U.S. Pat. No. 5,234,809). For the isolation process the
biological material is mixed with a solution containing the
denaturant. The resulting mix is brought into contact with the
solid phase material whereby nucleic acid molecules are bound to
the surface of the solid phase. Afterwards the solid material is
washed with solutions containing decreasing chaotropic salt
concentrations and increasing alcohol concentrations, in particular
ethanol, in order to further purify the bound nucleic acids from
other organic material and contaminating agents. In the last step
the solid material is brought into contact with a low salt solution
or water under alkaline pH in order to remove the bound nucleic
acid from the solid phase. The complete workflow comprises a sample
lysis step, a binding step, one or more washing steps, and an
elution (desorption) step.
[0009] The solid phase can be arranged in different conformations.
In a first design the solid phase is in fleece shape and embedded
in a plastic device. An example therefor is a micro spin column (EP
0 738 733). This design is preferentially used in workflows which
are performed manually. In a second design magnetic silica
particles are used as a solid phase (Bartl, K., et al., Clin. Chem.
Lab. Med. 36 (1998) 557-559). This design is preferentially used in
automated workflows.
[0010] A further improvement of this method was observed when
aliphatic alcohol (i.e. ethanol or isopropanol) or polyethylene
glycol is added to the solution at the binding step (U.S. Pat. No.
6,383,393).
[0011] U.S. Pat. No. 6,905,825 discloses addition of organic
solvents to the binding buffer. These organic solvents comprise the
aliphatic ethers ethylene glycol dimethyl ether, ethylene glycol
diethyl ether, propylene glycol dimethyl ether, propylene glycol
diethyl ether, diethylene glycol dimethyl ether, diethylene glycol
diethyl ether, tetrahydrofuran, and 1,4-dioxane, the aliphatic
esters propylene glycol monomethyl ether acetate, and ethyl
lactate, and the aliphatic ketones hydroxyacetone, acetone, and
methyl ethyl ketone.
[0012] US 2005/0079535 discloses the use of acetone, acetylacetone,
acetonitrile, dimethylsulfoxide, diethylketone, methylethylketone,
methylpropylketone, isobutylmethylketone, gamma-butyrolactone,
gamma-valerolactone, propylene carbonate, and
N-methyl-2-pyrrolidone as well as the use of the cyclic diether
dioxane in the binding buffer, in order to adsorb a nucleic acid to
a solid phase such as silica.
[0013] US 2006/0166368 A1 discloses a liquid solution comprising
Tetraethylene glycol dimethyl ether (TED) in a buffer containing
(1) a water-miscible organic component such as methanol, ethanol,
1- or 2-propanol, ethylene glycol, propylene glycol, glycerol,
acetonitrile, dimethyl sulfoxide, formamide, dimethylformamide,
diglyme, triglyme, or tetraglyme, at a concentration of up to 50%
(on a volume basis), (2) an acid component such as acetic acid,
formic acid, lactic acid, propionic acid, phosphoric acid,
trichloroacetic acid, trifluoroacetic acid, citric acid, oxalic
acid, or hydrochloric acid, at a concentration of up to 20% (on a
volume basis), (3) a buffer such as sodium phosphate, sodium
acetate, sodium formate, or sodium citrate, at a pH of from 1 to 6
and a concentration of from 5 to 200 mM, and (4) a detergent such
as sodium dodecyl sulfate, TRITON X-100, SB3-10, and TWEEN 20) at a
concentration of from 0.005% to 1% (on a weightivolume basis). The
liquid solution is used as a solvent of certain dyes which serve as
selective labels in protein biochemistry and particularly for
methods of protein detection.
[0014] The chemical properties of the reagents used in the nucleic
acid isolation/purification process determines the quality of the
nucleic acid (yield, purity and size) as well as their performance
in down-stream workflows, including polymerase or reverse
transcriptase based enzymatic reactions (Mullis, K. and Faloona F.
A., Methods Enzymol. 155 (1987) 335-350). Furthermore, additional
properties of the reagents like toxicity, as well as physical and
chemical aspects like flash point and vapor pressure are of major
importance.
[0015] Recently the analysis of small RNA molecules with 15 to 200
nucleotides gained strong interest. Especially microRNA (miRNA) and
small interfering RNA (siRNA), which have a strong effect on the
translation of specific messenger RNAs are investigated. Also for
other kinds of small RNA like tRNA, 5S and 5.8S rRNA, as well as
small nuclear RNA (snRNA) and small nucleolar RNA (snoRNA) involved
in mRNA and rRNA processing selective isolation procedures are
required.
[0016] Methods for isolating such small RNA molecules selectively
have been described in US 2005/0059024 by Conrad and in WO
2005/012487 by Madden et al. In order to isolate small RNA
molecules in both methods high concentrations of alcohol in the
order of 70% is needed to efficiently bind the small RNA to a solid
support. This increases the volume of a sample to be analysed
considerably. If a sample is to be adsorbed onto a solid support
such as a commonly available spin column, the amount of sample that
can be applied in one centrifugation run is limited to a small
volume. A higher amount of a more diluted sample can be applied
only in two consecutive centrifugation steps on the same column,
thereby increasing the number of handling steps and processing
time. It is therefore another need to improve the binding of small
DNA and RNA molecules without the need for diluting the sample with
high amounts of alcohol.
[0017] In view of the disadvantages of the state of the art it was
an object of the present invention to provide an alternative
organic compound to promote the adsorption of a nucleic acid to a
solid substrate.
[0018] The inventors have surprisingly found that adsorption of a
nucleic acid to a solid phase is effectively accomplished when
tetraethylene glycol dimethyl ether (TDE) is used in the adsorption
solution.
SUMMARY OF THE INVENTION
[0019] Therefore, a first aspect of the invention is a composition
comprising tetraethylene glycol dimethyl ether (TDE;
C.sub.10H.sub.22O.sub.5, MW: 178, CAS: 143-24-8), an aqueous
buffer, and a chaotropic agent. Another aspect of the invention is
the use of TDE for adsorbing a nucleic acid onto a solid phase.
Yet, a further aspect of the invention is a method of adsorbing a
nucleic acid from a sample onto a solid phase comprising the steps
of (a) providing the nucleic acid in a sample, whereby the sample
is dissolved in a liquid composition comprising TDE, an aqueous
buffer, and a chaotropic agent; followed by (b) providing the solid
phase and contacting the liquid composition of step (a) with the
solid phase, thereby adsorbing the nucleic acid on the solid phase.
Yet, a further aspect of the invention is a method for the
purification of a nucleic acid from a lysed sample, comprising the
steps of: (a) providing the nucleic acid in a sample, whereby the
sample is dissolved in a liquid composition comprising TDE, an
aqueous buffer, and a chaotropic agent; followed by (b) providing
the solid phase and contacting the liquid composition of step (a)
with the solid phase, thereby adsorbing the nucleic acid onto the
solid phase; followed by (c) separating the solid phase with the
adsorbed nucleic acid from the liquid phase; (d) optionally washing
with a washing solution the solid phase with the adsorbed nucleic
acid; followed by (e) contacting the solid phase with the adsorbed
nucleic acid with a desorption solution which preferably contains
solutes in a lower concentration compared to the composition of
step (a), thereby desorbing the nucleic acid from the solid phase
and dissolving the nucleic acid in the solution; followed by (f)
separating the solution with the nucleic acid from the solid phase,
thereby purifying the nucleic acid; and optionally (g)
precipitating the nucleic acid from the solution of step (f) and
isolating the precipitated nucleic acid, thereby further purifying
the nucleic acid. Yet, a further aspect of the invention is a
composition comprising TDE and magnetic particles with a silica
surface. Yet, a further aspect of the invention is a method for
purifying a nucleic acid with low molecular weight comprising the
steps of (a) providing the nucleic acid in a lysed sample, whereby
the sample is dissolved in a liquid composition comprising an
aqueous buffer, TDE at a concentration between 10% and 75%,
measured as volume by volume, a detergent and a chaotopic agent;
followed by (b) providing a solid phase and contacting the liquid
composition of step (a) with the solid phase; followed by (c)
separating the solid phase with the adsorbed nucleic acid from the
liquid phase; (d) washing with a washing solution the solid phase
of step (c), whereby the washing solution comprises an organic
solvent at a concentration of between 40% and about 100%; followed
by (e) contacting the solid phase with the adsorbed nucleic acid
with an aqueous desorption solution containing solutes in a lower
concentration compared to the composition of step (a), thereby
desorbing the nucleic acid from the solid phase and dissolving the
nucleic acid in the solution; followed by (f) separating the
solution with the nucleic acid from the solid phase, thereby
purifying the nucleic acid; and optionally (g) precipitating the
nucleic acid from the solution of step (f) and isolating the
precipitated nucleic acid, thereby further purifying the nucleic
acid. Yet, a further aspect of the invention is a method comprising
the steps of (a) providing the nucleic acid in a lysed sample,
whereby the sample is dissolved in a liquid composition comprising
an aqueous buffer, TDE at a concentration between 5% and 30%,
measured as volume by volume, a detergent and a chaotropic agent;
followed by (b) providing a first solid phase, contacting the
liquid composition of step (a) with the first solid phase, and
separating the liquid phase from the first solid phase; followed by
(c) mixing an additional amount of TDE with the liquid phase of
step (b), thereby adjusting the concentration of TDE in the liquid
phase of step (b) to between 20% and 70%, measured as volume by
volume, whereby the initial concentration of TDE in the liquid
phase is increased by a factor of 1.3 or more; followed by (d)
providing a second solid phase and contacting the liquid
composition of step (a) with the second solid phase; followed by
(e) washing with a washing solution the second solid phase of step
(d), whereby the washing solution comprises an organic solvent at a
concentration of between 50% and 100%; followed by (f) contacting
the second solid phase of step (e) with an aqueous desorption
solution containing solutes in a lower concentration compared to
the composition of step (a), thereby desorbing the nucleic acid
from the solid phase and dissolving the nucleic acid in the
solution; followed by (g) separating the solution with the nucleic
acid from the solid phase, thereby purifying the nucleic acid; and
optionally (h) precipitating the nucleic acid from the solution of
step (f) and isolating the precipitated nucleic acid, thereby
further purifying the nucleic acid. Yet, a further aspect of the
invention is a kit of parts, comprising packaging material,
containers, and (a) TDE, (b) a concentrated stock solution of a
buffer salt and a chaotropic agent is selected from the group
consisting of guanidine hydrochloride, guanidine thiocyanate,
guanidine isothiocyanate, urea, sodium acetate, an alkali
perchlorate, an alkali halogenide, and mixtures thereof; and (c)
chromatographic and filtering material comprising a material with a
surface capable of interacting with the phosphate residues in the
backbone of nucleic acids. Yet, a further aspect of the invention
is a kit of parts, comprising packaging material, containers, and
(a) a suspension of silica-coated magnetic particles in TDE; and
(b) a concentrated stock solution of a buffer salt and a chaotropic
agent is selected from the group consisting of guanidine
hydrochloride, guanidine thiocyanate, guanidine isothiocyanate,
urea, sodium acetate, an alkali perchlorate, and an alkali
halogenide. Yet, a further aspect of the invention is a method for
determining the presence of a nucleic acid in a sample, comprising
the steps of: (a) forming a composition containing (i) the sample,
(ii) an aqueous buffer, (iii) a chaotropic agent, (iv) TDE, whereby
the sample is dissolved in the liquid composition; (b) contacting
the composition of step (a) with a solid phase, thereby adsorbing
the nucleic acid onto the solid phase; (c) separating the solid
phase with the adsorbed nucleic acid from the liquid phase; (d)
optionally washing with a washing solution the solid phase with the
adsorbed nucleic acid; followed by (e) contacting the solid phase
with the adsorbed nucleic acid with an aqueous desorption solution
containing solutes in a lower concentration compared to the
composition of step (a), thereby desorbing the nucleic acid from
the solid phase and dissolving the nucleic acid in the solution;
followed by (f) separating the solution with the nucleic acid from
the solid phase, thereby purifying the nucleic acid; and (g)
detecting in the solution of step (f) the presence of the nucleic
acid, thereby determining the presence of the nucleic acid.
DESCRIPTION OF THE FIGURES
[0020] FIG. 1 Total nucleic acids isolated from EDTA whole blood
with different binding additives (see Example 1) after 1% agarose
gel electrophoresis and ethidium bromide staining. Similar volumes
of eluate were applied. Each individual lane is numbered. [0021]
lanes 1 and 18 show the size standard VI (catalogue no.
10236250001, Roche Diagnostics GmbH, Mannheim, Germany) [0022]
lanes 2 and 19 show the size standard II (catalogue no.
10236250001, Roche Diagnostics GmbH, Mannheim, Germany) [0023] lane
3: tetraethylene glycol dimethyl ether (TDE) [0024] lane 4:
glycerol formal [0025] lane 5: diethylene glycol diethyl ether
[0026] lane 6: methyl ethyl ketone [0027] lane 7: propylene glycol
dimethylether (dimethoxypropane) [0028] lane 8: ethylene glycol
diethylether (diethoxyethane) [0029] lane 9: propylene glycol
monomethyl ether acetate [0030] lane 10: tetrahydrofurane [0031]
lane 11: polyethylene glycol 1000 [0032] lane 12: 1,3 dioxolane
[0033] lane 13: hydroxyacetone [0034] lane 14: ethanol [0035] lane
15: isopropanol [0036] lane 16: ethyllactat [0037] lane 17:
acetone
[0038] FIG. 2 Effect of combinatorial use of TDE and glycerol
formal for the purification of total nucleic acids from K562 cells
(Example 2).
[0039] FIG. 3 Nucleic acids isolated/purified with ethanol and TDE
as additives in the washing buffer. Following desorption, equal
volumes of eluate were subjected to gel electrophoresis in 1%
agarose. The gel was stained with ethidium bromide. For further
details see Examples 3 (referring to lanes 1-6) and 4. (referring
to lanes 7-13). [0040] lane VI: DNA size standard VI (catalogue no.
10236250001, Roche Diagnostics GmbH, Mannheim, Germany) [0041] lane
1-3: nucleic acid preparation from k562 cells using ethanol in
washing buffers [0042] lanes 4-6: nucleic acid preparation from
k562 cells using TDE in washing buffers [0043] lane 7-9: recovered
DNA fragments using ethanol in washing buffers [0044] lane 10-12:
recovered DNA fragments using TDE in washing buffers [0045] lane
13: DNA fragments, untreated solution [0046] lane II: DNA size
standard II (catalogue no. 10236250001, Roche Diagnostics GmbH,
Mannheim, Germany)
[0047] FIG. 4 An eluate aliquot containing 500 ng of isolated
nucleic acids was subjected to electrophoresis in an 1% agarose gel
which was stained afterwards with ethidium bromide. [0048] lane 1:
size standard VI (catalogue no. 11062590001, Roche Diagnostics
GmbH, Mannheim, Germany) [0049] lane 2: size standard II (catalogue
no. 10236250001, Roche Diagnostics GmbH, Mannheim, Germany) [0050]
lanes 3-6: magnetic particles supplied as suspension in TDE [0051]
lanes 7-10: magnetic particles supplied as suspension in
diethyleneglycol diethyl ether [0052] lanes 11-14 magnetic
particles supplied as suspension in magnetic particles supplied as
suspension in isopropanol [0053] lane 15: size standard VI
(catalogue no. 11062590001, Roche Diagnostics GmbH, Mannheim,
Germany) [0054] lane 16: size standard II (catalogue no.
10236250001, Roche Diagnostics GmbH, Mannheim, Germany)
[0055] FIG. 5 Binding of low molecular weight nucleic acid
molecules of various sizes using different TDE concentrations in
the binding step. [0056] lane 1: 100 ng pre-purified miRNA16,
untreated [0057] lane 2: 50 ng pre-purified miRNA16, untreated
[0058] lane 3: adsorption in TDE 35% [v/v] [0059] lane 4:
adsorption in TDE 40% [v/v] [0060] lane 5: adsorption in TDE 45%
[v/v] [0061] lane 6: adsorption in TDE 50% [v/v] [0062] lane 7:
adsorption in TDE 55% [v/v]
[0063] FIG. 6 A Purification of low molecular weight nucleic acid
molecules using two consecutive separations with spin columns:
Different concentrations of TDE (as indicated below) were used for
adsorption to the first column. Lanes 4-10 indicate the nucleic
acids separated from the adsorption solution with the first column
and eluted therefrom. [0064] lane 1: 100 ng pre-purified miRNA16,
untreated [0065] lane 2: 50 ng pre-purified miRNA16, untreated
[0066] lane 3: 25 ng pre-purified miRNA16, untreated [0067] lane 4:
0% [v/v] TDE [0068] lane 5: 5% [v/v] TDE [0069] lane 6: 10% [v/v]
TDE [0070] lane 7: 15% [v/v] TDE [0071] lane 8: 20% [v/v] TDE
[0072] lane 9: 25% [v/v] TDE [0073] lane 10: 30% [v/v] TDE
[0074] FIG. 6 B Low molecular weight nucleic acid molecules were
adsorbed to a second spin column from the flow-through of the first
spin column using a final TDE concentration of 40% [v/v] in each
case. Lanes 4-10 indicate the purified nucleic acids of low
molecular weight which were eluted from the respective second
column subsequent to the separation with a given TDE concentration
for the first column. [0075] lane 1: 100 ng miRNA16 [0076] lane 2:
50 ng miRNA16 [0077] lane 3: 25 ng miRNA16 [0078] lane 4: eluate
from second column after adsorption with 0% [v/v] TDE on the first
column [0079] lane 5: eluate from second column after adsorption
with 5% [v/v] TDE on the first column [0080] lane 6: eluate from
second column after adsorption with 10% [v/v] TDE on the first
column [0081] lane 7: eluate from second column after adsorption
with 15% [v/v] TDE on the first column [0082] lane 8: eluate from
second column after adsorption with 20% [v/v] TDE on the first
column [0083] lane 9: eluate from second column after adsorption
with 25% [v/v] TDE on the first column [0084] lane 10: eluate from
second column after adsorption with 30% [v/v] TDE on the first
column
[0085] FIG. 7 Detection of mir17a in isolates from mouse tissues
using a specific RT-PCR assay and Light Cycler amplification.
[0086] Total Nucleic acids and small nucleic acid molecules of
sizes smaller than 150 nucleotides (i.e. microRNA) were isolated
from liver and kidney tissue with the one column or the two column
protocol, respectively, and detected using a Q-RT-PCR (quantitative
PCR following reverse transcription). The table shows at which
amplification cycle the miRNA 17a was detected.
[0087] FIG. 8 Optimizing binding enhancer concentration on column 1
for miRNA-purification from tissue [0088] Different concentrations
of TDE (as indicated below) were used for adsorption to the first
column. Lanes 8-11 indicate the nucleic acids separated from the
adsorption solution with the first column at the indicated TDE
concentrations and eluted therefrom. Small nucleic acid molecules
were adsorbed to a second spin column from the flow-through of the
first spin column using a final TDE concentration of 40% [v/v] in
each case. Lanes 4-7 indicate the purified nucleic acids of low
molecular weight which were eluted from the respective second
column subsequent to the separation with a given TDE concentration
for the first column. [0089] lane 1: 100 ng synthetic miRNA16
[0090] lane 2: 50 ng synthetic miRNA16 [0091] lane 3: 25 ng
synthetic miRNA16 [0092] lane 4: Eluate from second column after
adsorption with 10% [v/v] TDE on the first column [0093] lane 5:
Eluate from second column after adsorption with 15% [v/v] TDE on
the first column [0094] lane 6: Eluate from second column after
adsorption with 20% [v/v] TDE on the first column [0095] lane 7:
Eluate from second column after adsorption with 25% [v/v] TDE on
the first column [0096] lane 8: 10% [v/v] TDE [0097] lane 9: 15%
[v/v] TDE [0098] lane 10: 20% [v/v] TDE [0099] lane 11: 25% [v/v]
TDE [0100] lane 12: 50 ng synthetic miRNA16
DETAILED DESCRIPTION OF THE INVENTION
[0101] The present invention provides new compositions and methods
for the purification of nucleic acids. Certain terms are used with
particular meaning, or are defined for the first time, in this
description of the present invention. For the purposes of the
present invention, the terms used are defined by their art-accepted
definitions, when such exist, except that when those definitions
conflict or partially conflict with the definitions set forth
below. In the event of a conflict in definition, the meaning of a
terms is first defined by any of the definitions set forth
below.
[0102] The term "comprising" is used in the description of the
invention and in the claims to mean "including, but not necessarily
limited to".
[0103] The articles "a" and "an" are used herein to refer to one or
to more than one (i.e. to at least one) of the grammatical object
of the article. By way of example, "a compound" means one compound
or more than one compound.
[0104] When designating a range of numerical values such as a
concentration range, the range is indicated by the word "between",
followed by a first value n1 and a second value n2. The lower
boundary of the designated range is understood as being the value
equal to or higher than the first value. The higher boundary of the
designated range is understood as being the value equal to or lower
than the second value". Thus, a value x the designated range is
given by n1.ltoreq.x.ltoreq.n2.
[0105] Further, it is understood that the term "about" in
combination with a numerical value n indicates a value x in the
interval given by the numerical value .+-.5% of the value, i.e.
n-0.05*n.ltoreq.x.ltoreq.n+0.05*n. In case the term "about" in
combination with a numerical value n describes a preferred
embodiment of the invention, the value of n is most preferred, if
not indicated otherwise.
[0106] The term "water-miscible" indicates that at room temperature
and normal atmospheric pressure a water-miscible compound can be
dissolved in water at a ratio equal or greater than 1% (percent)
volume by volume, to form a homogeneous aqueous liquid phase. An
unlimited water-miscible compound, when mixed with water, forms a
homogeneous liquid phase at any water/compound ratio. In case the
solubility of the water-miscible compound is limited in water, the
compound may form a separate phase in addition to the aqueous
phase. The compound may also form an emulsion, especially in the
presence of a surfactant.
[0107] A compound or a composition is a "liquid" if at room
temperature and normal atmospheric pressure the compound is in the
"liquid" state and forms a liquid phase.
[0108] The terms "aqueous", "aqueous" phase and "aqueous" solution
describe a liquid phase of which the solvent portion comprises
water. However, other solvents such as a water-miscible organic
solvent can be present in the solvent portion, too. In view of the
presence of other solvents a solution is considered "aqueous" when
between 30% and 100%, measured as volume by volume [v/v], of the
solvent portion is water.
[0109] A "chaotropic agent" is a compound which weakens hydrophobic
interactions of the components in an aqueous solution. Certain ions
in water will tend to increase hydrophobic interactions, while
other ions will decrease hydrophobic interactions. Which ions have
a tendency to which effect is described by what is called a
Hofmeister series. The series is as follows:
Cations:
[0110]
NH.sub.4.sup.+>Rb.sup.+>K.sup.+>Na.sup.+>Cs.sup.+>L-
i.sup.+>Mg.sup.2+>Ca.sup.2+>Ba.sup.2+>guanidine
Anions:
[0111]
PO.sub.4.sup.3->SO.sub.4.sup.2->HPO.sub.4.sup.2->acetate&-
gt;citrate>tartrate>Cl.sup.->Br.sup.->NO.sub.3.sup.->ClO.su-
b.3.sup.->ClO.sub.4.sup.->I.sup.->SCN.sup.-
[0112] Ions on the left are said to be "kosmotropic" and increase
the strength of hydrophobic interactions and thus will precipitate
or "salt out" proteins at a high concentrations. Ions on the right
are "chaotropic" and tend to weaken hydrophobic interactions. The
Hofmeister series explains why a guanidine salt is a protein
denaturant. It weakens hydrophobic interactions causing proteins to
denature. In addition to the above, there are also chaotropic
compounds which are non-ionic. An example therefor is urea.
[0113] In the present document it is understood that the term "a
nucleic acid" denotes at least one nucleic acid. Furthermore, the
term "a nucleic acid" also may indicate a mixture of nucleic acids.
The term "nucleic acid" encompasses RNA, DNA, or both.
[0114] The term "solid phase" to which a nucleic acid is adsorbed
is understood as being a substrate which is insoluble in the
compositions according to the invention. A preferred solid phase is
a substrate with a surface capable of interacting with the
phosphate groups of the backbone of nucleic acids. The solid phase
may be in the form of porous or non-porous particles, powdered
particles, or fibers. A solid phase consisting of fleece material
which comprises a plurality of non-woven fibers is also
encompassed. Preferred solid phases consist of glass. Preferred
solid phases are porous or non-porous mineral substrates such as
silica, quartz, celites or other materials with oxidic surfaces
(including, e.g. zirconium oxide, aluminum oxide, and other metal
oxides) or mixtures thereof. Also, the term "solid phase"
encompasses magnetically attractable particles coated with silica,
glass, quartz, or celites. Further, it is understood that a
substrate in the form of "powder" or "powdered" material refers to
finely divided material which, when dispersed in a liquid
composition according to the invention, produces a suspension. The
term "powder" or "powdered" material is intended to include
tablets, in which the powdered material has been aggregated, but
still yields a suspension when combined with a liquid phase.
[0115] The term "silica" as used within this application denotes
materials which are mainly build up of silicon and oxygen. These
materials comprise silica, silicon dioxide, silica gel, fumed
silica gel, diatomaceous earth, celite, talc, quartz, glass, glass
particles including all different shapes of these materials. Glass
particles, for example, may comprise particles of crystalline
silica, soda-lime glasses, borosilicate glasses, and fibrous,
non-woven glass.
[0116] The term "magnetic particle" denotes a particle with
paramagnetic or superparamagnetic properties. That is to say, the
particle is magnetically displaceable but does not retain any
magnetisation in the absence of an externally applied magnetic
field.
[0117] The term "sample" (or "sample material") as used herein
refers to a complex sample, more preferred a biological sample. A
complex sample may contain a plurality of organic and inorganic
compounds which are desired to be separated from the nucleic acid.
The term "sample" also encompasses an aqueous solution containing
nucleic acids derived from other origins, e.g. from chemical or
enzymatic reaction mixtures, or from a previous purification of
biological sample material. The term biological sample, from which
nucleic acids are purified, encompasses samples comprising viruses
or bacterial cells, as well as isolated cells from multicellular
organisms such as human and animal cells as well as tissues and
cell cultures. Particularly, the sample can contain leucocytes, and
other immunologically active cells, chemical compounds with a low
and/or a high molecular weight such as haptens, antigens,
antibodies and nucleic acids. The sample can be whole blood, blood
serum, blood plasma, cerebral fluid, sputum, stool, biopsy
specimens, bone marrow, oral rinses, tissues, urine or mixtures
thereof. The present invention also encompasses biological samples
such as a fluid from the human or animal body; preferably the
biological sample is blood, blood plasma, blood serum or urine. The
blood plasma is preferably EDTA, heparin or citrate blood plasma.
In an embodiment of the invention the biological sample comprises
bacterial cells, eukaryotic cells, viruses or mixtures thereof. A
biological sample as exemplified above, preferably in a processed
form such as a lysate, can be part of the composition from which
the (target) nucleic acid is adsorbed to the substrate. Also
encompassed by the term "biological sample" are cells from plants,
and fungi as well as single cell organisms.
[0118] A preferred sample according to the invention is a lysate. A
"lysate" or a "lysed sample" can be obtained from a complex sample
and/or biological sample material comprising tissue, cells,
bacteria or viruses, whereby the structural integrity of the
material is disrupted. To release the contents of cells, tissue or,
more generally, from the particles which make up a biological
sample, the material may be treated with enzymes or with chemicals
to dissolve, degrade or denature the cellular walls and cellular
membranes of such organisms. This process is encompassed by the
term "lysis". It is common to use chaotropic agents such as a
guanidine salt and/or anionic, cationic, zwitterionic or non-ionic
detergent when nucleic acids are set free in the lysis process. It
is also an advantage to use proteases which rapidly degrade enzymes
with nucleolytic activity and other unwanted proteins. In case
there remains particulate, i.e. undissolved matter of the sample
material following the lysis process, the particulate matter is
usually separated from the lysate to result in a cleared lysate.
This can be done, e.g., by way of filtering or centrifugation. In
such a case the cleared lysate is processed further, e.g. by a
method according to the invention. Thus, the term "lysed sample"
encompasses a cleared lysate.
[0119] Nucleic acids which are set free can be purified by way of
binding (adsorbing) to a solid phase, washing said solid phase with
the bound nucleic acids and releasing, i.e. desorbing said nucleic
acids from said mineral support.
[0120] Hazardous substances used as binding enhancers often bear
environmental risks and cause high costs for waste management.
Their use can be restricted based on the required technical and/or
operational safety measures to be taken. The hazardous potential of
buffers used in the isolation/purification of nucleic acids is
chiefly influenced by the choice of the organic compound which
promotes adsorption of the nucleic acid to the solid phase. With
respect to the environmental burden it is desired to reduce the use
of toxic or harmful agents as far as possible. Also, the flash
point of a flammable organic compound, that is the lowest
temperature at which it can form an ignitable mixture with oxygen,
is desired to be high. This parameter particularly influences the
costs of production of kits for nucleic acid purification. An
organic compound with a flash point below room temperature has to
be handled in specially equipped production facilities which
prevent the development of explosive vapor. In addition,
restrictions apply to the transport of such organic compounds. A
low flash point is usually correlated with a high vapor pressure.
As a consequence, certain organic compounds, particularly lower
alcohols, tend to evaporate from solutions and therefore lead to
variations in concentration over time. This effect also influences
stability during storage as well as the handling of liquids with a
high vapor pressure in an automated pipetting instrument. Avoiding
substances with low flash points and a tendency to evaporate in the
isolation/purification process would make the production of
solutions for the purification of nucleic acids simpler and more
economical. In addition, compounds with a low vapor pressure are
desired as they increase the utility of nucleic acid isolation kits
by eliminating a major source of pipetting error, thereby
increasing the reliability of such kits.
[0121] The inventors have surprisingly found that adsorption of a
nucleic acid to a solid phase is effectively accomplished when
tetraethylene glycol dimethyl ether (TDE; C.sub.10H.sub.22O.sub.5,
MW: 178, CAS: 143-24-8) is used in the adsorption solution. TDE,
like butyl diglyme, is a very safe and effective solvent for a wide
ranges of uses. TDE boils at 275.degree. C. and has a flash point
of 141.degree. C. TDE is completely water soluble. Table 1 provides
a comparison of TDE with some organic compounds which can also be
used as additives for adsorbing a nucleic acid to a solid
phase.
TABLE-US-00001 TABLE 1 hazard vapor clas- flash pres- sifi- point
sure cation Aliphatic Ether Ethylene Glycol Dimethyl Ether
-2.degree. C. 48 F, T Ethylene Glycol Diethyl Ether 35.degree. C.
9.4 Xi Propylene Glycol Dimethyl Ether 1.degree. C. 40 F Diethylene
Glycol Dimethyl Ether 56.degree. C. 3 T Diethylene Glycol Diethyl
Ether 82.degree. C. 0.5 Xi Tetrahydrofuran -24.degree. C. 143 F, Xi
1,4-Dioxane 12.degree. C. 27 F, Xn Tetraethylene Glycol Dimethyl
Ether (TDE) 141.degree. C. <0.01 Aliphatic Polyether Polyethylen
Glycol 1000 n/a n/a Aliphatic Ester Propylene Glycol Monomethyl
Ether Acetate 43.degree. C. 3.7 Xi Ethyl Lactate 52.degree. C. 2 Xi
Acetal Glycerolformal 93.degree. C. n/a 1,3 Dioxolane -3.degree. C.
70 F, Xi 1,3 Dioxane 5.degree. C. n/a F, Xn Aliphatic Ketone
Acetone -15.degree. C. 184 F, Xi Metyl Ethyl Ketone -7.degree. C.
71 F, Xi Hydroxyacetone 56.degree. C. 5.6 Aliphatic Alcohol
Isopropanol 12.degree. C. 33 F, Xi Ethanol 13.degree. C. 44.6 F
[0122] Hazard classifications are given as generally known: "F"
(=flammable), T (=toxic), Xi (=irritant)
[0123] Flash point, vapor pressure as well as information about
hazard classification was obtained from the material safety data
available from a suppliers' internet pages (Sigma Aldrich).
[0124] By virtue of its high flash point the use of TDE for
adsorbing a nucleic acid to a solid support reduces a great deal of
hazardous potential. At the same time, a superior performance in
the nucleic acid isolation process was observed compared with
additives known so far. The present invention increases the
convenience and reduces the costs for nucleic acid
isolation/purification for the producer of nucleic acid isolation
kits as well as for the user of such kits. The reduced use of toxic
or harmful substances lowers the environmental risks. The reduction
or even replacement of flammable substances lowers production and
shipping costs. Increased convenience is achieved due to the fact
that the end user of a kit can be provided with ready-made kits
without the need to add components (e.g. ethanol) to a buffer
component of the kit. Also, lesser evaporation in TDE-containing
buffers is observed, thereby making automated liquid handling more
reliable.
[0125] According to the invention, the binding of a nucleic acid to
a solid phase can be performed with a composition comprising
tetraethylene glycol dimethyl ether (TDE), an aqueous buffer, and a
chaotropic agent with the exception of acetate at a pH of from 1 to
6 and a concentration of from 5 to 200 mM.
[0126] According to the invention, the preferred concentration of
TDE in the composition is between 10% and 75%, measured as volume
by volume, also referred to as [v/v]. Even more preferred, the
concentration of TDE is between 20% [v/v] and 55% [v/v]. Even more
preferred, the concentration of TDE is between 30% [v/v] and 45%
[v/v]. Most preferred, the TDE concentration in the composition is
about 40% [v/v].
[0127] According to the invention, addition of a further
water-miscible liquid organic solvent as an additive has proved to
be advantageous for the process of adsorbing the nucleic acid to
the solid phase. A preferred additive is a C1-5 aliphatic alcohol.
A very much preferred aliphatic alcohol is ethanol or isopropanol.
However, such compounds are flammable and vaporize rather easily.
Thus, more preferred is a liquid water-miscible acetal or ketal
which have a reduced tendency to evaporate. Such carbonyl
derivatives are characterized by their stability and lack of
reactivity in neutral to strongly basic environments. This is
particularly the case for cyclic acetals or ketals which are formed
by reaction of diols with aldehydes or ketones, respectively. Thus,
very much preferred, the composition according to the invention
additionally comprises a compound selected from the group
consisting of 1,3-dioxolan, 1,3-dioxan, 5-hydroxy-1,3-dioxane and
4-hydroxymethyl-1,3-dioxolane, and a mixture thereof. A mixture of
5-hydroxy-1,3-dioxane and 4-hydroxymethyl-1,3-dioxolane is also
known as "glycerol formal". Most preferred, the composition
according to the invention additionally comprises glycerol formal.
However, one or more of the above acetals can also be used as a
preferred additive in a composition comprising magnetic particles
or in a washing buffer (see below).
[0128] The preferred pH value of the composition according to the
invention is between 4 and 7.5. Even more preferred, the pH value
is between 5.5 and 7.5. It is obvious for the artisan to produce
suitable aqueous buffered solutions. In order to stabilize the pH
value, a buffer is present in the composition according to the
invention. Buffer systems which suitable for molecular biology
purposes may be found e.g. in Sambrook, Fritsch & Maniatis,
Molecular Cloning, A Laboratory Manual, 3rd edition, CSHL Press,
2001. Preferred buffer substances are acetic acid, citric acid,
phosphoric acid, N-(Carbamoylmethyl)-2-aminoethanesulfonic acid
(ACES), N-(2-Acetamido)iminodiacetic acid (ADA),
N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES),
Tris-(hydroxymethyl)-aminomethane (TRIS),
2-Bis(2-hydroxyethyl)amino-2-(hydroxymethyl)-1,3-propanediol
(BIS-TRIS), N-(2-Hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid)
(HEPES), 2-(N-Morpholino)ethanesulfonic acid (MES),
3-(N-Morpholino)propanesulfonic acid (MOPS),
3-(N-Morpholinyl)-2-hydroxypropanesulfonic acid (MOPSO),
piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), salts thereof,
or other suitable substances.
[0129] In detail, the procedure for binding a nucleic acid (also
referred to as target nucleic acid) to a solid phase such as, e.g.,
glass particles can be described as follows. It is preferably
performed in the presence of a chaotropic agent with a
concentration of between 0.5 M and 10 M, and preferably between 1 M
and 5 M. Most preferred, the concentration of the chaotropic agent
is between 2 M and 4 M. A preferred chaotropic agent is selected
from the group consisting of a guanidine salt such as guanidine
hydrochloride, guanidine thiocyanate and guanidine isothiocyanate,
furthermore urea, an alkali acetate salt such as sodium acetate and
potassium acetate, furthermore an alkali perchlorate, an alkali
iodide, lithium chloride, potassium chloride, and sodium chloride.
Mixtures comprising one or more of the listed agents are also
possible.
[0130] When lysing a biological sample in order to set free the
nucleic acids or when binding the nucleic acid to the solid phase
it is further preferred to use a detergent in the procedures, that
is to say an anionic, cationic, zwitterionic or non-ionic
detergent. Such detergents are well known to the person skilled in
the art. Generally, a "detergent" is a surface active agent, also
known as a surfactant. A detergent is capable of lowering the
surface tension of the medium in which it is dissolved, and/or the
interfacial tension with other phases, and, accordingly, is
positively adsorbed at the liquid/vapor and/or at other interfaces.
Thus, detergents are amphipathic molecules with polar (water
soluble) and nonpolar (hydrophobic) domains. They are capable of
binding to hydrophobic molecules or molecular domains to confer
water solubility. Depending on its ionic characteristics, a
detergent can be categorized as an ionic detergent, a non-ionic
detergent, and a zwitterionic detergent. Ionic detergents can be
further classified into either anionic detergents such as SDS
(sodium dodecyl sulfate) LiDS (lithium dodecyl sulfate), sodium
lauroyl sarcosine, 1-octanesulfonic acid, cholic acid, or
deoxycholic acid, and cationic detergents such as cetyl
trimethylammonium bromide (CTAB),
trimethyl(tetradecyl)ammoniumbromide, lauryl trimethylammonium
chloride (LTAB), lauryl trimethylammonium schloride (LTAC) or
stearyl trimethylammonium chloride (STAC). Thus, these are usually
highly protein denaturant. Non-ionic detergents such as Nonidet
P40, TWEEN 20, TRITON X-100, BRIJ 35 P (ICI Americas Inc.),
saponin, N,N-dimethyldodecylammine-N-oxide,
N,N-dimethyldodecylamine-N oxide, or nonaethylene glycol
monododecyl ether are usually less protein denaturant. This is also
true for zwitterionic detergents such as
3-(N,N-dimethylpalmitylammonio) propanesulfonate,
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS),
3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate
(CHAPSO) or Sulphobetaine 14. Zwitterionic compounds, also known as
zwitterions, inner salts or dipolar ions are neutral compounds
having formal unit electrical charges of opposite sign.
[0131] The composition according to the invention may thus also
comprise a detergent. It is preferred that the composition
comprises an anionic, cationic, zwitterionic or non-ionic
detergent. It is even more preferred that the detergent in the
composition is selected from the group consisting of Sodium dodecyl
sulfate, Lithium dodecyl sulfate, Cetyltrimethylammoniumbromide,
Deoxycholic acid, Sodium lauroyl sarcosine, TRITON-X100, TWEEN 20,
Octyl beta-D-glucoside, Nonidet P40, BRIJ 35 P or Sulphobetaine 14.
However, other detergents are possible.
[0132] Moreover, the composition may contain a protease. Generally,
when using the combination of a chaotropic agent, a detergent and a
protease for lysing a biological sample, the skilled artisan
selects e chaotropic agent and the detergent and their
concentrations in the composition according to the invention on the
basis that proteolytic activity is preserved in the
composition.
[0133] The composition according to the invention which
additionally contains a nucleic acid is also referred to as an
"adsorption solution" because the composition provides the
conditions necessary for adsorbing the nucleic acid to a solid
phase. Thus, another aspect of the invention is the use of TDE for
adsorbing a nucleic acid onto a solid phase. Yet, a further aspect
of the invention is a method of using TDE and a nucleic acid in a
sample comprising the steps of (a) providing the nucleic acid in a
sample, whereby the sample is dissolved in a liquid composition
comprising TDE, an aqueous buffer, and a chaotropic agent; followed
by (b) providing the solid phase and contacting the liquid
composition of step (a) with the solid phase, thereby adsorbing the
nucleic acid on the solid phase. Preferably, the sample has been
treated in order to obtain a lysed sample.
[0134] To bring the sample in contact with the solid phase, i.e.
the material with an affinity to nucleic acids, the sample is mixed
with the material and incubated for a period of time sufficient for
the binding to occur. Experts are usually familiar with the
duration of the incubation step. This step can be optimized by
determining the quantity of immobilized biological material on the
surface at different points in time. Incubation times of between 1
second (s) and 30 minutes (min) can be appropriate for nucleic
acids. After incubation, the solid phase with the adsorbed nucleic
acid(s) is separated from the liquid. This may be achieved in
general by gravity in the case a suspension of a pulverized solid
phase such as glass powder is used. In the convenient case of
nucleic acids bound to magnetic glass particles separation can be
achieved by immobilizing the magnetic particles with a magnetic
field and removing the liquid phase. For instance, the magnetic
particles can be pulled to the wall of the vessel in which
incubation was performed. The liquid containing the sample contents
that are not bound to the magnetic particles can then be removed.
The removal procedure used depends on the type of vessel in which
incubation was performed. Suitable steps include removing the
liquid via pipetting or aspiration. Another example is binding the
nucleic acid in the adsorption solution to a glass fleece.
Commercial kits often provide such a fleece at the bottom of a
column. The adsorption solution containing the nucleic acid is
transferred to the column and passed through the fleece by applying
force. The term "force" includes gravitational force and,
preferred, centrifugal force. Very much preferred is the "spin
column" procedure wherein the adsorption solution is passed through
the filter due to force being applied by way of centrifugation.
Other ways to pass the adsorption solution through the fleece
include the application of pressure or suction.
[0135] According to the invention, a preferred solid phase
comprises a porous or non-porous silica substrate. More preferred,
the solid phase comprises a substrate selected from the group
consisting of glass fibers and quartz fibers. Also very much
preferred, the solid phase comprises magnetic particles with a
silica surface. Magnetizable particulate adsorbents are a very much
preferred solid phase because they are suitable for automatic
sample preparation. Ferrimagnetic and ferromagnetic as well as
superparamagnetic particles are used for this purpose. Very much
preferred magnetic glass particles are those described in WO
01/37291. It is very convenient to provide the magnetic particles
as a suspension in an aqueous solution of TDE. Also preferred, the
magnetic particles are provided as a suspension in a solution
comprising a water-miscible cyclic acetal and TDE. The solution may
additionally comprise water. Therefore, another aspect of the
invention is a composition comprising TDE and magnetic particles
with a silica surface. liquid composition comprising TDE and
magnetic particles with a silica surface. Preferably the particles
are provided as powdered material. The magnetic glass particles
used in the present invention may be provided in different
formulations. It is possible to provide them in the form of a
tablet, as a powder or as a suspension which is preferred.
Preferably, these suspensions contain between 5 to 60 mg/ml
magnetic glass particles. Also preferred, the silica-containing
material is suspended in an aqueous buffered solution which may
optionally contain a chaotropic agent in a concentration of between
1 M and 10 M, and preferably between 2 M and 6 M.
[0136] Yet, a further aspect of the invention is a method for the
purification of a nucleic acid from a lysed sample, comprising the
steps of: (a) providing the nucleic acid in a sample, whereby the
sample is dissolved in a liquid composition comprising TDE, an
aqueous buffer, and a chaotropic agent; followed by (b) providing
the solid phase and contacting the liquid composition of step (a)
with the solid phase, thereby adsorbing the nucleic acid onto the
solid phase; followed by (c) separating the solid phase with the
adsorbed nucleic acid from the liquid phase; (d) optionally washing
with a washing solution the solid phase with the adsorbed nucleic
acid; followed by (e) contacting the solid phase with the adsorbed
nucleic acid with a desorption solution which preferably contains
solutes in a lower concentration compared to the composition of
step (a), thereby desorbing the nucleic acid from the solid phase
and dissolving the nucleic acid in the solution; followed by (f)
separating the solution with the nucleic acid from the solid phase,
thereby purifying the nucleic acid; and optionally (g)
precipitating the nucleic acid from the solution of step (f) and
isolating the precipitated nucleic acid, thereby further purifying
or concentrating the nucleic acid. The purification effect results
from the behavior of DNA or RNA to bind to material with a glass
surface under the conditions provided by the composition of the
invention in the adsorption solution containing the nucleic acid to
be purified.
[0137] The washing step may not always be necessary and therefore
represents a non-mandatory option. Performing the washing step is
opted for by the skilled person depending on the sample material
from which the nucleic acid is to be purified. The purpose of the
washing step(s) is to remove contaminants, i.e. undesired
components of the sample material from the adsorbed nucleic acid. A
washing solution is used that does not cause nucleic acid(s) to be
released from the surface of the solid phase but that washes away
the undesired contaminants as thoroughly as possible.
[0138] A washing step is preferably performed by incubating the
material with the adsorbed nucleic acid(s) with the washing
solution. The solid phase material is preferably resuspended during
this step. Also preferred, in case the material is a glass fleece
or a packing in a column, the washing step takes place by rinsing
the column with the washing solution. Preferably, the washing
solution is passed through the column by applying pressure,
suction, centrifugal force or gravitational force. The contaminated
washing solution is preferably removed just as in the step
described above for binding the nucleic acid to the solid phase.
After the last washing step, the material can be dried briefly in a
vacuum, or the fluid can be allowed to evaporate. Prior to
desorption, a pretreatment step using acetone may also be
performed.
[0139] Preferably, the washing solution contains an organic
compound selected from the group consisting of TDE, a C1-5
aliphatic alcohol and a liquid water-miscible cyclic acetal,
furthermore a chaotropic agent at a concentration between 0.5 M and
10 M, whereby the pH value of the washing solution is between pH 4
and pH 7.5. The preferred concentration of the organic compound in
the washing solution is between 10% and 90% [v/v]. A very much
preferred aliphatic alcohol is ethanol or isopropanol.
[0140] Under the conditions provided by the washing solution
preferably greater than 40%, more preferred greater than 50%, more
preferred greater than 70%, more preferred greater than 80%, even
more preferred greater than 90%, even more preferred greater than
95%, even more preferred greater than 99% of the nucleic acids
remain adsorbed to the solid phase.
[0141] Using a narrow concentration range for TDE the inventors
surprisingly found a way how to isolate small nucleic acids such as
miRNA, siRNA, etc. Thus, another aspect of the invention is
directed to a method of using TDE and a nucleic acid of low
molecular weight in a sample. The invention encompasses a method
for purifying a nucleic acid with low molecular weight comprising
the steps of (a) providing the nucleic acid in a lysed sample,
whereby the sample is dissolved in a liquid composition comprising
an aqueous buffer, TDE at a concentration between 10% and 75%,
measured as volume by volume, a detergent and a chaotopic agent;
followed by (b) providing a solid phase and contacting the liquid
composition of step (a) with the solid phase; followed by (c)
separating the solid phase with the adsorbed nucleic acid from the
liquid phase; (d) washing with a washing solution the solid phase
of step (c), whereby the washing solution comprises an organic
solvent at a concentration of between 40% and about 100%; followed
by (e) contacting the solid phase with the adsorbed nucleic acid
with an aqueous desorption solution containing solutes in a lower
concentration compared to the composition of step (a), thereby
desorbing the nucleic acid from the solid phase and dissolving the
nucleic acid in the solution; followed by (f) separating the
solution with the nucleic acid from the solid phase, thereby
purifying the nucleic acid; and optionally (g) precipitating the
nucleic acid from the solution of step (f) and isolating the
precipitated nucleic acid, thereby further purifying or
concentrating the nucleic acid.
[0142] The liquid composition of step (a) may additionally comprise
a detergent, preferably a non-ionic detergent or sodium lauroyl
sarcosine. Also preferred, in step (a) the concentration of TDE in
the composition is between 30% and 50%, and most preferred about
40%, measured as volume by volume.
[0143] Very much preferred, the chaotropic agent in the composition
of step (a) is in a concentration between 0.5 M and 10 M. Also
preferred, the chaotropic agent comprises guanidine isothiocyanate.
Furthermore, the composition of step (a) including the sample is
preferably buffered to a pH between 4.0 and 7.0, very much
preferred a pH between 4.5 and 6.5, and also very much preferred a
pH between 5.5 and 7.
[0144] Alternatively, a nucleic acid with low molecular weight can
be purified, according to the invention by a method comprising the
steps of (a) providing the nucleic acid in a lysed sample, whereby
the sample is dissolved in a liquid composition comprising an
aqueous buffer, TDE at a concentration between 5% and 30%, measured
as volume by volume, a detergent and a chaotropic agent; followed
by (b) providing a first solid phase, contacting the liquid
composition of step (a) with the first solid phase, and separating
the liquid phase from the first solid phase; followed by (c) mixing
an additional amount of TDE with the liquid phase of step (b),
thereby adjusting the concentration of TDE in the liquid phase of
step (b) to between 20% and 70%, measured as volume by volume,
whereby the initial concentration of TDE in the liquid phase is
increased by a factor of 1.3 or more; followed by (d) providing a
second solid phase and contacting the liquid composition of step
(a) with the second solid phase; followed by (e) washing with a
washing solution the second solid phase of step (d), whereby the
washing solution comprises an organic solvent at a concentration of
between 50% and 100%; followed by (f) contacting the second solid
phase of step (e) with an aqueous desorption solution containing
solutes in a lower concentration compared to the composition of
step (a), thereby desorbing the nucleic acid from the solid phase
and dissolving the nucleic acid in the solution; followed by (g)
separating the solution with the nucleic acid from the solid phase,
thereby purifying the nucleic acid; and optionally (h)
precipitating the nucleic acid from the solution of step (f) and
isolating the precipitated nucleic acid, thereby further purifying
or concentrating the nucleic acid.
[0145] In step (a), a concentration of TDE at about 20% [v/v] is
most preferred. Basically, in this method nucleic acids which are
not desired due to their larger size are adsorbed onto the first
solid phase and removed from the liquid phase. However, the
conditions applied still retain nucleic acids of the desired size
in solution. Nucleic acids of the desired size are adsorbed to the
second solid phase upon mixing further additive to the solution.
Preferably, further TDE is added. The final TDE concentration in
the adsorption solution for the second solid phase is preferably in
the range of between 30% [v/v] and less than 100% [v/v]. A more
preferred range for the final concentration of TDE in this step is
between 30% [v/v] and 80% [v/v], even more preferred between 30%
[v/v] and 60% [v/v], between 30% [v/v] and 50% [v/v]; most
preferred the final TDE concentration in step (c) is about 40%,
measured as volume by volume.
[0146] According to the invention, in step (c) the initial TDE
concentration [i.e the TDE concentration in the liquid composition
of step (a)] is increased by a factor of 1.3 or more to result in a
final TDE concentration of one the above specified preferred
concentration ranges. For example, if the initial concentration of
TDE is 20%, the final TDE concentration in the liquid composition
of step (c) has to be 20% [v/v]*1.3=26% [v/v] at minimum. If, by
way of another example, the initial TDE concentration is 5%, the
final TDE concentration in the liquid composition of step (c) is
calculated with a factor higher than 1.3, in order to reach the
minimally required TDE concentration of 20%. Accordingly, the
factor does not exceed 14 since the highest final TDE concentration
in the composition of step (c) is 70%. Thus, the value of the
factor is between 1.3 and 14.
[0147] The washing solution may additionally comprise an aqueous
buffer which buffers the pH of the washing solution at a value
between 4.0 and 7.5.
[0148] In view of the invention, a nucleic acid of low molecular
weight is preferably characterized in that (a) the nucleic acid is
single-stranded and the size of the purified nucleic acid is
between 10 bases and 150 bases; or (b) the nucleic acid is
double-stranded and the size of the purified nucleic acid is
between 5 bases and 75 bases. Very much preferred, the nucleic acid
of low molecular weight is DNA or RNA. Even more preferred, the
nucleic acid of low molecular weight is single-stranded or
double-stranded RNA.
[0149] The detergent which can be used in the adsorption solution
aids in the process of releasing the nucleic acids. E.g., cells and
tissues are lysed by detergents which disintegrate cellular
membranes. In addition, detergents enhance the dissociation of
nucleic acids from concomitant sample constituents such as protein.
In addition, a detergent increases the binding of nucleic acids to
solid phases, and when using a porous solid phase the detergent
facilitates access of the liquid phase to the pore compartments of
the solid phase.
[0150] The solid phases which can be preferably used for the
purification of low molecular weight nucleic acids are generally
the same as for the general nucleic acid purification procedure
according to the invention. A solid phase with a silica surface is
most preferred, however. A very much preferred pH range of an
adsorption solution for a low molecular weight nucleic acid is
between 4.0 and 7.5.
[0151] In order to reverse the conditions for adsorption, the
concentration of the chaotropic agent and/or TDE is decreased
resulting in desorption of the nucleic acid(s) bound to the solid
material. Thus, the invention also encompasses the method
comprising the step of releasing the adsorbed nucleic acid
(=desorbing) from the solid phase. Preferably, the process of
separating the substrate, e.g. the magnetic glass particles, from
the rest of the sample is done by pelleting the immobilized
biological material, e.g. by gravity force or by the use of a
magnet in the case of magnetic glass particles and removal of the
supernatant. Then the magnetic glass particles with the immobilized
biological material are resuspended in an aqueous solution with no
or only a low amount of chaotropic agent and/or TDE. Alternatively,
the suspension can be diluted with a solution with no or only a low
amount of chaotropic agent and/or TDE. Buffers of this nature are
known from DE 37 24 442 and Jakobi, R., et al., Anal. Biochem. 175
(1988) 196-201. An elution buffer, i.e. a desorption solution, has
a low salt content and preferably a pH greater than 7.5, a pH of
about 8 being most preferred. Preferably the desorption solution
contains solutes in a lower concentration compared to the
adsorption solution. Particularly preferred, the solutes are one or
more buffer salts with a content of less than 0.2 M of dissolved
matter. Thus, the preferred concentration of solutes in the
desorption solution is in between 0 M and 0.2 M. In addition, the
preferred desorption solution does not contain a chaotropic agent
or an organic solvent such as TDE. Preferably, the elution buffer
contains the substance TRIS for buffering purposes. Also very much
preferred, the elution buffer is demineralized water. The solution
containing the purified nucleic acid(s) can now be used for other
reactions. Optionally, the nucleic acid(s) can be precipitated from
the solution using, e.g., ethanol or isopropanol. The precipitate
can also be subjected to further washing steps. Methods of this
kind are well known to the skilled artisand are described in detail
in Sambrook, Fritsch & Maniatis, Molecular Cloning, A
Laboratory Manual, 3rd edition, CSHL Press, 2001.
[0152] For the desorption step conditions are chosen by the skilled
artisan, under which the nucleic acids are released from the
mineral support. Preferably, greater than 40%, more preferred
greater than 50%, more preferred greater than 70%, more preferred
greater than 80%, even more preferred greater than 90%, even more
preferred greater than 95%, even more preferred greater than 99% of
the nucleic acids are released from the mineral support.
[0153] Purification of a nucleic acid by way of adsorbing the same
to a substrate such as a mineral substrate in the presence of a
composition according to the invention can also applied to other
complex mixtures. Examples therefor are known to the person skilled
in the art of molecular biology and include reaction mixtures
following, e.g., in-vitro synthesis of nucleic acids such as PCR,
restriction enzyme digestions, ligation reactions, etc. Another
application for purification of a nucleic acid by way of adsorbing
the same to a solid phase in the presence of a composition
according to the invention is the removal of pyrogenic contaminants
which may have copurified with the nucleic acid.
[0154] With great advantage, the method according to the present
invention is suitable for the purification of nucleic acids, i.e.
RNA or DNA, from complex mixtures with other biological substances
containing them. Thereby also mixtures of different nucleic acids
may be purified, even mixtures containing a nucleic acid of
interest in low abundance. Thus, the present invention also
encompasses the purification of mixtures of specific nucleic acids
in which the target nucleic acid(s) may be a minor component in
terms of concentration (or may be present in low abundance).
[0155] The procedure described can also be used to isolate native
or modified nucleic acids. Native nucleic acids are understood to
be nucleic acids, the structure of which was not irreversibly
changed compared with the naturally-occurring nucleic acids. This
does not mean that other components of the sample can not be
modified, however. Modified nucleic acids include nucleic acids
that do not occur in nature, e.g., nucleic acids that are modified
by attaching to them groups that are reactive, detectable or
capable of immobilization. An example of this are biotinylated
nucleic acids.
[0156] The invention also contemplates kits. Such kits known to the
art comprise plasticware useful in the sample preparation
procedure. Examples therefor are microwell plates in the 96 or 384
well format or just ordinary reaction tubes manufactured e.g. by
Eppendorf, Hamburg, Germany. The kits of the invention also
comprise some or all other reagents for carrying out the methods
according to the invention. Therefore, a kit can additionally
contain a solid phase, i.e. a material with an affinity to nucleic
acids. Preferably the solid phase comprises a material with a
silica surface. Very much preferred, the solid phase comprises
glass or quartz fibers. Also very much preferred, the solid phase
is a composition comprising magnetic glass particles, i.e.
magnetically attractable particles coated with glass. Another
preferred material with an affinity to nucleic acids is anion
exchanger. The kit can further or additionally comprise a lysis
buffer containing e.g. a chaotropic agent, a detergent or mixtures
thereof. These components of the kit according to the invention may
be provided separately in tubes or storage containers. Depending on
the nature of the components, these may be even provided in a
single tube or storage container. The kit may further or
additionally comprise a washing solution which is suitable for the
washing step of the solid phase when DNA or RNA is bound thereto.
This washing solution may contain TDE according to the invention
and/or a chaotropic agent in a buffered solution or solutions with
an acidic pH without TDE and/or a chaotropic agent as described
above. Often the washing solution or other solutions are provided
as stock solutions which have to be diluted before use. The kit may
further or additionally comprise a desorption solution, i.e. an
elution buffer, that is to say a solution for desorbing the nucleic
acid from the solid phase. A preferred desorption solution can be a
buffer (e.g. 10 mM Tris, 1 mM EDTA, pH 8.0) or pure water. Further,
additional reagents or buffered solutions may be present which can
be used for the purification process of a nucleic acid, i.e. DNA or
RNA.
[0157] A further aspect of the invention is a kit of parts,
comprising packaging material, containers, and (a) TDE, (b) a
concentrated stock solution of a buffer salt and a chaotropic agent
is selected from the group consisting of guanidine hydrochloride,
guanidine thiocyanate, guanidine isothiocyanate, urea, sodium
acetate, an alkali perchlorate, an alkali halogenide, and mixtures
thereof; and (c) chromatographic and filtering material comprising
a material with a surface capable of interacting with the phosphate
residues in the backbone of nucleic acids.
[0158] A preferred embodiment of the present invention is to use
the methods or the kits of the present invention in automatable
methods as e.g. described in WO 99/16781. Automatable method means
that the steps of the method are suitable to be carried out with an
apparatus or machine capable of operating with little or no
external control or influence by a human being. Automated method
means that the steps of the automatable method are carried out with
an apparatus or machine capable of operating with little or no
external control or influence by a human being. Only the
preparation steps for the method may have to be done by hand, e.g.
the storage containers have to be filled up and put into place, the
choice of the samples has to be done by a human being and further
steps known to the expert in the field, e.g. the operation of the
controlling computer. The apparatus or machine may e.g. add
automatically liquids, mix the samples or carry out incubation
steps at specific temperatures. Typically, such a machine or
apparatus is a robot controlled by a computer which carries out a
program in which the single steps and commands are specified.
Preferred automated methods are those which are carried out in a
high-throughput format which means that the methods and the used
machine or apparatus are optimized for a high-throughput of samples
in a short time. In another embodiment of the invention the methods
or the kits according to the present invention are used in a
semi-automated process which means that some reaction steps may
have to be done manually. In a preferred embodiment of the
invention, a suspension containing magnetic glass particles
according to the present invention is taken from a storage
container and partial volumes are added to different reaction
vessels. Reaction vessels may be reaction tubes made from plastics
eventually in microwell plate format contain 96 or 384 or more
wells where a reaction can be carried out. However, these vessels
may be made from other material, e.g. from steel.
[0159] A further aspect of the invention is a kit of parts,
comprising packaging material, containers, and (a) a suspension of
silica-coated magnetic particles in TDE; and (b) a concentrated
stock solution of a buffer salt and a chaotropic agent is selected
from the group consisting of guanidine hydrochloride, guanidine
thiocyanate, guanidine isothiocyanate, urea, sodium acetate, an
alkali perchlorate, and an alkali halogenide.
[0160] Some of the organic compounds contemplated by the invention
might be capable of dissolving certain plastic materials. Thus,
when determining the nature of suitable storage or reaction
vessels, the skilled artisan will determine in a limited number of
obvious experiments the material which is suited best for executing
the methods of the invention or for producing kits according to the
invention.
[0161] In preferred embodiments of the invention the kits according
to the invention are used for the purification of nucleic acids in
research, bioanalytics or diagnostics. In preferred embodiments
according to the invention the kits according to the invention or
the methods according to the invention are used in a
high-throughput format, i.e. in an automated method which allows
the analysis of a high number of different samples in a very short
time.
[0162] The nucleic acids isolated using the methods according to
the invention can be used further as necessary. For instance, they
can be used as a substrate for various enzymatic reactions. The
nucleic acids can be used for a large number of purposes including
sequencing, radioactive or non-radioactive labelling, amplification
of one or more of the sequences they contain, transcription,
hybridization with labelled probe nucleic acids, translation or
ligation.
[0163] Yet, a further aspect of the invention is a method for
determining the presence of a nucleic acid in a sample, comprising
the steps of: (a) optionally lysing the sample; (b) forming a
composition containing (i) the sample or the lysed sample of step
(a), (ii) an aqueous buffer, (iii) a chaotropic agent, and (iv)
TDE, whereby the sample is dissolved in the liquid composition; (c)
contacting the composition of step (b) with a solid phase, thereby
adsorbing the nucleic acid onto the solid phase; (d) separating the
solid phase with the adsorbed nucleic acid from the liquid phase;
(e) optionally washing with a washing solution the solid phase with
the adsorbed nucleic acid; followed by (f) contacting the solid
phase with the adsorbed nucleic acid with an aqueous desorption
solution containing solutes in a lower concentration compared to
the composition of step (b), thereby desorbing the nucleic acid
from the solid phase and dissolving the nucleic acid in the
solution; followed by (g) separating the solution with the nucleic
acid from the solid phase, thereby purifying the nucleic acid; and
(h) detecting in the solution of step (g) the presence of the
nucleic acid, thereby determining the presence of the nucleic
acid.
[0164] It is preferred that the sample is a biological sample.
Preferably, the nucleic acid is determined by amplification of the
nucleic acid by means of the polymerase chain reaction using
specific primers, a specific detection probe, and an amplification
mixture, whereby amplification is monitored in real time. Also
preferred is to determine the nucleic acid by hybridizing the
nucleic acid to a hybridization probe and detecting and/or
quantifying the hybrid. The skilled artisan is aware of the fact
that not only a nucleic acid can serve as a hybridization probe but
also a nucleic acid comprising one or more nucleoside analogues can
be used. In addition, nucleic acid analogues such as PNA are known
to the art as being capable of forming detectable hybrids with
nucleic acids. It is understood that the nucleic acid to be
determined is DNA or RNA. Very much preferred is the above method,
whereby the nucleic acid is RNA and step (h) comprises (i) reverse
transcribing the RNA to form a cDNA, (ii) subsequently amplifying,
by means of the polymerase chain reaction, the cDNA, (iii)
detecting the presence of the cDNA, thereby determining the
presence of the nucleic acid.
[0165] The following examples and figures are provided to aid the
understanding of the present invention, the true scope of which is
set forth in the appended claims. It is understood that
modifications can be made in the procedures set forth without
departing from the spirit of the invention.
[0166] All Examples given below were performed as variations of the
standard workflow of the HIGH PURE (Roche Diagnostics Operations,
Inc.) PCR Template Preparation Kit, user manual version April 2005,
Roche Diagnostics GmbH, Mannheim, Germany, Catalogue No.
11796828001. Unless indicated otherwise, all working steps of the
workflow were performed as indicated in said user manual.
Example 1
Purification of Total Nucleic Acids from Blood Samples
[0167] The workflow for purification of the nucleic acids (NAs)
includes the following steps: Lysis of the sample in order to make
the nucleic acids accessible for the purification process.
Adsorption of the NAs onto the solid phase, separating the solid
phase from the liquid phase and washing the solid phase with the
bound NAs, and desorbing the NAs from the solid phase.
[0168] Each sample consisted of a volume of 200 .mu.l of EDTA whole
blood. The solid phase used was silica fleece present in HIGH PURE
spin columns (Roche Diagnostics GmbH, Mannheim, Germany). Compounds
tested for binding enhancement to the silica fleece were selected
from Table 1, except those that show toxic properties. Toxic
compounds were excluded.
[0169] EDTA blood was pooled and aliquots were subjected to nucleic
acid isolation according to the following protocol: 200 .mu.l whole
EDTA blood was mixed with 200 .mu.l Binding Buffer (6 M guanidine
HCl, 100 mM MES, 18.5% [v/v] TRITON X-100, pH 5.7) and 40 .mu.l
Proteinase K (20 .mu.g/.mu.l dissolved in bidestilled water)
solution. The mixture was incubated for 10 min at 70.degree. C. to
effect lysis. Afterwards, 100 .mu.l of one of the substances listed
in Table 2 was added to a lysed sample and mixed. The mixture was
applied to a spin column (HIGH PURE spin column, Roche Diagnostics
GmbH, Mannheim, Germany) for further processing. Handling of the
columns as well as washing and elution was performed according to
the package insert of the HIGH PURE PCR Template Preparation Kit,
version April 2005 (Catalogue No. 11796828001 Roche Diagnostics
GmbH, Mannheim, Germany).
TABLE-US-00002 TABLE 2 Yield and purity of nucleic acids obtained
from 200 .mu.l EDTA whole blood hazard yield classi- Substance (ng)
purity fication tetraethylene glycol dimethyl ether (TDE) 3975.32
1.88 glycerolformal 3856.59 1.84 diethylene glycol diethyl ether
3697.14 1.87 Xi methyl ethyl ketone 3606.98 1.87 F, Xi propylene
glycol dimethylether 3590.64 1.87 F (dimethoxypropane) ethylene
glycol diethyl ether (diethoxyethane) 3530.66 1.88 Xi propylene
glycol monomethyl ether acetat 3526.20 1.85 Xi tetrahydrofuran
3399.51 1.88 F, Xi polyethylene glycol 1000 3307.32 1.93 1,3
dioxolane 2988.81 1.88 F, Xi hydroxyacetone 2601.50 1.87 ethanol
2572.52 1.90 F isopropanol 2561.75 1.90 F, Xi ethyllactate 2403.28
1.83 Xi acetone 1884.73 1.89 F, Xi
[0170] Yield was determined by measuring OD at 260 nm and
multiplying the extinction value with the factor of 50 (for
double-stranded nucleic acids) or 40 (for single-stranded nucleic
acids).
[0171] Purity was assessed by measuring the extinction of the
eluate at 260 nm and 280 nm and calculating the 260/280 nm
quotient.
[0172] Integrity of the nucleic acids isolated by this method is
shown in FIG. 1. In conclusion, use of Tetraethylene Glycol
Dimethyl Ether (TDE) as well as glycerolformal lead to superior
yield of nucleic acids isolated from 200 .mu.l of whole EDTA blood
compared to other substances. At the same time, these two compounds
are particularly advantageous due to their very low or even lacking
toxicity.
Example 2
Purification of Total Nucleic Acids from Tissue Culture Cells
[0173] The workflow for purification of the nucleic acids (NAs)
includes the following steps: Lysis of the sample in order to make
the nucleic acids accessible for the purification process.
Adsorption of the NAs onto the solid phase, separating the solid
phase from the liquid phase and washing the solid phase with the
bound NAs, and desorbing the NAs from the solid phase.
[0174] Total nucleic acids were purified from 1.times.10.sup.6 K562
cells. Sedimented cells were resuspended in 200 .mu.l PBS buffer.
Afterwards 200 .mu.l binding buffer (6 M guanidine HCl, 100 mM MES,
18.5% [v/v] TRITON X-100, pH 5.7) and a measured amount of TDE
(final concentration of TDE: 10%, 20% and 40% [v/v]) were added and
mixed. Each sample was applied to a spin column (HIGH PURE spin
column, [CATALOG #] Roche Diagnostics GmbH, Mannheim, Germany) and
processed according to standard procedure (HIGH PURE PCR Template
Preparation Kit, user manual version April 2005, Roche Diagnostics
GmbH, Mannheim, Germany, Catalogue No. 11796828001). For
combinatorial use of TDE and glycerol formal a similar experimental
workflow was used. Only the binding buffer was changed to 6 M
guanidine HCl, 100 mM MES, 10% [v/v] glycerol formal, 18.5% [v/v]
TRITON X-100, pH 5.7. Results of this experiment are shown in FIG.
2. By using a combination of TDE and glycerolformal in the
purification process the nucleic acid yield could be increased
compared with the use of TDE only.
Example 3
Purification of Nucleic Acids from Tissue Culture Cells
[0175] Nucleic acids were purified from 1.times.10.sup.6 K562
cells. Sedimented cells were resuspended in 200 .mu.l PBS buffer.
Afterwards 200 .mu.l binding buffer (6 M guanidine HCl, 100 mM MES,
18.5% [v/v] TRITON X-100, pH 5.7) and 100 .mu.l TDE were added and
mixed. Each sample was applied to a spin column (HIGH PURE spin
column, Roche Diagnostics GmbH, Mannheim, Germany) and processed
according to standard procedure (HIGH PURE PCR Template Preparation
Kit, user manual version April 2005, Roche Diagnostics GmbH,
Mannheim, Germany, Catalogue No. 11796828001). A first and a second
washing buffer were used consecutively in each
isolation/purification process. To prepare the first washing buffer
(inhibitor removal buffer) a volume of 20 ml TDE or ethanol is
added to a volume of 33 ml concentrated stock solution of washing
buffer 1 to form a buffer in which the final concentration of TDE
or ethanol is about 39% [v/v], along with the remaining ingredients
5 M guanidine HCl, 20 mM Tris HCl, pH 6.6 (25.degree. C.) (final
concentrations after the addition of TDE/ethanol). To prepare the
second washing buffer (desalting buffer) ethanol or TDE were added
to a stock solution of washing buffer 2 to form a buffer in which
the final concentration of TDE or ethanol is about 80% [v/v], along
with the remaining ingredients 20 mM NaCl, 2 mM Tris HCl, pH 7.5
(25.degree. C.) (final concentrations after the addition of
TDE/ethanol).
[0176] Comparisons were made, whereby three washing steps were
applied to each sample, either with ethanol-containing or with
TDE-containing wash buffers. The first washing step was performed
with the first washing buffer, followed by two washing steps with
the second washing buffer. Subsequently, the nucleic acids were
desorbed from the solid phase. Except for the details given above,
all steps further steps of the workflow were performed according to
the standard procedure provided by the manufacturer (see manual for
the HIGH PURE PCR Template Preparation Kit, user manual version
April 2005, Roche Diagnostics GmbH, Mannheim, Germany).
TABLE-US-00003 TABLE 3 Comparison of TDE and ethanol as additives
in the washing buffers sample yield [.mu.g] Nucleic acid from K562
cells using ethanol in washing buffers 22.43 Nucleic acid from K562
cells using TDE in washing buffers 22.62
[0177] Yield was determined as described in Example 1.
[0178] FIG. 3 depicts an agarose gel which was run with the
isolated nucleic acids in order to demonstrate their integrity.
Example 4
Recovery of dsDNA of a Size Between 50 bp and 400 bp from an
Aqueous Phase
[0179] In order to specifically monitor the recovery of dsDNA
fragments of a size between 50 bp and 400 bp a solution containing
1.5 .mu.g of a 50 bp and 1.5 .mu.g of a 400 bp fragment was used in
the experiment. 100 .mu.l of the DNA solution was mixed with 200
.mu.l binding buffer (3 M guanidine SCN, 6.25% [v/v] Dioxolan 200
mM BES, 7.5% [v/v] TRITON, pH 7.0,) and 200 .mu.l TDE. After
binding the washing procedure was performed as described in Example
3, Again, ethanol was compared to TDE in the washing buffers. Table
4 indicates the results.
TABLE-US-00004 TABLE 4 Isolation of small DNA molecules, comparison
of TDE and ethanol as additives in the washing buffers yield
recovery sample [.mu.g] [%] Purified DNA fragments using ethanol in
2.98 87.86 washing buffers Purified DNA fragments using 3.13 92.27
TDE in washing buffers
[0180] Yield was determined as described in Example 1.
[0181] The integrity of the nucleic acids isolated/purified with
the above procedure is shown in FIG. 3.
Example 5
Evaporation of Additives from Liquid Compositions
[0182] The effect of evaporation of organic solvents as additives
in liquid compositions for the isolation/purification of nucleic
acids was investigated. Several compositions were tested, whereby
the compositions included organic solvents with differences in
vapor pressure indicating a different evaporation rate. Evaporation
affects stability of a reagent as well as reproducibility of the
process in which the reagent is used. This is especially true in
the case of automated systems for nucleic acid purification.
[0183] The evaporation rate of buffers with different compositions
was determined. A volume of 200 .mu.l of water was mixed with 200
.mu.l binding buffer containing 6 M guaninidine-HCl, 10 mM urea, 10
mM Tris HCl, 20% TRITON X-100 (v/v), pH4.4 (25.degree. C.) and 100
.mu.l of a substance listed below in Table 5. Incubation of the
mixtures was carried out for 30 min at 30.degree. C. in a standard
1.5 ml eppendorf tube, whereby the lid was left open. Evaporation
was assessed by weighing each tube before and after incubation and
tabulating weight loss.
TABLE-US-00005 TABLE 5 Evaporation of different substances after 30
min from 20% [v/v] solutions vapor pressure weight at 20.degree. C.
loss Substance [in mm Hg] [in .mu.g] Tetraethylene Glycol Dimethyl
Ether (TDE) <0.01 3.3 Diethylene Glycol Diethyl Ether 0.5 3.3
Ethyllactat 2 3.6 Hydroxyaceton 5.6 4.7 Glycerolformal n/a 5.2
Diethylene Glycole Dimethyl Ether 3 6.0 Polyethylene Glycol 1000
n/a 8.2 Propylene Glycol Monomethyl Ether Acetate 3.7 8.3 Ethanol
44.6 9.3 Isopropanol 33 14.0 Ethylene Glycol Diethyl Ether 9.4 14.4
1,3 Dioxolane 70 21.1 Propylene Glycol Dimethyl Ether 40 21.5 Metyl
Ethyl Ketone 71 28.6 Acetone 184 32.3 Tetrahydrofuran 143 44.4
[0184] When analyzing the amounts of weight loss one has to
appreciate that the evaporated matter may also comprise water,
apart from the organic solvent tested. The highest loss was
observed for the Tetrahydrofuran containing buffer. Approximately
44.4 .mu.g were evaporated from the 500 .mu.l volume after 30
minutes. However, from the TDE containing buffer only approximately
3.3 .mu.g evaporated. This example demonstrates the superior
properties of substances with low vapor pressure, particularly that
of TDE, compared with substances displaying a high evaporation rate
(i.e. Tetrahydrofuran, Acetone, etc.).
Example 6
Automated Nucleic Acid Isolation/Purification Workflow
[0185] For the demonstration of an automated workflow a MagnaPure
LC instrument (Roche Diagnostics GmbH, Mannheim, Germany) was used
for the evaluation. Total nucleic acids were purified from 10.sup.6
cultured K562 cells. For the procedure the MAGNA PURE LC (Roche
Diagnostics Operations, Inc.) DNA Isolation Kit--Large Volume
(Roche Diagnostics GmbH, Mannheim, Germany) was used. The standard
procedure according to the instructions by the supplyer using
magnetically attractable particles dissolved in isopropanol was
compared to a procedure with a similar amount of magnetic
particles, however dissolved in TDE or diethylene glycol diethyl
ether. Apart from this variation, the workflow was performed
exactly according to the package insert of the MAGNA PURE LC DNA
Isolation Kit--Large Volume (Version May 2006), Cat No
03310515001.
TABLE-US-00006 TABLE 6 Yield and purity of nucleic acid isolated
from 10.sup.6 K562 cells using the MAGNA PURE instrument and
various additives in the adsorption solution Magnetic particles
dissolved in Yield (.mu.g) Tetraethylene Glycol Dimethyl Ether 18.4
Isopropanol 10.8 Diethylene Glycol Diethyl Ether 8.1
[0186] Yield was determined as described in Example 1.
[0187] Table 5 displays average values obtained in 4 experimental
runs each. In the automated workflow of the MAGNA PURE instrument
tetraethylene glycol dimethyl ether showed a better performance
compared with isopropanol and diethylene glycol diethyl ether.
Integrity of the isolated nucleic acids was tested by size
separation using agarose gel electrophoresis and ethidium bromide
staining (see FIG. 4).
Example 7
Binding of Nucleic Acid Molecules of Various Sizes Using Different
TDE Concentrations in the Binding Step
[0188] Nucleic acids of various sizes from a biological sample were
mixed with small RNA molecules (in this case with microRNA,
=miRNA). The experimental setting was designed to determine under
which conditions small nucleic acids below 150 bases can be
isolated together with nucleic acids of larger sizes. Samples
consisting of either (i) 10.sup.6 K562 cells or (ii) 1 .mu.g of
pre-purified miRNA 16 (supplied by Metabion, Martinsried, Germany)
were dissolved separately in 300 .mu.l binding buffer "C45G45T"
containing 200 mM sodium citrate pH 4.5, 4.5 M guanidine SCN, and
2.5% [v/v] TRITON X-100. Then the two samples were mixed in equal
volumes. TDE was added to the solution to a final concentration of
between 35% [v/v] and 55% [v/v], and mixed for 10 s on a Vortex
mixer. Each mixture was subsequently applied to a HIGH PURE column
from the HIGH PURE PCR Product Purification Kit (Roche Diagnostics
GmbH, Mannheim, Germany, 11 732 668 001). The columns were
centrifuged for 30 s at 13,100.times.g. The flow-throughs were
discarded. The columns were then washed with 500 .mu.l of the
ethanol reconstituted washing buffer supplied with the HIGH PURE
PCR Product Purification Kit. After adding ethanol, the
reconstituted washing buffer consisted of 80% [v/v]ethanol, 20 mM
NaCl, 2 mM Tris-HCl pH 7.5. Elution was subsequently performed with
100 .mu.l desorption buffer containing 10 mM Tris-HCl pH 8.5. A 10
.mu.l aliquot of each fractions was electrophoresed on 15%
acrylamide gels with TBE/Urea runnig buffer (Invitrogen).
[0189] FIG. 5 shows that a concentration of 40% TDE was necessary
for optimum binding of the miRNA, whereas higher molecular weight
nucleic acids were bound also at a lower TDE concentration.
Example 8
Purification of Low Molecular Weight Nucleic Acid Molecules Using
Two Consecutive Separations with Spin Columns
[0190] Small nucleic acids with sizes lower than 150 nucleotides
(i.e. microRNA) were separated from a biologigal sample containing
nucleic acid molecules of various sizes. Samples consisting of
either 10.sup.6 K562 cells or 1 .mu.g of chemically synthesized
miRNA 16 were dissolved separately in 300 .mu.l binding buffer
"C45G45T" containing 200 mM sodium citrate pH 4.5, 4.5 M guanidine
SCN, and 2.5% [v/v] TRITON X-100. Then the two samples were mixed
in equal volumes. TDE was added to the solution to result in a
final concentration of between 0% [v/v] and 30% [v/v], and mixed
for 10 sec. Each mixture was subsequently applied to a HIGH PURE
column from the HIGH PURE PCR Product Purification Kit (Roche
Diagnostics GmbH, Mannheim, Germany, 11 732 668 001). The columns
were centrifuged for 30 at 13,100.times.g. The flow-throughs were
collected and TDE was added to a final concentration of 40% TDE in
each sample. After mixing for by vortexing for 10 s the mixtures
were applied onto a second set of HIGH PURE columns and centrifuged
for 30 s at 13,100.times.g. The first and second set of columns
were then washed two times with each 500 .mu.l of washing buffer
consisting of 80% [v/v]ethanol, 20 mM NaCl, 2 mM Tris-HCl pH 7.5
from the HIGH PURE PCR Product Purification Kit. Elution was
subsequently performed with 100 .mu.l desorption buffer containing
10 mM Tris-HCl pH 8.5.
[0191] 10 .mu.l aliquots of all fractions (eluates from the first
and second columns) were electrophoresed in 15% acrylamide gels
with TBE/Urea runnig buffer (Invitrogen).
[0192] FIG. 6 A shows that on the first column miRNA is not bound
efficiently at concentrations of less than 30% TDE concentration,
in contrast to nucleic acids with higher molecular weight. miRNA is
therefore not apparent in eluates from the first column. It is
bound however at a concentration of 40% TDE onto the second column.
FIG. 6 B shows that miRNA elutes from the second column.
Particularly, it can be seen that in lane 10 (30% TDE on first
column) only miRNA is eluted from the second column. The miRNA is
essentially purified from larger nucleic acid species.
Example 9
Nucleic Acid Isolation and Subsequent Detection
[0193] Small nucleic acid molecules of sizes smaller than 150
nucleotides (i.e. microRNA) were isolated from liver and kidney
tissue and detected using a Q-RT-PCR (quantitative-reverse
transcription-polymerase chain reaction) protocol.
[0194] Samples of small pieces of mouse liver or kidney tissue were
frozen in liquid nitrogen and pulverized in a cooled mortar.
Aliquots of 10 mg samples were then dissolved in 300 .mu.l of
Binding buffer "M55G45T" containing 500 mM MES pH 5.5, 4.5 M
guanidine SCN, 2.5% [v/v] TRITON X-100.
[0195] Then either a single column procedure was applied to isolate
total nucleic acids or a procedure with two consecutive columns was
followed, in order to purify miRNA from total nucleic acids.
Single Column Protocol:
[0196] To a volume of 300 .mu.l of each lysed sample (prepared as
descried above) TDE was added to result in a final concentration of
40% [v/v], and mixed for 10 sec. The whole mixtures were then
applied onto HIGH PURE columns from the HIGH PURE PCR Product
Purification Kit (Roche), 11 732 668 001 and spun for 30 s at
13,100.times.g. The flow-throughs were discarded. The columns were
washed two times with each 500 .mu.l washing buffer consisting of
80% [v/v]ethanol, 20 mM NaCl, 2 mM Tris-HCl pH 7.5 from the HIGH
PURE PCR Product Purification Kit. Elution was subsequently
performed with a volume of 100 .mu.l desorption buffer per column,
containing 10 mM Tris-HCl pH 8.5.
Procedure with Two Consecutive Columns:
[0197] To a volume of 300 .mu.l of each lysed sample (prepared as
descried above) TDE was added to result in a final concentration of
20% [v/v], and mixed for 10 sec. The whole mixtures were then
applied onto HIGH PURE columns from the HIGH PURE PCR Product
Purification Kit (Roche), 11 732 668 001 and spun for 30 s at
13,100.times.g. The flow-throughs were collected. TDE was added to
each flow-through sample, to result in a final TDE concentration of
40% [v/v] in each sample. After mixing by vortexing for 10 s each
mixture was applied onto a second HIGH PURE column. Each column was
then centrifuged for 30 s at 13,100.times.g. Each column of the
first and second set was washed two times with a volume of 500
.mu.l of washing buffer consisting of 80% [v/v]ethanol, 20 mM NaCl,
2 mM Tris-HCl pH 7.5 from the HIGH PURE PCR Product Purification
Kit. Elution was subsequently performed with a volume of 100 .mu.l
desorption buffer per column, containing 10 mM Tris-HCl pH 8.5.
[0198] 10 ng of total RNA nucleic acids as purified with the one
column protocol and the same aliquot of the original sample from
the purified small nucleic acids fraction (containing miRNA) from
the two column protocol (see Example 8) were reverse transcribed
and PCR amplified on the LightCycler 480 from Roche using the
hsa-let-7a miRNA Kit with the TaqMan MicroRNA Assay Protocol of
Applied Biosystems. The primer for the reverse transcription and
the PCR primers were contained in the Applied Biosystems Kit.
[0199] In FIG. 7 the result of the PCR amplification on the
LightCyler 480 is shown. It can be seen that the 1 column protocol
as well as the 2 column protocol yield signals at similar
amplification cycles (CP values) for the liver tissue CP 21.89
(with the single column protocol, see Example 7) as compared to CP
21.55 (with two column protocol). With kidney tissue the cp for the
purified miRNA sample was earlier CP 20.89 (with two column
protocol). as compared to the sample with the total nucleic acid CP
21.72 (with one column protocol). The negative controls without
Reverse Transcriptase and no template control yielded no signals
for all isolated RNA samples, as expected. This demonstrates, that
the purification protocol is compatible with the subsequent reverse
Transcription and the detection in the PCR amplification steps.
Example 10
Isolation of Small Nucleic Acids from Tissue, Including Proteinase
K Digestion
[0200] Samples of small pieces of mouse liver tissue were frozen in
liquid nitrogen and pulverized in a cooled mortar. Aliquots of 10
mg samples were then dissolved in 100 .mu.l Tissue Lysis Buffer
[consisting of 4 M Urea, 100 mM NaCl, 260 mM EDTA, 200 mM Tris-HCl,
pH 7.3-7.4] from the HIGH PURE RNA Paraffin Kit (Roche), catalog
no. 03 270 289 001. Then 16 .mu.l 10% SDS and 40 .mu.l Proteinase K
working solution from the HIGH PURE RNA Paraffin Kit (Roche),
catalog no. 03 270 289 001) were added to each sample.
[0201] The samples were then vortexed three times, each time for 5
s, and incubated for 1 h at 55.degree. C. Then 325 .mu.l of Binding
Buffer [consisting of 5 M GuSCN, 50 mM Tris-HCl, 20% TRITON X-100
(w/v), 1% DTT (w/v), pH 6.0] from the HIGH PURE RNA Paraffin Kit
(Roche), catalog no. 03 270 289 001 and 325 .mu.l TDE were added
and vortexed for 3.times.5 s. The final pH of that mixture was
7.3.
[0202] The mixtures were then applied onto HIGH PURE columns from
the HIGH PURE PCR Product Purification Kit (Roche), catalog no. 11
732 668 001 and spun for 30 s at 13,100.times.g. The flow-throughs
were collected. TDE was added to each flow-through sample, to
result in a final TDE concentration of 40% [v/v] in each sample.
After mixing by vortexing for 10 s each mixture was applied onto a
second HIGH PURE column. Each column was then centrifuged for 30 s
at 13,100.times.g. Each column of the first and second set was
washed two times with a volume of 500 .mu.l of washing buffer
[consisting of 80% [v/v]ethanol, 20 mM NaCl, 2 mM Tris-HCl pH 7.5]
from the HIGH PURE PCR Product Purification Kit. Elution was
subsequently performed with a volume of 100 .mu.l desorption buffer
per column, containing 10 mM Tris-HCl pH 8.5.
[0203] A 10 .mu.l aliquot of each fraction was electrophoresed on
15% acrylamide gels with TBE/Urea runnig buffer (Invitrogen).
[0204] FIG. 8 shows that a concentration of 20% TDE was necessary
on the first column to bind all larger nucleic acid species (lanes
6 and 10), while at lower TDE concentrations larger nucleic acids
copurified with the miRNA (lanes 4 and 5). At 25% TDE concentration
on the first column already some miRNA was bound to the first
column (lane 11).
Example 11
Recovery of Small Nucleic Acid Molecules from Silica Following
Adsorption at Different pH Values
[0205] Samples were prepared with different pH values adjusted.
Each sample consisted of an adsorption solution containing 2 .mu.g
dsDNA with a size of about 400 bp and 2 .mu.g dsDNA with a size of
about 50 bp (resulting in a total of 4 .mu.g of nucleic acid
molecules in each sample), 1.6 M guanidine isothiocyanate, 20 mM
MES, and 40% [v/v] TDE. Samples were identically prepared with the
exception that different pH values were adjusted. The adsorption
solutions were applied to HIGH PURE columns from the HIGH PURE PCR
Product Purification Kit (Roche), catalog no. 11 732 668 001 and
spun for 30 s at 13,100.times.g. Each column was washed once with a
volume of 500 .mu.l of washing buffer [consisting of 80%
[v/v]ethanol, 20 mM NaCl, 2 mM Tris-HCl pH 7.5] from the HIGH PURE
PCR Product Purification Kit. Elution was subsequently performed
with a volume of 100 .mu.l desorption buffer per column, containing
10 mM Tris-HCl pH 8.5.
[0206] The yield of nucleic acids obtained at different pH values
is given in Table 7. As shown, good yields were obtained at all pH
values given.
TABLE-US-00007 TABLE 7 DNA yield in relation to pH in the
adsorption solution pH value of adsorption solution yield [.mu.g]
pH 4.5 3.52 pH 5.0 3.41 pH 5.5 3.13 pH 6.5 3.31 pH 7.0 3.68
* * * * *