U.S. patent application number 11/155811 was filed with the patent office on 2005-11-03 for isolation of nucleic acids on surfaces.
Invention is credited to Bastian, Helge, Fuhrmann, Guido, Gauch, Simone, Oelmuller, Uwe, Schorr, Joachim, Ullmann, Susanne, Weber, Martin.
Application Number | 20050244882 11/155811 |
Document ID | / |
Family ID | 7846424 |
Filed Date | 2005-11-03 |
United States Patent
Application |
20050244882 |
Kind Code |
A1 |
Gauch, Simone ; et
al. |
November 3, 2005 |
Isolation of nucleic acids on surfaces
Abstract
New processes and equipment to isolate and purify nucleic acids
on surfaces are provided. The invention focuses on processes which
use surfaces, for example, porous membranes, on which the nucleic
acids are immobilized in a simple manner from the sample containing
the nucleic acids and can be released again by way of simple
procedural steps, whereby the simple performance of the process
according to the invention makes it possible to perform the
processes specifically in a fully automatic manner. An additional
aspect of the present invention focuses on binding the nucleic
acids to an immobile phase, especially to a membrane, in such a way
and manner, that they can be released without difficulty during an
additional reaction stage from this phase and, if desired, can be
used in other applications, such as restriction digestion, RT, PCR
or RT-PCR, or in any of the suitable analyses or enzyme reactions
mentioned in the disclosure. Special isolation devices are provided
that can be used to carry out the processes according to the
invention.
Inventors: |
Gauch, Simone; (Pasadena,
CA) ; Bastian, Helge; (Dusseldorf, DE) ;
Ullmann, Susanne; (Erkrath, DE) ; Oelmuller, Uwe;
(Erkrath, DE) ; Weber, Martin; (Leichlingen,
DE) ; Fuhrmann, Guido; (Erkelenz, DE) ;
Schorr, Joachim; (Hilden, DE) |
Correspondence
Address: |
LEON R. YANKWICH
YANKWICH & ASSOCIATES
201 BROADWAY
CAMBRIDGE
MA
02139
US
|
Family ID: |
7846424 |
Appl. No.: |
11/155811 |
Filed: |
June 17, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11155811 |
Jun 17, 2005 |
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10300111 |
Nov 20, 2002 |
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10300111 |
Nov 20, 2002 |
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09536735 |
Mar 28, 2000 |
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09536735 |
Mar 28, 2000 |
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PCT/EP99/02664 |
Apr 20, 1999 |
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09536735 |
Mar 28, 2000 |
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PCT/EP98/06756 |
Oct 23, 1998 |
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Current U.S.
Class: |
435/6.16 ;
536/25.4 |
Current CPC
Class: |
C12N 15/1006 20130101;
C12Q 1/6806 20130101; C12Q 1/6806 20130101; C12Q 1/6806 20130101;
C12Q 1/6806 20130101; C12N 15/1017 20130101; Y10T 436/143333
20150115; C12Q 2527/125 20130101; C12Q 2527/125 20130101; C12Q
2523/113 20130101; C12Q 2565/518 20130101; C12Q 2523/308
20130101 |
Class at
Publication: |
435/006 ;
536/025.4 |
International
Class: |
C12Q 001/68; C07H
021/04 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 23, 1997 |
DE |
DE 19746874.8 |
Claims
1-76. (canceled)
77. A process for isolating nucleic acids comprising the following
steps: (a) providing an isolation device with at least one membrane
located therein; (b) applying a nucleic acid-containing sample to
the isolation device; (c) precipitating the nucleic acids contained
in the sample with isopropanol, so that the nucleic acids are bound
to said membrane, and (d) releasing the bound nucleic acids from
said membrane with an elution agent; characterized in that said
membrane comprises cellulose and has pores that are is equal to or
larger than 0.2 micrometer.
78. The process according to claim 77, characterized in that the
isopropanol alcohol is added to the nucleic acid-containing sample
prior to adding the sample to the isolation device.
79. The process according to claim 77, characterized in that the
isopropanol is added to the nucleic acid-containing sample after
adding the sample to the isolation device.
80. The process according to claim 77, characterized in that the
surface of the membrane is selected so that all the nucleic acids
contained in the solution can be bound to the membrane.
81. (canceled)
82. The process according to claim 77, wherein said nucleic acids
precipitated are DNA and/or RNA.
83. (canceled)
84. The process according to claim 77, wherein the volume ratio of
the nucleic acids-containing sample to isopropanol is 2:1 to
1:1.
85-92. (canceled)
93. The process according to claim 77, wherein the membrane
consists of cellulose acetate or cellulose nitrate.
94. The process according to claim 93, wherein the membrane has a
pore size equal to or more than 0.45 .mu.m.
95. The process according to claim 93, wherein the membrane has a
pore size of more than 0.6 .mu.m.
96-120. (canceled)
121. The process according to any one of claims 77-80, 82, 84, and
93-95, wherein said elution agent is a solution of a compound
selected from the group consisting of water, morpholinopropane
sulfonic acid, tris(hydroxymethyl)aminomethane,
2-[4-(2-hydroxymethyl)piperazino]ethane sulfonic acid, an alkaline
metal salt, an alkaline-earth metal salt, a carboxylic acid salt, a
dicarboxylic acid salt, and combinations thereof.
122. The process according to any one of claims 77-80, 82, and 84,
wherein said isolation device is provided with one or more
membranes selected from the group consisting of a cellulose nitrate
membrane, a cellulose acetate membrane, and combinations of
cellulose nitrate and cellulose acetate membranes.
123. The process according to claim 122, wherein said isolation
device is provided with one or more membranes having a pore size
selected from the group consisting of 0.45 .mu.m, 0.65 .mu.m, and
combinations thereof.
124. The process according to any one of claims 77-80, 82, 84, and
93-95, wherein nucleic acids are released from said membrane in
step (d) at a final yield of 80% or greater.
125. The process according to any one of claims 77-80, 82, 84, and
93-95, wherein nucleic acids are released from said membrane in
step (d) at a final yield in a range of 80% to 90%.
126. The process according to any one of claims 77-80, 82, 84, and
93-95, wherein nucleic acids are released from said membrane in
step (d) at a final yield of 90% or greater.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of pending
International application no. PCT/EP99/02664, filed Apr. 20, 1999
and designating the United States, and of pending International
application no. PCT/EP98/06756, filed Oct. 23, 1998 and designating
the United States and claiming priority to German application DE
19746874.8, filed Oct. 23, 1997.
FIELD OF THE INVENTION
[0002] This invention concerns new processes for the isolation and
purification of nucleic acids on surfaces.
BACKGROUND OF THE INVENTION
[0003] It has been known for a long time that the genetic origin
and functional activity of a cell can be determined and studied by
examination of its nucleic acids. Methods of analyzing nucleic
acids permit direct access to the cause of cell activity. Such
methods are therefore potentially superior to indirect conventional
methods such as detecting metabolic products. For that reason a
large expansion in the number of nucleic acid analyses can be
expected in the future. For instance, molecular biological analyses
are already used in many areas, for example, in medical and
clinical diagnostics, in pharmacology for the development and
evaluation of medications, in analysis of foodstuffs as well as
monitoring food manufacturing and food inspection, in the
agricultural business for breeding useful plants and animals, in
environmental analysis, and in many research areas, including, for
example, paternity analyses, tissue typing, identification of
genetic diseases, genome analyses, molecular diagnostics, such as
the identification of infectious diseases, transgenic research,
basic research in the area of biology and medicine, as well as in
numerous related areas.
[0004] Through RNA analysis, especially mRNA in cells, gene
activity can be determined directly. The quantitative analysis of
transcript patterns (mRNA patterns) in cells, by way of modem
molecular biology methods, such as, e.g., real-time reverse
transcriptase PCR ("Real-Time RT-PCR") or gene expression chip
analyses, permit for example the recognition of defectively
expressed genes, through which many types of disorders, e.g.,
metabolic diseases, infections or the generation of cancer, may be
recognized. Analysis of DNA from cells by way of molecular
biological methods, such as, e.g., PCR, RFLP, AFLP or sequencing,
permits for example the assessment of genetic defects in or the
determination of the HLA type as well as of other genetic
markers.
[0005] The analysis of genomic DNA and RNA is also utilized to
directly prove the existence of infectious stimuli, such as
viruses, bacteria, etc.
[0006] In this connection, a general difficulty exists in the fact
that biological and/or clinical samples must be prepared in such a
way that the nucleic acids contained therein can be utilized
directly in the analytical method in question. It is especially
important that the nucleic acids be provided in good yield, that
the recovered nucleic acids be of high quality, and that there be
high reproducibility, in particular where there are a greater
number of samples, in which case the analysis should be capable of
being conducted automatically.
[0007] The state of the art already includes many processes for the
purification of DNA. For example, it is known how to purify plasmid
DNA for the purpose of cloning and other experimental processes.
See, e.g., the method of Bimboim, Methods in Enzymology, 100:243
(1983). In this process, a cleared lysate of bacterial origin is
exposed to a cesium chloride gradient and centrifuged for a period
of 4 to 24 hours. This step is usually followed by the extraction
and precipitation of the DNA. This process is associated with the
disadvantages that it is very apparatus-intensive, and it takes a
great deal of time, is expensive to run and cannot be
automated.
[0008] Other methods in which cleared lysates are used to isolate
DNA are based on ion-exchange chromatography (e.g., Colpan et al.,
J. Chromatog., 296:339 (1984)) and gel filtration (e.g., Moreau et
al., Analyt. Biochem., 166:188 (1987)). These processes are
primarily alternatives to the cesium chloride gradients; however
they require an extensive solvent supply system, and a
precipitation of the DNA fractions is necessary, since these
usually contain salts in high concentrations and are extremely
diluted solutions.
[0009] Marko et al., Analyt. Biochem., 121:382 (1982), and
Vogelstein et al., Proc. Nat. Acad. Sci., 76:615 (1979), have found
that if the DNA from extracts containing nucleic acids is exposed
to high concentrations of sodium iodide or sodium perchlorate, only
DNA will adhere to glass scintillation tubes, fiberglass membranes
or fiberglass sheets that have been finely ground by mechanical
means, while RNA and proteins will not. The DNA that has been bound
in this manner can be eluted, for example, with water.
[0010] For example, in international publication WO 87/06621, the
immobilization of nucleic acids on a PVDF membrane is described.
However, the nucleic acids bound to the PVDF membrane are not
eluted in the next step; instead the membrane, together with all
the bound nucleic acids is introduced directly into a PCR reaction.
Finally, in this international patent application and in the other
literature, it is stated that hydrophobic surfaces or membranes
must in general be wetted beforehand with water or alcohol, in
order to be able to immobilize the nucleic acids with yields that
are satisfactory.
[0011] On the other hand, for a number of modern applications, such
as, for example, the PCR, reversed transcription PCR, SunRise, LCR,
branched-DNA, NASBA, or TaqMan technologies and similar real-time
quantification methods for PCR, SDA, DNA and RNA chips and arrays
for gene expression and mutation analyses, differential display
analyses, RFLP, AFLP, cDNA synthesis or substractive hybridization,
it is absolutely necessary to be able to release the nucleic acids
directly from the solid phase. In this connection, WO 87/06621
teaches that, while the nucleic acids can indeed be recovered from
the membranes used in the process, this recovery is fraught with
problems and is far from suited to the quantitative isolation of
nucleic acids. In addition, the nucleic acid obtained in this
manner is, comparatively, extremely diluted, which makes subsequent
isolation and concentration steps absolutely necessary.
SUMMARY OF THE INVENTION
[0012] According to the present invention, all aqueous or other
solutions of nucleic acids, as well as all materials and all
samples containing nucleic acids, as well as biological samples and
materials, foodstuffs, etc. are defined as "nucleic acid samples".
In the sense of the present invention, a sample or a material
containing a nucleic acid is defined as a nucleic acid sample
and/or a sample preparation which contains the nucleic acids in
question. Biological material and/or biological samples in this
connection include, e.g., cell-free sample material, plasma, body
fluids--such as for example, blood, sputum, urine, feces, sperm,
cells, serum, leucocyte fractions, crusta phlogistica, smears;
tissue samples of any type, tissue parts and organs; foodstuff
samples which contain free or bound nucleic acids or nucleic
acid-containing cells; environmental samples which contain free or
bound nucleic acids or nucleic acid-containing cells, plants and
parts of plants, bacteria, viruses, yeasts and other funghi, other
eukaryotes and prokaryotes, etc., as they are published, e.g., in
the European patent publication No. EP 743 950 A1, which is
incorporated herein by reference, or free nucleic acids as well. In
the sense of the present invention, nucleic acids comprise all
types of nucleic acids, such as, e.g., ribonucleic acids (RNA) and
desoxyribonucleic acids (DNA), in all lengths and configurations,
such as double strands, single strand, circular and linear,
branched, etc.; monomer nucleotides, oligomers, plasmids, viral and
bacterial DNA and RNA, as well as genomic or other non-genomic DNA
and RNA from animal and plant cells or other eukaryotes, tRNA, mRNA
in processed and non-processed form, hn-RNA, rRNA and cDNA as well
as all other nucleic acids that can be envisioned.
[0013] For the reasons stated above, the processes known from the
state of the art do not constitute--particularly with regard to
automation of the process for obtaining nucleic acids--a suitable
starting point for an isolation of nucleic acid that is as simple
and quantitative as possible from the point of view of process
engineering. The purpose of this invention is therefore to overcome
the disadvantages of the processes known from the state of the art
for the isolation of nucleic acids and to provide a process and
method which are capable of being applied or carried out without
substantial technical expenditure.
[0014] According to the present invention, the aforementioned
disadvantages are solved by the processes, isolation and/or
reaction devices uses, automatic apparatus kits according to the
description, drawings and claims below.
[0015] In addition, the invention focuses on processes which make
use of surfaces, e.g., porous membranes, on which the nucleic acids
can be easily immobilized from the sample containing the nucleic
acids, and can again be released by way of similarly easy steps of
the process, whereby the simple performance of the process
according to the invention makes it possible to specifically carry
out the process in a fully automated manner.
[0016] Another purpose of this invention is, in particular, to bind
nucleic acids to an immobile solid phase--especially to a
membrane--in such a manner that in a subsequent reaction step they
can be released immediately from this phase and, if desired, used
in other applications, such as, for example, restriction digest,
RT, PCR or RT-PCR, as well as any other suitable analytical or
enzymatic reaction named above.
[0017] Within the scope of the present invention, a surface is
defined as any microporous separating layer. This may also directly
rest on a substratum and therefore only be accessible from one side
or be standing freely in space. Within the meaning of the present
invention a membrane is defined as a separating layer which is
accessible from both sides when it does not rest with its entire
surface area on an impenetrable substratum but is entirely free or
is only supported at single points.
[0018] Within the meaning of the present invention, isolation is
defined as any accumulation of nucleic acids, in which the
concentration of nucleic acids is increased and/or the portion of
non-nucleic acids in a sample preparation and/or sample is
reduced.
[0019] The invention provides a process to isolate nucleic acids
including the following steps:
[0020] applying at least one nucleic acid sample to a membrane;
[0021] immobilizing the nucleic acids on the membrane;
[0022] releasing the immobilized nucleic acids from the membrane;
and
[0023] removing the released nucleic acids through the
membrane,
[0024] whereby the membrane contains nylon, polysulfone,
polyethersulfone, polycarbonate, polyacrylate, acrylic copolymer,
polyurethane, polyamide, polyvinylchloride, polyfluorocarbonate,
polytetrafluoroethylene, polyvinylidene fluoride,
polyethylene-tetrafluoroethylene-copolymerisate, polybenzimidazole,
polyethylene-chlorotrifluoroethylene-copolymerisate, polyimide,
polyphenylene sulfide, cellulose, cellulose-mix-ester,
cellulose-nitrate, cellulose-acetate, polyacrylnitrile,
polyacrylnitrile-copolymers, nitrocellulose, polypropylene and/or
polyester.
[0025] Other membranes also, such as those mentioned below in the
present description, may be used for processes according to the
invention.
[0026] Preferably the loading process takes place from the top and
the removal process is carried out in a downward direction;
however, flow-through processes, for example, can be envisioned in
which a horizontal column is loaded from one side with a solution
containing nucleic acid, which, after immobilization of the nucleic
acids, penetrates through the membrane and can be removed at the
other end of the column.
[0027] Preferably, the membrane is situated in a container, e.g.,
the column mentioned above or any elongated container having an
inlet and an outlet, wherein the membrane stretches across the
entire diameter of the container.
[0028] The membrane may be coated so as to render it hydrophobic or
hydrophilic.
[0029] Isolation processes to date, especially in isolation
columns, function with relatively thick membranes and/or fleeces in
order to achieve a complete isolation of the nucleic acids. When
the solution is suctioned through the membrane, however, a
relatively large, so-called dead-space-volume, i.e., the volume of
the membrane, is generated from which the nucleic acids can only be
recovered by way of a larger quantity of an elution buffer. This,
however, causes the nucleic acids to be more diluted after the
elution, which is undesirable or disadvantageous for many
applications. For this reason, a preferred embodiment of the
invention uses a membrane which is less than 1 mm thick, preferably
less than 0.5 mm, and most preferably less than 0.2 mm, e.g., 0.1
mm thick.
[0030] The invention furthermore involves a process to isolate
nucleic acids with the following steps:
[0031] applying at least one nucleic acid sample to a surface;
[0032] immobilizing the nucleic acids on the surface; and
[0033] releasing the immobilized nucleic acids from the surface
with an elution agent.
[0034] This process is characterized in that the release takes
place at a temperature whose upper limit is 10.degree. C. or less
and whose lower limit is at the freezing point of the elution agent
to be used for such release, so that the elution agent does not
freeze. Therefore the following inequation applies: 10.degree.
C..gtoreq.T.gtoreq.T.sub.S, EM, in which T is the release
temperature and T.sub.S, EM is the freezing point of the elution
agent. We have discovered that, contrary to widespread opinion, a
release of the nucleic acids near the freezing point of the elution
agent is quite possible. Such an elution at low temperature even
has the unexpected advantage that the nucleic acids are treated
more gently and that the activity from any nucleases (DNases or
RNases) still present in the sample drops practically to nothing
near the freezing point, so that degradation of the nucleic acids
is reduced or completely prevented.
[0035] Accordingly, the temperature during elution should
preferably be even lower, e.g., at less than 5.degree. C. The lower
limit may also be at 0.degree. C. or -5.degree. C., if the specimen
is still liquid at this temperature, based on its ion content. The
upper temperature limit should if possible also be low, e.g., at
about 5.degree. C.
[0036] The process according to the invention therefore requires
cooling of the elution buffer and may require cooling of any
additional solutions used, as well as cooling of the isolation
device if necessary. Since cooling cannot always be guaranteed in a
reliable manner, especially during examinations performed in the
field, e.g., when screening human samples in developing countries,
the present invention also provides an isolation device which
allows isolation of nucleic acids at low temperatures independent
from any external cooling. For such situations, the instant
invention provides an isolation device to isolate nucleic acids
having at least an upper part with a top opening, a bottom opening
and a membrane, which is located at the bottom opening and which
fills the entire diameter of the upper part; a bottom part with an
absorbent material; and a collar surrounding the upper part, at
least in the area of the membrane, which contains a coolant. The
collar containing the coolant allows cooling of the membrane and
the solutions placed on the membrane such as the lysate, washing
buffer and elution buffer at low temperatures, so that the final
elution can take place in a reliable manner within the desired
temperature range near the freezing point of the elution
buffer.
[0037] In an embodiment of this isolation device, the collar has
two compartments, which are separated from one another by a
mechanically destructible or frangible separation wall, with each
of the compartments containing a solution and in which upon mixing
of both solutions after destruction of the separating wall, the
coolant is generated. The separating wall can be destroyed by the
user, e.g., by pressing against the external collar wall, e.g., at
points provided for such purpose, and thus causing the separating
wall to tear. Suitable solutions to fill the compartments are
familiar to practitioners in the area of chemical cooling
technology. These may be adjusted to the desired temperatures and
to the outside temperatures expected when using the isolation
device.
[0038] When recovering nucleic acids from biological samples, such
as the samples indicated above, it is often necessary to make a
lysate the cells or secretions first, in order to be able to reach
the nucleic acid. The lysates thus produced may also contain large
amounts of undesirable substances in addition to the nucleic acids,
such as proteins or fats. If the content of such substances in a
lysate is too high, the membrane may become clogged when the lysate
is applied, which reduces the efficiency of the nucleic acid
isolation and which reduces the permeability of the membrane during
washing or elution. In order to avoid this undesirable effect, the
invention provides a process in which undesirable substances are
removed before they reach the membrane.
[0039] In preferred embodiments, the process according to the
invention to isolate nucleic acids comprises the following
steps:
[0040] adjusting at least one nucleic acid sample to binding
conditions which allow immobilization of the nucleic acids
contained in at least one of the nucleic acid samples on a
surface;
[0041] applying at least one nucleic acid sample to the surface;
and
[0042] immobilization of the nucleic acids on the surface,
characterized in that before and/or after adjusting the binding
conditions, a pretreatment is applied.
[0043] The pre-treatment may, for instance, take place by salting
out or by filtration, centrifugation, enzymatic treatment,
temperature effect, precipitation and/or extraction of the nucleic
acid solution and/or binding contaminants of the nucleic acid
solution to surfaces. The pre-treatment may also involve mechanical
disruption or homogenizing the nucleic acid solution, if it is for
example the lysate of a biological sample.
[0044] The binding conditions that were adjusted may permit the
immobilization of RNA and/or DNA in this case.
[0045] A pre-treatment may be necessary especially in cases when
one intends to isolate biological samples with severe contaminants.
The biological sample may consist of any conceivable material which
is used either immediately or can be recovered from another
biological sample. For instance, this may be blood, sputum, urine,
feces, sperm, cells, serum, leukocyte fractions, crusta
phlogistica, smears, tissue samples, plants, bacteria, funghi,
viruses and yeasts, as well as all other types of biological
samples mentioned above.
[0046] The process according to the invention may be used to its
greatest advantage if the biological sample contains a large amount
of undesirable substances.
[0047] After immobilization of the nucleic acids from the
pre-treated nucleic acid sample, the usual isolation steps can be
followed, i.e.:
[0048] releasing the immobilized nucleic acids from the
surface;
[0049] recovering the nucleic acids released from the surface.
[0050] A special advantage of the isolation process according to
the invention concerns the fact that it may be connected with
chemical reactions, to which the nucleic acids are subjected
directly on the surface. A variety of analytical techniques for
nucleic acids may therefore be used with the nucleic acids isolated
on the surface. In this case it is possible to again release the
nucleic acids from the surface prior to the reaction in order to
guarantee their free accessibility. Alternatively, a suitable
reaction may also be performed with the nucleic acids which are
directly bound on the surface.
[0051] Accordingly, one aspect of the invention involves a process
with a pre-treatment, as outlined above, which is characterized in
that the following step preferably takes place at least once after
the release stage:
[0052] performing at least one chemical reaction with the nucleic
acids.
[0053] A special advantage of this process lies in the fact that
prior to the chemical reaction, no loss resulting from transfer of
the nucleic acids from the isolation device to a reaction device
occurs, because the isolation and chemical reaction can take place
in the same device.
[0054] In an additional aspect not related to pre-treatment, the
invention involves a process to carry out a nucleic acid
amplification reaction with the following steps:
[0055] applying at least one nucleic acid sample to a surface;
[0056] immobilizing the nucleic acids on the surface; and
[0057] performing an amplification reaction with the nucleic
acids.
[0058] Especially with the small quantities of material commonly
used in amplification reactions or available for use in
amplification reactions, it is generally advantageous if the whole
reaction sample of nucleic acids can be used in the reaction
without any loss from transfer. This is especially advantageous for
an automated process since all steps can be carried out in one
device. Furthermore, the amount of waste is reduced and the process
is faster and more cost-effective.
[0059] The amplification reaction may be an isothermal or a
non-isothermal reaction.
[0060] The amplification reaction may, e.g., consist of an
SDA-reaction ("strand displacement amplification"), a PCR, RT-PCR,
LCR or a TMA or a rolling circle amplification.
[0061] A NASBA-reaction is also possible with this process
according to the invention.
[0062] Prior to carrying out the amplification reaction, the
nucleic acids may be released from the surface with a reaction
buffer, whereby the eluate is located on or in the membrane.
Alternatively, the amplification reaction may be carried out in a
reaction buffer that does not produce a release of the nucleic
acids from the surface.
[0063] This process preferably produces these additional steps:
[0064] if necessary, release of the reaction products from the
surface (to the extent these were still bound during the reaction);
and
[0065] removal of the released reaction products from the
surface.
[0066] Another aspect involves a process to perform chemical
reactions with nucleic acids by way of the following steps:
[0067] applying at least one nucleic acid sample to a surface;
[0068] immobilizing nucleic acids on the surface;
[0069] releasing the immobilized nucleic acids from the
surface;
[0070] performing at least one chemical reaction with the nucleic
acids; and
[0071] removal of the nucleic acids from the surface without prior
immobilization.
[0072] In this process the nucleic acids are no longer bound
(immobilized) to the membrane after the chemical reaction, but
removed without binding. Although the elimination of such an
additional step may compromise the purity of the removed specimen,
it may be preferred because it saves time in critical applications
and it also simplifies certain application methods. A wide range of
chemical reactions is available as a result of the process
according to the invention. Within the meaning of the invention
"chemical reaction" should be defined in this connection as any
interaction of the nucleic acids with other substances (with the
exception of the surface, since this "reaction" occurs in all
processes described herein), i.e., enzymatic modifications,
hybridization with probes, chemical sequencing reactions,
pH-value-changes, e.g., for basic depurination of RNA and acid
depurination of DNA, as well as antibody binding and protein
binding. Generally, each reaction, whether it concerns the changing
of covalent bonds or hydrogen bonds, is included.
[0073] One advantage of the process according to the invention is
the permanent, spatial combination of a volume chamber, in which a
great variety of processes can take place, and a membrane to which
nucleic acids can be bound. Simply put, this combination allows the
manipulation of nucleic acids followed by binding to a membrane.
This is especially advantageous for automated processes. After
binding to the membrane, the nucleic acids are available for
additional treatment steps, e.g., as mentioned above, for isolation
of highly pure nucleic acids or for performing chemical reactions
with the nucleic acids. An additional aspect of the invention makes
it also possible to immediately subject the nucleic acids still
bound to the membrane to further analysis, in order to determine
certain properties of the nucleic acids.
[0074] For that reason the invention also involves a process to
analyze nucleic acids in an isolation device with the following
steps:
[0075] making available an isolation device with a membrane located
therein;
[0076] applying at least one nucleic acid sample to the isolation
device;
[0077] immobilizing the nucleic acids on the membrane;
[0078] leading the fluid components of the sample through the
membrane; and
[0079] analyzing at least one property of the nucleic acids on the
membrane located in the isolation device.
[0080] After passing the fluid components through the membrane, at
least one chemical reaction as mentioned above can be performed
with the nucleic acids in an additional embodiment. This may serve,
e.g., to allow the subsequent analysis of the nucleic acids.
Examples of reactions in this context are the hybridization of
probes, the radioactive labeling of nucleic acids bound to the
membrane or the binding of specific antibodies. Auxiliary reactions
such as staining nucleic acids, e.g., with intercalating substances
such as ethidium bromide should also be considered as a chemical
reaction.
[0081] Various properties of nucleic acids are open to an analysis
while they are bound to the membrane. They have already been
described for conventional membranes without a combined reaction
device. Some of the properties that can be analyzed are the
radioactivity of nucleic acids or their binding affinity for
molecules, in which the molecules for example may be antibodies or
dye molecules that bind nucleic acids or are bound to nucleic acids
or proteins.
[0082] This process represents a considerable simplification of the
analysis of nucleic acids, since the manipulation of the free
membrane is no longer necessary. This is now located in the
isolation device.
[0083] An irreversible bond of the nucleic acids to the membrane,
e.g., for subsequent analytical steps is also within the scope of
the present invention. This long-lasting or irreversible bond
permits the manipulation of the membrane and the nucleic acids
bound thereon to an extent that is not possible for reversibly
bound nucleic acids.
[0084] An additional aspect of the invention focuses on the
quantitative precipitation of nucleic acids.
[0085] In previously known methods based on anion-exchange
chromatography for purification of 100 .mu.g and more plasmid-DNA
(hereinafter indicated as "large scale" DNA purification), the
plasmid-DNA is eluted in a high saline buffer from the column
during the last step. In order to separate the plasmid-DNA from the
salt on the one hand, and to concentrate it on the other, it is
precipitated with the aid of alcohol (e.g., isopropanol) and
centrifuged in a suitable device. The centrifugation pellet thus
obtained is washed with 70% ethanol, in order to remove the
residual traces of salt and is then again subjected to
centrifugation. The pellet from the second centrifugation is
typically dissolved in a small amount of low saline buffer and the
plasmid-DNA is processed further in this form.
[0086] In addition, the state of the art has proposed processes in
which DNA is added in such a form by adding chaotropic salts to the
high saline buffer so as to cause binding to silica membranes.
After a corresponding washing step, the DNA can again be released
from the membrane by way of a low saline buffer.
[0087] A similar application is described in a publication (Ruppert
et al., Analytical Biochemistry, 230: 130-134 (1995)) in which on a
small scale (isolation of less than 100 .mu.g of plasmid-DNA) DNA
precipitated with isopropanol is bound to PVDF-membranes with pore
sizes of less than 0.2 .mu.m, subsequently washed with ethanol and
then eluted with TE (Tris-EDTA). However, there is no description
of such a method for the large scale process.
[0088] The DNA precipitation described with subsequent
centrifugation is extremely time-consuming (approx. 1 hour), and
furthermore requires the use of centrifuges. In addition to the
time factor for this procedure, the last step described for plasmid
preparation is particularly prone to errors. A partial or complete
loss of the DNA-pellet also occurs occasionally. A decisive roll
appears to be the type (material) of the centrifugation device
used.
[0089] The use of chaotropic salts (also described) and the
subsequent binding of nucleic acids to silica membranes is also
time-consuming; moreover, because of the introduction of chaotropic
salts to the preparation there is the risk of contamination of the
finally isolated DNA.
[0090] The filtration of alcoholic precipitates on a small scale as
described above has the disadvantage that the operation cannot be
transferred linearly to a large scale process.
[0091] Conventional membranes only permit the isolation of small
amounts of nucleic acids, as the membranes are quickly saturated
with nucleic acids and no longer absorb anything. When the
precipitate buffer is removed and washed, a large portion of the
nucleic acids is frequently lost again. In order to avoid this
loss, the invention also involves a process to precipitate nucleic
acids by way of the following steps:
[0092] making available an isolation device with at least one
membrane situated therein;
[0093] applying a nucleic acid sample to the isolation device;
[0094] precipitation of the nucleic acids contained in the sample
with alcohol, so that the nucleic acids are at least bound to a
membrane. The process is characterized in that the pore size of at
least one membrane is the same or greater than 0.2 micrometers.
[0095] Alcohols considered to perform the process according to the
invention are first of all hydroxyl derivates of aliphatic or
acyclical saturated or unsaturated hydrocarbons. Among the
aforementioned hydroxyl compounds, the C.sub.1-C.sub.5 alkanols,
such as methanol, ethanol, n-propanol, n-butanol, tert.-butanol,
n-pentanol or mixtures thereof are preferred. Especially preferred
is the use of isopropanol to carry out the process according to the
invention.
[0096] In this process, the alcohol can be mixed with this solution
before or after loading the isolation device with the solution
containing the nucleic acid. The volume ratio of the nucleic
acid-containing solution to alcohol, especially isopropanol,
preferably is 2:1 to 1:1, most preferably 1.67:1 to 1:1, and for
example 1.43:1.
[0097] The surface of the membrane is preferably chosen so that all
the nucleic acids contained in the solution can be bound to the
membrane.
[0098] The invention also involves the use of membranes with a pore
size of equal or larger than 0.2 .mu.m to bind the
alcohol-precipitated nucleic acids, which may consist of DNA and/or
RNA. Especially advantageous is the use of a 0.45 .mu.m cellulose
acetate or cellulose nitrate filter and/or the use of various
layers of a 0.65 .mu.m cellulose acetate or cellulose nitrate
filter. The procedure can both be used as vacuum filtration and as
pressure filtration.
[0099] The process according to the invention permits a time-saving
transfer of nucleic acids from a high-salt buffer system to a
low-salt buffer system, which is possible without use of complex
apparatus. It is suitable as a substitute for the classical
alcoholic precipitation of DNA from a high-salt buffer, which is
typically by centrifugation steps. Because of the great
effectiveness of the method (minor loss of yield) it is especially
suitable as a preparation for a large scale process. Furthermore
the process according to the invention does not introduce any
additional substances in the already purified nucleic acids. In
addition, compared to the classical method, susceptibility to
errors is less (loss of the centrifugation sediment during the
washing cycle is not possible using the process of the
invention).
[0100] Preferably, applying the solution should take place from the
top in the various processes explained above. In principle, a wide
range of methods are available which pass various solutions such as
nucleic acid-containing immobilization buffers, washing buffers,
eluate, etc. through the membranes.
[0101] This may be achieved through gravity, centrifugation,
vacuum, positive pressure (on the loading side), and capillary
forces.
[0102] Between the immobilization and the separation step, the
immobilized nucleic acids may be washed with at least one washing
buffer. The washing preferably consists of the following steps for
each washing buffer:
[0103] applying a predetermined quantity of washing buffer to the
surface, and
[0104] passing the washing buffer through the surface.
[0105] The application and immobilization of the nucleic acids may
again consist of the following steps:
[0106] mixing of the nucleic acid sample with an immobilization
buffer; applying the nucleic acid sample with the immobilization
buffer on the surface, and
[0107] passing the liquid components through the surface in
essentially the direction of the loading step.
[0108] The processes have the major advantage that they can easily
be automated, so that at least one of the steps can be fully
automated in an automatic device. It is also possible to have all
steps of the processes performed in a pre-arranged sequence by an
automatic apparatus. Especially in these cases, but also for manual
handling, it is possible that a majority of nucleic acids are
simultaneously subject to isolation. For example, multi-isolation
devices may be used in the form of commonly available "multi-well"
devices with 8, 12, 24, 48, 96 or more single isolation wells.
[0109] The removal of the nucleic acids may take place in two
roughly different directions. On the one hand it is possible to
feed (pass) the (eluted) nucleic acids that were removed through
the membrane and to remove them toward the membrane's side, that is
located opposite the side on which the nucleic acid-containing
solution and/or the lysate was placed. In this case the nucleic
acid is removed in the direction of its passing through the
membrane. The other possibility consists of removing the nucleic
acids from the membrane and/or from the surface on the side where
they were introduced. The removal then takes place in the direction
opposite to their introduction or "in the same direction", in which
they were introduced; in other words, on the side where they were
introduced. In this case the nucleic acids do not pass through the
membrane. In some of the processes according to the invention,
removal of the nucleic acids takes place through the membrane in
the direction they were introduced. In the event a process is
carried out with a surface that does not have a non-permeable
substratum, e.g., a synthetic layer, the removal can of course only
take place in the direction of introduction (hence in the opposite
direction). For a few processes, however, the substance can be
removed in both directions.
[0110] If the nucleic acids are eluted (released) from the surface
essentially in the opposite direction from the direction in which
they were introduced and immobilized, "the same direction" is
essentially considered each direction with an angle equal or
smaller than 180.degree., compared to the direction of
introduction, so that upon elution, the nucleic acids under no
circumstances permeate the surface, e.g., a membrane, but are
removed from the surface in the direction opposite from the loading
direction in which they were introduced to the surface. In
preferred embodiments, on the other hand, the other buffers, i.e.,
those buffers which contain nucleic acids during the loading
process, and if required a washing buffer, are suctioned through
the surface or otherwise transferred. If the isolation takes place
on a membrane located in a device, whereby the membrane fills the
entire diameter of the device, the preferred loading method is from
the top. In this case the removal step again occurs upward. FIG. 2
shows an example of a funnel-shaped isolation device, which is
loaded from the top and in which the removal of the nucleic acids
takes place in an upward direction. It is understood that, in the
case of removal in a direction opposite to introduction, other
configurations are also imaginable, e.g., removal of the nucleic
acids from below. It is possible, for example, to suction a buffer
containing nucleic acids, such as a lysate buffer from a reaction
device directly into an isolation device by way of a suction
installation, so that the nucleic acids will be bound to the bottom
of a membrane in the isolation device. In such a case, the removal
of the nucleic acids from the surface can be carried out, in such a
way that an elution buffer is suctioned up from below and is
drained again downward into a device after separation of the
nucleic acids. The removal of the nucleic acids therefore also
takes place in a downward direction.
[0111] A lateral removal of the nucleic acids is also possible,
e.g., if a horizontal column with a membrane located therein is
loaded with a lysate during the flow-through process and the
horizontal column is subsequently washed with elution buffers on
the side of the membrane to which the nucleic acids are bound.
[0112] An example for the maximum possible angle of 180.degree. is
a slope with a surface suitable to bind nucleic acids along which
surface the various solutions and/or buffers flow. Like all
buffers, the elution buffer also arrives from one side and is
drained on the other side. In this case, the inflow direction of
the buffer and the draining direction of the buffer with the
nucleic acids included therein make an angle of 180.degree.; the
removal, however, continues to take place on the same side of the
surface as the immobilization.
[0113] Following the process according to the invention, the sample
containing nucleic acids described above is added to a solution
which contains the appropriate salts and/or alcohol(s);
[0114] subsequently the sample is lysed, if necessary, and the
mixture obtained in this manner is led by way of a vacuum,
centrifugation, positive pressure, capillary forces or by way of
other appropriate processes, through a porous surface, whereby the
nucleic acids are immobilized on the surface.
[0115] Suitable salts for the immobilization of nucleic acids on
membranes or other surfaces and/or for the lysis of nucleic acid
samples are salts of metal cations, such as alkaline or alkaline
earth metals, with mineral acids; especially alkaline or
alkaline-earth halides and/or sulfates or phosphates, including the
halides of sodium, lithium or potassium or magnesium sulfate, which
are most preferable. Other metal cations, e.g., Mn, Cu, Cs or Al,
or the ammonium cation can be used, preferably as salts of mineral
acids.
[0116] Furthermore to carry out the process according to the
invention, salts having one or more basic functions or even
polyfunctional organic acids with alkaline or alkaline-earth metals
are suitable. These especially include sodium, potassium or
magnesium salts with organic dicarboxylic acids, such as e.g.,
oxalic, malonic or succinic acids, or with hydroxy and/or
polyhydroxycarboxylic acids, such as, e.g., with citric acids,
preferably.
[0117] The substances indicated above to immobilize the nucleic
acids on surfaces and/or for the lysis of nucleic acid samples may
be used separately or in mixtures, if this should prove to be more
suitable for certain applications.
[0118] In this connection the use of so-called chaotropic agents
has proved to be particularly effective. Chaotropic substances are
able to disrupt the three-dimensional structure of hydrogen bonds.
This also weakens the intramolecular binding forces which are
involved in the formation of spatial structures, such as, e.g.,
primary, secondary, tertiary or quaternary structures, in
biological molecules. Suitable chaotropic agents are well known to
those skilled in the art (see, Rompp, Lexikon der Biotechnologie,
Publisher H. Dellweg, R. D. Schmid and W. E. Fromm, Thieme Verlag,
Stuttgart 1992).
[0119] According to this invention preferred chaotropic substances
are salts from the group of trichloroacetates, thiocyanates,
perchlorates, iodides or guanidinium hydrochloride and urea. The
chaotropic substances are then used in a 0.01 to 10 molar aqueous
solution, preferably in a 0.1 to 7 molar aqueous solution, and most
preferably in a 0.2 to 5 molar aqueous solution. In this connection
the aforementioned chaotropic agents can be used individually or in
combination. Most preferably 0.01 to 10 molar aqueous solutions, or
0.1 to 7 molar aqueous solutions, or 0.2 to 5 molar aqueous
solutions of sodium perchlorate, guanidinium hydrochloride,
guanidinium isothiocyanate, sodium iodide and/or potassium iodide
are used.
[0120] The salt solutions used in the processes according to the
invention for lysis, binding, washing and/or for elution are
preferably buffered. All suitable buffer systems can be considered
as buffer substances, such as, e.g., carboxylic acid buffers,
especially citrate buffers, acetate buffers, succinate buffers,
malonate buffers as well as glycine buffers,
morpholino-propane-sulfone-acids (MOPS) or Tris (hydroxymethyl)
aminomethane (Tris) in concentrations of 0.001 to 3 mol/liter,
preferably 0.005 to 1 mol/liter, and most preferably 0.01 to 0.5
mol/liter, and particularly preferred 0.01 to 0.2 mol/liter.
[0121] To carry out the process according to the invention, first
all hydroxyl derivates of aliphatic or acyclical saturated or
unsaturated hydrocarbons are eligible as alcohols. It is irrelevant
whether these compounds contain one, two, three or more hydroxyl
groups--such as polyvalent C.sub.1-C.sub.5 alkanols, e.g., ethylene
glycol, propylene glycol or glycerin.
[0122] In addition, the alcohols that can be used according to the
invention also include sugar derivates, the so-called aldites, as
well as phenols, e.g., polyphenols.
[0123] Among the aforementioned hydroxy compounds,
C.sub.1-C.sub.5-alkanol- s, such as methanol, ethanol, n-propanol,
tert.-butanol and pentanols, or mixtures of such alcohols, are most
preferred.
[0124] Within the meaning of this invention, such substances and/or
membranes which by their chemical nature easily mix with water or
absorb water are considered hydrophilic.
[0125] Within the meaning of this invention, such substances and/or
membranes which by their chemical nature do not penetrate water or
vice-versa and which cannot stay dissolved in water are considered
hydrophobic.
[0126] Within the meaning of this invention, any microporous
separating layer is understood to be a surface. In the case of a
membrane the surface consists of a film made of polymer material.
The polymer preferably consists of monomers with polar groups.
[0127] In a further embodiment of the process according to the
invention, the concept of surface furthermore also comprises a
layer of particles and/or a granulate as well as fibers such as
silica gel fleece.
[0128] When hydrophobic membranes are used in the practice of this
invention, membranes are preferred which consist of a hydrophilic
basic material and which are made hydrophobic by a corresponding
chemical post-treatment which is known from the state of the art.
Membranes such as commercially available hydrophobic nylon
membranes are preferably used.
[0129] According to the invention membranes that are hydrophobic
are generally defined as those membranes which are originally
hydrophilic membranes that have been coated with hydrophobical
coating agents mentioned below. Such hydrophobical coating agents
coat the hydrophilic substances with a thin film of hydrophobic
groups, which, e.g., include longer alkyl chains or siloxane
groups. Many suitable hydrophobic coating agents are known and
include, e.g., paraffins, waxes, metallic soaps, etc., if necessary
with additions of aluminum, zirconium salts, quaternary organic
compounds, ureic derivates, lipid-modified melamine resins,
silicones, zinc organic compounds, glutaric dialdehydes, and
similar compounds.
[0130] According to the invention suitable hydrophobic membranes
also are those membranes which are by themselves hydrophobic or
which have been made hydrophobic and whose basic material may
contain polar groups. According to these criteria, e.g., especially
hydrophobic materials from the following group are suitable for use
according to the invention: Nylon, polysulfones, polyether
sulfones, cellulose nitrate, polypropylene, polycarbonates,
polyacrylates as well as acrylic copolymers, polyurethanes,
polyamides, polyvinyl-chloride, polyfluorocarbonates,
polytetrafluoroethylene, polyvinylidene fluoride,
polyethylene-tetra-fluoroethylene copolymerisates,
polyethylene-chlorotrifluoro-ethylene-copolymerisates, or
polyphenylene sulfide, as well as cellulose and cellulose-mix
esters, cellulose acetate or nitrocellulose as well as
polybenzimidazoles, polyimides, polyacryl nitrites,
polyacrylnitril-copolymers, hydrophobisized glass fiber membranes,
including hydrophobisized nylon membranes which are most
preferable.
[0131] Preferred hydrophilic surfaces include hydrophilic materials
per se and also hydrophobic materials which have been
hydrophilisized. For instance the following substances can be used:
hydrophilic nylon, hydrophilic polyether-sulfones, hydrophilic
polycarbonates, hydrophilic polyesters, hydrophilic
poly-tetra-fluoroethylenes on polypropylene tissues, hydrophilic
polytetrafluoroethylenes on polypropylene fleece, hydrophilisized
polyvinylidene fluoride, hydrophilisized polytetrafluoroethylenes,
hydrophilic polyamides, nitrocellulose, hydrophilic
polybenzimidazoles, hydrophilic polyimides, hydrophilic
polyacryl-nitriles, hydrophilic polyacrylnitril-copolymers,
hydrophilic polypropylene, cellulose nitrate, cellulose-mix-ester
and cellulose acetate.
[0132] The membranes described above are already known in the art,
partially for their use in nucleic acid binding, but not yet in the
context of the invention. A series of materials for this particular
use is, however, not known from the state of the art. The extensive
trials disclosed herein have demonstrated that there are additional
membranes that are suitable to bind nucleic acids.
[0133] The present invention therefore also involves the use of
cellulose acetate, non-carboxylized, hydrophobic polyvinylidene
fluoride, or massive hydrophobic poly-tetra-fluorethylene as a
material on which to precipitate and isolate nucleic acids. In this
context, the term "massive" denotes a material which generally
consists of the corresponding compound and is neither coated nor
applied as a coating on a carrier material.
[0134] The material may be used as a membrane, as granulate, as
fibers or in other suitable forms. The fibers may, e.g., be
configured as fleece and the granulate may be pressed as a
grid.
[0135] The membranes used in the process described above according
to the invention (with the exception of isopropanol precipitate)
for instance have a pore diameter of 0.001 to 50 .mu.m, preferably
0.01 to 20 .mu.m, and most preferably a pore size of 0.05 to 10
.mu.m. In case the nucleic acids are precipitated with isopropanol
according to the process described above, the pore size must be
greater than 0.2 .mu.m.
[0136] The salts or alcohols described above or the phenols or
polyphenols may also be considered as washing buffers. Detergents
and natural substances in the broadest sense of the word, such as
albumin, or milk powder may also be used for the washing steps. The
addition of chaotropic substances is also possible. Polymers as
well as detergents with dissolving abilities and similar materials
may also be added. The washing buffers and the substances contained
therein should at any rate generally be able to bind undesirable
contaminants, to dissolve them or to react with them, so that these
contaminants or their decomposition products can be removed jointly
with the washing buffer.
[0137] The temperatures during the washing stage typically range
from about 10.degree. to 30.degree. C., preferably at room
temperature, although higher or lower temperatures may also be
applied successfully. When elution is performed at a low
temperature, e.g. 2.degree. C., one should not forget to also cool
the washing buffer in order to pre-cool the temperature of the
isolation device and the surface and/or membrane to the desired
temperature. One application for low temperatures is cytoplasmatic
lysis, during which the cell nuclei remain undamaged. Higher
temperatures of the washing buffers on the other hand cause better
dissolution of the contaminants to be washed out.
[0138] Suitable eluting agents for the purposes of the invention
are water or aqueous salt solutions. Buffer solutions that are
known from the state of the art are used as salt solutions, such as
morpholinopropane sulfonic acid (MOPS),
tris(hydroxymethyl)aminomethane (TRIS),
2-[4-(2-hydroxyethyl)piperazino]ethane sulfonic acid (HEPES) in a
concentration from 0.001 to 0.5 moles/liter, preferably 0.01 to 0.2
moles/liter, most preferably 0.01 to 0.05 molar solutions. Also
preferred for use are aqueous solutions of alkaline or
alkaline-earth metal salts, in particular, their halogenides, for
example, including 0.001 to 0.5 molar (preferably 0.01 to 0.2
molar, most preferably 0.01 to 0.05 molar) aqueous solutions of
sodium chloride, lithium chloride, potassium chloride or magnesium
chloride. Also preferred for use are solutions of salts of the
alkaline or alkaline-earth metals with carboxylic or dicarboxylic
acids, e.g., oxalic acid or acetic acid, or solutions of sodium
acetate or sodium oxalate in water, e.g., in the concentration
range mentioned above, such as 0.001 to 0.5 molar, preferably 0.01
to 0.2 molar, most preferably from 0.01 to 0.05 molar.
[0139] The addition of subsidiary compounds such as detergents or
DMSO is also possible. If a chemical reaction must be carried out
with the eluted nucleic acids, either directly on the membrane or
in another reaction device, it is also possible to add such
substances or other subsidiary compounds which are to be used in
the reaction to the elution buffer. For instance, the addition of
DMSO in low concentrations is customary in many reactions.
[0140] After a chemical reaction with the nucleic acids, these can
also be eluted with the reaction buffer. For instance, the nucleic
acids can be eluted with the reaction buffer or the reaction master
mix after a SDA- or a NASBA-reaction.
[0141] Most specifically, pure water is the preferred elution
agent, e.g., demineralized, bi-distilled, or ultra pure millipore
water.
[0142] The elution can, for example, be carried out successfully at
temperatures from below 0.degree. C. to 90.degree. C., e.g., from
10.degree. to 30.degree. C. or at higher temperatures. It is also
possible to elute with water vapor. The lower limit of the elution
temperature is, as explained above, the freezing point of the
elution buffer.
[0143] Based on the smooth executability of the processes according
to the invention which can also be performed "in the field", i.e.,
outside of established laboratory installations and therefore
without extensive electrically powered equipment, the invention
also involves the preparation of isolation devices with which the
process according to the invention can be carried out with a
minimum of additional subsidiary materials. For this, a reaction
device can be used which contains a membrane. This can be brought
into contact with an absorbent material, such as a sponge, in order
to absorb the various buffers used through the membrane. The sponge
acts therefore as a combination vacuum pump or centrifuge in
conjunction with a waste collector. In order to recover the eluate,
contact of the absorbent material with the membrane is eliminated,
so that the eluate cannot be lost, but instead can be removed or
studied further.
[0144] In this aspect, the invention specifically involves an
isolation device to isolate nucleic acids with at least a
cylindrical upper part with a top opening, a bottom opening and a
membrane which is located at the bottom opening and fills the
entire diameter of the upper part; is equipped with a bottom part
containing an absorbent material; and a mechanism for the
connection between the upper and lower parts, in which, after the
connection has been made, the membrane is in contact with the
absorbent material, and when the connection is not made, the
membrane is not in contact with the absorbent material.
[0145] Preferably, the bottom or lower part is a cylinder with the
same diameter as the upper part. In this manner, a simple tube is
obtained having essentially a constant diameter, which can be
handled in the same way as traditional reaction devices. Especially
if the upper part or the upper part plus lower part create a tube
which can be placed in reaction device holders, such as those used
in laboratories, this effect can be achieved. The mechanism can be
a connection which allows a spatial separation of the upper and
lower parts, for example a bayonet socket, a plug-in socket or a
threaded end. A bayonet socket has the advantage that it is easier
to lock and unlock, whereas the threaded connection allows for a
better, more watertight connection of the upper and lower parts.
Alternatively, a pre-determined breaking point can be provided
between the upper and lower parts, which at least allows for the
one-time separation of both parts and which can be manufactured at
a very low price. Alternatively, the connection can also be a
sliding mechanism which can be slid between the absorbent material
and the membrane. In this embodiment, a separation of membrane and
absorbent material can be achieved as well.
[0146] To increase the processing capacity and to be able to carry
out the process according to the invention even more economically,
it is also possible to modify the isolation device according to the
invention described above in such a way that various upper parts
are placed on a bottom part. The bottom part can serve
simultaneously as a holder of the assembly and in addition have
such dimensions that a variety of isolation processes, at least
more than mere connections for the upper and lower parts, are
available, and can be carried out before the suction capacity of
the absorbent material in the bottom part is exhausted.
[0147] The absorbent material in the lower part may contain a
sponge and/or a granulate. The granulate can consist of a
superabsorbent material, as is known by those skilled in the art of
absorption technology (e.g., for hygiene-related items).
[0148] The invention similarly involves utilization of this
isolation device according to the invention for the analysis of
properties of nucleic acids and to isolate nucleic acids.
[0149] With respect to the separate stages, the processes according
to the invention are typically carried out as follows:
[0150] When starting from biological samples, they must first be
subjected to lysis in the appropriate buffers. Additional processes
to achieve lysis may be needed, e.g., a mechanical action, such as
homogenization or ultrasound, enzymatic reaction, temperature
changes or additives. In case it is required or desirable, a
pre-treatment can follow this lysis in order to remove debris from
the lysate. Subsequently, in case this has not happened yet, the
conditions in the lysate are adjusted, so that immobilization of
the nucleic acids on the surface can take place. Even after
adjustment of the binding conditions, a pre-treatment step can
follow cumulatively or alternatively to the above pre-treatment
step.
[0151] This pretreated lysate of the sample used for the recovery
of nucleic acids or the originally free nucleic acid(s)-if one did
not start from a biological sample is/are pipetted, for example, in
a (plastic) column, in which the hydrophobic membrane is fastened,
for example, on the floor. It is more efficient if the membrane is
fastened to a grid, which serves as a mechanical support. The
lysate is then conducted through the membrane, which can be
achieved by applying a vacuum at the outlet of the column. The
transport can also be accomplished by applying positive pressure to
the lysate. In addition, as mentioned above, the transport of the
lysate can take place by centrifugation or by the effect of
capillary forces. The latter can be produced, for example, with a
sponge-like material which is introduced below the membrane and is
in contact with the lysate or filtrate. In the case of
centrifugation, the isolation device open at the bottom may be used
in a collection tube for the flowthrough liquid.
[0152] The washing stage included in the preferred embodiments can
take place if the washing buffer is transported through the surface
of the membrane or is remaining on the same side of the surface as
the nucleic acids. If the washing buffer is transported or
suctioned through, this can take place in different ways, e.g., by
a sponge located on the other side of the membrane, a suction or
positive pressure mechanism or by centrifugation or gravity.
[0153] The advantage of a configuration utilizing an absorbent,
possibly spongy material is that it provides a simple, secure and
handy means for disposing of the filtrate, in this case only the
sponge, which by that time is more or less saturated with the
filtrate and needs to be replaced. At this point it is clear that
the column can be operated continuously or also in a batch-like
manner, and that both modes of operation can be fully automated,
until the membrane is saturated with nucleic acids. In the last
stage, if required, the elution of the nucleic acids takes place,
which for example can be pipetted or lifted from the membrane or
can be removed upward in another way, if no in situ analysis of the
nucleic acids that are still bound is to be performed.
[0154] The desired nucleic acids are present in very small volumes
of buffers with no or low salt concentrations, which is a great
advantage for all molecular biological analyses, since it is always
desirable to have pure nucleic acids in high concentrations and in
the smallest volumes possible. In order to obtain the smallest
possible volumes of eluate, it is especially preferred to use as
surfaces those membranes that are as thin as possible, so that only
very little liquid can accumulate in them.
[0155] Furthermore, the present invention offers the advantage that
in the case of a vertical configuration of the device (where the
membrane is placed in a horizontal direction) the volume located
above the membrane can be used as a reaction chamber. Hence, it is
possible, for example, after isolation and removal of the nucleic
acids recovered according to the process of the invention, to not
remove them immediately but to leave them in the isolation device
and to subject them to a molecular biological application, such as
restriction digest, RT, PCR, RT-PCR, in vitro transcription, NASBA,
rolling circle, LCR (ligase chain reaction), SDA (strand
displacement amplification) or enzyme reactions, such as RNase- and
DNase-digestion for the complete removal of any of the nucleic
acids that are not wanted, to bind the nucleic acids resulting from
these reactions again to the membrane according to the process
according to the invention or to leave them in the supernatant, if
necessary to wash them as described, and subsequently to elute
them, to isolate and/or analyze them, e.g., by way of
chromatography, spectroscopy, fluorometry, electrophoresis, or
similar measurements.
[0156] The nucleic acids isolated according to the invention are
free of enzymes that degrade the nucleic acids and have such a high
purity that they can immediately be used and processed in the
greatest variety of ways.
[0157] The nucleic acids produced according to the invention can be
used for cloning and as substrates for a great variety of enzymes,
such as, e.g., DNA-polymerases, RNA-polymerases such as, e.g.,
T7-polymerase or T3-polymerase, DNA-restriction enzymes,
DNA-ligase, reverse transcriptase and others.
[0158] The nucleic acids produced by the processes of the invention
are especially suitable for amplification, especially for PCR,
strand displacement amplification, rolling circle processes, ligase
chain reaction (LCR), SunRise, NASBA and similar processes.
[0159] The processes according to the invention are furthermore
extremely suitable to produce nucleic acids for their use in
diagnostics, e.g., in food analysis, in toxicological examinations,
in medical and clinical diagnostics, in diagnostics of germs, gene
expression analysis, and in environmental analysis. The processes
are especially suitable for a diagnostic process, which is
characterized in that the nucleic acids purified by way of the
processes according to the invention are amplified in a subsequent
step, and the nucleic acids that are thus amplified are detected
subsequently and/or simultaneously (see, e.g., Holland et al.,
1991, Proc. Natl. Acad. Sci., 88: 7276-7280; Livak et al., 1995,
PCR Methods Applic., 4: 357-362; Kievits et al., 1991, J. Virol.
Meth., 35: 273-286; Uyttendaele et al., 1994, J. Appl. Bacteriol.,
77: 694-701).
[0160] Moreover, the processes according to the invention are
especially suitable to produce nucleic acids which, in a subsequent
step, are subjected to a signal amplification step based on a
hybridization reaction, which is specifically characterized by the
fact that the nucleic acids produced in the process according to
the invention are brought into contact with "branched nucleic
acids", especially branched DNA and/or branched RNA and/or
corresponding dendritic nucleic acids and the signal that is
generated is detected, as described in the following literature
(e.g., Bresters et al., 1994, J. Med. Virol., 43(3): 262-286;
Collins et al., 1997, Nucl. Acids Res., 25(15): 2979-2984).
[0161] An example of automation of a process according to the
invention is explained below and examples to perform the process
with different surfaces and nucleic acids are also described. In
this description reference is made to the attached figures which
illustrate the following.
BRIEF DESCRIPTION OF THE DRAWINGS
[0162] FIG. 1 shows automatic equipment suitable to perform the
process according to the invention in a stylized graph.
[0163] FIG. 2 shows a first embodiment of an isolation device and
collector to perform the process according to the invention.
[0164] FIG. 3 shows a second embodiment of an isolation device and
collector to perform the process according to the invention.
[0165] FIG. 4 shows a third embodiment of an isolation device and
collector to perform the process according to the invention.
[0166] FIG. 5 shows embodiments of isolation devices with an upper
part according to the invention.
[0167] FIG. 6 shows the ethidium bromide stained gel of an
electrophoretic separation of various samples according to the
process of the invention.
[0168] FIG. 7 shows another ethidium bromide stained gel of an
electrophoretic separation of various samples according to the
process of the invention.
[0169] FIG. 8 shows another ethidium bromide stained gel of an
electrophoretic separation of various samples according to the
process of the invention.
[0170] FIG. 9 shows the ethidium bromide stained gel of an
electrophoretic separation of various samples according to the
process of the invention.
[0171] FIG. 10 shows another ethidium bromide stained gel of an
electrophoretic separation of various samples according to the
process of the invention.
[0172] FIG. 11 shows another ethidium bromide stained gel of an
electrophoretic separation of various samples according to the
process of the invention.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0173] The processes according to the invention are preferably
performed in an automatic manner either partially or completely, in
other words, in all stages. An example for suitable automatic
equipment is illustrated in FIG. 1, in which a main part 1 is
equipped with control electronics and driving engines with a work
platform 3 and a movable arm 2. Various elements are positioned on
the work platform, such as area 4 to hold various devices. A vacuum
manifold 5 serves to absorb liquids from isolation devices which
are placed above it and are open at the bottom, or otherwise with
the devices connected to the vacuum manifold. A shaker 6 is also
provided, which can be used, e.g., for the lysis of biological
samples. The isolation device assemblies used are, e.g.,
injection-molded parts with integrated isolation devices, in which
the surfaces according to the invention are included. Typically 8,
12, 24, 48, 96 or up to 1536 isolation devices can be used as these
are available for example in the formats of modem
multi-well-plates. Even higher numbers of isolation devices might
be possible in one plate, if standards are available. With the aid
of Luer-adapters it is, however, also possible to make separate
bottoms of the assembly available and to equip these with one or
more isolation devices as needed. Isolation devices used
individually without Luer-adapters are also included in the
invention.
[0174] Under a vacuum and dispensing mechanism 8 the isolation
devices are placed in the automatic apparatus and with these,
liquids can be taken up and dispensed. In this assembly several
single vacuum units may be provided, so as to make the simultaneous
processing of an isolation or reaction device possible. The vacuum
and dispensing mechanism 8 therefore acts as a pipet. Vacuum and
pressure are fed to the vacuum and dispensing mechanism 8 via tube
9.
[0175] To isolate the nucleic acids, reaction devices with cells
may for example be placed in the shaker/holder 6, into which lysis
buffers are introduced with the help of the dispensing mechanism.
After mixing, the cell lysates are transferred to isolation
devices. The lysis buffer is subsequently passed through the
surfaces in the isolation devices. Subsequently, the surfaces may
be washed with a washing buffer in order to remove cell lysate
residues, in which also the washing buffer is drained off downward.
Finally, an elution buffer is dispensed into the isolation devices
and after repeated shaking the separated nucleic acids are removed
from above and transferred to collection microtubes.
[0176] Usually, disposable tips are used on the vacuum and
dispensing mechanism 8 to prevent contamination of the samples.
[0177] FIGS. 2 through 4 show different schematic examples for
suitable isolation devices to be used according to the present
invention.
[0178] In FIG. 2, a funnel-shaped isolation device 10 is provided
with a surface 11, e.g., a membrane, which is placed on a collector
12, which contains a sponge-like material 13 that serves to absorb
the lysis and washing buffers. Under the sponge-like material 13 a
superabsorbent layer 14 may be placed to improve the suction
performance. Alternatively, layer 14 may also contain a material
which is chemically able to react with water, e.g., acrylate. The
water is therefore also removed from the process. Lysate or another
preparation of nucleic acids is placed in the funnel. The
sponge-like material 13 absorbs the applied liquid through membrane
11. Prior to the addition of the elution buffer, the sponge is
moved some distance from the membrane, e.g., by a mechanism inside
a collector 12 (not visible in the drawing). This will prevent the
elution buffer in the last stage from being also suctioned through
membrane 11. This buffer, however, stays on the surface (FIG. 2b)
and can be removed together with the nucleic acids from above. When
using this assembly, the vacuum mechanism 5 in the automatic
apparatus is no longer necessary.
[0179] FIG. 3 shows another example of an isolation device, which
is connected to a collector 16 via a Luer-connection located at the
bottom via a Luer-adapter 17, which in this case does not contain a
sponge, but is connected to a vacuum mechanism via a muff 18. Lysis
and washing buffers may in this case be suctioned through membrane
11 by creating a vacuum (FIG. 3a). When the eluate buffer is
introduced, the vacuum remains turned off, so that the eluate can
be removed from above (FIG. 3b). With the use of a Luer-connection,
individual isolation devices can be removed from the isolation
device assembly. It will be understood, however, that the vacuum
collector can also be combined with fixed isolation devices, e.g.,
multi-well devices containing 8, 12, 24, 48, 96 or more single
devices.
[0180] FIG. 4 finally shows an embodiment which provides a
collector, into which the buffers are suctioned through the
membrane or surface 11 by way of gravity or centrifuged. The eluate
buffer, which is used in small volumes, is not heavy enough itself
to penetrate membrane 11 and can again be removed from above (FIG.
4b).
[0181] FIG. 5 shows embodiments of the isolation devices according
to the invention.
[0182] In FIG. 5A, an isolation device with a cylindrical upper
part 20 has been illustrated. This upper part is connected to a
bottom part 22 by way of a threaded connection 25. Instead of the
threaded connection other types of connections may also be used, to
the extent these permit a watertight connection of the upper and
bottom parts and provide a possibility of introducing membrane 11.
In this embodiment, membrane 11 is applied directly to the bottom
opening of upper part 20. It may, however, also be moved inward or
be placed at an angle other than 90.degree. with respect to the
upper part's wall. The bottom part also has a cylindrical shape,
but may be of a different design in other embodiments. For example,
a quadrangular shape may be used, which improves the stability of
the upper part 20 on a surface. The widening of bottom part 22
compared to upper part 20 is also possible, for example in case a
larger cavity is required in bottom part 22 in certain embodiments
of the process according to the invention in order to fully absorb
the solutions used in the absorbent material 13.
[0183] An alternative embodiment to the embodiment shown in FIG. 5A
is illustrated in FIG. 5B. In this case upper part 20 and bottom
part 22 are fixed to one another or may also be built in one piece.
Between the absorbing material 13 and membrane 11, a sliding
mechanism 27 may be slid via an opening 26 into the isolation
device to separate membrane 11 and absorbent material 13 from one
another. In this example sliding mechanism 27 is equipped with an
additional handle 28, which facilitates pulling out sliding
mechanism 27. The sliding mechanism can, however, also be designed
without this handle. As shown in FIG. 5B, the absorbent material 13
expands slightly, to be able to bridge the space taken up by the
sliding mechanism and to make contact with the membrane.
[0184] FIG. 5C shows another embodiment of the isolation device
according to the invention. In this case the bottom part 23 is
equipped with several connections 30 to accommodate the upper parts
20, thus permitting the simultaneous processing of a multiplicity
of samples. The upper parts 20 in this example are connected with
bottom part 23 by way of threaded connections 31. Although shown
smaller in the illustration than the upper parts 20 of FIGS. 5A and
5B, it is understood that the upper parts can be the same size (or
can be larger or smaller) as indicated in those embodiments.
[0185] Finally, FIG. 5 D shows an isolation device according to the
invention with a collar 32 with coolant, which surrounds membrane
11 on the outside. In this embodiment, upper part 20 and bottom
part 24 are connected to one another by way of a plug-in socket.
Another type of connection or a one-piece version are, however,
also possible. Collar 32 consists of two compartments, 33 and 34,
which can be connected with one another by destroying the
separating wall 35. Both compartments 33, 34 are loaded with
substances, e.g. solutions, which, when mixed after destruction of
the separating wall 35, causes the temperature of the entire
mixture to drop.
[0186] The invention described above will be further explained in
the following examples. Different and alternative designs of the
devices and processes will become clear to the skilled practitioner
from the description above and from the following examples. It
should expressly be pointed out, however, that these examples and
the description accompanying these examples only serve as an
illustration of the invention and are not to be considered a
limitation of the invention.
EXAMPLE 1
Isolation of Total RNA from HeLa Cells
[0187] Commercially available nylon membranes (for example, a
material from MSI, "Magna SH" with a pore diameter of 1.2 .mu.m, or
a material from Pall GmbH, "Hydrolon" with a pore diameter of 1.2
.mu.m), which are chemically post-treated and to be hydrophobic,
were placed as a single layer in a plastic column. The membranes
were placed on a polypropylene grid which served as a mechanical
support. The membranes were fixed in the plastic column with a
ring. The column prepared in this manner was connected by means of
a Luer connection to a vacuum chamber. All the isolation steps were
carried out through the application of a vacuum.
[0188] For the isolation, 5.times.10.sup.5 HeLa cells were
harvested by centrifugation and the supernatant removed. The cells
were lysed by the addition of 150 .mu.l of a commercial guanidium
isothiocyanate buffer (e.g., RLT buffer from QIAGEN GmbH, Hilden,
Del.), in a manner thoroughly familiar to those skilled in the art.
Lysis was promoted by roughly mixing by pipetting or vortexing for
5 seconds. Then 150 .mu.l of 70% ethanol were added and mixed in by
repeatedly pipetting or by vortexing for about 5 seconds.
[0189] The lysate was transferred into the plastic column and
suctioned through the membrane by evacuating the vacuum chamber.
Under these conditions, the RNA remained bound to the membrane.
Next, washing was performed using a first commercial washing buffer
containing guanidium isothiocyanate (e.g., with RW1 buffer from
QIAGEN GmbH) and, after that, with a second washing buffer
containing TRIS or TRIS and alcohol (e.g., with the RPE buffer from
QIAGEN GmbH). The washing buffers in each case were suctioned
through the membrane by evacuation of the vacuum chamber. After the
final washing step, the vacuum was maintained for a period of about
10 minutes, in order to dry the membrane, after which the vacuum
was switched off.
[0190] For the elution, 70 .mu.l RNase-free water was pipetted onto
the membrane in order to dissolve the purified RNA from the
membrane. After incubation for one minute at a temperature in the
range from 10.degree. to 30.degree. C., the eluate was pipetted
from the membrane from above and the elution step was repeated in
order to make sure that the elution was complete.
[0191] The quantity of isolated total RNA obtained in this manner
was determined by spectrophotometric measurement of the light
absorption at 260 nm. The ratio between the absorbance values at
260 and 280 nm gives an estimate of RNA purity.
[0192] The results of the two isolations with hydrophobic nylon
membranes (Nos. 1 and 2) are shown in Table 1, compared with
experiments in which on the one hand a hydrophilic nylon membrane
(Nyaflo) (No. 3) and a silica membrane (No. 4) were used. The
values reported in the table provide convincing support for the
impressive isolation yield and separation effect of the materials
used in accordance with this invention. They also show that silica
gel-fleece clearly produces a lower yield, which presumably can be
attributed to its fleecelike structure and the ensuing absorption
of a large portion of the eluate buffer.
1TABLE 1 RNA yield and purity of total RNA isolated according to
Example 1. Yield of Total-RNA Absorbance Sample No. Type of
Membrane (.mu.g) E.sub.260/E.sub.280 1 Magna SH 1.2 .mu.m 6.0 1.97
(hydrophobic nylon) 2 Hydrolon 1.2 .mu.m 7.1 2.05 (hydrophobic
nylon) 3 Nylaflo (hydrophilic <0.2 Not nylon) Determined 4
hydrophilic silica <0.2 Not membrane Determined
[0193] The isolated RNA can also be analyzed on agarose gels that
have been stained with ethidium bromide. For this purpose, for
example, 1.2% formaldehyde agarose gels were prepared. The result
is shown in FIG. 6. In FIG. 6, Lane 1 is the total RNA that was
isolated on a hydrophobic nylon membrane (Magna SH, Sample no. 1)
with a pore diameter of 1.2 .mu.m. Lane 2 s total RNA that was
isolated by means of a hydrophobic nylon membrane (Hydrolon, Sample
no. 2) with a pore diameter of 1.2 .mu.m. Lane 3 represents the
chromatogram of a total RNA that was isolated by means of a silica
membrane (Sample no. 4). In each case, 50 .mu.l of the total RNA
eluate was analyzed. FIG. 6 provides convincing evidence that when
a silica membrane was used, no measurable proportion of the total
RNA can be isolated.
EXAMPLE 2
Isolation of Free RNA by Binding the RNA to Hydrophobic Membranes
by Means of Various Salt-Alcohol Mixtures
[0194] In this example, the lysate and washing solutions are
conducted through the hydrophobic membrane by applying a
vacuum.
[0195] Hydrophobic nylon membranes (e.g., 1.2 .mu.m Hydrolon from
Pall) were introduced into plastic columns connected to a vacuum
chamber, in a manner similar to that of Example 1. To 100 .mu.l
aliquots of an aqueous solution containing total RNA were added 350
.mu.l of a commercially available lysis buffer containing guanidium
isothiocyanate (e.g., RLT buffer from QIAGEN), 350 .mu.l of 1.2 M
sodium acetate solution, or 350 .mu.l of 4 M lithium chloride
solution, respectively, and the resulting solutions were mixed by
pipetting.
[0196] Next, 250 .mu.l of ethanol were added to each mixture and
mixed, likewise by pipetting. After that, the solutions containing
RNA were transferred into the plastic columns and suctioned through
the membrane by evacuating the vacuum chamber. Under the conditions
described, the RNA remains bound to the membranes. The membranes
were then washed, as described in Example 1. Finally, the RNA, also
as described in Example 1, was removed from the membrane by
pipetting from above.
[0197] The quantity of isolated total RNA was determined by
spectrophotometric measurement of the light absorption at 260 nm.
The ratio between the absorbance values at 260 and 280 nm gives an
estimate of RNA purity. The results are set forth in Table 2
below.
2TABLE 2 Isolation of RNA from aqueous solution by binding the RNA
to hydrophobic membranes using various salt-alcohol mixtures. Sam-
Yield of ple Total RNA Absorbance No. Salt/Alcohol mixture (.mu.g)
E.sub.260/E.sub.280 1 RLT-Buffer QIAGEN/35% Ethanol 9.5 1.92 2 0.6
M Sodium Acetate/35% Ethanol 8.5 1.98 3 1 M Sodium Chloride/35%
Ethanol 7.9 1.90 4 2 M Lithium Chloride/35% Ethanol 4.0 2.01
EXAMPLE 3
Isolation of Total RNA from HeLa Cells
[0198] Following the procedures of Example 1, plastic columns were
assembled with different hydrophobic membranes. Each column thus
prepared was placed in a collection tube, and the following
isolation steps were performed by way of centrifugation.
[0199] For the isolation, 5.times.10.sup.5 HeLa cells were
harvested by centrifugation and the supernatant removed. The cells
were lysed by the addition of 150 .mu.l of a commercially available
guanidinium isothiocyanate buffer, such as, e.g., RLT-buffer from
QIAGEN, using well known procedures. In this connection, lysis is
encouraged by multiple pipetting or by vortexing for 5 seconds.
Subsequently, 150 .mu.l of 70% ethanol was added and mixed by
multiple pipetting or by vortexing for 5 seconds.
[0200] The lysate was subsequently transferred into a plastic
column and passed through the membrane by centrifugation at
10000.times.g for 1 minute. Subsequently, washing was performed
with a commercially available washing buffer containing guanidinium
isothiocyanate, e.g., with the RW1-buffer of QIAGEN, followed by a
second washing step using a buffer containing TRIS and alcohol,
e.g., RPE-buffer from QIAGEN. The washing buffers were passed
through the membrane by centrifugation. The last washing step takes
place at 20000.times.g for 2 minutes to dry the membrane.
[0201] For elution, 70 .mu.l of RNase-free water were pipetted onto
the membrane to release the purified RNA from the membrane. After a
1-2 minute incubation at a temperature between
10.degree.-30.degree. C., the eluate was taken from above by
pipetting from the membrane. The elution step was repeated once to
achieve complete elution.
[0202] The quantity of isolated total RNA was determined by
spectrophotometric measurement of the light absorption at a
wavelength of 260 nm. RNA quality is determined by
spectrophotometric determination of the light absorption ratio
compared at 260 nm and at 280 nm. The isolation results with
different hydrophobic membranes are listed in Table 3 below. The
data represent the average of 3-5 parallel tests per membrane.
Using a silica membrane, no measurable quantity of total RNA could
be isolated, where the eluate was recovered by removing it from
above from the membrane.
3TABLE 3 Yield of total RNA isolated by binding to hydrophobic
membranes. Manufacturer Membrane Material RNA(.mu.g)
E.sub.260/E.sub.280 Pall Hydrolon, 1.2 .mu.m Hydrophobic Nylon 6.53
1.7 Pall Hydrolon, 3 .mu.m Hydrophobic Nylon 9.79 1.72 Pall Fluoro
Trans G Hydrophobic Polyvinylidene 6.16 1.72 Fluoride Pall Fluoro
Trans W Hydrophobic Polyvinylidene 5.4 1.9 Fluoride Pall Bio Trace
Hydrophobic Polyvinylidene 4.3 1.97 Fluoride Pall Supor-450 PR
Hydrophobic Polyethersulfone 3.96 1.76 Pall V-800 R Hydrophobic
Acryliccopolymer 6.26 1.72 Pall Versapor-1200R Hydrophobic
Acryliccopolymer 6.23 1.68 Pall Versapor-3000R Hydrophobic
Acryliccopolymer 3.54 1.74 Gore-Tex OH 9335 Hydrophobic Poly- 1.59
1.72 Tetrafluoroethylene Gore-Tex OH 9336 Hydrophobic Poly- 2.15
1.65 Tetrafluoroethylene Gore-Tex OH 9337 Hydrophobic Poly- 3.6
1.59 Tetrafluoroethylene Gore-Tex QH 9316 Hydrophobic Poly- 3.61
1.69 Tetrafluoroethylene Gore-Tex QH 9317 Hydrophobic Poly- 2.87
1.70 Tetrafluoroethylene Millipore Mitex Membrane Hydrophobic Poly-
1.98 1.62 Tetrafluoroethylene Millipore Durapore Hydrophobic
Polyviylidene 7.45 1.72 Fluoride MSI Magna-SH, 1.2 .mu.m
Hydrophobic Nylon 4.92 1.69 MSI Magna-SH, 5 .mu.m Hydrophobic Nylon
10.2 1.71 MSI Magna-SH, 10 .mu.m Hydrophobic Nylon 7.36 1.76 MSI
Magna-SH, 20 .mu.m Hydrophobic Nylon 7.04 1.65 Sartorius Type 118
Hydrophobic Poly- 7.6 1.61 Tetrafluoroethylene Mupor PM12A
Hydrophobic Poly- 6.7 1.77 Tetrafluoroethylene Mupor PM3VL
Hydrophobic Poly- 6.6 1.77 Tetrafluoroethylene
EXAMPLE 3
Isolation of Total RNA from HeLa-Cells by Binding to Hydrophilic
Membranes
[0203] Using the procedures of Example 1, plastic columns were
assembled using different hydrophilic membranes. Each column thus
prepared was placed in a collection tube, and the following
isolation steps were performed by centrifugation.
[0204] For the isolation, 5.times.10.sup.5 HeLa cells were used.
The isolation steps and elution of the nucleic acids were carried
out as described above in Example 3 for hydrophobic membrane
columns.
[0205] The quantity of isolated total RNA was determined by
spectrophotometric measurement of the light absorption at a
wavelength of 260 nm. RNA quality was determined by the
spectrophotometric determination of the ratio of the light
absorption compared at 260 nm and at 280 nm. The isolation results
with various hydrophilic membranes are listed in Table 3b below.
The data represent the average of 2-5 parallel tests per membrane.
Using a silica membrane, no measurable quantity of total RNA could
be isolated, where the eluate was recovered by removing it from
above from the membrane.
4TABLE 3b Yield of total RNA isolated by binding to hydrophilic
membranes. Manufacturer Membrane Material RNA (.mu.g)
E.sub.260/E.sub.280 Pall Loprodyne Hydrophilic Nylon 3.1 1.8 Pall
Loprodyne Hydrophilic Nylon 3.1 1.78 Pall Biodyne A Hydrophilic
Nylon 3.1 1.8 Pall Biodyne A Hydrophilic Nylon 3.6 1.83 Pall
Biodyne B Hydrophilic Nylon 2.6 1.84 Pall Biodyne B Hydrophilic
Nylon 4.2 1.84 Pall Biodyne C Hydrophilic Nylon 6.1 1.88 Pall
Biodyne C Hydrophilic Nylon 5.2 1.91 Pall Biodyne plus Hydrophilic
Nylon 3.3 1.87 Pall I.C.E.-450 Hydrophilic Polyethersulfone 6.36
1.8 Pall I.C.E.-450sup Hydrophilic Polyethersulfone 3.07 1.71 Pall
Supor-800 Hydrophilic Polyethersulfone 4.12 1.7 Pall Supor-450
Hydrophilic Polyethersulfone 4.69 1.69 Pall Supor-100 Hydrophilic
Polyethersulfone 3.25 1.71 Pall Hemasep V Hydrophilic Polyester
4.16 1.74 Pall Hemasep L Hydrophilic Polyester 6.67 1.65 Pall
Leukosorb Hydrophilic Polyester 1.5 1.84 Pall Premium Release
Hydrophilic Polyester 1.66 1.63 Membrane Pall Polypro-450
Hydrophilic Polypropylene 5.09 1.78 Gore-Tex OH 9339 Hydrophilic
Poly- 1.08 1.65 Tetrafluoroethylene Gore-Tex OH 9338 Hydrophilic
Poly- 3.97 1.67 Tetrafluoroethylene Gore-Tex QH 9318 Hydrophilic
Poly- 3.61 1.69 Tetrafluoroethylene Millipore Durapore
Polyvinylidene Fluoride made 5.6 1.69 Hydrophilic Millipore
Durapore Polvinylidene Fluoride made 3.12 1.68 Hydrophilic
Millipore LCR Poly-Tetrafluoroethylene 3.14 1.66 made Hydrophilic
Sartorius Type 250 Hydrophilic Polyamide 4.3 1.66 Sartorius Type
113 Hydrophilic Cellulose Nitrate 1.8 1.86 Sartorius Type 113
Hydrophilic Cellulose Nitrate 1.9 1.74 Infiltec Polycone, 0.01
Hydrophilic Polycarbonate 0.17 1.64 Infiltec Polycone, 0.1
Hydrophilic Polycarbonate 0.73 1.68 Infiltec Polycone, 1
Hydrophilic Polycarbonate 3.33 1.86
EXAMPLE 4
Isolation of Free RNA from an Aqueous Solution
[0206] Using the procedures according to Example 1, plastic columns
were assembled with different hydrophobic membranes. 100 .mu.l of
an aqueous solution containing total RNA were mixed with 350 .mu.l
of a commercially available lysis buffer containing
guanidinium-isothiocyanate, e.g., RLT-buffer from QIAGEN.
Subsequently, 250 .mu.l of ethanol were added and mixed by
pipetting. This mixture was then introduced to the column and
passed through by centrifugation (10000.times.g; 1 minute) through
the membrane. The membranes were subsequently washed twice with a
washing buffer, e.g., RPE from QIAGEN. The buffer was passed
through the membranes by centrifugation. The last washing step was
carried out at 20000.times.g to dry the membranes.
[0207] Next, the RNA, as described in Example 1, was eluted with
RNase-free water and removed from the membrane from above by
pipetting. The quantity of isolated total RNA was determined by
spectrophotometric measurement of light absorption at a wavelength
of 260 nM. RNA quality was determined by the spectrophotometric
determination of the ratio of the light absorption at 260 nm to 280
nm. The isolation results with various hydrophobic membranes are
listed in Table 4 below. The data represent the average of 3-5
parallel tests per membrane. Using a silica membrane, no measurable
quantity of total RNA could be isolated, where the eluate was
recovered by removing it from above from the membrane.
5TABLE 4 Isolation of free RNA from an aqueous solution by binding
to hydrophobic membranes. Manufacturer Membrane Material RNA
(.mu.g) E.sub.260/E.sub.280 Pall Hydrolon, 1.2 .mu.m Hydrophobic
Nylon 5.15 1.75 Pall Hydrolon, 3 .mu.m Hydrophobic Nylon 0.22 1.79
Pall Fluoro Trans G Hydrophobic Polyvinylidene Fluoride 5.83 1.79
Pall Fluoro Trans W Hydrophobic Polyvinylidene Fluoride 5.4 1.84
Pall Bio Trace Hydrophobic Polyvinylidene Fluoride 4.0 1.79 Pall
Emflon Hydrophobic Poly-Tetrafluor-Ethylene 0.2 1.7 Pall Supor-450
PR Hydrophobic Polyethersulfone 5.97 1.71 Pall Supor-200 PR
Hydrophobic Polyethersulfone 2.83 1.66 Pall V-800 R Hydrophobic
Acrylatecopolymer 2.74 1.77 Gore-Tex OH 9335 Hydrophobic
Poly-Tetrafluor-Ethylene 4.35 1.63 Gore-Tex OH 9336 Hydrophobic
Poly-Tetrafluor-Ethylene 7.43 1.71 Gore-Tex OH 9337 Hydrophobic
Poly-Tetrafluor-Ethylene 5.96 1.62 Gore-Tex QH 9316 Hydrophobic
Poly-Tetrafluor-Ethylene 5.92 1.67 Gore-Tex QH 9317 Hydrophobic
Poly-Tetrafluor-Ethylene 8.7 1.66 Millipore Fluoropore Hydrophobic
Poly-Tetrafluor-Ethylene 8.46 1.70 Millipore Durapore, 0.65 .mu.m
Hydrophobic Polyvinylidene Fluoride 4.23 1.8 MSI Magna-SH, 1.2
.mu.m Hydrophobic Nylon 1.82 1.76 MSI Magna-SH, 5 .mu.m Hydrophobic
Nylon 0.6 1.78 Sartorius Type 118 Hydrophobic
Poly-Tetrafluor-Ethylene 0.9 1.82 Sartorius Type 118 Hydrophobic
Poly-Tetrafluor-Ethylene 5.4 1.74 Mupor PM12A Hydrophobic
Poly-Tetrafluor-Ethylene 1.1 1.98
EXAMPLE 4B
Isolation of Free RNA from an Aqueous Solution by Binding to
Hydrophilic Membranes
[0208] Following the procedures of Example 1, plastic columns were
assembled using different hydrophilic membranes.
[0209] 100 .mu.l of an aqueous solution containing total RNA were
mixed with 350 .mu.l of a commercially available lysis buffer
containing guanidinium-isothiocyanate, e.g., RLT-buffer from
QIAGEN. Subsequently 250 .mu.l of ethanol were added and mixed by
pipetting back and forth. This mixture was then introduced to the
column, passed through the membrane, washed and dried according to
the procedure used in Example 4, above.
[0210] Finally, the RNA, as described in Example 1, was eluted with
RNase-free water and removed from the membrane using a pipette.
[0211] The quantity of isolated total RNA was determined by
spectrophotometric measurement of the light absorption at a
wavelength of 260 nm. RNA quality was determined by the
spectrophotometric determination of the ratio of the light
absorption compared at 260 nm and at 280 nm. The isolation results
with various hydrophilic membranes are listed in Table 4b below.
The data represent the average from 2-5 parallel tests per
membrane. Using a silica membrane, no measurable quantity of total
RNA could be isolated, where the eluate was recovered by removing
it from above from the membrane.
6TABLE 4b Isolation of free RNA from an aqueous solution by binding
to hydrophilic membranes. Manufacturer Membrane Material RNA
(.mu.g) E.sub.260/E.sub.280 Pall Loprodyne Hydrophilic Nylon 2 1.8
Pall Loprodyne Hydrophilic Nylon 1.4 1.87 Pall Biodyne A
Hydrophilic Nylon 4.5 1.93 Pall Biodyne A Hydrophilic Nylon 3.1 1.9
Pall Biodyne B Hydrophilic Nylon 1.7 1.94 Pall Biodyne B
Hydrophilic Nylon 1.2 1.94 Pall Biodyne C Hydrophilic Nylon 3.7
1.93 Pall Biodyne C Hydrophilic Nylon 3.1 1.93 Pall Biodyne plus
Hydrophilic Nylon 1.1 1.87 Pall I.C.E.-450 Hydrophilic
Polyethersulfone 1.92 1.82 Pall I.C.E.- Hydrophilic
Polyethersulfone 0.87 1.67 450sup Pall Supor-800 Hydrophilic
Polyethersulfone 3.93 1.74 Pall Supor-450 Hydrophilic
Polyethersulfone 1.78 1.74 Pall Supor-100 Hydrophilic
Polyethersulfone 1.04 1.68 Pall Hemasep V Hydrophilic Polyester 4
1.79 Pall Hemasep L Hydrophilic Polyester 0.47 2.1 Pall Polypro-450
Hydrophilic Polypropylene 5.09 1.78 Gore-Tex OH 9339 Hydrophilic
Poly-Tetrafluor- 0.43 1.48 Ethylene Gore-Tex OH 9338 Hydrophilic
Poly-Tetrafluor- 3.63 1.64 Ethylene Gore-Tex QH 9318 Hydrophilic
Poly-Tetrafluor- 5.92 1.67 Ethylene Millipore Durapore
Polyvinylidene Fluoride made 1.18 1.79 Hydrophilic Millipore LCR
Poly-Tetrafluor-Ethylene made 2.84 1.72 Hydrophilic Sartorius Type
250 Hydrophilic Polyamide 2.7 1.7 Sartorius Type 111 Hydrophilic
Cellulose Acetate 1.6 1.85 Sartorius Type 111 Hydrophilic Cellulose
Acetate 2.2 2.1 Sartorius Type 111 Hydrophilic Cellulose Acetate
0.3 2.01 Sartorius Type 113 Hydrophilic Cellulose Nitrate 4 1.88
Sartorius Type 113 Hydrophilic Cellulose Nitrate 3.8 1.87
EXAMPLE 5
Isolation of Total RNA from HeLa-Cells Depending on the Pore Size
of the Membranes
[0212] Following the procedures of Example 1, plastic columns were
assembled with different hydrophobic membranes with different pore
sizes.
[0213] As in Example 3, a cell lysate was made from
5.times.10.sup.5 HeLa cells and transferred to the columns.
Subsequently the membranes were washed with the commercially
available buffers RW1 and RPE from QIAGEN. The last centrifugation
step was carried out at 20000.times.g for 2 minutes to dry the
membrane. The elution was carried out as described in Example
1.
[0214] The results are listed in Table 5 below. 3-5 parallel tests
per membrane were performed and the average value calculated for
each.
7TABLE 5 Yield of isolated total RNA using hydrophobic membranes
with different pore sizes. Pore Size RNA E.sub.260/ Manufacturer
Membrane Material (.mu.m) (.mu.g) E.sub.280 Infiltec Polycon 0.01
Hydrophilic 0.01 0.17 1.64 Polycarbonate Pall Fluoro Hydrophobic
0.2 6.16 1.72 Trans G Polyvinylidene Fluoride Pall Supor-450
Hydrophobic 0.45 3.96 1.76 PR Polyethersulfone Millipore Durapore
Hydrophobic 0.65 7.45 1.72 Polyvinylidene Fluoride MSI Magna-SH
Hydrophobic Nylon 1.2 4.92 1.69 MSI Magna-SH Hydrophobic Nylon 5
10.2 1.71 MSI Magna-SH Hydrophobic Nylon 10 7.36 1.76 MSI Magna-SH
Hydrophobic Nylon 20 7.04 1.65
EXAMPLE 6
Stability and Quality of Isolated Total RNA from HeLa Cells
[0215] According to procedures of Example 1, plastic columns were
assembled with a commercially available membrane (Pall, Hydrolon
with a 3 .mu.m pore size).
[0216] According to the procedures of Example 3, a cell lysate was
made from 5.times.10.sup.5 HeLa cells and transferred to the
columns. Subsequently, the membranes were washed with the
commercially available buffers RW1 and RPE from QIAGEN. The last
centrifugation step was carried out at 20000.times.g for 2 minutes
to dry the membrane. The elution was carried out as described in
Example 1.
[0217] The isolated total RNA was left to incubate for 16 hours at
37.degree. C. and subsequently placed on a denaturating agarose gel
and analyzed. It was demonstrated that the RNA did not suffer
degradation. The RNA isolated with the method described above shows
no contaminants with enzymes that degrade nucleic acids and
therefore is of high quality.
EXAMPLE 7
Isolation of Free RNA from an Aqueous Solution by Binding to a
Hydrophilic Membrane in a 96-Well Plate
[0218] A 96-well plate with a hydrophilic Polyvinylidene Fluoride
membrane (Durapore, 0.65 .mu.m by Millipore) was used. 5.3 ml of an
aqueous solution containing total RNA were mixed with 18.4 ml of a
commercially available lysis buffer containing guanidinium
isothiocyanate, e.g., RLT buffer from QIAGEN. Subsequently 13.1 ml
ethanol were added and mixed by pipetting back and forth. For each
well, 350 .mu.l of this mixture were introduced and passed through
the membrane by applying a vacuum. The membranes were subsequently
washed twice with a buffer, e.g., RPE from QIAGEN. The buffer was
passed through the membrane each time by applying a vacuum. After
the last washing step, the plate was dabbed once with a paper towel
and subsequently dried for 5 minutes by applying a vacuum.
[0219] The RNA was eluted as described in Example 1, with
RNase-free water and removed from the membrane by way of a pipette.
The quantity of isolated total RNA was determined by
spectrophotometric measurement of the light absorption at a
wavelength of 260 nm and the average value as well as the standard
deviation for the entire plate was calculated. The average value is
8.4 .mu.g with a standard deviation of 0.7 .mu.g.
EXAMPLE 8
Isolation of Total RNA by Way of Capillary Forces
[0220] A 96-well plate with a hydrophilic Polyvinylidene Fluoride
membrane (Durapore, 0.65 .mu.m by Millipore) was used. 33 .mu.l of
an aqueous solution containing total RNA were mixed with 110 .mu.l
of a commercially available lysis buffer containing guanidinium
isothiocyanate, e.g., RLT buffer from QIAGEN. Subsequently 78 .mu.l
ethanol were added and mixed by pipetting. 45 .mu.l of this mixture
were introduced into each well. An absorbent household sponge was
moistened with water, and the 96-well plate was placed with the
membrane's bottom side on the sponge. The RNA mixture was passed
through the membrane by way of capillary forces. The membranes were
subsequently washed twice with a buffer, e.g., RPE from QIAGEN. The
wash buffer was also passed through the membrane by placing the
plate on the sponge. After the last washing step, the plate was
air-dried for 5 minutes.
[0221] The RNA, as described in Example 1, was eluted with
RNase-free water and removed from the membrane by way of a
pipette.
[0222] The quantity of isolated total RNA is subsequently
determined by spectrophotometric measurement of the light
absorption at a wavelength of 260 nm, and the average value as well
as the standard deviation is calculated. The average value is 5.9
.mu.g with a standard deviation of 0.7 .mu.g.
EXAMPLE 9
Isolation of Genomic DNA from an Aqueous Solution by Way of a
Buffer Containing Guanidinium Hydrochloride
[0223] According to Example 1, plastic columns were assembled with
hydrophobic membranes (e.g., Magna-SH, 5 .mu.m by the MSI Company).
Purification is carried out with commercially available buffers
from QIAGEN.
[0224] 200 .mu.l of an aqueous solution of genomic DNA from liver
tissue were introduced in PBS buffers. 200 .mu.l of a buffer
containing guanidinium hydrochloride, e.g. QIAGEN's AL, were added
to and mixed with this solution. Subsequently 210 .mu.l of ethanol
were added and mixed through vortexing. The mixture was introduced
to the column according to Example 3 and passed through the
membrane by way of centrifugation. The membrane was then washed and
dried with an alcohol containing buffer, e.g., QIAGEN's AW. The
elution was performed as described in Example 1. Three parallel
tests were carried out and the average value calculated. The amount
of isolated DNA is subsequently determined by spectrophotometric
measurement of the light absorption at a wavelength of 260 nm and
is approx. 30% of the starting amount. The absorption ratio at 260
nm to 280 nm is 1.82.
EXAMPLE 10
Isolation of Genomic DNA from an Aqueous Solution by Binding to
Hydrophobic Membranes by Way of a Buffer Containing Guanidinium
Isothiocyanate
[0225] According to Example 1, plastic columns were assembled with
different membranes. 100 .mu.l of an aqueous solution containing
total DNA were mixed with 350 .mu.l of a lysis buffer containing
guanidinium isothiocyanate (4 M GITC, 0.1 M MgSO.sub.4, 25 mM
Na-Citrate, pH 4). Subsequently 250 .mu.l ethanol were added and
mixed by pipetting. This mixture was then transferred to the column
and passed through the membrane by way of centrifugation
(10000.times.g; 1 minute). The membranes were subsequently washed
twice with a buffer, e.g., RPE by QIAGEN. The buffer was passed
through the membranes by way of centrifugation. The last washing
step was carried out at 20000.times.g to dry the membranes.
[0226] The elution was performed as described in Example 1. Three
parallel tests were carried out per membrane and the average value
is calculated each time. The results are listed in Table 6.
8TABLE 6 DNA-yield from an aqueous solution by binding to
hydrophobic membranes Manufacturer Membrane Material DNA (.mu.g)
Pall Hydrolon, 1.2 .mu.m Hydrophobic Nylon 1.3 Pall Supor-450 PR
Hydrophobic 2.2 Polyethersulfon Millipore Fluoropore Hydrophobic
1.1 Poly-Tetrafluor-Ethylene Millipore Durapore Hydrophobic 1.2
Polyvinylidene Fluoride
EXAMPLE 11
Isolation of Genomic DNA from Tissue
[0227] According to Example 1, plastic columns were assembled with
hydrophobic membranes (e.g., Magna-SH, 5 .mu.m by MSI).
Purification was carried out with the commercially available
buffers from QIAGEN.
[0228] 180 .mu.l of ATL-buffer were added to 10 mg of kidney tissue
(mouse) and ground in a mechanical homogenizer. Subsequently
proteinase K (approx. 0.4 mg dissolved in 20 .mu.l of water) were
added and incubated for 10 minutes at 55.degree. C. After adding
200 .mu.l of a buffer containing guanidinium hydrochloride, e.g.,
AL by QIAGEN, and after a 10-minute incubation at 70.degree. C.,
200 .mu.l of ethanol were added and mixed with this solution. This
mixture was transferred on to the column and passed through the
membrane by centrifugation. The membrane was then washed with
alcohol containing buffers, e.g., AW1 and AW2 from QIAGEN, and
subsequently dried by way of centrifugation. The elution was
carried out as described in Example 1. Three parallel tests were
carried out and the average value calculated.
[0229] The amount of isolated DNA, determined by spectrophotometric
measurement of the light absorption at a wavelength of 260 nm, was
on average 9.77 .mu.g. The absorption ratio at 260 nm to 280 nm was
1.74.
EXAMPLE 12
Isolation of Genomic DNA from Blood
[0230] According to the procedures of Example 1, plastic columns
were assembled with hydrophobic membranes (e.g., Magna-SH, 5 .mu.m
by MSI). Purification was carried out with the commercially
available buffers from QIAGEN.
[0231] 200 .mu.l of AL buffer and 20 .mu.l of QIAGEN protease were
added to 200 .mu.l of blood, thoroughly mixed, and left to incubate
for 10 minutes at 56.degree. C. After adding 200 .mu.l of ethanol,
the solution was mixed, transferred onto the column, and passed
through the membrane by way of centrifugation. The membrane was
then washed with alcohol containing buffers, e.g., AW1 and AW2 from
QIAGEN, and subsequently dried by way of centrifugation. The
elution was carried out as described in Example 1.
[0232] The amount of isolated DNA, determined by spectrophotometric
measurement of the light absorption at a wavelength of 260 nm, was
1.03 .mu.g. The absorption ratio at 260 nm 280 nm is 1.7.
EXAMPLE 13
Isolation of Total RNA from an RNA-DNA-Mixture
[0233] Following the procedures of Example 1, plastic columns were
assembled with hydrophobic membranes (e.g., Hydrolon 1.2 .mu.m by
the Pall Company). 275 .mu.l of an aqueous solution containing
total RNA and genomic DNA were mixed with 175 .mu.l of a
commercially available lysis buffer containing guanidinium
isothiocyanate, e.g., the RLT buffer from QIAGEN. 250 .mu.l of
ethanol were added and mixed by pipetting. The mixture was
transferred to the column and passed through the membrane, washed
and dried according to Example 4. The flow-through from the first
centrifugation step was placed on a commercially available
mini-spin column (e.g., QIAamp Mini-Spin Column from QIAGEN) and
passed through the membrane via centrifugation. The remaining
washing steps were performed as described in Example 4.
[0234] After this, the nucleic acids were eluted with 140 .mu.l of
RNase-free water by way of centrifugation (10000.times.g, 1 minute)
and analyzed in non-denaturing agarose gel (see FIG. 7). The major
part of the total RNA can be separated from the genomic DNA with
the use of the method described above.
[0235] FIG. 7 shows an ethidium-bromide stained gel of an
electrophoretic separation of two different eluates.
[0236] Lane 1: Isolation of total RNA by way of a hydrophobic nylon
membrane.
[0237] Lane 2: Isolation of genomic DNA from the flow-through by
way of a QIAamp mini-spin column of the QIAGEN company.
EXAMPLE 14
Isolation of Plasmid DNA from an Aqueous Solution by Binding to
Hydrophobic and Hydrophilic Membranes
[0238] Following the procedures of Example 1, plastic columns were
assembled utilizing different membranes.
[0239] 100 .mu.l of an aqueous solution (pCMV.beta. from Clontech)
containing plasmid were mixed with 350 .mu.l of lysis buffer
containing guanidinium isothiocyanate (4 M GITC, 0.1 M MgSO.sub.4,
25 mM sodium-acetate, pH 4). Subsequently, 250 .mu.l of isopropanol
were added and mixed by pipetting. This mixture was then
transferred onto one of the columns and passed through the
membrane, washed and dried according to the procedures described in
Example 4. Finally the plasmid DNA, as described previously in
Example 1, was eluted with RNase-free water and removed from the
membrane by pipetting.
[0240] The amount of isolated plasmid DNA was determined by
spectrophotometric measurement of the light absorption at a
wavelength of 260 nm. The isolation results using various membranes
are listed in Table 7 below. Three parallel tests per membrane were
carried out and each time the average value is calculated.
9TABLE 7 Plasmid DNA-yield from an aqueous solution by binding to
membranes Plasmid DNA Manufacturer Membrane Material (.mu.g) Pall
Hydrolon, 1.2 .mu.m Hydrophobic Nylon 1.9 Pall Fluoro Trans G
Hydrophobic Polyvinylidene Fluoride 2.2 Pall I.C.E.-450 Hydrophilic
Polyethersulfone 0.8 Pall I.C.E.-450sup Hydrophilic
Polyethersulfone 1.5 Pall Supor-450 PR Hydrophobic Polyethersulfone
4.7 Pall Supor-200 PR Hydrophobic Polyethersulfone 4 Pall Supor-800
Hydrophilic Polyethersulfone 0.5 Pall Supor-450 Hydrophilic
Polyethersulfone 0.9 Pall Supor-100 Hydrophilic Polyethersulfone 1
Pall V-800 R Hydrophobic Acrylic Copolymer 1.5 Pall Versapore-1200R
Hydrophobic Acrylic Copolymer 0.2 Pall Polypro-450 Hydrophilic
Polypropylene 1.4 Gore-Tex QH 9318 Hydrophilic
Poly-Tetrafluoro-Ethylene 4.9 Gore-Tex OH 9335 Hydrophobic
Poly-Tetrafluoro- 4.3 Ethylene Millipore Durapore, 0.65 .mu.m
Polyvinylidene Fluoride made 1.8 Hydrophobic Millipore Durapore,
0.65 .mu.m Hydrophobic Polyvinylidene Fluoride 1.7 MSI Magna-SH,
1.2 .mu.m Hydrophobic Nylon 1.1
EXAMPLE 15
Immobilization of Total RNA from an Aqueous Solution with the Use
of Different Chaotropic Agents
[0241] Following the procedures of Example 1, plastic columns were
assembled utilizing different hydrophobic membranes.
[0242] 100 .mu.l of an aqueous solution containing total RNA were
mixed with 350 .mu.l of different lysis buffers, which contain
guanidinium isothiocyanate (GITC) or guanidinium hydrochloride
(GuHCl) in different concentrations. 250 .mu.l ethanol were added
and mixed by pipetting. This mixture was then placed on one of the
columns and passed through the membrane by way of centrifugation
(10000.times.g; 1 minute). The membranes were subsequently washed
twice with an alcohol containing buffer, e.g., RPE from QIAGEN. The
buffer was passed through the membrane by centrifugation. The last
washing step was performed at 20000.times.g to dry the membrane.
The elution was carried out as described in Example 1. Two tests
were carried out to determine the average value. The results are
listed in Table 8.
10TABLE 8 RNA-yield from an aqueous solution by way of chaotropic
agents Chaotropic Agents, Concentration in Binding Membrane
Solution Yield of Total RNA (.mu.g) Hydrolon, 1.2 .mu.m GITC, 500
mM 2.3 Hydrolon, 1.2 .mu.m GITC, 1 M 0.8 Hydrolon, 1.2 .mu.m GITC,
3 M 0.9 Fluoro Trans G GITC, 500 mM 0.4 Fluoro Trans G GITC, 1 M
1.25 Fluoro Trans G GITC, 3 M 0.6 Hydrolon, 1.2 .mu.m GuHCI, 500 mM
2.6 Hydrolon, 1.2 .mu.m GuHCI, 1 M 6.7 Hydrolon, 1.2 .mu.m GuHCI, 3
M 2.9 Fluoro Trans G GuHCI, 500 mM 0.4 Fluoro Trans G GuHCI, 1 M
1.25 Fluoro Trans G GuHCI, 3 M 0.6
EXAMPLE 16
Immobilization of Total RNA from an Aqueous Solution Using
Alcohols
[0243] Following the procedures of Example 1, plastic columns were
assembled utilizing different hydrophobic membranes. 100 .mu.l of
an aqueous solution containing total RNA are mixed with 350 .mu.l
of a lysis buffer containing guanidinium isothiocyanate
(concentration 4 M). Different amounts of ethanol and isopropanol
were added and filled with RNase-free water up to 700 .mu.l and
mixed. This mixture was then introduced to a column and passed
through the membrane and washed according to the procedures of
Example 4. The elution took place as in Example 1. Two tests were
carried out to determine the average yield. The results are listed
in Table 9.
11TABLE 9 RNA-yield from an aqueous solution with different
alcohols in a binding solution Alcohol, Concentration in Yield of
Membrane Binding Solution Total RNA (.mu.g) Hydrolon, 1.2 .mu.m
Ethanol, 5% 0.7 Hydrolon, 1.2 .mu.m Ethanol, 30% 2.85 Hydrolon, 1.2
.mu.m Ethanol, 50% 4.5 Durapore, 0.65 .mu.m Ethanol, 5% 0.4
Durapore, 0.65 .mu.m Ethanol, 30% 1.25 Durapore, 0.65 .mu.m
Ethanol, 50% 0.6 Hydrolon, 1.2 .mu.m Isopropanol, 5% 0.35 Hydrolon,
1.2 .mu.m Isopropanol, 30% 4.35 Hydrolon, 1.2 .mu.m Isopropanol,
50% 3.2 Durapore, 0.65 .mu.m Isopropanol, 10% 1.35 urapore, 0.65
.mu.m Isopropanol, 30% 4.1 Durapore, 0.65 .mu.m Isopropanol, 50%
3.5
EXAMPLE 17
Immobilization of Total RNA from an Aqueous Solution with Various
pH-Values
[0244] Using the procedures described in Example 1, plastic columns
were assembled utilizing various hydrophobic membranes. 100 .mu.l
of an aqueous solution containing total RNA were mixed with 350
.mu.l of a lysis buffer containing guanidinium isothiocyanate
(concentration 4 M). The buffer contained 25 mM of sodium citrate
and was adjusted to different pH-values with HCl or NaOH.
Subsequently, 250 .mu.l of ethanol were added and mixed. This
mixture was then introduced to the column and passed through the
membrane and washed according to the procedures of Example 4. The
elution took place as in Example 1. Two tests are carried out to
determine an average value. The results are listed in Table 10.
12TABLE 10 RNA-yield from an aqueous solution with various
pH-values in a binding solution pH of Yield of Membrane Binding
Solution Total RNA (.mu.g) Hydrolon, 1.2 .mu.m pH 3 0.15 Hydrolon,
1.2 .mu.m pH 9 1.6 Hydrolon, 1.2 .mu.m pH 11 0.05 Fluoro Trans G pH
1 0.45 Fluoro Trans G pH 9 2.85 Fluoro Trans G pH 11 0.25
EXAMPLE 18
Immobilization of Total RNA from an Aqueous Solution with Various
Salts
[0245] According to Example 1, plastic columns are assembled with
hydrophobic membranes. 100 .mu.l of a total RNA containing aqueous
solution were mixed with 350 .mu.l of a salt containing lysis
buffer (NaCl, KCL, MgSO.sub.4). 250 .mu.l of H.sub.2O or ethanol
were then added and mixed. This mixture was then transferred to a
column and passed through the membrane, washed and eluted according
to the procedures of Example 4. Two tests were carried out to
determine the average value. The results are listed in Table
11.
13TABLE 11 RNA-yield from an aqueous solution with various salts in
the binding solution Salt Yield of Total Membrane Concentration in
Binding Solution RNA (.mu.g) Hydrolon, 1.2 .mu.m NaCl, 100 mM;
without ethanol 0.1 Hydrolon, 1.2 .mu.m NaCl, 1 M; without ethanol
0.15 Hydrolon, 1.2 .mu.m NaCl, 5 M; without ethanol 0.3 Hydrolon,
1.2 .mu.m KCl, 10 mM; without ethanol 0.2 Hydrolon, 1.2 .mu.m KCl,
1 M; without ethanol 0.1 Hydrolon, 1.2 .mu.m KCl, 3 M; without
ethanol 0.25 Hydrolon, 1.2 .mu.m MgSO.sub.4, 100 mM; without
ethanol 0.05 Hydrolon, 1.2 .mu.m MgSO.sub.4, 750 mM; without
ethanol 0.15 Hydrolon, 1.2 .mu.m MgSO.sub.4, 2 M; without ethanol
0.48 Hydrolon, 1.2 .mu.m NaCl, 500 mM; with ethanol 2.1 Hydrolon,
1.2 .mu.m NaCl, 1 M; with ethanol 1.55 Hydrolon, 1.2 .mu.m NaCl,
2.5 M; with ethanol 1.35 Hydrolon, 1.2 .mu.m KCl, 500 mM; with
ethanol 1.6 Hydrolon, 1.2 .mu.m KCl, 1 M; with ethanol 2.1
Hydrolon, 1.2 .mu.m KCl, 1.5 M; with ethanol 3.5 Hydrolon, 1.2
.mu.m MgSO.sub.4, 10 mM; with ethanol 1.9 Hydrolon, 1.2 .mu.m
MgSO.sub.4, 100 mM; with ethanol 4.6 Hydrolon, 1.2 .mu.m
MgSO.sub.4, 500 M; with ethanol 2
EXAMPLE 19
Immobilization of Total RNA from an Aqueous Solution Using Various
Buffer Conditions
[0246] Following the procedures of Example 1, plastic columns were
assembled using different hydrophobic membranes.
[0247] 100 .mu.l of an aqueous solution containing total RNA were
mixed with 350 .mu.l of a lysis buffer containing guanidinium
isothiocyanate (concentration 2.5 M). The lysis buffer was mixed
with various concentrations of sodium citrate, pH 7, or sodium
oxalate, pH 7.2. Subsequently 250 .mu.l of ethanol were added and
mixed. This mixture was then transferred to a column and passed
through the membrane and eluted according to the process described
in Example 4. The results are listed in Table 12. Two tests were
carried out to determine the average value.
14TABLE 12 RNA-yield from an aqueous solution with various buffer
concentrations in a binding solution Na-Citrate/Na-Oxalate, Yield
of Total RNA Membrane Conc. in Lysis Buffer (.mu.g) Hydrolon, 1.2
.mu.m Na-Citrate, 10 mM 2.2 Hydrolon, 1.2 .mu.m Na-Citrate, 100 mM
2.4 Hydrolon, 1.2 .mu.m Na-Citrate, 500 mM 3.55 Supor-450 PR
Na-Citrate, 10 mM 1.1 Supor-450 PR Na-Citrate, 100 mM 1.15
Supor-450 PR Na-Citrate, 500 mM 0.2 Hydrolon, 1.2 .mu.m Na-Oxalate,
1 mM 1.5 Hydrolon, 1.2 .mu.m Na-Oxalate, 25 mM 1.05 Hydrolon, 1.2
.mu.m Na-Oxalate, 50 mM 0.9 Supor-450 PR Na-Oxalate, 1 mM 1.9
Supor-450 PR Na-Oxalate, 25 mM 1.3 Supor-450 PR Na-Oxalate, 50 mM
1.7
EXAMPLE 20
Immobilization of Total DNA from an Aqueous Solution Using Various
Buffers
[0248] According to the procedures of Example 1, plastic columns
were assembled with hydrophobic membranes (for example Hydrolon 1.2
.mu.m from the Pall Company).
[0249] 100 .mu.l of an aqueous solution containing total DNA were
mixed with 350 .mu.l of a lysis buffer containing guanidinium
isothiocyanate (4 M GITC, 0.1 M MgSO.sub.4). To this lysis buffer
various buffer substances were added (concentration 25 mM) and
adjusted to different pH-values. Subsequently, 250 .mu.l of ethanol
were added and mixed. The mixture was then introduced to the column
and passed through the membrane, washed and eluted as in Example
4.
[0250] The results are set forth in Table 13. Triple tests are
carried out and average values determined.
15TABLE 13 DNA-yield from an aqueous solution with various buffer
substances in a binding solution pH in the Yield of Buffer
Substance Lysis Buffer DNA (.mu.g) Sodium Citrate pH 4 1.3 Sodium
Citrate pH 5 0.6 Sodium Citrate pH 6 1.4 Sodium Citrate pH 7 0.5
Sodium Acetate pH 4 0.9 Sodium Acetate pH 5 1 Sodium Acetate pH 6
0.6 Sodium Acetate pH 7 0.5 Potassium Acetate pH 4 0.6 Potassium
Acetate pH 5 0.9 Potassium Acetate pH 6 1.2 Potassium Acetate pH 7
1.4 Ammonium Acetate pH 4 0.7 Ammonium Acetate pH 5 0.3 Animonium
Acetate pH 6 5.7 Ammonium Acetate pH 7 1.5 Glycine pH 4 0.5 Glycine
pH 5 1.1 Glycine pH 6 1.6 Glycine pH 7 1.1 Malonate pH 4 1.5
Malonate pH 5 0.3 Malonate pH 6 3.1 Malonate pH 7 1.6 Succinate pH
4 2.8 Succinate pH 5 2.3 Succinate pH 6 2.5 Succinate pH 7 4.7
EXAMPLE 21
Immobilization of Total RNA from an Aqueous Solution Using
Phenol
[0251] According to the procedures of Example 1, plastic columns
were assembled with hydrophobic membranes (e.g., Hydrolon, 1.2
.mu.m from the Pall Company).
[0252] An aqueous solution containing RNA was mixed with 700 .mu.l
of phenol and passed through the membranes using centrifugation.
The membranes were washed and the RNA eluted as in Example 4. Two
tests were carried out and an average value determined.
[0253] The amount of isolated RNA was subsequently determined by
spectrophotometric measurement of the light absorption at a
wavelength of 260 nm and is on average 10.95 .mu.g. The absorption
ratio at 260 nm to the one at 280 nm is 0.975.
EXAMPLE 22
Washing of Immobilized Total RNA under Different Salt
Concentrations
[0254] Following the procedures of Example 1, plastic columns were
assembled with hydrophobic membranes.
[0255] 100 .mu.l of an aqueous solution containing total RNA were
mixed with 350 .mu.l of a lysis buffer containing guanidinium
isothiocyanate (concentration 4 M). Subsequently, 250 .mu.l of
ethanol were added and mixed. This mixture was then transferred to
the column and passed through the membrane and washed according to
Example 4. The membranes were then washed twice with a buffer
containing various concentrations of NaCl and 80% ethanol. The
buffer was passed through the membrane by centrifugation. The last
washing step was carried out at 20000.times.g in order to dry the
membranes. The elution takes place according to the procedure of
Example 1. Two tests were carried out and an average value
determined. The results are listed in Table 14.
16TABLE 14 RNA-yield from an aqueous solution with NaCl in the
washing buffer NaCl in the Yield of Total Membrane Washing Buffer
RNA (.mu.g) Hydrolon, 1.2 .mu.m NaCl, 10 mM 1.4 Hydrolon, 1.2 .mu.m
NaCl, 50 mM 3.15 Hydrolon, 1.2 .mu.m NaCl, 100 mM 3 Durapore, 0.65
.mu.m NaCl, 10 mM 2.7 Durapore, 0.65 .mu.m NaCl, 50 mM 2.85
Durapore, 0.65 .mu.m NaCl, 100 mM 2.7
EXAMPLE 23
Elution of Immobilized Total RNA under Different Salt and Buffer
Conditions
[0256] According to the procedures of Example 1, plastic columns
were assembled with hydrophobic membranes.
[0257] 100 .mu.l of an aqueous solution containing total RNA were
mixed with 350 .mu.l of a lysis buffer containing guanidinium
isothiocyanate (concentration 4 M). Subsequently, 250 .mu.l of
ethanol were added and mixed. This mixture was then introduced to
the column and passed through the membrane and washed according to
the procedures of Example 4.
[0258] For elution, 70 .mu.l of a NaCl-containing solution, a
Tris/HCl buffer (pH 7) or a sodium oxalate solution (pH 7.2) were
pipetted onto the membrane, in order to elute the purified RNA from
the membrane. After 1 to 2 minutes of incubation at a temperature
of 110.degree. C.-30.degree. C., the eluate was pipetted from above
from the membrane. The elution step was repeated once in order to
achieve complete elution. Two tests were carried out and an average
value determined. The results are summarized in Table 15.
17TABLE 15 RNA-yield from an aqueous solution with NaCl, Tris/HCl
or sodium oxalate in the elution buffer NaCl or Tris in Yield of
Total Membrane the Elution Buffer RNA (.mu.g) Hydrolon, 1.2 .mu.m
NaCl, 1 mM 1.35 Hydrolon, 1.2 .mu.m NaCl, 50 mM 1.2 Hydrolon, 1.2
.mu.m NaCl, 250 mM 0.45 Durapore, 0.65 .mu.m NaCl, 1 mM 0.9
Durapore, 0.65 .mu.m NaCl, 50 mM 0.35 Durapore, 0.65 .mu.m NaCl,
500 mM 0.15 Hydrolon, 1.2 .mu.m Tris/HCl, 1 mM 0.35 Hydrolon, 1.2
.mu.m Tris/HCl, 10 mM 0.75 Durapore, 0.65 .mu.m Tris/HCl, 1 mM 1.5
Durapore, 0.65 .mu.m Tris/HCl, 50 mM 1 Durapore, 0.65 .mu.m
Tris/HCl, 250 mM 0.1 Hydrolon, 1.2 .mu.m Na-Oxalate, 1 mM 0.45
Hydrolon, 1.2 .mu.m Na-Oxalate, 10 mM 0.65 Hydrolon, 1.2 .mu.m
Na-Oxalate, 50 mM 0.3 Durapore, 0.65 .mu.m Na-Oxalate, 1 mM 2
Durapore, 0.65 .mu.m Na-Oxalate, 10 mM 0.155 Durapore, 0.65 .mu.m
Na-Oxalate, 50 mM 0.15
EXAMPLE 24
Elution of the Immobilized RNA at Different Temperatures
[0259] Following the procedure of Example 1, plastic columns were
assembled using a hydrophobic membrane (e.g., Hydrolon, 3 .mu.m
from the Pall Company).
[0260] For isolation, 5.times.10.sup.5 HeLa-cells were used. The
following isolation steps were carried out as described in Example
3.
[0261] For elution, 70 .mu.l of RNase-free water of a different
temperature were pipetted onto the membrane in order to elute the
purified RNA from the membrane. After an incubation of 1-2 minutes
at the corresponding elution temperature, the eluate was pipetted
off the membrane from above. The elution step was repeated once in
order to achieve complete elution. Triple tests were carried out
and an average value determined. The results are summarized in
Table 16.
18TABLE 16 RNA-yield at different elution temperatures Elution
Yield of Membrane Temperature Total RNA (.mu.g) Hydrolon, 3 .mu.m
Ice cold 2.2 Hydrolon, 3 .mu.m 40.degree. C. 3.2 Hydrolon, 3 .mu.m
50.degree. C. 3.9 Hydrolon, 3 .mu.m 60.degree. C. 3.7 Hydrolon, 3
.mu.m 70.degree. C. 3.7 Hydrolon, 3 .mu.m 80.degree. C. 2.9
EXAMPLE 25
Elution of Immobilized RNA by Way of Centrifugation
[0262] Following the procedures of Example 1, plastic columns were
assembled with a hydrophobic membrane (e.g., Hydrolon 1.2 .mu.m
from the Pall Company).
[0263] 100 .mu.l of an aqueous solution containing total RNA were
mixed with 350 .mu.l of a commercially available lysis buffer
containing guanidinium isothiocyanate (e.g., RLT buffer from
QIAGEN). 250 .mu.l of ethanol were then added and mixed by
pipetting. This mixture was then transfered onto the column and
passed through the membrane using centrifugation (10000.times.g; 1
minute). The membranes were subsequently washed twice with a buffer
(e.g., RPE buffer from QIAGEN). Each time the buffer was passed
through the membranes by way of centrifugation. The last washing
step was carried out at 20000.times.g in order to dry the
membrane.
[0264] For elution, 70 .mu.l of RNase-free water were pipetted onto
the membrane in order to elute the RNA from the membrane. After an
incubation of 1 minute at a temperature of 10.degree. C.-30.degree.
C., the eluate was passed through the membrane by centrifugation
(10000.times.g, 1 minute). In order to achieve complete elution,
the elution step was repeated once and the eluates joined together.
Five parallel tests were carried out and the average value
calculated.
[0265] The amount of isolated total RNA was subsequently determined
by spectrophotometric measurement of the light absorption at a
wavelength of 260 nm and was on average 6.4 .mu.g. The absorption
ratio at 260 nm to 280 nm was 1.94.
EXAMPLE 26
Use of Total RNA in a `Real Time` Quantitative RT-PCR Using 5'
nuclease PCR Assay to Amplify and Detect .beta.-actin mRNA
[0266] Following the procedures of Example 3, plastic columns were
assembled using a commercially available membrane (Hydrolon from
Pall, with a pore size of 3 .mu.m).
[0267] To isolate RNA, 1.times.10.sup.5 HeLa cells were used, and
the purification of total RNA was carried out as described in
Example 1. The elution was carried out with 2.times.70 .mu.l of
H.sub.2O as described in Example 1. For the complete removal of
remaining amounts of DNA, the sample was treated with a DNase prior
to analysis.
[0268] A "one-device `Real Time` quantitative RT-PCR" was carried
out with the use of the commercially available reaction system of
Perkin-Elmer (TaqMan.TM. PCR Reagent Kit) by using a M-MLV reverse
transcriptase. By using a specific primer and a specific TaqMan
probe for .beta.-actin (TaqMan.TM. .beta.-actin Detection Kit, made
by Perkin Elmer) the .beta.-actin mRNA molecules in the total RNA
sample were first converted into .beta.-actin cDNA and subsequently
the total reaction was amplified and detected immediately, without
interruption, in the same reaction device. The reaction specimens
were produced according to the manufacturer's instructions. Three
different amounts of isolated total RNA are used (1, 2, 4 .mu.l of
eluate) and triple determination tests were carried out. As a
control, three specimens without RNA were also tested.
[0269] The cDNA synthesis was carried out at 37.degree. C. for one
hour, immediately followed by a PCR which comprised 40 cycles. The
reactions and the analyses were carried out on an ABI PRISM.TM.
7700 Sequence Detector manufactured by Perkin Elmer Applied
Biosystems. Every amplicon generated during a PCR-cycle produces a
light emitting molecule, which is generated by splitting from the
TaqMan-probe. The total light signal that is generated is directly
proportional to the amplicon quantity that is being generated and
hence to the original amount of transcript available in the total
RNA sample. The emitted light is measured by the instrument and
evaluated by a computer program. The PCR cycle, during which the
light signal must first be detected over the background noise, will
be designated as the "Threshold Cycle" (ct). This value is a
measure for the amount of specifically amplified RNA available in
the sample.
[0270] For the 1 .mu.l RNA eluate, isolated with the process
described here, an average ct-value of 17.1 was calculated; for 2
.mu.l in total RNA the ct-value was 16.4 and for 4 .mu.l of total
RNA the ct-value was 15.3. This resulted in a linear dependency
between the total RNA and the ct-value, indicating that the total
RNA was free of substances that might inhibit the amplification
reaction. The control specimens containing no RNA did not produce
any signals.
EXAMPLE 27
Use of Total RNA in an RT-PCR for Amplification and Detection of
.beta.-actin mRNA
[0271] According to Example 1, plastic columns were assembled with
commercially available membranes (Pall, Hydrolon with a pore size
of 1.2 or 3 .mu.m; Sartorius, Sartolon with a pore size of 0.45
.mu.m).
[0272] For isolation of RNA, two different starting materials were
used: (1) total RNA from liver (mouse) in an aqueous solution;
purification, elution carried out as described in Example 4; and
(2) 5.times.10.sup.5 HeLa-cells, the purification of total RNA and
the elution are carried out as described in Example 3.
[0273] For each test, 20 ng of isolated total RNA were used. As a
control, RNA which was purified by way of RNeasy-Kits (QIAGEN) and
a sample without RNA were used.
[0274] A RT-PCR was performed with these samples under standard
conditions. For amplification two different primer pairs were used
for the .beta.-actin-mRNA. A 150 bp-sized fragment serves as proof
of sensitivity, a 1.7 kbp-sized fragment assesses the integrity of
the RNA. From the RT-reaction, 1 .mu.l was removed and introduced
to the subsequent PCR. 25 cycles were performed for the small
fragment and 27 cycles for the large fragment. The annealing
temperature was 55.degree. C. The amplified samples were
subsequently placed on a non-denaturing gel and analyzed (FIG.
8).
[0275] For the 20 ng quantity used of total RNA isolated in the
process described above, the corresponding DNA-fragments can be
demonstrated in the RT-PCR. When using total RNA from mouse liver,
no transcript can be demonstrated, as the conditions used here are
adjusted to human .beta.-actin mRNA. The control specimens which
contain no RNA do not produce any signals.
[0276] FIG. 8 shows ethidium bromide stained agarose gels of an
electrophoretic separation of RT-PCR reaction products.
[0277] FIG. 8A: Lanes 1 to 8: RT-PCR of the 150 bp fragment:
[0278] Lanes 1 & 2: RNA from mouse liver in an aqueous solution
purified with the Hydrolon 1.2 .mu.m membrane;
[0279] Lanes 3 & 4: RNA from HeLa-cells purified with the
Sartolon membrane;
[0280] Lanes 5 & 6: RNA from HeLa-cells purified with the
Hydrolon 3 .mu.m membrane;
[0281] Lane 7: RNA purified using the RNeasy-Mini-Kit;
[0282] Lane 8: Control without RNA.
[0283] FIG. 8B: Lanes 1 to 8: RT-PCR of the 1.7 kbp fragment:
[0284] Lanes 1 & 2: RNA from mouse liver in an aqueous solution
purified with the Hydrolon 1.2 .mu.m membrane;
[0285] Lanes 3 & 4: RNA from HeLa-cells purified with the
Sartolon membrane;
[0286] Lanes 5 & 6: RNA from HeLa-cells purified with the
Hydrolon 3 .mu.m membrane;
[0287] Lane 7: RNA purified using the RNeasy-Mini-Kit;
[0288] Lane 8: Control without RNA.
EXAMPLE 28
Use of Total RNA in a NASBA-Reaction (Nucleic Acid Sequence Based
Amplification) for the Amplification and Detection of .beta.-Actin
mRNA
[0289] Following the procedures described in Example 1, plastic
columns were assembled with commercially available membranes (Pall,
Hydrolon with a pore size of 1.2 or 3 .mu.m; Sartorius, Sartolon
with a pore size of 0.45 .mu.m).
[0290] For isolation of RNA, two different starting materials were
used: (1) total RNA from liver (mouse) in an aqueous solution;
purification, elution carried out as described in Example 4; and
(2) 5.times.10.sup.5 HeLa-cells, the purification of total RNA and
the elution are carried out as described in Example 3.
[0291] A NASBA-reaction is performed under standard conditions
(Fahy, E. et al., 1991, PCR Methods Amplic., 1:25-33). For
amplification, .beta.-actin specific primers were used.
[0292] For each test 20 ng of isolated total RNA are used. As a
control, RNA which was purified by way of RNeasy-Kits (QIAGEN) and
a sample without RNA, were used. First they were incubated for 5
minutes at 65.degree. C. and for 5 minutes at 41.degree. C.
Following this step, an enzyme mixture consisting of RNaseH,
T7-polymerase and AMVV-RT was added and incubated for 90 minutes at
41.degree. C. The amplified samples were subsequently placed on a
non-denaturing gel and analyzed. For the 20 ng of total RNA
isolated in the process described above, a specific transcript can
be demonstrated (FIG. 9).
[0293] FIG. 9 shows an ethidium-bromide stained agarose gel of an
electrophoretic separation of the NASBA-reactions.
[0294] Lanes 1 to 8: NASBA-Reactions:
[0295] Lanes 1 & 2: RNA from mouse liver purified from an
aqueous solution with the 1.2 .mu.m Hydrolon membrane;
[0296] Lane 3 & 4: RNA from HeLa-cells purified with the
Sartolon membrane;
[0297] Lane 5 & 6: RNA from HeLa-cells purified with the 3
.mu.m Hydrolon membrane;
[0298] Lane 7: RNA purified using the RNeasy-Mini-Kit;
[0299] Lane 8: Control without RNA.
EXAMPLE 29
NASBA-Reaction for Amplification and Detection of .beta.-Actin mRNA
on Hydrophobic Membranes
[0300] According to the procedures of Example 1, plastic columns
were assembled with commercially available membranes (Pall,
Hydrolon with a pore size of 3 .mu.m; Supor-450 PR with a pore size
of 0.45 .mu.m; Millipore, Fluoropore with a pore size of 3
.mu.m).
[0301] For the isolation of RNA, different quantities of HeLa cells
were used, the purification of total RNA was carried out as
described in Example 3. The elution was performed by adding 20
.mu.l NASBA-reaction buffer. The NASBA-reaction is subsequently
performed on the membrane.
[0302] A NASBA-reaction is performed under standard conditions
(Fahy, E. et al., 1991, PCR Methods Amplic., 1:25-33). For
amplification, .beta.-actin specific primers were used.
[0303] The reaction device was first incubated for 5 minutes at
41.degree. C. in a water bath. Following this step, an enzyme
mixture consisting of RNaseH, T7-Polymerase and AMVV-RT was added
and incubated for 90 minutes at 41.degree. C. The amplified samples
subsequently were placed on a non-denaturing gel and analyzed. For
the quantity of RNA used and isolated from 5.times.10.sup.5 to
3.times.10.sup.4 HeLa cells, a specific transcript can be observed
for the total RNA isolated by the process described here.
[0304] FIG. 10 shows an ethidium-bromide stained agarose gel of an
electrophoretic separation of the NASBA-reactions.
[0305] FIG. 10A: Lanes 1 to 4: RNA from HeLa-cells purified with
the 3 .mu.m Hydrolon membrane:
[0306] Lane 1: 2.5.times.10.sup.5 cells;
[0307] Lane 2: 1.25.times.10.sup.5 cells;
[0308] Lane 3: 6.times.10.sup.4 cells;
[0309] Lane 4: 3.times.10.sup.4 cells.
[0310] FIG. 10B: Lanes 1 to 3: RNA purified from HeLa-cells:
[0311] Lane 1: RNA from 2.5.times.10.sup.5 HeLa-cells purified with
the 3 .mu.m Hydrolon membrane;
[0312] Lane 2: RNA from 5.times.10.sup.5 HeLa-cells purified with
the Supor-450 PR membrane;
[0313] Lane 3: RNA from 5.times.10.sup.5 HeLa-cells purified with
the 3 .mu.m Fluoropore membrane;
EXAMPLE 30
Restriction of Plasmid DNA with the Ava I Enzyme on a Hydrophobic
Membrane
[0314] According to the procedures of Example 1, plastic columns
were assembled with hydrophobic membranes (e.g., Supor-200 PR from
Pall).
[0315] 100 .mu.l of a plasmid-containing aqueous solution
(pCMV.beta. by Clontech) were mixed with 350 .mu.l of a lysis
buffer containing guanidinium isothiocyanate (4 M GITC, 0.1 M
MgSO.sub.4, 25 mM sodium acetate, pH 4). Subsequently, 250 .mu.l of
isopropanol were added and mixed by pipetting. This mixture was
then introduced to the column and passed through the membrane,
washed and dried according to Example 4.
[0316] 100 .mu.l of a 1.times. buffer for the restriction enzyme
Ava I were placed on the membrane and either: (1) removed,
transferred to a new reaction device and subsequently treated with
the restriction enzyme (i.e., Ava I by Promega); or (2) a
restriction enzyme (i.e., Ava I by Promega) was added directly to
the eluate in the column.
[0317] The reaction mixtures were incubated for 1 hour at
37.degree. C. and subsequently placed on a non-denaturing gel and
analyzed (see FIG. 11).
[0318] FIG. 11 shows an ethidium-bromide stained agarose gel of an
electrophoretic separation of pCMV.beta.-plasmid after restriction
with Ava I
[0319] Lane 1: uncut plasmid;
[0320] Lanes 2 & 3: elution with the reaction buffer for Ava I,
restriction reaction in a separate device;
[0321] Lane 4 & 5: restriction with Ava I on the membrane.
EXAMPLE 31
Pressure Filtration for Isopropanol Precipitation of DNA
[0322] The isolation of plasmid DNA was performed according to
standard protocols including the elution step via anion exchange
chromatography. The DNA was eluted from the column in a high saline
buffer.
[0323] Subsequently, 0.7 volume of isopropanol was added to this
DNA solution, the sample was mixed and incubated for 1-5 minutes at
room temperature. A 0.45 .mu.m cellulose acetate filter with a 5
cm.sup.2 surface in a filtration cartridge (standard installation
for sterile filtration, e.g., Minisart by Sartorius) was used as a
filtration installation. This filter was connected to a syringe
from which the plunger has been removed first. The syringe was then
filled with the DNA/isopropanol mixture and pressed through the
filter with the syringe plunger. A high percentage of the DNA in
this precipitate stays on the filter (i.e., cannot pass the
pores).
[0324] The plunger was again removed from the syringe, was inserted
again, and air was pressed through the filter. This step was
repeated once or twice and serves to dry the membrane.
[0325] Subsequently, elution was performed with a corresponding
volume of a low saline buffer, whereby the buffer fills the syringe
and was pressed through the filter with the plunger. To increase
the yield, this first eluate was again put into the syringe and
pressed through the filter with the plunger. In this test
configuration, the yields obtained typically range from 80 to 90%
(see Example 34).
EXAMPLE 32
Vacuum Filtration for the Isopropanol Precipitation of DNA
[0326] As with pressure filtration, first plasmid DNA was isolated
and mixed with 0.7 volume isopropanol. An apparatus designed for
vacuum filtration was used as a filtration installation, in which a
0.45 .mu.m cellulose acetate filter with a surface of 5 cm.sup.2
was placed. 0.45 .mu.m cellulose nitrate filters or several layers
of 0.65 .mu.m cellulose acetate or cellulose nitrate filters may be
used. The isopropanol-DNA mixture was incubated for 1-5 minutes and
placed on the filter assembly. By creating a vacuum, the solution
was suctioned through the filter. The DNA-precipitates on the
filter were mixed with a corresponding volume of 70% ethanol and
washed by creating a vacuum. The elution of the DNA from the filter
takes place by adding a low salt buffer, a short incubation and
renewed creation of a vacuum. The yield can either be obtained by
repeated elution from the filter with a second volume of low saline
buffer or by elution with the eluate from the first elution step.
Here also, typical yields range from 80%-90% of the DNA.
EXAMPLE 33
[0327] The method used is the vacuum filtration method described in
Example 32. The filter device used is the vacuum filter apparatus,
Sartorius 16315. pCMV.beta. was used as the plasmid DNA, which was
isolated from DH5.alpha. cells.
[0328] Procedure: In each test, 15 ml of QF-buffer (high saline
buffer) are mixed with 500 .mu.g of plasmid. 10.5 ml of isopropanol
are added and this is mixed again. Then the mixture is left to
incubate for 5 minutes. The plasmid DNA thus precipitated is
deposited on the membrane in the filter assembly. Next a vacuum is
created and the filtration takes place. The membranes are washed
with 5 ml of 70% ethanol (by creating another vacuum), then 1 ml
TE-buffer is pipetted onto the membranes, left to incubate for 5
minutes, and the DNA is eluted by creating a vacuum. Subsequently a
post-elution is performed with 1 ml TE-buffer. Total DNA amounts
are measured in the flow-through, in the washing stage and in the
combined eluate (OD260). The following results were obtained:
19 Test Flow- Washing Flow Membrane Number through Stage Eluate
Speed PVDF 0.2 .mu.m 1 0 .mu.g DNA 0 .mu.g DNA 131 .mu.g Very slow
DNA Cellulose Nitrate 2 0 .mu.g DNA 0 .mu.g DNA 418 .mu.g Fast 0.65
.mu.m DNA Cellulose Acetate 3 0 .mu.g DNA 0 .mu.g DNA 469 .mu.g
Fast 0.65 .mu.m DNA
[0329] Calculated on the basis of 500 .mu.g of DNA starting
quantity, the following yields are obtained with this method:
20 PVDF 0.2 .mu.m 26% Cellulose Acetate 0.65 .mu.m 94% Cellulose
Nitrate 0.65 .mu.m 84%
EXAMPLE 34
[0330] The pressure filtration method indicated in Example 31 was
used. The filter assembly used was a commercially available 0.45
.mu.m cellulose acetate filter (Minisart, Sartorius). pCMV.beta. is
used as plasmid DNA, which was isolated from DH5.alpha. cells.
[0331] Procedure: For each test, 15 ml of QF-buffer (high salt
buffer) are added to and mixed with 100, 200, 300, etc., up to 900
.mu.g of plasmid. 10.5 ml isopropanol are added and again mixed.
Subsequently, there is a 5-minute incubation period. The plasmid
DNA thus precipitated is transferred to a syringe, to which the
filter had been previously fitted. Pressure filtration takes place
with the aid of the syringe. The filter is then washed with 2 ml of
70% ethanol and, as described, dried twice. The elution is
performed with 2 ml of TE-buffer. A second elution is performed
with the eluate. The total amount of DNA is measured in the
combined eluate (OD260).
[0332] Following the above procedure, the following results were
obtained:
21 DNA-quantities DNA-quantities % used eluted Yield 100 .mu.g 100
.mu.g 100% 200 .mu.g 176 .mu.g 88% 300 .mu.g 257 .mu.g 86% 400
.mu.g 361 .mu.g 90% 500 .mu.g 466 .mu.g 93% 600 .mu.g 579 .mu.g 97%
700 .mu.g 671 .mu.g 96% 800 .mu.g 705 .mu.g 88% 900 .mu.g 866 .mu.g
96%
EXAMPLE 35
[0333] The vacuum filtration method indicated in Example 32 was
used. The filter assembly used was a commercially obtained 0.45
.mu.m cellulose acetate filter (Minisart, Sartorius), that had been
attached to a filtration chamber (QIAvac). As buffer reservoir, a
syringe was attached to the other end of the filter pCMV.beta. was
used as plasmid DNA, which was isolated from DH5.alpha. cells.
[0334] Procedure: 15 ml of QF-buffer (high saline buffer) are added
to and mixed with 500 .mu.g of plasmid. 10.5 ml isopropanol are
added and again mixed. Subsequently, there is a 5-minute incubation
period. The plasmid DNA thus precipitated is then transferred to
the filter assembly syringe. Now a vacuum is created and filtration
takes place. The filter is not washed with 70% ethanol. Rather,
elution with 2 ml of EB buffer (QIAGEN) follows immediately.
Post-elution is performed with the eluate. The total DNA quantity
in the combined eluate is measured (OD260). The following result
was obtained:
22 Test % Number Eluted DNA Yield 1 434 .mu.g 87% 2 437 .mu.g
87%
[0335] Although a number of embodiments have been described above,
it will be understood by those skilled in the art that
modifications and variations of the described devices and methods
may be made without departing from concept of the invention as
defined in the appended claims. The articles and other publications
cited herein are incorporated by reference.
* * * * *