Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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Processes and Means for the Isolation and Purification of Nucleic Acids at
Surfaces
The present invention refers to new processes and equipment for the isolation
and purification of
nucleic acids on surfaces.
That the genetic origin and functional activity of a cell can be determined
and researched by
studying their nucleic acids has been known for a long time. The analysis of
nucleic acids enable
the direct access to the cause of activities of cells. Thus they are
potentially superior to indirect,
conventional methods such as e.g. verification of metabolic products. A strong
increase in
analyses of nucleic acids is therefore to be anticipated for the future.
Molecular biological
analyses are already employed in many areas, e.g. in the medical and clinical
diagnostics, in the
pharmaceutical industry with the development and evaluation of medicines, in
food analysis as
well as with the supervision of food production and the control of foodstuffs,
in agriculture with
the cultivation of plants and breeding of animals as well as in environmental
analysis and many
other areas of research. Examples include paternity analysis, tissue typing,
identification of
hereditary diseases, genome analysis, molecular diagnostics e.g. the
identification of infectious
diseases, transgenetic research, basic research in areas of biology and
medicine as well as
multiple related areas.
The activities of genes can be determined directly through analysis of the
RNA, especially the
mRNA in cells. Metabolic diseases, infections or the development of cancer
through recognition
of faulty exprimated genes has become possible through the quantitative
analysis of transcript
patterns (mRNA patterns) in cells by means of modem molecular biological
methods such as e.g.
real time - reverse transcriptase PCR ( "real time RT-PCR" ) or gene
expression chip analysis.
For example, proof of genetic defects or the determination of the HLA type as
well as other
genetic markers has become possible through the analysis of the DNA from cells
through
molecular biological methods such as e.g. PCR, RFLP, AFLP or sequencing.
The analysis of genomic DNA and RNA is also used for the direct verification
of infectious
pathogenes, such as viruses, bacteria etc.
In this, the general difficulty lies in the preparation of biological and
clinical samples such that
the nucleic acids contained can be employed directly in the respective
analytical method.
Precisely the direct utilization of nucleic acids with good yield and high
quality and
simultaneously high reproducibility is however important with high numbers of
samples when
the analysis is to be run automatically.
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 as well -- according to the method of Birnboim [Methods in
Enzymology 100 (1983)
p 243]. In this process, a clarified 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
disadvantage that a
very expensive apparatus is required on the one hand and, on the other hand,
it takes a great deal
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of time, is cost-intensive and cannot be automated.
Other techniques in which clarified lysates are used to isolate DNA consist of
ion-exchange
chromatography [Colpan et al., J. Chromatog. 296 (1984) p 339] and gel
filtration [Moreau et al.,
Analyt. Biochem. 166 (1987) 188]. These processes are offered primarily as
alternatives to the
cesium chloride gradients, but require an elaborate apparatus for the solvent
supply as well as the
precipitation of the DNA fractions such obtained, since these usually contain
salts in high
concentrations and are in extremely-diluted solutions.
Marko et al. [Analyt. Biochem., 121 (1982) p.382] and Vogelstein et al. [Proc.
Nat. Acad. Sci. 76
(1979) p 615] recognized that if the DNA from extracts containing nucleic
acids is exposed to
high concentrations of sodium iodide or sodium perchlorate, the DNA alone will
adhere to small
glass scintillation tubes, fiberglass membranes of fiberglass sheets that have
been finely
particulated by mechanical means, while RNA and proteins do not. The DNA that
has been
bound in this manner can be eluted, for example, with water.
For example, in WO 87/06621, the immobilization of nucleic acids on a PVDF
membrane is
described. However, the nucleic acids bound to the PVDF membrane are
subsequently not
eluted; instead the membrane, together with all the bound nucleic acids is
introduced directly into
a PCR preparation. Finally, in this international patent application and in
the other literature, it is
revealed 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 halfway
satisfactory.
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 TagMan technologies
and similar
real-time quantification techniques for PCR, SDA, DNA- and RNA-chips and -
arrays for the
gene expression- and mutation analysis, differential display analysis, RFLP,
AFLP, cDNA-
synthesis, or subtractive 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 acids obtained in this manner are, comparatively,
extremely dilute -a
circumstance that makes subsequent steps to concentrate and isolate them
absolutely necessary.
All aqueous and other solutions of nucleic acids as well as all substances and
samples, such as
biological samples and materials, foodstuffs etc: shall be included in the
term nucleic acid
sample in the sense of the present invention. In the sense of the present
invention, a sample
containing nucleic acid or a substance shall be defined by a sample or sample
preparation that
contains nucleic acids. By biological substance or biological sample is meant,
e.g. sample
substance void of cells, plasma, body fluids - such as blood, saliva, urine,
feces, sperm, cells,
serum, fractions of leucocytes, crust phlogistica, smears, tissue samples of
any kind, tissue parts
and organs, food samples containing free or bonded nucleic acids or cells
containing nucleic
acids, plants or plant particles, bacteria, viruses, yeasts and other fungi,
other eucaryotes and
*Trade-mark
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procaryotes etc. as they are set forth by the European
Patent Application No.: 95909684.3, to which content is
herewith referred - or free nucleic acids as well. By
nucleic acids, in the sense of the present invention, are
meant all possible kinds of nucleic acids, as, e.g.,
ribonucleic acids (RNA) and deoxyribonucleic acids (DNA) in
all lengths and configurations as double-stranded,
single-stranded, circular and linear, branched, etc.,
monomer nucleotides, oligomers, plasmides, 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, t-RNA, mRNA in processed and unprocessed form,
hn-RNA, rRNA, and cDNA, as well as all other imaginable
nucleic acids.
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 a quantitative
isolation of nucleic acids that is as simple as possible as
far as process engineering is concerned.
In one aspect, the invention relates to a process
for the precipitation of plasmid nucleic acid, comprising:
providing an isolation container comprising a membrane located
therein, said membrane comprising pores having a pore size
larger than or equal to 0.45 micrometers and wherein said
membrane comprises hydrophilized nylon, polyethersulfone,
polycarbonate, polyacrylate, acrylatecopolymers, polyurethane,
polyamide, polyvinylchloride, polyfluorocarbonate,
polytetrafluoroethylene, polyvinylidendifluoride,
polyethylenetetrafluoroethylene-copolymerisate, one
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polyethylenechlorotrifluoroethylene- copolymerisate, cellulose
acetate, cellulose nitrate or polyphenylene sulfide; charging
said isolation container with a solution containing plasmid
nucleic acids; and precipitating said plasmid nucleic acids
contained in said solution with iso-propanol, wherein the
volume ratio of the solution containing plasmid nucleic acids
to iso-propanol is 2:1 to 1:1, whereby said plasmid nucleic
acids bond with said membrane.
In another aspect, the invention relates to use of a
membrane comprising pores having a pore size larger or equal
to 0.45 micrometers and wherein said membrance comprises
hydrophilized nylon, polyethersulfone, polycarbonate,
polyacrylate, acrylatecopolymers, polyurethane, polyamide,
polyvinylchloride, polyfluorocarbonate,
polytetrafluoroethylene, polyvinylidendifluoride,
polyethylenetetrafluoroethylene-copolymerisate, one
polyethylenechlorotrifluoroethylene- copolymerisate, cellulose
acetate, cellulose nitrate or polyphenylene sulphide to bind
plasmid nucleic acids precipitated with iso-propanol, wherein
the volume ratio of the solution containing nucleic acids to
iso-propanol is 2:1 to 1:1.
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Further advantageous aspects and embodiments of the invention are evident from
the dependent
claims, the description, and the attached figures.
In this connection, the invention involves processes which use surfaces, e.g.,
porous membranes,
on which the nucleic acids can be immobilized in a simple way from the test
sample containing
the nucleic acids and can again be released by means of simple procedural
steps. In particular, the
simple procedure, according to the invention, makes it possible to carry out
the processes
completely automatically.
A further aspect of the present invention is aimed at binding nucleic acids to
an immobile phase,
a membrane in particular, in such a way, that they can again be released from
that phase, without
complications, in the following reaction step and can, if applicable, come to
additional use in
e.g. restriction digestion, RT, PCR or RT-PCR or in any other of the above
mentioned analytical
or enzyme reactions.
A surface in the sense of the present invention is defined through any micro-
porous separation
layer. This can for example rest directly upon a substrate and as such be
accessible only from one
side or stand freely. A separation layer, accessible from both sides,
therefore not resting with its
entire surface upon an impermeable substrate but completely free or only
supported at a few
points, shall be referred to as a membrane in accordance with the present
invention.-
3b
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Isolation in the sense of the present invention is defined as any enrichment
of nucleic acids
during which the concentration of the nucleic acids is increased and/or the
portion of the non-
nucleic acid in a preparation or sample is decreased.
The invention provides for a process to isolate nucleic acids according to the
following steps:
- charging of a membrane with at least one sample of nucleic acids;
- immobilization of the nucleic acids at the membrane;
- release of the immobilized nucleic acids from the membrane; and
- removal of the released nucleic acids by transfer through the membrane
whereby the membrane comprises nylon, polysulfone, polyethersulfone,
polycarbonate,
polyacrylate, acrylate- copolymer, polyurethane, polyamide, polyvinylchloride,
polyfluorocarbonate, polytetrafluorethylene, polyvinylidene difluoride,
polyethylene
tetrafluorethylene-copolymerisate, polybenzimidazoles,
polyethylenechlorotrifluorethylene-
copolymerisate, polyimides, polyphenylene sulfide, cellulose, cellulose mixed
esters, cellulose
nitrate, cellulose acetate, polyacrylnitriles, polyacrylnitrile-copolymers,
nitrocellulose,
polypropylene and/or polyester.
Other membranes, for example those mentioned in addition within this
description, can be used
for the process according to this invention.
Preferable the charging occurs from the top and the removal downwards, it
could however be
imagined that, for example, liquid transfer processes, for which a
horizontally arranged column is
charged with a solution containing nucleic acids from one side, penetrating
the membrane after
the immobilization of the nucleic acids at the membrane and is then taken off
at the other end of
the column.
The membrane can preferably be placed inside a container, e.g. the above
mentioned or another
column, fitted with a supply and take-off and cover the entire cross section
of the container.
The membrane can be coated, whereby it can be rendered hydrophobic or
hydrophilic by the
coating.
To achieve a complete isolation of the nucleic acids, isolation processes
known so far,
particularly within columns, operate with relatively thick membranes or
fleeces. This however,
during the suction transfer of the solution through the membrane, leads to a
relatively large, so-
called dead space volume, namely the volume of the membrane, from which the
nucleic acids
can only be obtained with larger amounts of elution buffer. As a result, after
the elution, the
nucleic acids are present in a more diluted form which , for many
applications, is undesirable or
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disadvantageous. For this reason a preferred embodiment for carrying out the
invention utilizes a
membrane which is less than 1 mm, preferably less than 0.5 and particularly
preferred less than
0.2, e.g. 0. lmm, thick.
The invention furthermore concerns a process for the isolation of nucleic
acids according to the
following steps:
- charging of a surface with at least one sample of nucleic acids;
immobilizing the nucleic acids at the surface; and
releasing of the immobilized nucleic acids from the surface with an elution
medium.
This process is characterized in that the release is carried out at a
temperature where the upper
limit is 10 C or below and the lower limit is at the freezing point of the
elution medium such that
the elution medium does not freeze. Thus the inequation 10 C >= T >= TS,EM
applies, whereby T
describes the temperature of the release and Ts,EM the freezing; point of the
elution medium. This
is because it has been shown, against common belief, that the release of
nucleic acids is definitely
possible near the freezing point of the elution medium. An elution at such a
low temperature
even has the unexpected advantage that the nucleic acids are being treated
gentler and that ,
where there are still signs of the presence of nucleases (DNases or RNases),
these come to a
virtual stand still near the freezing point, so that the degradation of the
nucleic acids are reduced
or completely suppressed.
Accordingly, during elution, the temperature should preferably be even lower,
e.g. below 5 C.
The lower limit can also be at 0 C or -5 C if the preparation, due to its ion
content, is still liquid
at that temperature. The upper limit should also be low if possible, e.g.
approximately 5 C.
This process according to the present invention, sometimes demands a cooling
of the elution
buffer and can demand a cooling of further solutions, if applicable, cooling
of the isolation
container as well. Since cooling cannot always be assured reliably,
particularly during
examinations in the field, such as screening of persons in developing
countries, the present
invention is furthermore directed at an isolation container which permits an
isolation of nucleic
acids at low temperatures, independent of external cooling.
Thus the invention is furthermore directed at an isolation container for the
isolation of nucleic
acids comprising at least one upper part with one top opening, one bottom
opening and one
membrane which is located at the bottom opening and covers the entire cross
section of the upper
part; one lower part with an absorbant material; and a jacket to contain a
coolant which surrounds
the upper part at least in the area of the membrane. The jacket which contains
the coolant permits
the cooling of the membrane and the solutions to lower temperatures, such as
the lysate, the
washing buffers and the elution buffer which are brought onto the membrane so
that the final
elution can be conducted reliably within the desired temperature range, close
to the freezing point
of the elution buffer.
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With one of the embodiments of the isolation container, the jacket comprises
two compartments
which are separated by a mechanically destructible partition, whereby a
solution is contained in
each of the compartments and the coolant is formed through the mixture of the
both solutions
after the destruction of the partition.
The partition can be destroyed by the experimenter in that he e.g. presses
against the provided
points of the outer wall of the jacket and thus ruptures the partition.
Solutions suitable for the
containment inside the compartments are known to specialists in the area of
chemical
refrigeration techniques. These can be adjusted to the desired temperatures
and to the ambient
temperatures expected during the utilization of the isolation container.
During the extraction of nucleic acids from biological samples, e.g. the
samples mentioned
above, it is frequently necessary to firstly lyse cells or secretions, to
access the nucleic acids. The
lysates thus obtained may contain, besides nucleic acids, large amounts of
undesirable
substances, e.g. proteins or lipids. A blockage of the membrane may result
from a charge if the
amount of undesirable substances within the lysate is too large which reduces
the efficiency of
the isolation of the nucleic acids and reduces the permeability of the
membrane during washing
or the elution. To avoid this undesirable effect, the invention is therefore
also directed at a
process during which undesirable substances are removed before they reach the
membrane.
This process for the isolation of nucleic acids according to the present
invention comprises the
following steps:
standardization of at least one sample of nucleic acids to bonding conditions
which
permit the immobilization of nucleic acids, contained in the at least one
sample of nucleic
acids, at a surface;
charging of the surface with at least one sample of nucleic acids; and
immobilization of the nucleic acids at the surface,
characterized in that prior and/or after the adjustment of the bonding
conditions, a pre-
purification is carried out.
The pre-purification can be carried out , for example, by demineralization or
through filtration,
centrifugation, enzymatic treatment, temperature influence, precipitation,
and/or extraction of the
nucleic acids solution and/or bonding of contaminants of the nucleic acids
solution to surfaces.
The pre-purification can also be carried out by mechanical part:iculation or
homogenization of the
nucleic acids solution where a lysate of an organic sample is concerned.
The standardized bonding conditions can hereby enable an immobilization of the
RNA and/or
DNA.
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A pre-purification may be particularly necessary if an isolation is carried
out on biological
samples with severe contamination. The biological sample may comprise any
imaginable
substance which may be used directly or is in turn obtained from other
biological samples. These
can for example be blood, saliva, urine, feces, sperm, cells, serum, leucocyte
fractions, crusta
phlogistica, smears, tissue samples, plants, bacteria, fungi, viruses and
yeasts as well as all other
types of biological samples mentioned above.
If the biological sample contains a high proportion of undesirable substances,
the process
according to the present invention can naturally be employed to a particular
advantage.
After immobilization of the nucleic acids out of the pre-purified sample of
nucleic acids, the
common isolation steps follow, i.e.:
release of the immobilized nucleic acids from the surface;
removal of the released nucleic acids from the surface.
A particular advantage of the processes according to the present invention
lies in the fact that
these can be combined with the chemical reactions to which the nucleic acids
are subjected at the
surface. A multitude of analytical techniques can thus be carried out on the
nucleic acids isolated
at the surface. To achieve free access, in doing so, it is possible to release
the nucleic acids from
the surface prior to the reaction. Alternatively however, a suitable reaction
can also be carried out
on nucleic acids bonded directly to the surface.
In one aspect, accordingly, the invention is directed at a process with pre-
purification, as
illustrated above, characterized in that the following step is carried out
after the release step,
preferably at least once:
carrying out of at least one chemical reaction on the nucleic acids.
A particular advantage of this process lies in the fact that prior to the
chemical reaction no
decanting from the isolation container to a reaction container is required
which might cause
losses, but that isolation and reaction can occur in the same container.
In a further aspect, independent from pre-purification, the invention is
directed at a process to
carry out a nucleic acid amplification reaction with the following steps:
charging of a surface with at least one sample of nucleic acids;
immobilization of the nucleic acids at the surface; and
carrying out of an amplification reaction with the nucleic acids.
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Particularly in case of the small amounts of substance usually or necessarily
worked with in
amplification reactions, it is an advantage if the entire preparation of
nucleic acids can be
employed without losses due to decantation. This is also of particular
advantage for an
automation as all processes can be carried out in one container. In addition
the amount of waste
is reduced and the process becomes faster and more cost efficient.
With this the amplification reaction can be an isothermal or a non-isothermal
reaction.
With this the amplification reaction can for example be a SDA reaction (strand
displacement
amplification), a PCR, RT-PCR, LCR or a TMA or a Rolling Circle Amplification.
A NASBA amplification reaction is also possible with this process according to
the present
invention.
Prior to the amplification reaction the nucleic acids can be released from the
surface by means of
a reaction buffer, where the elution medium is located on or within the
membrane. Alternatively
the amplification reaction can be carried out inside a reaction buffer that
does not lead to a
release of the nucleic acids from the surface.
This process preferably shows these further steps:
if applicable, the release of the reaction products from the surface (as far
as these were
still bound during the reaction); and
removal of the released reaction products from the surface.
A further aspect contains a process for the carrying out of chemical reactions
on nucleic acids
with the following steps:
- charging a surface with at least one sample of nucleic acids;
- immobilization of the nucleic acids at the surface;
- release of the immobilized nucleic acids from the surface;
- carrying out of at least one chemical reaction on the nucleic acids; and
- removal of the nucleic acids from the surface without prior immobilization.
With this process the nucleic acids are not bound to the membrane
(immobilized) after the
chemical reaction but are removed without bond. Although saving such an
additional step might
deteriorate the purity of the preparation but might be preferred due to time
savings in time critical
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applications and the simplification in certain forms of application. The
process according to the
invention offers a wide selection of chemical reactions. Chemical reaction in
the sense of this
invention shall be understood as any interaction of the nucleic acids with
further substances
(except with the surface, as this "reaction" occurs with all processes
described herein), i.e.
enzymatic modifications, hybridization with probes, chemical sequencing
reactions, pH value
modifications e.g. the basic depurination of RNA and acidic depurination of
DNA, as well as
bonding anti-bodies and protein additions. Generally any reaction, be it aimed
at a modification
of covalent bonds or hydrogen bonds, shall be included.
One advantage of the process according to the present invention is to be seen
in the permanent
spatial unification of a volume space in which different processes occur and a
membrane to
which nucleic acids may bond. In the simplest way this unification permits a
manipulation of
nucleic acids with an immediate subsequent membrane bond. This is of great
advantage
particularly for automated processes. Once bonded to the membrane , the
nucleic acids are
available for further treatment steps, e.g. - as illustrated above - for the
isolation of potentially
pure nucleic acids or for the carrying out of chemical reactions with nucleic
acids. In a further
aspect of the invention it is however also possible to subject the nucleic
acids, bound to the
membrane, immediately to another analysis to determine specific properties of
the nucleic acids.
Thus the invention is furthermore directed at a process for the analysis of
nucleic acids inside an
isolation container with the following steps:
- providing an isolation container with a membrane located inside;
- charging of the isolation container with at least one sample of nucleic
acids;
- immobilization of the nucleic acids on the membrane;
- transfer of the liquid components of the sample through the membrane; and
- analysis of at least one property of the nucleic acids on the membrane
located inside the
isolation container.
In a further process embodiment at least one chemical reaction, as described
above, can be
carried out on the nucleic acids, after the transfer of the liquid components.
This can, for
example, serve the purpose to enable the subsequent analysis of the nucleic
acids. Examples for
this application are hybridization with probes, the radioactive marking of
nucleic acids bound to
the membrane or the bond of specific anti-bodies. Auxiliary reactions such as
the coloring of
nucleic acids, e.g. with intercalating substances such as ethidium bromide
shall also be
understood as chemical reactions.
Different properties of the nucleic acids are open to a membrane bound
analysis and have
already been described for conventional membranes without a combined reaction
container.
Some of the properties that can be analyzed are the radioactivity of the
nucleic acids or their
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ability to bond with molecules, whereby the molecules cart be e.g. anti-
bodies, nucleic acid
bonding colorant molecules or colorant molecules bonding to nucleic acids or
proteins.
This process presents a considerable simplification of the analysis of nucleic
acids as a
manipulation of the exposed membrane is no longer necessary. Rather, it is
located within the
isolation container.
An irreversible bond of the nucleic acids to the membrane, e.g. for the
subsequent analysis steps,
is also contained within the frame of the present invention. The durable or
irreversible bond
allows the manipulation of the membrane and the nucleic acids bound hereto in
a manner not
open to reversibly bound nucleic acids.
In a further aspect the invention is aimed at the quantitative precipitation
of nucleic acids.
With previously known methods for the pure-preparation of 100 g or more
(hereafter referred
to as "large scale") of plasmid-DNA, based on anion exchange chromatography,
the plasmid
DNA is eluted from the column with a high salt concentration buffer in the
last step. To separate
the Plasmid DNA from the salt for one and to concentrate it for the other, it
is precipitated by
means of alcohol (e.g. iso-propanol) and centrifuged in a suitable container.
The centrifuge pellet
is washed with a 70% ethanol to remove the remaining traces of the salt and
then subjected to a
centrifugation again. The pellet of the second centrifugation is typically
dissolved in a small
amount of a low salt concentration buffer and the plasmid-DNA is processed
further in this form.
Furthermore, processes have been suggested in the state of the art, which
transform the DNA ,
through an addition of chaotropic salts to the high salt concentration buffer,
into a state which
bonds on silica membranes. After washing accordingly, the DNA is released from
the membrane
with a low salt concentration buffer.
In a publication ( Ruppert, A. et al., Analytical Biochemistry (1995), 230:
130-134) a similar
application is described with which, on a small scale (isolation of less than
100 g plasmid-
DNA) , a DNA which was precipitated with iso-propanol, bonds to PVDF membranes
with a
pore size smaller than 0.2 m, thereafter washed with ethanol and subsequently
eluted with TE
(tris-EDTA). A description of such a method on a large scale is however non-
existent.
The described precipitation of DNA with subsequent centrifugation is extremely
time
consuming (approximately 1 hour), in addition, the use of centrifuges is
necessary. Besides the
high time requirement for this procedure, the described last step in the
plasmid preparation is
particularly susceptible to faults. Every now and then a partial or complete
loss of the DNA-
pellet occurs. It appears that the kind (material) of which the centrifugation
container is made
plays a decisive role.
The also described application of chaotropic salts and subsequent bonding of
nucleic aids to
silica membranes is also time consuming, compounded by the fact that the
introduction of
chaotropic salts into the preparation might pose the danger of a contamination
of the lastly
CA 02348054 2001-04-20
isolated DNA.
The described filtration of alcoholic precipitates on a small scale has the
disadvantage of not
being able to be transferred to the large scale in a linear fashion. Membranes
employed according
to the state of the art only permit the isolation of small amounts of nucleic
acids because the
membranes are rapidly saturated and no longer absorbant. Frequently a large
part of the nucleic
acids is thus lost during the removal of the precipitation buffer and during
washing. To avoid
such a loss, the invention is also aimed at a process for the precipitation of
nucleic acids with the
following steps:
providing an isolation container in which at least one membrane has been
placed;
charging of the isolation container with one sample of nucleic acids;
- precipitation, with alcohol, of the nucleic acids contained in the sample,
such, that the
nucleic acids bond with at least the one membrane. The process is
characterized in that
the pore size of the at least one membrane is larger or equal to 0.2
micrometer.
Firstly the alcohols considered for the carrying out of the process according
to the present
invention, are all hydroxyl derivatives of aliphatic or acyclic saturated or
unsaturated
hydrocarbons.
Among the hydroxyl compounds, the Cl - C5 alcanoles such as methanol, ethanol,
n-propanol,
n-butanol, tert.-butanol, n-pentanol or their mixtures are preferred. For the
carrying out of the
process according to the present invention iso-propanol is particularly
preferred.
In this the alcohol can be mixed with this solution prior or after the
charging of the isolation
container with the solution containing nucleic acid. The volume ratio of the
nucleic acid
containing solution to alcohol, particularly iso-propanol, is preferably 2:1
to 1:1, particularly
preferred 1.67:1 to 1:1 and e.g. 1.43:1.
The surface area of the membrane is preferably selected such that the total of
the nucleic acids
contained in the solution bonds with the membrane.
For the bonding of alcohol precipitated nucleic acids, which can be DNA and/or
RNA, the
invention is also aimed at the use of membranes with a pore size larger or
equal to 0.2 m.
The use of a 0.45 m cellulose acetate - or cellulose nitrate filter or the
use of several layers of
0.65 m cellulose acetate - or cellulose nitrate filters is regarded as
particularly advantageous.
The procedure can be used for vacuum filtration as well as pressure
filtration.
The process according to the present invention enables a time saving transfer
of nucleic acids
from a high salt concentration buffer system to a low salt concentration
buffer system which is
possible without extensive apparatus requirements. It is suitable as a
substitute for the classical
11
CA 02348054 2001-04-20
alcoholic precipitation of DNA from a high salt concentration buffer which is
typically
performed with the aid of centrifugation steps. Due to the high efficiency of
the method (low loss
of yield) it is particularly suitable for large scale preparations.
Furthermore, through the use of
the process according to the present invention, no foreign substances are
introduced into the
already purified nucleic acids. In addition, the susceptibility to faults
(loss of centrifugation
sediment during the washing step is not possible here), in comparison to the
classical method, is
reduced.
The charging during the various processes described above is preferably
carried out from above.
In principal, diverse methods are available to transfer the different
solutions, i.e. the
immobilization buffers containing nucleic acids, washing buffers, eluates etc
through the
membrane. This can be gravitation, centrifugation, vacuum, positive pressure
(from the charging
side) and capillary forces.
Between the steps of immobilization and release, a washing step of the
immobilized nucleic
acids with at least one washing buffer can be carried out. The washing
comprises the following
steps for each washing buffer:
application of the pre-determined amount of washing buffer onto the surface;
and
- transfer of the washing buffer through the surface.
The charging and immobilization of the nucleic acids can again include the
following steps:
- mixing of the sample of nucleic acids with an immobilization buffer;
- charging of the sample of nucleic acids together with the immobilization
buffer onto the
surface; and
transfer of the liquid components through the surface, essentially in the
direction of
charging.
The procedures have the great advantage that they can be easily automated,
with the result that at
least one of the steps can be carried out completely automatically by means of
an automated
apparatus. It is also possible that all the steps of the procedures can be
carried out in a controlled
series of steps by an automated apparatus.
Particularly in such cases, but also during manual processing, it is possible
to subject a multitude
of nucleic acids to the isolation simultaneously. For example multiple
isolation containers in the
form of commercially available "Multi-well" containers with 8, 12, 24, 48, 96
or more isolation
recesses can be used.
The removal of the nucleic acids can occur in two fundamentally different
directions. For one, it
is possible to transfer the removed (eluted) nucleic acids through the
membrane and remove them
12
CA 02348054 2001-04-20
to that side of the membrane, which lies opposite to the side onto which the
solution, containing
the nucleic acids or lysate, was added. In this case, the nucleic acid is
removed in the direction of
the addition through the membrane. To remove the nucleic acids from the side
of the addition
onto the membrane or surface is the other possibility. The removal then occurs
in the direction
opposite to that of the addition or "to the same direction" from which was
added; being the side
of the addition. In that case the nucleic acids do not have to pass the
membrane.With some of the
processes according to the present invention, the removal of the nucleic acids
occurs always
through the membrane in the direction of the addition. Should the process be
carried out with a
surface placed upon a substrate impermeable to liquids, e.g. a plastic wall,
the removal can
obviously only occur in the direction towards the direction of the addition
(being the opposite
direction). In the case of some processes however, a removal in both
directions is possible.
If the nucleic acids are eluted (released) from the surface essentially in a
direction opposite to the
one in which they were added and immobilized, the "same direction" is actually
referred to for
any direction with an angle smaller or equal to 180 compared. to the
direction of the addition, so
that, in any event, during the elution, the nucleic acids do not penetrate the
surface, e.g. a
membrane, but are removed from the surface in a direction opposite to the
direction of charging
onto the surface. In preferred forms of carrying out the process, the other
buffers, therefore that
buffer in which the nucleic acids are located during charging, and, if
applicable, a washing
buffer, are transferred through the surface by means of suction or otherwise.
If the isolation
occurs on a membrane located inside a container, where the membrane covers the
entire cross-
section of the container, the charging preferably occurs from above. In this
case the removal step
occurs upwards again. Fig. 2 for example shows a funnel type isolation
container which is
charged from above and the removal of the nucleic acids occurs upwards.
It is to be understood, however, that also with a removal in a direction
opposite to that of the
addition, other arrangements are conceivable, i.e., a removal of the nucleic
acids from below. It
is, for example, conceivable that a buffer containing nucleic acids, such as a
lysate buffer, can be
drawn from a reaction container directly into an isolation device by means of
a vacuum
apparatus, so that the nucleic acids are bound to the underside of a membrane
in the isolation
device. In such a case, the removal of the nucleic acids from the surface
takes place by means of
an elution buffer drawn from below, which after release of the nucleic acids
is then drained
downward into a container. In this case, the removal of the nucleic acids
therefore takes place in
a downward direction.
Also, a lateral removal of the nucleic acids is possible, for example, when a
column placed
horizontally with a membrane located inside is charged in a flow-through
process with a lysate
and the horizontally placed column is subsequently rinsed with an elution
buffer on the side of
the membrane on which the nucleic acids are bound.
An example for the maximum angle of 180 degrees possible is an inclined
surface with a surface
suitable for the bonding of nucleic acids over which the various solutions or
buffers flow
downwards. Like all buffers, the elution buffer, too, comes from one side and
flows down to the
other side. In this case, the direction of the entering stream of the buffer
and the exiting stream of
13
CA 02348054 2001-04-20
the buffer containing the nucleic acids form an angle of 180 degrees; the
removal, however,
always takes place on the same side of the surface as the immobilization.
In the process according to this invention, the sample containing nucleic
acids described above is
introduced into a solution which contains the appropriate salts or alcohol(s),
then, in appropriate
cases, solubilizes the preparation and passes the mixture achieved in this
way, by means of a
vacuum, through the use of a centrifuge, by means of positive pressure, by
capillary forces, or by
other appropriate procedures through a porous surface, by which process the
nucleic acids are
immobilized on the surface.
Suitable salts for the immobilization of nucleic acids on membranes or other
surfaces and/or
lysates of samples of nucleic acids include salts of metal cations such as the
alkaline or alkaline
earth metals with mineral acids, in particular alkaline or alkaline earth
halogenides or sulfates or
phosphates, with the halogenides of sodium, lithium or potassium or magnesium
sulfate being
especially preferred. Other metal cations, e.g. Mn, Cu, Cs or Al or the
Ammonia cation can be
used, preferably as salts of mineral acids.
Also suited for carrying out the process according to the invention are salts
of mono- or polybasic
acids or polyfunctional organic acids with alkaline or alkaline earth metals.
These include, in
particular, salts of sodium, potassium or magnesium with organic dicarboxylic
acids -- such as
oxalic, malonic or succinic acid -- or with hydroxy- or polyhydroxycarboxylic
acids -- for
example, preferably, with citric acid.
If it turns out to be more suitable for certain applications, the above listed
substances for the
immobilization of nucleic acids on the surfaces and/or to lyse samples of
nucleic acids can with
this be used on their own or in mixtures.
The use of so-called chaotropic agents has proved to be especially effective.
Chaotropic
substances are capable of disturbing the three-dimensional structure of
hydrogen bonds. This
process also weakens the intramolecular binding forces that participate in
forming the spatial
structures -- including primary, secondary, tertiary or quaternary structures -
- in biological
molecules. Chaotropic agents of this kind are known to the expert from the
state of the art
(Rompp, Lexikon der Biotechnologie, published by H. Dellweg, R.D. Schmid and
W.E. Fromm,
Thieme Verlag, Stuttgart 1992).
The preferred chaotropic substances for use with this invention are, for
example, salts from the
trichloroacetate, thiocyanate, perchlorate or iodide group or guanidinium
hydrochloride and urea.
The chaotropic substances are 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. The
chaotropic agents mentioned above can be used alone or in combinations. In
particular, a 0.01- to
10-molar aqueous solution, preferably a 0.1- to 7-molar aqueous solution and
most preferably a
0.2- to 5-molar aqueous solution of sodium perchlorate, guanidinium
hydrochloride, guanidinium
isothiocyanate, sodium iodide, and/or potassium iodide is used.
14
1,
CA 02348054 2001-04-20
The salt solutions used for the lysis, bonding, washing and /or elution in the
process according to
the invention preferably are buffered. Suitable as buffer substances are the
following buffer
systems, e.g. carboxylic acid buffers, in particular, citrate buffer, acetate
buffer, succinate buffer,
malonate buffer as well as glycine buffer, morpholinopropansulfonic acid
(MOPS) or
Tris(hydroxymethyl)aminomethane (Tris) in a concentration of 0.001 - 3 mol/l,
preferably
0.005- 1 mol/1, particularly preferred 0.01 - 0.5 mol/l, especially preferred
0.01 - 0.2 mol/l.
The suitable alcohols for the conduct of the process according to the
invention include, first of
all, all the hydroxyl derivatives of aliphatic or acyclic saturated or
unsaturated hydrocarbons. It is
initially unimportant whether these compounds contain one-, two, three or more
hydroxyl groups
-- such as polyvalent C 1-C5 alcanols, including ethylene glycol, propylene
glycol or glycerine.
In addition, the usable alcohols according to the invention include the sugar
derivates, the so-
called aldites, as well as the phenols, such as polyphenols.
Among the hydroxyl compounds mentioned previously, the ('11-C5 alkanols, such
as methanol,
ethanol, n-propanol, tertiary butanol and the pentanols are especially
preferred.
In the sense of the present invention, the term hydrophilic applies to such
materials or
membranes which by virtue of their chemical nature mix easily with water or
absorb water.
In the sense of the present invention, the term hydrophobic applies to such
materials or
membranes which by virtue of their chemical nature do not penetrate into water
-- or vice versa --
and which are not able to remain in it.
By the word surface, in the sense of the present invention, is meant any
microporous-separating
layer. In the case of a membrane, the surface consists of a film of a polymer
material. The
polymer will preferably be composed of monomers with polar groups.
In another embodiment of the process according to the invention, the concept
of surface in the
broader sense includes a layer of particles or a granulate or even fibers such
as e.g. silica gel
fleeces.
In connection with the use of hydrophobic membranes, in the sense of the
present invention,
those membranes are preferred which consist of a hydrophilic substance and
which can be
rendered hydrophobic by a subsequent chemical treatment which is well known
from the state of
the art, such as hydrophobized nylon membranes which are commercially
available.
For the purposes of this invention, hydrophobized membranes include, in
general, those
membranes which may or may not have been hydrophilic to begin with and are
coated with the
hydrophobic coating agents mentioned below. Hydrophobic coating agents of this
kind cover
hydrophilic substances with a thin layer of hydrophobic groups, such as fairly
long alkyl chains
or siloxane groups. Suitable hydrophobic coating agents are known in great
number from the
CA 02348054 2001-04-20
state of the art; for purposes of the invention, these include paraffins,
waxes, metallic soaps etc.,
if necessary with additives of aluminum or zirconium salts, quaternary organic
compounds, urea
derivatives, lipid-modified melamine resins, silicones, zinc-organic
compounds, glutaric
dialdehydes and similar compounds.
In addition, the hydrophobic membranes that can be used for purposes of the
invention are
membranes which are hydrophobic per se and those that have been made
hydrophobic and whose
basic material contains polar groups. According to these criteria, for
example, materials from the
following group -- particularly hydrophobized ones -- are suitable for use
with the invention:
Nylon, polysulfones, polyether sulfones, cellulose nitrate, polypropylene,
polycarbonates,
polyacrylates and acrylate copolymers, polyurethanes, polyamides, polyvinyl
chloride,
polyfluorocarbonates, polytetrafluorethylene, polyvinylidene difluoride,
polyethylenetetrafluorethylene copolymerisates, polyethylene
chlorotrifluorethylene
copolymerisates or polyphenylene sulfide as well as cellulose and cellulose
mixed esters,
cellulose acetate or nitric celluloses and polybenzimidazoles, polyimides,
polyacrylnitriles,
polyacrylnitrile copolymers, hydrophobized glass fibre membranes amongst which
hydrophobized nylon membranes are particularly preferred.
Preferred hydrophilic surfaces include hydrophilic materials per se and also
hydrophobic
materials, which have been made hydrophilic. For instance the following
substances can be used:
hydrophilic nylon, hydrophilic polyether-sulfones, hydrophilic polycarbonates,
hydrophilic
polyesters, hydrophilic polytetrafluoro-ethylenes on polypropylene tissues,
hydrophilic
polytetrafluorethylenes on polypropylene fleeces, hydrophilized polyvinylidene
difluorides,
hydrophilized polytetrafluorethylenes, hydrophilic polyamides, nitric
cellulose, hydrophilic
polybenzimidazoles, hydrophilic polyimides, hydrophilic polyacrylnitriles,
hydrophilic
polyacrylnitrile copolymers, hydrophilic polypropylene, cellulose nitrate,
cellulose mixed esters
and cellulose acetate.
In connection with processes for bonding of nucleic acids the membranes
mentioned above are
partially known, although not in the context of this invention, from the
present state of the art. A
selection of materials for this process is however not yet known from the
state of the art. The
extensive experiments by the inventors have shown, however, that there are
further membranes
suitable for the bonding of nucleic acids.
The present invention is thus also aimed at the use of cellulose acetate, non
carboxylated,
hydrophobic polyvinylidene difluoride or solid, hydrophobic
polytetrafluorethylene as material
for adhesion and isolation of nucleic acids.
The term "solid" herein refers to a material which consists throughout of the
relevant chemical
compound and is neither coated nor placed as a coating on a substrate.
16
CA 02348054 2001-04-20
The material can be used in the form of a membrane, as granules, in the form
of fibers or in any
other suitable form. The fibers for example can be arranged as a fleece and
the granules as a
compressed frit.
The membranes that are used in the processes according to the invention
described above (with
the exception of iso-propanol precipitation) have, for example, a pore
diameter of 0.001 to 50
m, preferably 0.01 to 20 m and most preferably 0.05 to 10 m. In the case of
the precipitation
of nucleic acids with iso-propanol according to the process described above,
the pore size has to
be above 0.2 gm.
As washing buffers, the salts or alcohols described above, or phenols or
polyphenols, can be
considered. Also detergents and natural substances in the widest sense, such
as albumin and milk
powder, can be used for the washing steps. The addition of chaotropic
substances is also
possible. Polymers as well as detergents and similar substances can be added.
In any event, the
washing buffer and the substances contained therein should generally be able
to bind, solubilize
or react with the undesirable contaminants such, that these contaminants or
their decomposite
products are removed together with the washing buffer. The temperatures in the
washing step
will usually be within the range from 10 to 30 C, preferably room
temperature, whereas higher
or lower temperatures can also be used successfully. As such it might for
example be indicated to
cool the washing buffer for elutions performed at low temperatures, e.g. 2 C,
to pre-cool the
isolation container and the surface or membrane to the desired temperature
value. An application
for low temperatures is the cytoplasmic lysis, during which the cell nuclei
initially remain
undamaged. Higher temperatures of the washing buffer, on the other hand, cause
a better
solubilization of the contaminants that are to be washed out.
Suitable eluting agents for the elution of bound nucleic acids for the
purposes of the invention are
water or aqueous salt solutions. As salt solutions, buffer solutions that are
known from the
present state of the art are used, such as morpholinopropane sulfonic acid
(MOPS), Tris
(hydroxymethyl) aminomethane (TRIS), 2-[4-(2-hydroxyethyl) -1- piperazino]
ethane sulfonic
acid (HEPES) in a concentration from 0.001 to 0.5 mol/liter, preferably 0.01
to 0.2 mol/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,
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 dichloride. Also
preferred for use
are solutions of salts of the alkaline or alkaline earth metals with
carboxylic or dicarboxylic
acids, such as oxalic acid or acetic acid, solutions of sodium acetate or -
oxalate in water, for
example in the range of concentrations mentioned previously , for example
0.001 to 0.5 molar,
preferably 0.01 to 0.2 molar, most preferably 0.01 to 0.05 molar.
The addition of auxiliary substances like detergents and DMSO is also
possible. If a chemical
reaction is to be carried out with the eluted nucleic acids, be it directly at
the membrane or in
another reaction container, it is also possible to add such substances or
other auxillary substances
to the elution buffer that is to be used in the reaction preparation. Thus the
addition of DMSO in
low concentrations is common for many reaction preparations.
17
CA 02348054 2001-04-20
. 'f
After a chemical reaction has been performed on nucleic acids those can also
be eluted with the
reaction buffer. After a SDA or a NASBA reaction the nucleic acid can for
example be eluted
with the reaction buffer or the reaction master mix.
Pure water is especially preferred as a means of elution, e.g., demineralized,
double distilled, or
ultrapure Millipore water.
Elution can be carried out successfully at temperatures from below 00 to 90
Celsius, for
example, between 10 to 30 Celsius, and even at higher temperatures. Elution
with steam is also
possible. The lower limit of the elution temperature is, as described above,
to be seen in the
freezing point of the elution medium.
Because the process according to the invention can be carried out without
problems even in the
"field", i.e. away from laboratory installations and thus without extensive,
electrically powered
equipment, the invention is also aimed at the provision of isolation
containers with which the
process, according to the invention, can be carried out with a minimum of
additional accessories.
For this a reaction container, which contains a membrane, can be used. This
can be brought into
contact with an absorbant material , such as a sponge, to draw the various
used buffers through
the membrane. The sponge thus functions like a combination of a vacuum pump or
a centrifuge
in conjunction with a waste container. To obtain the eluate, the contact of
the absorbant material
with the membrane is interrupted such, that the eluate is not lost but removed
and/or inspected
further.
In this aspect the invention is specifically aimed at an isolation container
for the isolation of
nucleic acids with at least one cylindrical upper part with a top opening , a
bottom opening and a
membrane which is located at the bottom opening and covers the entire cross-
section of the upper
part; a lower part with an absorbant material; and a mechanism for the
coupling of the upper part
and the lower part, where, with established connection, the membrane is in
connection with the
absorbant material and with non-established connection the membrane is not in
contact with the
absorbant material.
The lower part is preferably a cylinder with a diameter equal to that of the
upper part. In this way
a simple tube with essentially constant diameter is obtained which can be
manipulated like
conventional reaction containers. This effect is achieved when the upper part
or the upper part
plus the lower part form a tube which can be placed inside a reaction
container holder as utilized
in laboratories. The mechanism can be a connection which permits a physical
separation of upper
part and lower part for example a bayonet coupling, a plug coupling or a screw
coupling. Here a
bayonet coupling has the advantage that it is easier to lock and unlock while
the screw coupling
permits a better, more water tight connection of upper and lower parts.
Alternatively a
predetermined breaking point between upper and lower parts can be provided for
to permit at
least a once-off separation of both parts and can be manufactured particularly
cost efficient.
18
CA 02348054 2001-04-20
The mechanism can however also be a slide which can be inserted between the
absorbant
material and the membrane. This design also permits a separation of membrane
and absorbant
material.
To increase the process capacitiy and to make the process according to the
invention even more
economical it is furthermore possible to modify the isolation container
described above in such a
manner that several upper parts are located on top of the lower part. The
lower part can at the
same time serve as the support of the arrangement and be dimensioned such that
a multitude of
isolation processes can be carried out, in any event more than the connections
for upper parts
available in the lower part, before the drawing capacity of the absorbant
material inside the
lower part is exhausted.
The absorbant material in the lower part can show a sponge and/or granules.
The granules can for
example consist of superabsorbant material as it is known to specialists in
the area of absorption
technology (such as e.g. articles for hygiene).
The invention is simultaneously aimed at the use of the isolation container
according to the
present invention for the analysis of properties of nucleic acids and the
isolation of nucleic acids.
With regard to the individual steps, the processes according to the invention
are performed as
follows:
If biological samples are the origin they firstly are to be lysed with a
suitable buffer. Here
additional processes can be introduced for the lysis, e.g. mechanical
influence such as
homogenization or ultra sound, enzymatic influence, change in temperature or
additives. If
necessary or desired, a pre-purification step can follow the lysis to remove
any debris from the
lysis. Subsequently, if not already carried out, the conditions, under which
the immobilization of
the nucleic acids at the surface is to be carried out, are standardized. Even
after standardization of
the bonding conditions a pre-purification step can follow either cumulative or
alternative.
The pre-treated lysate of the sample used for the recovery of the nucleic
acids or the originally
free nucleic acid(s) (unless the original sample is biological) is/are
pipetted, for example, into a
(plastic) column, in which the membrane is fastened -- for example, on the
bottom section. It is
more efficient if the membrane is fastened to a frit, which serves as a
mechanical support. The
lysate is then transferred through the membrane, which can be achieved by
applying a vacuum at
the outlet of the column. On the other side the transfer can be accomplished
by applying positive
pressure from the lysate side. 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 brought in contact with the
lysate or filtrate below
the membrane. For a centrifugation the isolation container, open at the
bottom, can be used inside
a collection container for the transferred liquid.
The added washing step in the preferred embodiments of the invention can take
place by having
the washing buffer transferred through the surface or membrane, or by having
it remain on the
19
CA 02348054 2001-04-20
same side of the surface as the nucleic acids. If the washing buffer is passed
or drawn through,
this can take place in a variety of ways, e.g., by a sponge mounted on the
other side of the
membrane, by a vacuum or positive pressure apparatus, or by a centrifuge or
gravity.
The advantage of an arrangement using an absorbant material such as a sponge
lies in a simple,
safe and easy way to dispose of the filtrate -- in this case, only the sponge,
which is now more or
less saturated with the filtrate, needs to be changed. It should be clear at
this point that the
column can be operated continuously or in batches and that both these methods
of operation can
be carried out fully automated, until the membrane is completely saturated
with nucleic acid.
During the last step, if applicable, the elution of the nucleic acid occurs ,
which can for example
be drawn off or removed by a pipette or removed upwards in some other way if
no in situ
analysis of the still bonded nucleic acids is to be carried out.
The desired nucleic acids are obtained in weak or no salt containing solutions
in very small
volumes which is of great advantage for all processes of molecular biological
analysis as it is
desirable to employ as small a volume as possible at simultaneously high
concentration. To
obtain as small a volume of eluate as possible, membranes that are as thin as
possible are used as
surfaces because only little liquid collects in them.
Moreover, the present invention has the advantage that, when the device is
placed in a vertical
position (the membrane then being in horizontal position), the space above the
membrane can be
used as a reaction area. Thus, it is possible, for example, that after the
isolation and release of the
nucleic acids produced by the process which is fundamental to this invention,
not only not to
remove them, but also to leave them in the isolation device and subject them
to a molecular-
biological application -- like restriction digestion, RT, PCR, RT-PCR, in
vitro transcription,
NASBA, rolling circle, LCR (ligase chain reaction), SDA (strand displacement
amplification) or
enzyme reactions like Rnase and Dnase-digestion for the complete removal of
the nucleic acids
not desired from time to time, to again bind the nucleic acids, which were
produced by these
reactions, to the membrane in accordance with the procedures on which this
invention is based,
or to retain them in the supernatant liquid, if applicable to wash them as
described and
subsequently to elute them, to isolate or analyze them, by means of, e.g.,
chromatography,
spectroscopy, fluorometry, electrophoresis or similar measuring techniques.
The nucleic acids isolated pursuant to this invention are free from enzymes
which decompose
nucleic acids, and have a level of purity which is so high that they can
immediately be treated and
processed in the most varied ways.
The nucleic acids, which are produced according to this invention, can be used
for cloning, and
can serve as substrates for the most varied enzymes, such as DNA polymerases,
RNA
polymerases, e.g. T7 polymerase or T3 polymerase, DNA restriction enzymes, DNA-
ligase,
reverse transcriptase and others.
CA 02348054 2001-04-20
The nucleic acids produced by the process in this invention are especially
well suited for
amplification, particularly for the PCR, strand displacement amplification,
the rolling circle
procedure, the ligase chain reaction (LCR), SunRise, NASBA and similar
procedures.
In addition, the processes according to this invention are particularly well
suited for the
preparation of nucleic acids for use in diagnostics, e.g. food analysis,
toxicological tests, in
medical and clinical diagnostics, pathogen diagnostics, gene expression
analysis and in
environmental analysis. The processes are particularly suitable for a
diagnostic procedure which
is characterized in that the nucleic acids purified by the process according
to the invention are
amplified in a subsequent step, and the nucleic acids amplified in this way
are then subsequently
or immediately detected (e.g. Holland, P.M. et al., 1991. Proc. Natl. Acad.
Sci. 88, 7276 - 7280.
Livak, K.J. et al., 1995. PCR Methods Applic. 4, 357 - 362; Kievits, T. et
al., 1991. J. Virol.
Meth. 35, 273 - 286; Uyttendaele, M. et al., 1994. J. Appl. Bacteriol. 77, 694
- 701).
Furthermore, the processes according to this invention are particularly well
suited for the
preparation of nucleic acids which can be subjected in a subsequent step to a
signal amplification
step based on a hybridization reaction, which is characterized especially in
that the nucleic acids
produced by the process according to the invention can be brought into contact
with "branched
nucleic acids," especially branched DNA and/or branched RNA and/or dendritic
nucleic acids, as
described in the following articles (e.g., Bresters, D. et al., 1994. J. Med.
Virol. 43 (3), 262-286;
Collins M.L. et al., 1997. Nucl. Acids Res. 25 (15), 2979-2984), and that the
arising signal can be
detected.
In the following an example for the ability to automate the process according
to the invention is
explained and examples are listed for the carrying out of the process with
various surfaces and
nucleic acids. Reference is made to the attached figures which illustrate the
following:
Fig. 1 shows automatic equipment suitable for the performance of the process
according to the
invention in a schematic diagram.
Fig. 2 shows a first embodiment of an isolation container and collection tube
for the
performance of the process according to the invention.
Fig. 3 shows a second embodiment of an isolation container and collection tube
for the
performance of the process according to the invention.
Fig. 4 shows a third embodiment of an isolation container and collection tube
for the
performance of the process according to the invention.
Fig. 5 shows embodiments of isolation containers with an upper part according
to the invention.
Fig. 6 shows the ethidium bromide stained gel of an electrophoretic separation
of various
samples according to the process of the invention.
21
CA 02348054 2001-04-20
Fig. 7 shows another ethidium bromide stained gel of an electrophoretic
separation of various
samples according to the process of the invention.
Fig. 8 shows another ethidium bromide stained gel of an electrophoretic
separation of various
samples according to the process of the invention.
Fig. 9 shows yet another ethidium bromide stained gel of an electrophoretic
separation of various
samples according to the process of the invention.
Fig. 10 again shows another ethidium bromide stained gel of an electrophoretic
separation of
various samples according to the process of the invention.
Fig. 11 finally shows another ethidium bromide stained gel of an
electrophoretic separation of
various samples according to the process of the invention.
The processes according to the invention are preferably performed
automatically, either partially
or completely, in other words, in all steps. An example for suitable automatic
equipment is
illustrated in Fig. 1, in which a main part 1 is equipped with control
electronics and driving
motors 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 containers. A vacuum manifold 5
serves to draw
liquids from isolation containers, positioned above and open at the bottom or
otherwise with the
containers connected to the vacuum manifold. A shaker 6 is also provided,
which e.g. can be
used to subject the biological samples to lysis. The isolation container
assemblies used are e.g.
injection-molded parts with integrated isolation containers, in which the
surfaces are placed
according to the invention. Typically 8, 12, 24, 48, 96 or up to 1536
isolation containers can be
used as these are for example seen in the formats of modern multi-well-trays.
Even higher
numbers of isolation containers might be possible on one assembly, if the
corresponding
standards are available. With the aid of Luer-adapters it is, however, also
possible to make
individual bottoms of the assembly available and to equip these with one or
more isolation
containers as needed. Isolation containers used individually without Luer-
adapters are also
included in the invention.
Under a vacuum and dispensing mechanism 8 the isolation container assemblies
are placed in the
automatic apparatus and via these, liquids can be taken in and drained off. In
this assembly
several single vacuum tubes can be used, so as to make the simultaneous
processing of more than
one isolation or reaction container possible. The vacuum and dispensing
mechanism 8 therefore
acts as a pipette. Vacuum and pressure are fed to the vacuum and dispensing
mechanism 8 via
tube 9.
To isolate the nucleic acids, reaction containers with cells may for example
be placed in the
shaker/holder 6, into which lysis buffers are introduced by means of the
dispensing mechanism 8.
After mixing, the cell lysates are transferred to isolation containers. The
lysis buffer is
subsequently drawn through the surfaces in the isolation containers.
Subsequently, the surfaces
may be washed with a washing buffer in order to remove cell lysate residue, in
which also the
22
CA 02348054 2001-04-20
washing buffer is drained off downwards. Finally an elution buffer is
dispensed into the isolation
devices and after possibly additional shaking, the released nucleic acids are
removed in an
upwards direction and transferred to collection tubes.
Usually, disposable tips are used on the vacuum and dispensing mechanism 8 to
prevent
contamination of the samples.
Fig. 2 to 4 show different schematic examples for suitable isolation
containers to be used in the
present invention.
In Fig. 2 a funnel-shaped isolation container 10 is provided with a surface
11, e.g. a membrane,
which is placed on a collection tube 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 able to convert water chemically, e.g. acrylate. The water
is thereby also
removed from the process. Lysate or another preparation of nucleic acids is
placed in the funnel.
The sponge-like material 13 draws the applied liquid through membrane 11.
Prior to the addition
of the elution buffer, the sponge is spaced some distance from the membrane,
e.g. by a
mechanism inside collection tube 12 (not visible in the fig.). This will
prevent that the eluate
buffer in the last step is also suctioned off through membrane 11. This buffer
rather stays on the
surface (Fig. lb) and can be removed together with the nucleic acids in an
upwards direction.
When using this assembly, the vacuum mechanism 5 is not required in the
automatic apparatus.
Fig. 3 shows another example of an isolation device, which, via a Luer-
connection located at its
bottom, is, by way of a Luer-adapter 17, connected to a collection container
16, which in this
case does not contain a sponge, but is connected to a vacuum mechanism with
the aid of a nozzle
18. Lysis and washing buffers may in this case be drawn through membrane 11 by
applying a
vacuum. When the eluate buffer is introduced, the vacuum remains turned off,
so that the eluate
can be removed in an upward direction. With the use of a Luer-connection,
individual isolation
containers can be removed from the isolation container assembly. It is to be
understood however,
that the vacuum collection container can also be combined with fixed isolation
devices, e.g.
multi-well containers with 8, 12, 24, 48, 96 or more individual containers.
Fig. 4 finally shows an embodiment which provides for a collection container,
into which the
buffers are drawn through gravity or centrifugation. The eluate buffer, which
is used in small
volumes, is not heavy enough in and of itself to penetrate membrane 11 and can
again be
removed in an upward direction.
Fig. 5 shows the embodiments of the isolation containers according to the
invention.
Fig. 5 A shows an isolation container with a cylindrical upper part 20. This
upper part is
connected to the lower part 22 by means of a screw coupling 25. In place of
the screw coupling,
other forms of connections may be used as long as these permit a liquid-tight
connection and
support the membrane 11. In this example of an embodiment the membrane 11 is
located directly
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CA 02348054 2008-01-04
29620-1
at the bottom opening of the upper part 20. It may however be placed inwards
or at an angle other
than 90 to the wall of the upper part. The lower part shows a cylindrical
form as well but may,
with other embodiments, be developed differently. As such a rectangular form
might be used
which improves the stability of the upper part 20 on the substrate. An
enlargement of the lower
part 22 in relation to the upper part 20 is also possible, for example, if,
with certain options of the
process according to the invention, a larger space is required in the lower
part 22 to completely
contain the used solutions in the absorbant material.
Part B shows an alternative embodiment to the embodiment shown in fig. 5 A. In
this the upper
part 20 and the lower part 22 are permanently connected with one another or
may also be made
as one single component. Between the absorbant material 13 and the membrane
11, a slide 27
can be inserted into the isolation container through an opening 26 to separate
the membrane 11
and the absorbant material 13. In this example slide 27 additionally shows a
handle section 28 to
ease the retraction of the slide 27. However, a slide may also be designed
without this handle
section. As shown in fig. 5 B, the absorbant material 13 slightly expands to
bridge the space
taken by the slide and to contact the membrane.
Fig. 5 C shows a further embodiment of the isolation container according to
the invention. Here
the lower part 23 is equipped with several sockets 30 to accommodate the upper
parts 20 which
permit the simultaneous processing of several preparations. In this example
the upper parts 20 are
connected to the lower part 23 by means of screw couplings 31. Although
depicted smaller than
the upper parts 20 in Fig. 5 A and B, it is to be understood that the upper
parts can be of the same
size (or larger or smaller) as or than these embodiments.
Fig. 5 D finally shows an isolation container with a jacket 32 containing
cooling liquid and
surrounding the membrane 11 on the outside. In this example upper part 20 and
lower part 24 are
plugged together. Another form of connection or a one-part embodiment are also
possible. The
jacket 32 comprises two compartments 33 and 34 which can be connected through
a destructible
partition 35. Both compartments 33, 34 are filled with substances, e.g.
solutions, whose
temperatures sink through mixing after the destruction of the partition 35.
The procedure described above is explained by the following examples.
Different and various
ways of using the procedures will be evident to the expert from the foregoing
description and
from the examples. Reference is explicitly made to the fact that, these
examples and the
corresponding descriptions are presented solely for the purpose of
illustration, and are not to be
regarded as limitations on the invention.
Example 1
Isolation of total RNA from HeLa-cells
Commercially available hydrophobic nylon membranes (for example, a material
from MSI:
Magna SH with a pore diameter of 1.2 m or a material from Pall GmbH:
Hydrolori with a pore
*Trade-mark
24
CA 02348054 2001-04-20
diameter of 1.2 m) which have been made hydrophobic by means of a chemical
post-treatment
are placed in a plastic column in a single layer. The membranes are placed on
a polypropylene
frit which serves as a mechanical support. The membranes are fixed in the
plastic column with a
ring.
The column prepared in this manner is connected by means of a Luer connection
to a vacuum
chamber. All the isolation steps are conducted through the application of a
vacuum.
For the isolation, 5 x 105 HeLa-cells are pelletized by centrifugation and the
supernatant liquid is
removed. The cells are lysed by the addition of 150 p.1 of a commercial
guanidinium
isothiocyanate buffer -- for example RLT buffer from the Qiagen Company -- in
a manner
familiar from the present state of the art. The lysis is promoted through
roughly mixing by
pipetting or vortexing over a period of about 5 s. Then 150 l of 70% ethanol
are added and
mixed in by pipetting or by vortexing over a period of about 5 s.
The lysate is then pipetted into the plastic column and suctioned through the
membrane by
evacuating the vacuum chamber. Under the conditions thus created, the RNA
remains bound to
the membrane. Next, washing takes place with a first commercial washing buffer
containing
guanidinium isothiocyanate -- for example, with the RW1 buffer of QIAGEN --
and, after that,
with a second washing buffer containing TRIS or TRIS and alcohol -- for
example, with the RPE
buffer of QIAGEN. In each case the washing buffers are suctioned through the
membrane by
evacuation of the vacuum chamber. After the final washing step, the vacuum is
maintained for a
period of about 10-min, in order to dry the membrane, after which the vacuum
chamber is
airated.
For the elution, 70 l of RNase-free water is pipetted onto the membrane in
order to release the
purified RNA from the membrane. After incubation for one minute at a
temperature in the range
from 10 to 30 C, the eluate is pipetted off the membrane from the top and the
elution step is
repeated in order to make sure that the elution is complete.
The amount of isolated total RNA obtained in this manner is then determined by
photometric
measurement of the light absorption with a wavelength of 260 rim. The
photometric
determination of the ratio between the absorbance values at 260 and 280 nm
measures the quality
of the RNA such obtained.
The results of the two isolations with hydrophobic nylon membranes (Nos. 1 and
2) are
compared in the following Table 1, comparative experiments, in which on the
one hand a
hydrophilic nylon (Nylaflo) (No. 3) as well as 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 the invention. They also show
that silica-gel
fleeces produce clearly less yield, which presumably can be attributed to its
fleece-like structure
and the ensuing absorption of the major portion of the eluate buffer.
CA 02348054 2001-04-20
Table 1. RNA yield and purity of the total RNA isolated in accordance with
Example 1
No. Type of membrane Yield of Total RNA ( g) E260/E280
1 Magna SH 1.2 m (hydrophobic nylon) 6.0 1.97
2 Hydrolon 1.2 m (hydrophobic nylon) 7.1 2.05
3 Nylaflo (hydrophilic nylon) <0.2 Not determined
4 Hydrophilic silica membrane <0.2 Not determined
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 are
produced. The
result is reproduced in Fig. 6.
In Fig. 6, line 1 embodies a total RNA that was isolated by means of a
hydrophobic nylon
membrane from Magna SH with a pore diameter of 1.2 m.
Line 2 depicts a total RNA that was isolated by means of a hydrophobic nylon
membrane from
Hydrolon with a pore diameter of 1.2 m.
Line 3 depicts the chromatogram of a total RNA that was isolated by means of a
silica
membrane.
In each case, 50 gl of the total RNA isolates were analyzed.
Fig. 6 provides distinct evidence that, when a silica membrane is 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.
In this example, the lysate and washing solutions are lead across the
hydrophobic membrane by
applying a vacuum.
Hydrophobic nylon membranes (for example, 1.2 gm Hydrolon from the Pall
Company) are
introduced into plastic columns that are connected to a vacuum chamber, in a
manner analogue
to that of Example 1.
100 gl of an aqueous solution containing total RNA is mixed, by pipetting,
with, respectively,
350 1 of a commercially available lysis buffer containing guanidium
isothiocyanate (for
example, the RLT buffer from QIAGEN), 350 gl of 1.2 M sodium acetate solution,
350 l 2 M
26
CA 02348054 2001-04-20
sodium chloride solution and 350 gl of 4 M lithium chloride solution,
respectively, and mixed by
pipetting.
Next, 250 gl of ethanol is added to each mixture and mixed in, likewise by
pipetting. After that,
the solutions containing RNA are pipetted 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 are then washed, as described in
Example 1.
Finally, the RNA - also as described in Example 1 - is removed from the
membrane by pipetting
in an upward direction.
The volume of isolated total RNA was determined by photometric measurement of
the light
absorption at 260 nm. The photometric determination of the ratio between the
light absorbance
values at 260 and 280 nm measures the quality of the RNA such obtained.
Table 2: Isolation of free RNA by binding the RNA to hydrophobic membranes by
means of
various salt-alcohol mixtures
No. Salt/Alcohol-mixture Yield of Total RNA (gg) E260/E280
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
According to example 1, plastic columns with various hydrophobic membranes are
assembled.
The column prepared in this manner is placed in a collection tube, the
following isolation steps
are conducted through centrifugation.
For the isolation, 5 x 105 HeLa-cells are pelletized by centrifugation and the
supernatant
substance is removed. The cells are lysated by the addition of 150 gl of a
commercial guanidium
isothiocyanate buffer - for example RLT buffer from QIAGEN - in a manner
principly familiar
from the state of the art. Lysis is promoted through repeatedly mixing by
pipetting or vortexing
over a period of about 5 s. Then 150 gl of 70% ethanol are added and mixed in
by pipetting or
by vortexing over a period of about 5 s.
27
CA 02348054 2001-04-20
The lysate is subsequently transferred into the plastic column and passed
through the membrane
by way of centrifugation at 10000 x g for 1 minute. Subsequently, washing
takes place with a
commercially available washing buffer containing guanidinium isothiocyanate-
e.g. with the
RW1-buffer of QIAGEN - followed by a second washing buffer containing Tris and
alcohol-
e.g. RPE-buffer by QIAGEN. The washing buffers are passed through the membrane
by
centrifugation. The last washing step takes place at 20000 x g for 2 minutes
to dry the membrane.
For the elution, 7O 1 RNase-free water is pipetted onto the membrane in order
to release the
purified RNA from the membrane. After incubation for 1 - 2 minutes at a
temperature in the
range of 10 to 30 C, the eluate is pipetted off the membrane from the top.
The elution step is
repeated once in order to achieve a complete elution.
The volume of isolated total RNA obtained in this manner is then determined by
photometric
measurement of the light absorption at a wavelength of 260 nun. The
photometric determination
of the ratio between the light absorbance values at 260 and 280 urn measures
the quality of the
RNA.
The results of the isolations with different hydrophobic membranes are shown
in Table 3.
3 - 5 parallel tests per membrane are carried out and the average value is
calculated. By using a
silica membrane, no measurable volume of total RNA can be isolated, if the
eluate is obtained by
removing it from the top of the membrane.
Table 3: RNA-yield of total RNA according to example 3 by binding to
hydrophobic membranes
Manufacturer Membrane Material RNA E260 /
( g) E280
Pall Hydrolon, 1.2 m hydrophobic nylon 6.53 1.7
Pall Hydrolon, 3 m hydrophobic nylon 9.79 1.72
Pall Fluoro Trans G hydrophobic polyvinylidene 6.16 1.72
difluoride
Pall Fluoro Trans W hydrophobic polyvinylidene 5.4 1.9
difluoride
Pall Bio Trace hydrophobic polyvinylidene 4.3 1.97
difluoride
Pall Supor-450 PR hydrophobic polyether sulfone 3.96 1.76
Pall V-800 R hydrophobic acrylate copolymer 6.26 1.72
Pall Versapor-1200R hydrophobic acrylate copolymer 6.23 1.68
Pall Sciences Versapor-3000R hydrophobic acrylate copolymer 3.54 1.74
GORE-TEX OH 9335 hydrophobic polytetrafluor-ethylene 1.59 1.72
GORE-TEX OH 9336 hydrophobic polytetrafluor-ethylene 2.15 1.65
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29620-1
GORE-TEX OH 9337 hydrophobic polytetrafluor-ethylene 3.6 1.59
GORE-TEX QH 9316 hydrophobic polytetrafluor-ethylene 3.61 1.69
GORE-TEX QH 9317 hydrophobic polytetrafluor-ethylene 2.87 1.70
Millipore Mitex Membrane hydrophobic polytetrafluor-ethylene 1.98 1.62
Millipore Durapore * hydrophobic polyvinylidene 7.45 1.72
difluoride
MSI Magna-SH, 1.2 p.m hydrophobic nylon 4.92 1.69
MSI Magna-SH, 5 pm hydrophobic nylon 10.2 1.71
MSI Magna-SH, 10 m hydrophobic nylon 7.36 1.76
MSI Magna-SH, 20 m hydrophobic nylon 7.04 1.65
Sartorius type 118 hydrophobic polytetrafluor- 7.6 1.61
ethylene
Mupor PM 12 A hydrophobic polytetrafluor- 6.7 1.77
ethylene
Mupor PM 3 VL hydrophobic polytetrafluor- 6.6 1.77
ethylene
Example 3b
Isolation of total RNA from HeLa-cells through bonding onto hydrophilic
membranes
According to example 1, plastic columns with various hydrophilic membranes are
assembled.
The column prepared in this manner is placed in a collection tube, the
following isolation steps
are conducted through centrifugation.
For the isolation, 5 x 105 HeLa-cells are used. The following isolation steps
and the elution of the
nucleic acid are carried out as described in example 3.
The volume of isolated total RNA obtained in this manner is then determined by
photometric
measurement of the light absorption at a wavelength of 260 nm. The photometric
determination
of the ratio between the light absorbance values at 260 and 280 nm measures
the quality of the
RNA.
The results of the isolations with different hydrophilic nylon membranes are
shown in Table 3b.
2 - 5 parallel tests per membrane are carried out and the average value is
calculated. By using a
silica membrane, no measurable volume of total RNA can be isolated, if the
eluate is obtained by
removing it from the top from the membrane.
*Trade-mark
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CA 02348054 2008-01-04
29620-1
Table 3b: RNA-yield of total RNA according to example 3b by binding to
hydrophilic
membranes
Manufacturer Membrane Material RNA ( ) E260/E280
Pall Lo rod e* hydrophilic nylon 3.1 1.8
Pall Lo rodyne 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 Biod e P* hydrophilic nylon 2.6 1.84
Pall Biodyne 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 of ether sulfone 6.36 1.8
Pall I.C.E.-450sw;* hydrophilic of ether sulfone 3.07 1.71
Pall Su or-800 * h dro hilic polyether sulfone 4.12 1.7
Pall Su or-450 * hydrophilic of ether sulfone 4.69 1.69
Pall Su or-100* hydrophilic of ether sulfone 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 *'j polyester membrane 1.66 1.63
Pall Pol ro-450', hydrophilic polypropylene 5.09 1.78
Gore-Tex OH 9339* hydrophilic polytetrafluor- ethylene 1.08 1.65
Gore-Tex OH 9338` hydrophilic polytetrafluor- ethylene 3.97 1.67
Gore-Tex H 9318' hydrophilic polytetrafluor- ethylene 3.61 1.69
Millipore Dura ore h dro hilized of in lidene difluoride 5.6 1.69
Milli ore Dura ore*~ h dro hilized of in lidene difluoride 3.12 1.68
Millipore LCR * h dro hilized polytetrafluoro ethylene 3.14 1.66
Sartorius Type 250 hydrophilic of amide 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 Polycon, 0.01 hydrophilic of carbonate 0.17 1.64
Infiltec Polycon, 0.: hydrophilic polycarbonatte 0.73 1.68
Infiltec Polycon, 1* hydrophilic polycarbonate 3.33 1.86
Example 4
Isolation of free RNA from an aqueous solution
According to Example 1 plastic columns with different hydrophobic membranes
are assembled.
100 d of an aqueous solution containing total RNA are mixed with 350 41 of a
commercially
available lysis buffer containing guanidinium-isothiocyanate - e.g. RLT-
buffers by QIAGEN.
*Trade-mark
CA 02348054 2008-01-04
29620-1
Subsequently 250 41 of ethanol are added and mixed by pipetting. This mixture
is then
transferred to the column and passed through the membrane by way of
centrifugation (10000 x g;
1 minute). The membranes are subsequently washed twice with a buffer - e.g.
RPE by QIAGEN.
Each buffer is passed through the membranes by way of centrifugation. The last
washing step is
carried out at 20000 x g to dry the membranes.
Subsequently, the RNA, as already described in Example 1, is eluted with RNase-
free water and
pipetted off the membrane from the top.
The volume of isolated total RNA- obtained in this manner is then determined
by photometric
measurement of the light absorption at a wavelength of 260 mn. The photometric
determination
of the ratio between the light absorbance values at 260 and 280 nm measures
the quality of the
RNA.
The results of the isolation with various hydrophobic membranes are listed in
Table 4 below.
3 - 5 parallel tests per membrane are carried out and in each case the average
value is calculated.
By using a silica membrane, no measurable volume of total RNA can be isolated,
if the eluate is
recovered by removing it off the membrane from the top.
Table 4: Isolation of free RNA from an aqueous solution by binding to
hydrophobic membranes
Manufacturer Membrane Material RNA E26o/Ezao
p
Pall Hydrolon, 1.2 m2 Hydrophobic nylon 5.15 1.75
Pall Hydrolon, 3 m' Hydrophobic nylon 0.22 1.79
Pall Fluoro Trans G* Hydrophobic polyvinylidene difluoride 5.83 1.79
Pall Fluoro Trans W * Hydrophobic polyvinylidene difluoride 5.4 1.84
Pall Bio Trace * Hydrophobic polyvinylidene difluoride 4 1.79
Pall Emflon* Hydrophobic polytetrafluor - ethylene 0.2 1.7
Pall Su or-450 PR* Hydrophobic of ether sulfone 5.97 1.71
Pall Su or-200 PR* Hydrophobic of ether sulfone 2.83 1.66
Pall V-800 R* Hydrophobic ac late co-polymer 2.74 1.77
Gore-Tex OH 9335 * Hydrophobic polytetrafluor - ethylene 4.35 1.63
Gore-Tex OH 9336* Hydrophobic polytetrafluor - ethylene 7.43 1.71
Gore-Tex OH 9337* Hydrophobic polytetrafluor - ethylene 5.96 1.62
Gore-Tex QH 9316 * Hydrophobic polytetrafluor - ethylene 5.92 1.67
Gore-Tex QH 9317* Hydrophobic polytetrafluor - ethylene 8.7 1.66
Millipore Fluoropore Hydrophobic polytetrafluor - ethylene 8.46 1.70
Millipore Durapore, 0.65 m * Hydrophobic polytetrafluor - ethylene 4.23 1.8
MSI Ma a-SH, 1.2 mW ' Hydrophobic nylon 1.82 1.76
MSI Ma a-SH, 5 gm * Hydrophobic nylon 0.6 1.78
Sartorius Typ 118* Hydrophobic polytetrafluor - ethylene 0.9 1.82
Sartorius Typ 118* Hydrophobic polytetrafluor - ethylene 5.4 1.74
Mu or PM12A* Hydrophobic polytetrafluor - ethylene 1.1 1.98
*Trade-mark
31
CA 02348054 2001-04-20
Example 4 b
Isolation of free RNA from an aqueous solution through binding to hydrophilic
membranes
According to Example 1 plastic columns with different hydrophilic membranes
are assembled.
100 l of an aqueous solution containing total RNA are mixed with 350 l of a
commercially
available lysis buffer containing guanidinium-isothiocyanate - e.g. RLT-
buffers by QIAGEN.
Subsequently 250 l of ethanol are added and mixed by pipetting. This mixture
is then
transferred to the column and, according to example 4, passed through the
membrane, washed
and dried.
Subsequently, the RNA, as already described in Example 1, is eluted with RNase-
free water and
pipetted off the membrane from the top.
The volume of isolated total RNA obtained in this manner is then determined by
photometric
measurement of the light absorption at a wavelength of 260 nm. The photometric
determination
of the ratio between the light absorbance values at 260 and 280 nm measures
the quality of the
RNA.
The results of the isolations with various hydrophilic membranes are listed in
Table 4b below.
2 - 5 parallel tests per membrane are carried out and in each case the average
value is calculated.
By using a silica membrane, no measurable volume of total RNA can be isolated,
if the eluate is
recovered by removing it off the membrane from the top.
Table 4b: Isolation of free RNA out of an aqueous solution through binding to
hydrophilic
membranes
Manufacturer Membrane Material RNA E260/E280
Pall Lo rod e hydrophilic U !on 2 1.8
Pall Lo rod e hydrophilic nylon 1.4 1.87
Pall Biodyne A hydrophilic a !on 4.5 1.93
Pall Biod e A hydrophilic a !on 3.1 1.9
Pall Biodyne B hydrophilic a !on 1.7 1.94
Pall Biodyne B hydrophilic n 'Ion 1.2 1.94
Pall Biodyne C hydrophilic nylon 3.7 1.93
Pall Biodyne C hydrophilic a !on 3.1 1.93
Pall Biodyne plus hydrophilic a !on 1.1 1.87
Pall I.C.E.-450 hydrophilic of ether sulfone 1.92 1.82
Pall I.C.E.-450sup hydrophilic of ether sulfone 0.87 1.67
Pall Su or - 800 hydrophilic polyether sulfone 3.93 1.74
Pall Su or - 450 hydrophilic olyether sulfone 1.78 1.74
Pall Su or - 100 hydrophilic polyether sulfone 1.04 1.68
Pall Hemasep V hydrophilic polyester 4 1.79
Pall Hemasep L hydrophilic of ester 0.47 2.1
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CA 02348054 2001-04-20
Pall Polypro - 450 hydrophilic polypropylene 5.09 1.78
Gore-Tex OH 9339 hydrophilic polytetrafluor- 0.43 1.48
ethylene
Gore-Tex OH 9338 hydrophilic polytetrafluor- 3.63 1.64
ethylene
Gore-Tex QH 9318 hydrophilic polytetrafluor- 5.92 1.67
ethylene
Millipore Durapore hydrophilized polyvinylidene 1.18 1.79
difluoride
Millipore LCR hydrophilized polytetrafluor- 2.84 1.72
ethylene
Sartorius Type 250 hydrophilic of amide 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 hilic cellulose acetate 0.3 2.01
Sartorius T e 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 membrane's pore size
According to Example 1 plastic columns have been assembled with hydrophobic
membranes
with different pore sizes.
According to Example 3, a cell lysate is made from 5x105 HeLa-cells and
transferred to the
columns. Subsequently the membranes are washed with the commercially available
buffers RW1
and RPE of QIAGEN by aid of centrifugation. The last centrifixgation step is
carried out at 20000
x g for 2 minutes to dry the membrane. The elution is carried out as described
in Example 1.
The results are listed in Table 5 below.
3 - 5 parallel tests are performed per membrane and the average value is
calculated for each.
33
CA 02348054 2001-04-20
Table 5: RNA-yield of isolated total RNA by binding to hydrophobic membranes
with different
pore sizes
Manufacturer Membrane Material Pore Size RNA E260/E280
( m) ( g)
Infiltec Polycon 0.01 Hydrophilic Polycarbonate 0.01 0.17 1.64
Pall Fluoro Trans G Hydrophobic 0.2 6.16 1.72
polyvinylidene difluoride
Pall Supor-450 PR Hydrophobic polyether 0.45 3.96 1.76
sulfone
Millipore Durapore Hydrophobic 0.65 7.45 1.72
polyvinylidene difluoride
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 Total-RNA isolated from HeLa cells
According to Example 1, plastic columns are assembled with hydrophilic
membranes (e.g.
Hydrolon, pore size 3 m by the Pall Company).
According to Example 3, a cell lysate is made from 5x105 HeLa-cells and
transferred to the
columns. Subsequently the membranes are washed with the commercially available
buffers RW1
and RPE of QIAGEN by aid of centrifugation. The last centrifugation step is
carried out at 20000
x g for 2 minutes to dry the membrane. The elution is carried out as described
in Example 1.
The isolated total-RNA is incubated for 16 hours at 37 C and subsequently
applied to a non-
denaturalizing agarose gel and analyzed. It becomes evident that the RNA was
not subjected to
any degradation. The RNA isolated according to the method described above
shows no
contamination with nucleic acid degrading enzymes and is thus of a high
quality.
Example 7
Isolation of free RNA out of aqueous solutions through binding at a
hydrophilic membrane inside
a 96-well-tray
A 96-well-tray with a hydrophilic polyvinylidene difluoride membrane
(Durapore, 0.65 gm, from
Millipore Company) is used.
5.3 ml of an aqueous solution containing total-RNA are mixed with 18.4 ml of a
commercially
34
i~'
CA 02348054 2001-04-20
1 ,
available lyse buffer containing guanidinium isothiocyanate e.g. RLT buffer
from QIAGEN
company. Subsequently 13.1 ml ethanol are added and mixed through pipetting.
350 gl of this
mixture are added to every well and transferred through the membrane by
applying a vacuum.
The membranes are subsequently washed twice with a buffer e.g. RPE of QIAGEN
company.
The buffer is transferred through the membrane through application of a vacuum
in every
instance. After the last washing step the tray is dabbed off with a paper
towel and then dried for 5
minutes through applying a vacuum.
Subsequently the RNA is eluted, as described in example 1, with RNase free
water and pipetted
off the membrane.
The volume of isolated total RNA obtained in this manner is then determined by
photometric
measurement of the light absorption at a wavelength of 260 rum and the average
value as well as
the standard deviation calculated for the entire tray. The average value is
8.4 pg with a standard
deviation of 0.7 pg.
Example 8
Isolation of Total-RNA through capillary forces
A 96-well-tray with a hydrophilic polyvinylidene difluoride membrane
(Durapore, 0.65 m, from
Millipore Company) is used.
33 gl of an aqueous solution containing total-RNA are mixed with 110 gl of a
commercially
available lyse buffer containing guanidinium - iso -cyanate e.g. RLT buffer
from QIAGEN
company. Subsequently 78 l ethanol are added and mixed through pipetting. 45
gl of this
mixture are added to every well. A household sponge with strong suction
properties is wetted
with water and the 96-well-tray placed with the bottom of the membrane on top
of the sponge.
The RNA mixture is transferred through the membrane by means of the capillary
forces. The
membranes are subsequently washed twice with a buffer e.g. RPE of QIAGEN
company. The
buffer is also transferred through the membrane by placing the tray on top of
the sponge. After
the last washing step the tray is dried for 5 minutes exposed to air.
Subsequently the RNA is eluted, as described in example 1, with RNase free
water and pipetted
off the membrane.
The volume of isolated total RNA obtained in this manner is then determined by
photometric
measurement of the light absorption at a wavelength of 260 mn and the average
value as well as
the standard deviation calculated for the entire tray. The average value is
5.9 pg with a standard
deviation of 0.7 pg.
Example 9
Isolation of genomic DNA from an aqueous solution by means of a buffer
containing
guanidinium hydrochloride.
According to Example 1, plastic columns are assembled with hydrophobic
membranes (e.g.
Magna-SH, pore size 5 m by the MSI Company). The purification is carried out
with
CA 02348054 2001-04-20
commercial buffers from QIAGEN company.
200 gl of an aqueous solution containing genomic DNA derived from liver tissue
are prepared in
PBS buffer. 200 l of a buffer containing guanidinium hydrochloride e.g. AL
from QIAGEN
company are added and mixed. Subsequently 210 gl ethanol are added and mixed
by vortexing.
The mixture is added to the column according to example 3 and transferred
throught the
membrane by centrifugation. Subsequently the membrane is washed and dried with
a buffer
containing alcohol, e.g. AW from QIAGEN company. The elution is carried out as
described in
example 1. Three parallel tests are carried out and the average value is
calculated.
The volume of isolated DNA is then determined by photometric measurement of
the light
absorption at a wavelength of 260 nm and amounts to approximately 30% of the
original volume.
The ratio of the absorption at 260nm to that at 280 nm amounts to 1.82.
Example 10
Isolation of genomic DNA out of an aqueous solution through binding at
hydrophobic
membranes by means of a buffer containing guanidinium-iso-thiocyanate.
According to the example 1 plastic columns with various membranes are
assembled.
100 gl of an aqueous solution containing total- DNA are mixed with 350 l of a
lysis buffer (4 M
GITC, 0.1 M MgSO4, 25 mM Na citrate, pH 4) containing guanidinium
isothiocyanate.
Subsequently 250 gl ethanol are added and mixed by pipetting. The mixture is
added to the
column and transferred through the membrane by centrifugation (10000 x g; 1
minute).
Subsequently the membranes are washed twice with a buffer e.g. RPE from QIAGEN
company.
The buffer is transferred through the membranes by centrifugation on each
occasion. The last
washing step is carried out at 20000 x g to dry the membranes. The elution is
carried out as
described in example 1. Three parallel tests are carried out per membrane and
for each the
average value is calculated.
The results are listed in table 6.
Table 6: Yield of DNA out of aqueous solution through binding to hydrophobic
membranes
Manufacturer Membrane Material DNA
Pall H drolon l.2 m Hydrophobic nylon 1.3
Pall Su or-450 PR Hydrophobic polyether sulfone 2.2
Millipore Fluoropore Hydrophobic polytetrafluor - ethylene 1.1
Millipore Durapore Hydrophobic of inylidene difluoride 1.2
36
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Example 11
Isolation of genomic DNA from tissue
According to Example 1, plastic columns are assembled with hydrophobic
membranes (e.g.
Magna-SH, 5 m by MSI). Purification is carried out with the commercially
available buffers of
QIAGEN.
180 l of ATL-buffer are added to 10 mg of kidney tissue (mouse) and ground in
a mechanical
homogenizer. Subsequently proteinase K (approx. 0.4 mg eluted in 20 l of
water) are added and
left to incubate for 10 minutes at 55 C. After adding 200 l of a buffer
containing guanidinium
hydrochloride - e.g. AL by QIAGEN - and after a 10 minute incubation at 70 C,
200 l of
ethanol are added and mixed with this solution. This mixture is placed in the
column and passed
through the membrane by centrifugation. The membrane is then washed with
alcohol containing
buffers, e.g. AW1 and AW2 from QIAGEN company - and subsequently dried by way
of
centrifugation. The elution is carried out as described in Example 1. Three
parallel tests are
carried out and the average value is calculated.
The amount of isolated DNA is subsequently determined by photometric
measurement of the
light absorption at a wavelength of 260 nm and is on average 9.77 g. The
absorption ratio at 260
nm to 280 nm is 1.74.
Example 12
Isolation of genomic DNA from blood
According to Example 1, plastic columns are assembled with hydrophobic
membranes (e.g.
Magna-SH, 5 m from MSI company). Purification is carried out with the
commercially
available buffers from QIAGEN.
To 200 d blood 200 l of AL and 20 l QIAGEN protease are added , mixed in
thoroughly and
incubated at 56 C for 10 minutes. After an addition of 200 l of ethanol the
preparation is mixed,
added to the column and transferred through the column by means of
centrifugation. The
membrane is washed with buffers containing alcohol - e.g.. AW 1 and AW2 from
QIAGEN
company - and dried through centrifugation. The elution is carried out as
described in example 1.
The amount of isolated DNA is subsequently determined by photometric
measurement of the
light absorption at a wavelength of 260 nm and amounts to 1.03 g. The
absorption ratio at 260
nm to 280 nm is 1.7.
37
CA 02348054 2001-04-20
Example 13
Isolation of total-RNA from a RNA-DNA-mixture
According to Example 1, plastic columns are assembled with hydrophobic
membranes (e.g.
Hydrolon 1.2 m by the Pall Company).
275 l of an aqueous solution containing total-RNA and genomic DNA are mixed
with 175 gl of
a lysis buffer, e.g. RLT buffer from QIAGEN company, containing guanidinium
isothiocyanate.
Subsequently 250 gl ethanol are added and mixed by pipetting. This mixture is
added to the
column and transferred through the membrane, washed and dried according to
example 4. The
penetrant from the first centrifugation step is transferred to a commercially
available Mini-Spin-
Column (e.g. QlAamp Mini-Spin-Column from QIAGEN company) and drawn through
the
membrane by centrifugation. The further washing steps are carried out as per
example 4.
Subsequently the nucleic acids are eluted with 140 1 RNase-free water by
means of
centrifugation (10000 x g, 1 minute) and analyzed in a non denaturizing
agarose gel (fig.7). The
method described here permits a separation of total-RNA and genomic DNA to a
great extent.
Figure 7 shows an ethidium bromide stained gel of an electrophoretic
separation of two different
eluates.
Line 1: Isolation of total-RNA by means of hydrophobic nylon membrane;
Line 2: Isolation of genomic DNA from the penetrant by means of a QlAamp Mini-
Spin-Column
from QIAGEN company.
Exam lpe14
Isolation of plasmid-DNA from aqueous solutions through binding to hydrophobic
and
hydrophilic membranes
According to example 1 plastic columns are assembled with various membranes.
100 gl of an aqueous solution (pCMVLJ from Clontech company) containing
plasmid are mixed
with 350 l of a lyse buffer (4 M GITC, 0.1 M MgSO4, 25 mM sodium acetate, pH
4) containing
guanidinium hydrochloride. Subsequently 250 l iso-propanol are added and
mixed by pipetting.
The mixture is added to the column according to example 4 and transferred
through the
membrane, washed and dried. Finally the plasmid-DNA is eluted with RNase-free
water as
already described in example 1 and pipetted off the membrane.
The volume of isolated plasmid-DNA is then determined by photometric
measurement of the
light absorption at a wavelength of 260 nm.
The results of the isolations with the various membranes are listed in the
following table. Three
parallel tests are conducted per membrane and the average value is calculated
for each.
38
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Table 7: Yield of Plasmid-DNA out of an aqueous solution through binding on
membranes
Manufacturer Membrane Material Plasmid-DNA
(Rg)
Pall Hydrolon 1.2 m Hydrophobic nylon 1.9
Pall Fluoro Trans G Hydrophobic of in li.dene difluoride 2.2
Pall I.C.E.-450 H do hilic of ether sulfone 0.8
Pall I.C.E.-450su H do hilic of ether sulfone 1.5
Pall Su or-450 PR Hydrophobic of ether sulfone 4.7
Pall Su or-200 PR Hydrophobic po ether sulfone 4
Pall Su or-800 Hydrophilic of ether sulfone 0.5
Pall Su or-450 Hydrophilic of ether sulfone 0.9
Pall Su or-100 Hydrophilic polyether sulfone 1
Pall V-800 R Hydrophobic ac late-copolymer 1.5
Pall Versa ore - 1200R Hydrophobic ac late copolymer 0.2
Pall Polypro - 450 Hydrophilic polypropylene 1.4
Gore-Tex QH 9318 Hydrophilic of etrafluor - ethylene 4.9
Gore-Tex OH 9335 Hydrophobic polytetrafluor - ethylene 4.3
Millipore Durapore, 0.65 m H dro hilized polyvinylidene difluoride 1.8
Millipore Durapore, 0.65 m Hydrophobic of in li.dene difluoride 1.7
MSI Magna-SH, 1.2 gm Hydrophobic nylon 1.1
Example 15
Immobilization of total-RNA from an aqueous solution with the use of different
chaotropic
agents
According to example 1 plastic columns are assembled with hydrophobic
membranes.
Each 100 f of an aqueous solution containing total RNA are mixed with 350 l
of different lysis
buffers, which contain guanidinium iso thiocyanate (GITC) or guanidinium
hydrochloride
(GuHC1) in different concentrations. Subsequently 250 l ethanol are added and
mixed by
pipetting. The mixture is then transferred to the column and passed through
the membrane by
way of centrifugation (10000 x g; 1 minute). The membranes are subsequently
washed twice with
an alcohol-containing buffer, e.g. RPE from QIAGEN company. The buffer is
passed through the
membrane by way of centrifugation. The last washing step is performed at 20000
x g to dry the
membranes. The elution is carried out as described in example 1. Twin tests
are carried out and
each average value is shown.
The results are listed in table 8.
39
CA 02348054 2001-04-20
Table 8: RNA yield out of an aqueous solution by way of chaotropic agents
Membrane Chaotropic agents and concentration in Binding Yield of Total RNA (
g)
Preparation
Hydrolon 1.2 m GITC, 500mM 2.3
Hydrolon 1.2 m GITC, 1M 0.8
Hydrolon 1.2 m GITC, 3M 0.9
Fluoro Trans G GITC, 500mM 0.4
Fluoro Trans G GITC,IM 1.25
Fluoro Trans G GITC, 3M 0.6
Hydrolon 1.2 m GuHCI, 500mM 2.6
Hydrolon 1.2 m GuHC1,1M 6.7
Hydrolon 1.2 m GuHC1, 3M 2.9
Fluoro Trans G GuHCI, 500mM 0.4
Fluoro Trans G GuHC1,1M 1.25
Fluoro Trans G GuHCI, 3M 0.6
Example 16
Immobilization of total RNA from an aqueous solution with the use of different
alcohols
According to Example 1, plastic columns are assembled with hydrophobic
membranes.
100 l of an aqueous solution containing total RNA are mixed with 350 l of a
lysis buffer
containing guanidinium isothiocyanate (concentration 4 M). Subsequently,
different amounts of
ethanol or isopropanol are added and loaded with RNase-free water up to 700 l
and mixed. This
mixture is then transferred to the column and passed through the membrane and
washed
according to Example 4. The elution took place as in Example 1.
Twin tests are carried out and each average value is shown.
The results are listed in Table 9.
Table 9: RNA-yield from an aqueous solution with different alcohols in a
binding preparation
Membrane Alcohol and Concentration Yield of Total RNA
in Binding Preparation ( g)
Hydrolon, 1.2 m Ethanol, 5% 0.7
Hydrolon, 1.2 m Ethanol, 30% 2.85
Hydrolon, 1.2 gm Ethanol, 50% 4.5
Durapore, 0.65 gm Ethanol, 5% 0.4
Durapore, 0.65 gm Ethanol, 30% 1.25
Durapore, 0.65 gm Ethanol, 50% 0.6
Hydrolon, 1.2 m Isopropanol, 5% 0.35
CA 02348054 2001-04-20
Hydrolon, 1.2 m Isopropanol, 30% 4.35
Hydrolon, 1.2 m Isopropanol, 50% 3.2
Durapore, 0.65 gm Isopropanol, 10% 1.35
Durapore, 0.65 m Isopropanol, 30% 4.1
Durapore, 0.65 m Isopropanol, 50% 3.5
Example 17
Immobilization of total RNA from an aqueous solution with various pH-values
According to Example 1, plastic columns are assembled with hydrophobic
membranes.
100 l of an aqueous solution containing total RNA are mixed with 350 l of a
lysis buffer
containing guanidinium isothiocyanate (concentration 4 M). The buffer contains
25 mM of
sodium citrate and is adjusted to different pH-values by way of HCl or NaOH.
Subsequently 250
1 of ethanol are added and mixed. This mixture is then transferred to the
column and passed
through the membrane and washed according to Example 4. The elution also took
place as in
Example 1. Twin tests are carried out and each average value is shown.
The results are listed in Table 10.
Table 10: RNA-yield from an aqueous solution with various pH-values in a
binding preparation
Membrane pH-Value in Binding preparation Yield of Total RNA
( g)
Hydrolon, 1.2 m pH 3 0.15
Hydrolon, 1.2 m pH 9 1.6
Hydrolon, 1.2 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
According to Example 1, plastic columns are assembled with hydrophobic
membranes.
100 pl of a total RNA containing aqueous solution are mixed with 350 l of a
lysis buffer
containing salts (NaCl, KCL, MgSO4). Subsequently 250 l of H 20 or ethanol
are added and
mixed. This mixture is then transferred to the column and passed through the
membrane, washed
and eluted according to Example 4. Twin tests are carried out and each average
value is shown.
The results are listed in Table 11.
41
CA 02348054 2001-04-20
Table 11: RNA-yield from an aqueous solution with various salts in a binding
preparation
Membrane Salt Concentration in Binding Preparation Yield of Total RNA
( g)
Hydrolon, 1.2 m NaCl. 100 mM; without ethanol 0.1
Hydrolon, 1.2 m NaCl. 1 M; without ethanol 0.15
Hydrolon, 1.2 m NaCl, 5 M; without ethanol 0.3
Hydrolon, 1.2 m KCI, 10 mM; without ethanol 0.2
Hydrolon, 1.2 m KCI, 1 M; without ethanol 0.1
Hydrolon, 1.2 m KCI, 3 M; without ethanol 0.25
Hydrolon, 1.2 m MgSO4, 100 mM; without ethanol 0.05
Hydrolon, 1.2 m MgSO4, 750 mM; without ethanol 0.15
Hydrolon, 1.2 m MgSO4, 2 M; without ethanol 0.48
Hydrolon, 1.2 m NaCl, 500 mM; with ethanol 2.1
Hydrolon, 1.2 m NaCl, 1 M; with ethanol 1.55
Hydrolon, 1.2 m NaCl, 2.5 M; with ethanol 1.35
Hydrolon, 1.2 m KCI, 500 mM; with ethanol 1.6
Hydrolon, 1.2 m KCI, 1 M; with ethanol 2.1
Hydrolon, 1.2 m KCl, 1.5 M; with ethanol 3.5
Hydrolon, 1.2 m MgSO4, 10 mM; with ethanol 1.9
Hydrolon, 1.2 m MgSO4, 100 mM; with ethanol 4.6
Hydrolon, 1.2 m MgSO4, 500 M; with ethanol (sic!) 2
Translated as per German original
Example 19
Immobilization of total RNA from an aqueous solution by way of various buffer
conditions
According to Example 1, plastic columns are assembled with hydrophobic
membranes.
100 l of an aqueous solution containing total RNA are mixed with 350 l of a
lysis buffer
containing guanidinium isothiocyanate (concentration 2.5 M). The lysis buffer
is mixed with
various concentrations of sodium citrate, pH 7, or sodium oxalate, pH 7.2.
Subsequently 250 gl
of ethanol are added and mixed. This mixture is then transferred to the column
and, according to
Example 4, passed through the membrane, washed and eluted.
The results are listed in Table 12. Twin tests are carried out and each
average value is shown.
42
CA 02348054 2001-04-20
Table 12: RNA-yield from an aqueous solution with various buffer
concentrations in a binding
preparation
Membrane Na-Citrate/Na-Oxalate in the Lysis Buffer Yield of Total RNA
(lug)
Hydrolon, 1.2 m Na-Citrate, 10 mM 2.2
Hydrolon, 1.2 m Na-Citrate, 100 mM 2.4
Hydrolon, 1.2 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 m Na-Oxalate, 1 mM 1.5
Hydrolon, 1.2 m Na-Oxalate, 25 mM 1.05
Hydrolon, 1.2 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 by way of various buffer
substances
According to Example 1, plastic columns are assembled with hydrophobic
membranes (e.g.
Hydrolon 1.2 m from Pall company).
100 l of an aqueous solution containing total-DNA are mixed with 350 l of a
lysis buffer
containing guanidinium isothiocyanate (4 M GITC, 0.1 M MgSO4). The lysis
buffer is mixed
with various buffer substances (concentration 25 mM) and standardized to
various pH values.
Subsequently 250 l of ethanol are added and mixed. This mixture is then
transferred to the
column and, according to Example 4, passed through the membrane, washed and
eluted.
The results are listed in Table 13. Triple tests are carried out and the
average value determined
each time.
Table 13: DNA-yield from an aqueous solution with various buffer
concentrations in a binding
preparation
Buffer Substance H in Lysis Buffer Yield of DNA
Sodium -citrate pH 4 1.3
Sodium -citrate pH5 0.6
Sodium -citrate H6 1.4
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CA 02348054 2001-04-20
Sodium -citrate pH7 0.5
Sodium -acetate pH4 0.9
Sodium -acetate pH5 1
Sodium -acetate H6 0.6
Sodium -acetate pH 7 0.5
Potassium - acetate pH 4 0.6
Potassium - acetate H5 0.9
Potassium - acetate pH6 1.2
Potassium - acetate H7 1.4
Ammonia - acetate H4 0.7
Ammonia - acetate H5 0.3
Ammonia - acetate H6 5.7
Ammonia - acetate pH7 1.5
Glycine H4 0.5
Glycine H5 1.1
Glycine H6 1.6
Glycine pH7 1.1
Malonate H4 1.5
Malonate H5 0.3
Malonate pH6 3.1
Malonate pH7 1.6
Succinate pH4 2.8
Succinate H5 2.3
Succinate H6 2.5
Succinate H7 4.7
Example 21
Immobilization of total RNA from an aqueous solution by means of phenol
As in Example 1, plastic columns with hydrophobic membranes (e.g., Hydrolon,
1.2 m from the
company Pall) are constructed.
An aqueous RNA solution is mixed with 700 l of phenol and distributed across
the membranes
by means of centrifugation. As in example 4, the membranes are washed and the
RNA eluted.
Twin measurements were carried out, and in each case the average value is
indicated.
The volume of isolated DNA (sic!) is then determined by photometric
measurement of the light
absorption at a wavelength of 260 nm and amounts to approximately 10.95 g.
The ratio of the
absorption at 260nm to that at 280 nm amounts to 0.975.
44
CA 02348054 2001-04-20
Example 22
Washing of immobilized total-RNA under different salt concentrations
According to Example 1, plastic columns are assembled with hydrophobic
membranes.
100 l of an aqueous solution containing total RNA are mixed with 350 gl of a
lysis buffer
containing guanidinium isothiocyanate (concentration 4 M). Subsequently, 250
l of ethanol are
added and mixed. This mixture is then transferred to the column and passed
through the
membrane as well as washed according to Example 4. The membranes are then
washed twice
with a buffer which contains various concentrations of NaCl and 80% ethanol.
The buffer is
passed through the membrane each time by way of centrifugation. The last
washing step is
carried out at 20000 x g in order to dry the membranes. The elution also takes
place according to
Example 1. Twin tests are carried out and in each case the average value is
shown.
The results are listed in Table 14.
Table 14: RNA-yield from an aqueous solution with NaCl in the washing buffer
Membrane NaCl in the Washing Buffer Yield of Total RNA ( g)
Hydrolon, 1.2 m NaCl, 10 mM 1.4
Hydrolon, 1.2 gm NaCl, 50 mM 3.15
Hydrolon, 1.2 m NaCl, 100 mM 3
Durapore, 0.65 m NaCl, 10 mM 2.7
Durapore, 0.65 gm NaCl, 50 mM 2.85
Durapore, 0.65 m NaCl, 100 mM 2.7
Example 23
Elution of immobilized total RNA under different salt and buffer conditions
According to Example 1, plastic columns are assembled with hydrophobic
membranes.
100 l of an aqueous solution containing total RNA are mixed with 350 l of a
lysis buffer
containing guanidinium isothiocyanate (concentration 4 M). Subsequently 250 gl
of ethanol are
added and mixed. This mixture is then transferred to the column and passed
through the
membrane and washed according to Example 4.
For the elution, 70 l of a solution containing NaCl, of a Tris/HC1-buffer (pH
7) or of a sodium
oxalate solution (pH 7.2) are pipetted onto the membrane, in order to elute
the purified RNA
from the membrane. After 1 to 2 minutes of incubation, at a temperature
between 10 - 30 C, the
eluate is pipetted from the top off the membrane. The elution step is repeated
once in order to
achieve complete elution. Twin tests are carried out and in each case the
average value is shown.
CA 02348054 2001-04-20
The results are summarized in Table 12.
Table 12: RNA-yield from an aqueous solution with NaCl, Tris/HCl or Na-oxalate
in the elution
buffer
Membrane NaCl, Tris or Na-oxalate in the Yield of Total RNA ( g)
Elution Buffer
Hydrolon, 1.2 pm NaCl, 1 mM 1.35
Hydrolon, 1.2 m NaCl, 50 mM 1.2
Hydrolon, 1.2 pm NaCl, 250 mM 0.45
Durapore, 0.65 m NaCl, 1 mM 0.9
Durapore, 0.65 m NaCl, 50 mM 0.35
Durapore, 0.65 m NaCl, 500 mM 0.15
Hydrolon, 1.2 m Tris 1 mM 0.35
Hydrolon, 1.2 m Tris 10 mM 0.75
Durapore, 0.65 m Tris 1 mM 1.5
Durapore, 0.65 m Tris 50 mM 1
Durapore, 0.65 m Tris 250 mM 0.1
Hydrolon, 1.2 m Na-Oxalate, 1 mM 0.45
Hydrolon, 1.2 m Na-Oxalate, 10 mM 0.65
Hydrolon, 1.2 m Na-Oxalate, 50 mM 0.3
Durapore, 0.65 gm Na-Oxalate, 1 mM 2
Durapore, 0.65 m Na-Oxalate, 10 mM 1.55
Durapore, 0.65 m Na-Oxalate, 50 mM 0.15
Example 24
Elution of immobilized RNA at various temperatures
According to Example 1, plastic columns are assembled with a hydrophobic
membrane (e.g.
Hydrolon, 3 gm from Pall company).
.For the isolation 5x105 HeLa cells are used. The following isolation steps,
as described in
example 3, are carried out.
For the elution 70 l of RNase-free water of various temperatures is pipetted
onto the membrane
to elute the RNA from the membrane. After an incubation of 1-2 minutes at the
appropriate
elution temperature, the eluate is pipetted off the membrane from above. The
elution step is
repeated once to achieve a complete elution.
Triple tests are carried out and in each case the average value is shown.
The results are summarized in table 16.
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CA 02348054 2001-04-20
Table 16: Yield of RNA at various elution temperatures
Membrane Elution Temperature Yield of total RNA (g g)
Hydrolon, 3 m Ice cold 2.2
Hydrolon, 3 m 40 C 3.2
Hydrolon, 3 m 50 C 3.9
Hydrolon, 3 m 60 C 3.7
Hydrolon, 3 m 70 C 3.7
Hydrolon, 3 m 80 C 2.9
Example 25
Elution of immobilized RNA by way of centrifugation
According to Example 1, plastic columns are assembled with a hydrophobic
membrane (e.g.
Hydrolon 1.2 gm from Pall company).
100 gl of an aqueous solution containing total-DNA are mixed with 350 l of a
lysis buffer
containing guanidinium isothiocyanate - e.g. RLT buffer from QIAGEN.
Subsequently 250 gl
ethanol are added and mixed by pipetting. This mixture is then transferred to
the column and,
through centrifugation (10000 x g; 1 minute), passed through the membrane.
Subsequently the
membranes are washed twice with a buffer - e.g. RPE from QIAGEN company. The
buffer is
transferred through the membrane by centrifugation in each case. The last
washing step is carried
out at 20000 x g to dry the membrane.
For the elution 70 gl of RNase-free water is pipetted onto the membrane to
release the RNA from
the membrane. After an incubation period of 1 minute at a temperature within a
range of 10 -
30 C the eluate is transferred through the membrane by way of centrifugation
(10000 x g, 1
minute). The elution step is repeated again to assure a complete elution and
the eluates are
combined. 5 parallel tests are carried out and in each case the average value
is shown.
The volume of isolated total RNA is then determined by photometric measurement
of the light
absorption at a wavelength of 260 nm and amounts, on average, to 6.4 g. The
ratio of the
absorption at 260 nm to that at 280 mn amounts to 1.94.
Example 26
Use of total RNA in a "Real Time" quantitative RT-PCR with the use of 5'-
nuclease PCR-assays
for the amplification and detection of R-actin mRNA.
According to Example 3, plastic columns are assembled. with a commercially
available
membrane (Pall, Hydrolon with a pore size of 3 m).
To isolate RNA, 1 x 105 HeLa-cells are used and the purification of total RNA
is carried out as
described in Example 1. The elution takes place with 2 x 70 l of H2O as
described in example 1.
47
CA 02348054 2001-04-20
4 c
For the complete removal of remaining slight amounts of DNA, the sample is
treated with a
DNase prior to analysis.
A "single-container `Real Time' quantitative RT-PCR" is carried out with the
use of the
commercially available reaction system of Perkin-Elmer (Taq:ManTM PCR Reagent
Kit) by using
an M-MLV reverse transcriptase. By using a specific primer and a specific
TaqMan-probe for 1-
actin (TagManTM (3-Actin Detection Kits made by Perkin Elmer) the R-actin mRNA-
molecules in
the total RNA-sample, are first transcripted to R-actin cDNA and subsequently
the total reaction
is amplified and detected immediately, without interruption, in the same
reaction container. The
reaction preparations are produced according to the manufacturer's
instructions. Three different
volumes of isolated total RNA are used (1, 2, 4 l of eluate) and triple tests
are carried out. As a
control, three preparations without RNA are also tested.
The cDNA synthesis takes place at 37 C for one hour, immediately followed by a
PCR which
comprises 40 cycles. The reactions and the analyses are carried out on an ABI
PRISMTM 7700
Sequence Detector supplied 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
volume 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 apparatus 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 õThreshold Cycle" (ct). This value
is a measure for
the amount of specifically amplified RNA available in the sample.
For the employed volume 1 l of total RNA, isolated with the process described
here, an average
ct-value of 17.1 is obtained; for 2 l of total RNA the ct-value is 16.4 and
for 4 l of total RNA
the ct-value is 15.3. This results in a linear correlation between the total
RNA employed and the
ct-value. This indicates that the total RNA is free of substances that might
inhibit the
amplification reaction. The control specimens containing no RNA do not produce
any signals.
Example 27
Use of total RNA in an RT-PCR for amplification and detection of (3-actin
mRNA.
According to Example 1, plastic columns are assembled with commercially
available membranes
(Pall company, Hydrolon with a pore size of 1.2 or 3 m; Sartorius company,
Sartolon with a
pore size of 0.45 m).
To isolate RNA, two different starting materials are used:
1) total RNA from liver (mouse) in an aqueous solution; purification and
elution are carried
out as described in example 4 and
2) 5 x 105 HeLa-cells, the purification of total RNA and the elution are
carried out as
described in example 3.
48
CA 02348054 2001-04-20
s
V"" e
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 preparation without RNA are used.
A RT-PCR is performed with these samples under standard conditions. For the
amplification two
different primer pairs are used for the (3-Actin-mRNA. A 150 Bp-sized fragment
serves to verify
the sensitivity, a 1.7 kBp-sized fragment assesses the integrity of the RNA.
From the RT-
reaction, 1 l is removed and introduced to the subsequent PCR. 25 cycles are
performed for the
small fragment and 27 cycles for the large fragment. The temperature for the
addition reaction is
55 C. The amplified preparations are subsequently placed on a non-denaturizing
gel and
analyzed.
For the employed 20 ng volume of total RNA isolated in the process described
above, the
corresponding DNA-fragments can be verified in the RT-PCR. When using total
RNA from
mouse liver, no transcript can be verified, as the conditions used here are
adjusted to human 13-
actin-mRNA. The control specimens which contain no RNA do not produce any
signals.
Fig. 8 shows ethidium bromide stained agarose gels of an electrophoretic
separation of RT-PCR
reaction products.
A: Line 1 to 8: RT-PCR of a 150 Bp-fragment;
Line 1, 2: RNA from mouse liver out of an aqueous solution purified with the
Hydrolon 1.2 .tm
membrane;
Line 3, 4: RNA from HeLa-cells purified with the Sartolon membrane;
Line 5, 6: RNA from HeLa-cells purified with the Hydrolon 3 pm membrane;
Line 7: RNA purified by way of RNeasy-Mini-Kit;
Line 8: Control without RNA.
B: Line 1 to 8: RT-PCR of a 1.7 kBp-fragment;
Line 1, 2: RNA from mouse liver out of an aqueous solution purified with the
Hydrolon 1.2 m
membrane;
Line 3, 4: RNA from HeLa-cells purified with the Sartolon membrane;
Line 5, 6: RNA from HeLa-cells purified with the Hydrolon 3,4m membrane;
Line 7: RNA purified by way of RNeasy-Mini-Kit;
Line 8: Control without RNA.
Example 28
Use of total RNA in a NASBA-reaction (nucleic acid sequence based
amplification) for
amplification and detection of R-actin mRNA.
According to Example 1, plastic columns are assembled with commercially
available membranes
(Pall company, Hydrolon with a pore size of 1.2 or 3 m; Sartorius company,
Sartolon with a
pore size of 0.45 m).
49
CA 02348054 2001-04-20
To isolate RNA, two different starting materials are used:
1) total RNA from liver (mouse) in an aqueous solution; purification and
elution are carried
out as described in example 4 and
2) 5 x 105 HeLa-cells, the purification of total RNA and the elution are
carried out as
described in example 3.
A NASBA-reaction is performed under standard conditions (Fahy, E. et al.,
1991. PCR Methods
Amplic. 1, 25 - 33). For the amplification (3-actin-specific primers are used.
20 ng each of the isolated total RNA are employed. As a control, RNA purified
by way of the
RNeasy-kit (QIAGEN company) and a preparation without RNA are carried out.
Firstly the
incubation takes place for 5 minutes at 65 C and for 5 minutes at 41 C.
Subsequent to this step
an enzyme mixture consisting of RNaseH, T7-polymerase and AMVV-RT are added
and
incubated for 90 minutes at 41 C. The amplified preparations are subsequently
placed on a non-
denaturizing gel and analyzed.
For the employed 20 ng volume of total RNA isolated in the process described
above, a specific
transcript can be verified (Fig. 9).
Fig. 9 shows an ethidium bromide stained agarose gel of an electrophoretic
separation of
NASBA-reactions.
Line 1 to 8: NASBA-reactions;
Line 1, 2: RNA from mouse liver out of an aqueous solution purified with the
Hydrolon 1.2 m
membrane;
Line 3, 4: RNA from HeLa-cells purified with the Sartolon membrane;
Line 5, 6: RNA from HeLa-cells purified with the Hydrolon 3 p.m membrane;
Line 7: RNA purified by way of RNeasy-Mini-Kit;
Line 8: Control without RNA.
Exam lp e 29
NASBA-reaction for the amplification and detection of (3-actin mRNA on
hydrophobic
membranes.
According to Example 1, plastic columns are assembled with commercially
available membranes
(Pall company, Hydrolon with a pore size of 3 m; Supor-450 PR with a pore
size of 0.45 m;
Millipore company, Fluoropore with a pore size 3 gm).
To isolate RNA, various amounts of HeLa-cells are used, the purification of
the total RNA is
carried out as described in example 3. The elution is carried out through the
addition of 20 gl
NASBA reaction buffer. The NASBA reaction is subsequently carried out at the
membrane.The
reaction takes place at standard conditions (Fahy, E. et al., 1991 PCR Methods
Amplic. 1, 25 -
33). For the amplification P-actin specific primers are employed.
The reaction container is firstly incubated for 5 minutes at 41 C in a
waterbath. Subsequent to
CA 02348054 2001-04-20
this step an enzyme mixture consisting of RNaseH, T7-polymerase and AMVV-RT is
added and
incubated for 90 minutes at 41 C. The amplified preparations are subsequently
placed on a non-
denaturizing gel and analyzed.
For the employed amount of total RNA, isolated from 5 x 105 to 3 x 104 HeLa-
cells, at the total
RNA isolated according to the process described here, a specific transcript
can be verified (Fig.
10).
Fig. 10 shows an ethidium bromide stained agarose gel of an electrophoretic
separation of
NASBA-reactions.
A: Line 1 to 4: RNA purified with the membrane Hydrolon 3 m out of HeLa-
cells;
Line 1: 2.5 x 105 cells;
Line 2: 1.25 x 105 cells;
Line 3: 6 x 104 cells;
Line 4: 3 x 104 cells;
B: Line 1 to 3: RNA purified out of HeLa-cells;
Line 1: RNA purified with the Hydrolon 3 m membrane out of 2.5 x 105 HeLa
cells;
Line 2: RNA purified with the Supor-450 PR membrane out of 5 x 105 HeLa cells;
Line 3: RNA purified with the Fluoropore 3 m membrane out of 5 x 105 HeLa
cells.
Example 30
Isolation of plasmid-DNA on a hydrophobic membrane with the enzyme Aval
According to example 1 plastic columns are assembled with hydrophobic
membranes (e.g.
Supor-200 PR from Pall company).
100 l of an aqueous solution (pCMV^ from Clontech company) containing plasmid
are mixed
with 350 gl of a lyse buffer (4 M GITC, 0.1 M MgSO4, 25 mM sodium acetate, pH
4) containing
guanidinium isothiocyanate. Subsequently 250 gl iso-propanol are added and
mixed by pipetting.
The mixture is added to the column according to example 4 and transferred
through the
membrane, washed and dried.
100 gl of a 1 x buffer for the restriction enzyme Aval are applied to the
membrane and
1) removed, transferred into a new reaction container and subsequently a
restriction enzyme
(e.g. AvaI from Promega company) is added;
2) a restriction enzyme (e.g. AvaI from Promega company) is added to the
eluate in the column.
The reactions are incubated for one hour at 37 C and subsequently applied to a
non-denaturizing
gel and analyzed (Fig. 11).
Fig. 11 shows an ethidium bromide stained agarose gel of an electrophoretic
separation of the
plasmid pCMV(3 after restriction with Aval.
Line 1: uncut plasmid;
Line 2, 3: elution with the reaction buffer for Aval, restriction in new
container;
Line 4, 5: Restriction with Aval on the membrane.
51
L._
CA 02348054 2001-04-20
C ry
Example 31
Pressurized filtration for the precipitation of DNA with isopropanol
The isolation of plasmid DNA including the elution step via anion exchange
chromatography, is
carried out according to standard protocols. The DNA is eluted from the column
in a high salt
concentration buffer. Subsequently 0.7 volume isopropanol is added to this DNA
solution, the
preparation is mixed and incubated for 1-5 minutes at room temperature.The
filtration equipment
used is a 0.45 gm cellulose acetate filter with a surface of 5 crn2 inside a
filtration cartridge
(standard equipment for sterile filtration, e.g. Minisart from Sartorius
company).This filter is
plugged onto a syringe from which the plunger has been removed.
The syringe is now filled with the DNA-isopropanol mixture and the mixture
pressed through the
filter with the plunger. In this form of precipitate a high percentage of the
DNA remains on the
filter (can not penetrate the pores).
Now the plunger is removed from the syringe again, replaced and air is pressed
through the filter.
This step is repeated 1-2 times and serves the drying of the membrane.
Subsequently a low salt buffer is used to elute such that the buffer is filled
into the body of the
syringe and is pressed through the filter with the aid of the plunger. To
increase the yield, this
first eluate is filled into the syringe body once more and pressed through the
filter with the aid of
the plunger. The yields obtained typically range from 80% to 90% with this
test arrangement
(refer example 34).
Example 32
Vacuum filtration for the precipitation of DNA with isopropanol
As with the pressure filtration the plasmid-DNA is firstly isolated and 0.7
vol isopropanol is
added. The filtration set-up is an apparatus constructed for vacuum filtration
into which a 0.45
gm cellulose acetate filter with a surface of 5 cm2 was fitted. 0.45 gm
cellulose nitrate filters or
several layers of 0.65 gm cellulose acetate or cellulose nitrate filters can
also be used.
The isopropanol-DNA mixture is incubated for 1-5 minutes and then transferred
onto the
filtration set-up. By connecting the vacuum the solution is drawn through the
filter. An
appropriate amount of 70% ethanol is given onto the filter with the DNA-
precipitates and washed
by connecting the vacuum again. The elution of the DNA from the filter is
carried out by adding
a low salt buffer, a short incubation and once more, connection of the vacuum.
The yield can be
obtained through either again eluting from the filter with a second volume of
a low salt buffer or
through a renewed elution with the eluate from the first elution step.
Here as well the yields obtained typically range from 80% to 90% of the
originally employed
DNA.
52
CA 02348054 2001-04-20
Example 33
As method the vacuum filtration indicated in example 32 is used. The vacuum
filtration
instrument Sartorius 16315 is used as the filtration container. pCMV(3, which
had been isolated
from DH5a, is used as plasmid DNA.
15 ml QF buffer (high salt concentration buffer) are added to 500 gg of
plasmid and mixed. 10.5
ml isopropanol are added and mixed again. This is incubated for 5 minutes. The
plasmid-DNA
thus precipitated is transferred onto the membrane fastened into the
filtration apparatus. Now the
vacuum is connected and filtration begins. The membranes are washed with 5 ml
of 70% ethanol
(renewed application of vacuum). Thereafter 1 ml TE-buffer is pipetted onto
the membrane,
incubated for 5 minutes and the DNA eluted by applying vacuum. Subsequently a
post-eluation is
carried out with 1 ml of TE-buffer. The total amounts of DNA are subsequently
measured each in
the penetrant, in the washing fraction and in the combined eluate (OD260). The
following results
are achieved:
Membrane Amount Penetrant Washing Fraction Eluate Speed
PVDF 0.2 gm 1 0 gg DNA 0 gg DNA 131 g DNA very slow
Cellulose- 3 0 gg DNA 0 gg DNA 469 gg DNA fast
Acetate
0.65 gm
Cellulose- 2 0 gg DNA 0 gg DNA 418 gg DNA fast
Nitrate
0.65 gm
Calculated for the 500 gg original amount, the following yields result in this
test:
PVDF 0.2 gm 26%
Cellulose acetate 94%
0.65 gm
Cellulose nitrate 84%
0.65 gm
Exam lp e 34
The pressure filtration as mentioned in example 34 is used as the method. The
filtration
apparatus employed is a commercial 0.45 gm cellulose acetate filter
(Minisart,Sartorius).
PCMV(3, which had been isolated from DH5a, is used as plasmid DNA.
15 ml QF buffer (high salt concentration buffer) are added to 100, 200, 300,
etc up to 900 gg of
plasmid and mixed. 10.5 ml isopropanol are added and mixed again. This is
subsequently
53
CA 02348054 2001-04-20
incubated for 5 minutes. The plasmid-DNA thus precipitated is transferred into
a syringe from
which the plunger has been removed. Now the pressure filtration occurs with
the aid of the
syringe. The filter is washed with 2 ml of 70% ethanol and dried twice as
described. The elution
is carried out with 2 ml TE-buffer. A post-eluation is carried out with the
eluate. The total
amounts of DNA are subsequently measured each with combined eluate (OD260).
The following
results are achieved:
Employed DNA-amount Eluted DNA-amount % yield
100 gg 100 gg 100%
200 jig 176 gg 88%
300 jig 257 gg 86%
400 gg 361 g 90%
500 g 466 g 93%
600 gg 579 gg 97%
700 g 671 gg 96%
800 gg 705 gg 88%
900 g 866 g 96%
Example 35
The pressure filtration as mentioned in example 32 is used as the method. The
filtration
apparatus employed is a commercial 0.45 gm cellulose acetate filter
(Minisart,Sartorius) which
is plugged onto a filtration chamber (QIAvac). As a reservoir the body of a
syringe is attached to
the other end of the filter. PCMV(3, which had been isolated from DH5a, is
used as plasmid
DNA. 15 ml QF buffer (high salt concentration buffer) are added to 500 g of
plasmid and
mixed. 10.5 ml isopropanol are added and mixed again. This is incubated for 5
minutes. The
plasmid-DNA thus precipitated is transferred onto the membrane fastened into
the filtration
apparatus. Now the vacuum is connected and filtration begins. The filter is
not washed with 5 ml
of 70% ethanol. The immediate elution is rather carried out with 2 ml buffer
EB (QIAGEN).
Post-eluation is carried out once with the eluate. The total amount of DNA is
measured (OD260).
The following result is obtained:
54
CA 02348054 2001-04-20
Test Number Eluted DNA % Yield
1 434 gg 87%
2 437 g 87%
