Methods for Obtaining Liquid from a Solid Phase
This invention relates to methods for obtaining a liquid from a porous solid phase,
and to kits and apparatus for performing the methods.
Methods for isolation of nucleic acids of a quality suitable for downstream
applications such as polymerase chain reaction (PCR) and sequencing by adsorption
and release from a solid phase are well-established (Vogelstein, B. and Gillespie, D,
1979; PNAS 76, 615). The methods use: (i) a lysis buffer to release nucleic acid from
biological samples, (ii) a lysis or a binding buffer to capture nucleic acid to a solid
phase, (iii) a wash buffer(s) to wash the captured nucleic acid, and (iv) an elution
buffer to release the captured nucleic acid from the solid phase. The quality of nucleic
acid isolated using such methods depends on the efficiency of buffer exchange
between the lysis, binding, wash and elution steps. Carry-over of lysis, binding or
wash buffer into the eluted sample inhibits many downstream applications such as
PCR, sequencing and cloning.
In some methods, efficient buffer exchange is accomplished by centrifugation.
Centrifugation is an extremely efficient method of removing buffer from the solid
phase, and is particularly advantageous for optimum recovery of elution buffer
containing released nucleic acid in the elution step.
In other methods, buffer exchange is accomplished by applying a positive pressure at
the top of the solid phase, for example by using a syringe or a piston pump to pass
air through the solid phase (Zymo-Spin V, Zymo Research), or by applying a negative
pressure at the bottom of the solid phase using a vacuum (Fastfilter system, OMEGA
Bio-tek). However, formation of a pressure differential across the solid phase to
mediate buffer exchange depends on the formation of a good seal. If an air channel
is formed through the solid phase before all of the buffer has been removed, the
pressure differential is disrupted and residual buffer remains trapped in the solid
phase. Residual buffer remaining after the elution step includes nucleic acid, so the
yield of nucleic acid obtained from the solid phase is reduced. To obtain high
extraction yield, it is necessary to remove the buffer by centrifugation (for example,
Zymo-Spin V, Zymo Research & Fastfilter system, OMEGA bio-tek).
The requirement for a centrifugation step to obtain high yield limits the usefulness of
pressure differential methods for nucleic acid isolation, and complicates the
extraction process. In particular, nucleic acid extraction processes that require a
centrifuge cannot be carried out in areas where such equipment is not available, for
example in a physician's office or in remote areas. The requirement for a centrifuge is
also a particular disadvantage for automated systems because the complexity of
such systems is increased. In particular, a robotic arm is required to perform relatively
complex actions, such as gripping and movement of sample tubes to transfer them to
the centrifuge. This increases the cost and complexity of automated systems, and
increases the likelihood of errors occurring.
There is a need, therefore, to provide nucleic acid extraction methods that achieve
high yield without requiring a centrifugation step. There is also a need to provide
simplified nucleic acid extraction methods that can more readily be automated.
According to the invention, there is provided a method for obtaining a liquid from a
porous solid phase, which comprises: forming a liquid seal at a first end of a porous
solid phase to which a liquid is bound, wherein liquid of the liquid seal is immiscible
with the liquid bound to the solid phase; and applying a pressure differential across
the porous solid phase to cause the immiscible liquid to move through the porous
solid phase towards a second end of the porous solid phase, thereby displacing the
liquid bound to the porous solid phase towards the second end and releasing this
liquid from the second end.
The term "porous solid phase" is used herein to mean a solid phase that is
permeable to liquid. The permeability may be due to pores or channels in the solid
phase material itself or, for example, because the solid phase comprises several
particles, such as beads, between which liquid can pass. Examples of porous solid
phases include columns that comprise particles, gels, membranes, or beads.
Particular examples include any chromatography column in which material bound to
the column is to be eluted by passing a liquid through the column. Preferred
examples include chromatography columns for liquid chromatography, ion-exchange
chromatography, affinity chromatography, reversed-phase chromatography. The solid
phase used in chromatography columns is usually a finely ground powder, a gel, or a
microporous material. Chromatography columns commonly comprise silica gel,
alumina, or cellulose powder. Other examples of porous solid phases suitable for use
in methods of the invention include magnetic beads, and devices (such as chips)
comprising microfluidic channels.
Typically, the porous solid phase will have affinity for a biomolecule, for example for
nucleic acids or proteins. In certain embodiments, the solid phase comprises material
to which nucleic acid is able to bind at a lower pH and from which the nucleic acid
can be eluted at a higher pH. Several suitable examples of such solid phases are
well known to the skilled person. In preferred embodiments, the solid phase
comprises an inorganic oxide, preferably silica. In other embodiments, the solid
phase comprises ion-exchange material for protein purification. Several suitable
examples of such solid phases are known to the skilled person.
Reference herein to liquid "bound" to the porous solid phase means liquid that is
associated with the porous solid phase, and which it is desired to release from the
porous solid phase.
The term "immiscible liquid" is used herein to refer to the liquid of the liquid seal.
Typically, the liquid bound to the porous solid phase will comprise an aqueous liquid,
and the immiscible liquid will comprise a hydrophobic liquid. A property of liquids that
are immiscible with each other is that they cannot be diluted in equal parts without
separation occurring. It will be appreciated that the immiscible liquid should be
substantially immiscible with the liquid bound to the solid phase at least for the period
during which the pressure differential is applied across the solid phase.
To form a liquid seal, the immiscible liquid should form a complete layer across the
surface of the liquid bound to the porous solid phase at the first end of the porous
solid phase.
In certain embodiments, the immiscible liquid is a mineral oil. Suitable mineral oil is
molecular biology grade mineral oil. An example is mineral oil that contains only
saturated hydrocarbons, for example 36% naphthene (saturated 5- or 6-carbon cyclic
paraffins), 64% paraffin. Suitable molecular biology grade mineral oil is available, for
example, from Sigma (catalogue no. M5904; density 0.84 g/mL at 25°C).
It is generally envisaged that the porous solid phase will be arranged such that the
pressure differential will cause the immiscible liquid to move down through the porous
solid phase. In such arrangements, it is preferred that the immiscible liquid is less
dense than the liquid bound to the porous solid phase, so that the immiscible liquid
will settle on top of the liquid bound to the porous solid phase. In alternative
embodiments, the immiscible liquid may be more dense than the liquid bound to the
porous solid phase. However, such liquid should be applied to an upper end of the
porous solid phase only when there is no layer of liquid bound to the solid phase
above the upper end of the porous solid phase. If necessary, a pressure differential
can be applied to move the liquid of any such layer into the porous solid phase before
the more dense immiscible liquid is applied. Examples of immiscible liquids that are
more dense than aqueous buffer solutions include an organic solvent such as phenol,
or a concentrated sucrose solution.
The liquid bound to the porous solid phase may comprise an elution buffer. The
content of the elution buffer will depend on the identity of the porous solid phase and
on the identity of the material that it is desired to release from the porous solid phase.
Examples of typical elution buffers for purification of nucleic acid include Tris-HCI
buffer, and Tris-EDTA (TE) buffer. A typical elution buffer for affinity purification of
proteins is Glycine-HCI buffer.
It will be appreciated that the liquid bound to the porous solid phase may comprise a
biomolecule, such as a protein or nucleic acid, which was bound to the porous solid
phase, and which has been released from the porous solid phase into the liquid.
The pressure differential may be applied by increasing the air pressure at the first
end of the porous solid phase, for example using a pump or syringe, or by reducing
the air pressure at the second end of the porous solid phase, for example by applying
a vacuum at the second end of the porous solid phase.
Methods of the invention provide several advantages, as explained below.
The volume of liquid that is obtained from the porous solid phase using methods of
the invention is higher than corresponding methods in which no liquid seal is formed.
If the liquid contains material, such as a biomolecule, which has been released from
the porous sold phase into the liquid, the yield of this material from the solid phase is
thereby also increased.
Without being bound by theory, there are believed to be a number of reasons for the
increase in the volume of liquid that is obtained from a porous solid phase using
methods of the invention.
In conventional methods in which liquid is forced through a column containing a
porous solid phase, as the liquid moves down through the column, the upper surface
of the liquid approaches the top of the porous solid phase, until only a thin layer of
liquid remains above the top of the solid phase. The pressure differential across the
solid phase causes disruption of the thin layer of liquid, allowing air to enter the
porous solid phase. Air channels are then formed through the porous solid phase,
thereby reducing the pressure differential across the solid phase. Residual liquid
becomes trapped in the porous solid phase because there is insufficient pressure
differential to force this out of the solid phase.
In methods of the invention, the liquid seal is believed to prevent the formation of air
channels in the porous solid phase after a pressure differential has been applied,
thereby preventing residual liquid from becoming trapped in the solid phase.
For example, in embodiments of the invention in which liquid is forced down through
a column containing a porous solid phase, an immiscible liquid may be used which is
less dense than the liquid bound to the porous solid phase. In such embodiments, the
immiscible liquid forms a layer on top of the liquid bound to the porous solid phase. A
pressure differential is applied across the porous solid phase to cause the immiscible
liquid, and the liquid bound to the solid phase, to move down through the column. As
the upper surface of the liquid bound to the solid phase approaches the top of the
solid phase, the layer of immiscible liquid prevents disruption of the liquid below it,
and thereby prevents the formation of air channels in the porous solid phase. The
pressure differential across the porous solid phase is maintained, thus increasing the
amount of liquid that is released from it.
The pores in a porous solid phase used for extraction of nucleic acids typically range
from 0.1 to 12 mM. The immiscible liquid enters the pores of the solid phase, and
displaces the liquid bound to the porous solid phase from the pores. If the solid phase
comprises a porous material contained within a column, liquid can remain trapped at
the interface between the solid phase and the column. The liquid seal formed by the
immiscible liquid ensures that this trapped liquid is also released from the porous
solid phase.
Methods of the invention reduce the variability in the volume of liquid recovered from
the porous solid phase, thereby providing consistent recovery and yield from the solid
phase. This is an important advantage because results obtained from subsequent
processing of different samples collected from the solid phase are more comparable.
When the liquid seal is formed at the first end of the porous solid phase, a semispherical
meniscus is formed at the interface between the liquid seal and the liquid
bound to the solid phase (as shown in Figure 5). When a pressure differential is
applied across the solid phase, the force at the semi-spherical meniscus is directed
towards the centre. This is believed to reduce the pressure differential required to
displace the liquid bound to the solid phase towards the second end of the solid
phase.
The liquid seal also prevents frothing of liquid in the porous solid phase when the
pressure differential is applied. Such frothing can inhibit downstream processing of
liquid samples collected from the porous solid phase, and reduce the yield of liquid
that can be obtained.
The liquid seal prevents evaporation from the first end of the porous solid phase. This
allows the solid phase to be heated without a need to cap the solid phase. This is
advantageous, particularly for isolation of nucleic acid, because heating of the solid
phase is commonly used to increase the amount of nucleic acid that is released from
the solid phase. Thus, in some embodiments of the invention, the porous solid phase
is heated. Heating can take place before, during, or after formation of the liquid seal
at the first end of the porous solid phase, but preferably after formation of the liquid
seal.
If the pressure differential is applied for sufficient time that at least some of the
immiscible liquid is also released from the porous solid phase, the released
immiscible liquid (provided this is less dense than the liquid bound to the solid phase)
will form a layer over the top of the liquid in the collected sample. This can be
particularly advantageous for subsequent processing of the collected sample. For
example, if the liquid seal is a mineral oil, and the liquid in the collected sample
contains nucleic acid released from the porous solid phase, the collected sample can
be used directly for downstream manipulations in which heating of the sample is
required. Examples of such manipulations include nucleic acid amplification reactions
(such as polymerase chain reaction, or transcription-mediated amplification), or
nucleic acid sequencing reactions.
In conventional methods in which no liquid seal is used when obtaining liquid from a
porous solid phase, downstream reactions in which the sample is heated are carried
out in capped collection tubes, or a layer of mineral oil is applied before heating the
sample, to minimise evaporation. Capped collection tubes, whilst preventing
evaporation from the tube, do not prevent evaporation within the tube. The sample
volume at the bottom of the tube is reduced, and this can adversely affect the
efficiency of the reaction. Addition of a layer of mineral oil before heating maintains
the sample volume, but has the potential to contaminate the sample. This can be a
serious problem, for example, if the sample is to undergo a nucleic acid amplification
reaction.
Methods of the invention in which at least some of the mineral oil is collected with the
liquid sample have several advantages. The layer of mineral oil prevents evaporation
of the liquid sample, allowing reactions to take place in uncapped collection tubes.
The oil layer also prevents changes in the sample volume due to evaporation as the
reaction takes place, thereby helping to maintain optimum conditions for the reaction.
A further advantage is that the oil layer minimises splashing of the sample liquid, and
therefore reduces the chances of cross-contamination between samples.
Methods of the invention allow higher volumes of liquid to be recovered from a
porous solid phase than conventional methods, without the need for a centrifugation
step. This simplifies methods for obtaining liquid from the solid phase, and allows the
methods to be more readily automated.
Methods of the invention may further comprise binding a component to the porous
solid phase and releasing the component from the solid phase into the liquid bound
to the solid phase prior to forming the liquid seal at the first end of the solid phase.
The component may be released from the solid phase by applying an elution buffer to
the solid phase prior to forming the liquid seal.
It will be appreciated that methods of the invention may be used with conventional
methods for purification in which a biological component, such as a nucleic acid or
protein, is bound selectively to a porous solid phase, and then eluted from the porous
solid phase. Suitable methods are well-known to the skilled person. Examples of
nucleic acid purification methods include methods that use chaotropic agents, such
as guanidinium thiocyanate, and organic solvents to lyse cells, and denature proteins
(including nucleases, which would otherwise degrade the nucleic acid). For example,
Boom et al. (Journal of Clinical Microbiology, 1990, Vol. 28(3): 495-503) describes a
method in which a sample containing human serum or urine is contacted with silica
particles in the presence of a lysis/binding buffer containing guanidinium thiocyanate.
Released nucleic acid binds to the silica particles, which are then washed with a
wash buffer containing guanidinium thiocyanate, then with ethanol, and then acetone.
The bound nucleic acid is subsequently eluted from the silica particles in an aqueous
low salt buffer (Tris-HCI, EDTA, pH 8.0).
Other methods avoid use of chaotropic agents and organic solvents, which are highly
inhibitory to enzymatic reactions. Residual amounts of these substances carried over
into the eluted sample can interfere with subsequent enzymatic processing of the
isolated nucleic acid, for example in nucleic acid sequencing or amplification. Use of
chaotropic agents and organic solvents is also undesirable because these reagents
are toxic and difficult to handle, and require special provision for their disposal. The
requirement for chaotropic salts and organic solvents is avoided in a method
described by Hourfar et al. (Clinical Chemistry, 2005, 51(7): 1217-1222). Plasma
sample is mixed with magnetic silica particles in the presence of a lysis/binding buffer
containing a kosmotropic salt (ammonium sulphate) before addition of proteinase K.
Following separation, the magnetic particles are washed with wash buffer containing
proteinase K, and eluted in elution buffer (Tris-HCI, pH 8.5) at 80°C.
Examples of protein purification methods include ion-exchange methods. Methods of
the invention may be particularly advantageous for membrane-based ion exchange
chromatography. Thermo Scientific Pierce Strong Ion Exchange Spin Columns use
membrane-adsorber technology as a chromatographic matrix to fractionate proteins
based on their charge differences. According to the manufacturer's protocol, liquid is
removed from the column by centrifugation following the application of elution buffer.
If instead, a liquid seal is formed according to the invention after the elution buffer
has been added to the column, protein purification may be performed using such
columns without the need for centrifugation.
According to the invention there is also provided a kit for obtaining a liquid from a
porous solid phase, which comprises: a porous solid phase; and a liquid which is
immiscible with the liquid to be obtained from the solid phase, and which can form a
liquid seal at an end of the porous solid phase.
There is further provided according to the invention a kit for obtaining a liquid from a
porous solid phase, which comprises: a porous solid phase; and instructions for
applying a liquid, which is immiscible with the liquid to be obtained from the porous
solid phase, to an end of the porous solid phase to form a liquid seal at the end of the
porous solid phase. The kit may further comprise the immiscible liquid.
There is also provided according to the invention apparatus for obtaining a liquid from
a porous solid phase, which comprises: a porous solid phase; a liquid which is
immiscible with the liquid to be obtained from the porous solid phase, and which can
form a liquid seal at an end of the porous solid phase; and a means for applying a
pressure differential across the porous solid phase.
The means for applying a pressure differential across the porous solid phase may
comprise a pump, for example a piston pump, a syringe, or a vacuum pump.
The immiscible liquid is preferably less dense than the liquid to be obtained from the
porous solid phase. The immiscible liquid may be a hydrophobic liquid, such as a
mineral oil.
Preferred embodiments of the invention are now described, by way of example only,
with reference to the accompanying drawings in which:
Figure 1 shows the volume of liquid remaining in a porous solid phase after different
volumes of air were pumped through the solid phase using a syringe in the absence
of a liquid seal;
Figure 2 shows the volume of liquid collected from a porous solid phase by pumping
air through the solid phase with and without a liquid seal (a layer of mineral oil) at an
upper end of the porous solid phase;
Figure 3 shows the strength of the assay signal obtained following amplification and
detection of HIV-1 RNA eluted from a porous solid phase with and without a liquid
seal (a layer of mineral oil) at an upper end of the porous solid phase;
Figure 4 shows photographs of the lower end of columns containing a silica-based
solid phase through which liquid containing a red coloured dye has been passed (a)
without, and (b) with a liquid seal (a layer of mineral oil) at an upper end of the solid
phase;
Figure 5 shows photographs of the lower end of columns containing a silica-based
solid phase with a buffer containing a red coloured dye bound to the solid phase. In
figure (a) there is no layer of mineral oil over the buffer bound to the solid phase. In
figure (b) there is a layer of mineral over the buffer bound to the solid phase. A semispherical
meniscus can be seen at the interface between the mineral oil and the
buffer;
Figure 6 shows a comparison of the results from detection of amplified HIV-1 RNA
eluted from porous solid phases in a capped column with no mineral oil, an uncapped
column with no mineral oil, and an uncapped column with a layer of mineral oil over
the solid phase; and
Figure 7 shows the yield of nucleic acid eluted from a porous solid phase using a
method of the invention compared with a conventional method in which liquid bound
to the column was removed by centrifugation. The black horizontal bars show the
average yield in each case.
Example 1
Use of mineral oil increases the recovery of liquid from a solid phase
A silica-based solid phase was loaded with 0.5ml lysis buffer (0.2M sodium citrate
buffer pH 4.3, 0.3 M ammonium sulphate, 0.4% Triton-X 100), and then 30ml, 100ml,
200ml, 300ml, or 400ml of air was passed through it using a syringe. The volume of
liquid left in the solid phase is recorded in Figure 1. The data shows that there is
always a volume of residual buffer retained in the solid phase, even after 400ml of air
has been pumped through it.
The recovery of liquid from the solid phase was increased by use of mineral oil
according to the invention. A silica-based solid phase was loaded with 150m I elution
buffer (10 mM Tris-HCI pH 8.5). Mineral oil was then applied to form a liquid seal at
the upper end of the solid phase, prior to pumping air through the solid phase with a
syringe. The volume of liquid collected from the solid phase was recorded, and
compared with the volume of liquid collected without application of mineral oil. The
results are shown in Figure 2. The data points represent six individual measurements
of liquid collected with and without mineral oil. The black bar indicates the average
volume collected, and "CV" is the coefficient of variation. The results show that
mineral oil increases the recovery of liquid from the solid phase and reduces the
variation in the amount of liquid recovered.
Example 2
Use of mineral oil increases the yield of nucleic acid from a solid phase
RNA was extracted from human plasma spiked with HIV-1 RNA using a silica-based
solid phase in a column. RNA was eluted from the solid phase with and without a
liquid seal formed by a layer of mineral oil at the upper end of the solid phase. HIV-1
RNA in the eluate was amplified, and specifically detected by dipstick assay using a
method as described in Dineva et al (Journal of Clinical Microbiology, 2005, Vol.
43(8): 4015-4021). The assay signals were scored from 0.5 to 5 using a scorecard,
with 5 being strongest and 0.5 weakest. The results, shown in Figure 3 , demonstrate
that the assay signal was increased by over 50% when mineral oil was used. It is
concluded that the yield of nucleic acid from the solid phase is increased by use of
mineral oil.
Example 3
Use of mineral oil to obtain liquid trapped at interfaces
Liquid containing a red coloured dye was applied to a column containing a porous
solid phase, and then removed from the solid phase by pumping air through the
column using a syringe. The liquid leaves the column through an nozzle at the lower
end of the column. A photograph of the lower end of the column with the nozzle is
shown in Figure 4(a). Liquid remains trapped in at an interface between the solid
phase and the column, and in the nozzle. Figure 4(b) shows the effect of applying a
layer of mineral oil over the solid phase before air is pumped through the column. No
liquid is trapped at the interface of the solid phase with the column, or in the nozzle.
It is concluded that use of mineral oil also increases the amount of liquid that can be
obtained from the solid phase by removing liquid from the interface of the solid phase
with the solid phase support.
Example 4
Formation of a semi-spherical meniscus at the interface between mineral oil and
aqueous liquid bound to the solid phase
Figure 5 shows that when mineral oil is layered at an upper end of a porous solid
phase to which an aqueous liquid is bound, a semi-spherical meniscus is formed at
the interface between the mineral oil and the aqueous liquid. When a pressure
differential is applied across the solid phase, the force at the semi-spherical meniscus
is directed towards the centre. It is believed that this reduces the pressure differential
required to displace the liquid bound to the solid phase towards the second end of
the solid phase.
Example 5
A procedure for isolation of nucleic acid from a plasma sample using a method of the
invention
In this example, a detailed procedure for isolation of nucleic acid from a plasma
sample using a method of the invention is described.
A plasma sample is lysed, digested with a proteinase, and then applied to a porous
solid phase contained within a column. Nucleic acid in the lysed plasma sample binds
to the solid phase, and is then washed with a wash buffer. Next, elution buffer is
applied to the column to release the nucleic acid from the solid phase. Elution buffer
containing the released nucleic acid is then obtained from the solid phase using a
method of the invention.
Buffers:
Lysis buffer: comprises a kosmotropic salt, and a non-ionic detergent at acidic pH;
Wash buffer: comprises Tris-HCI at acidic pH;
Elution buffer: comprises Tris-HCI at alkaline pH.
Procedure:
Mix 240m I plasma with 760m I lysis buffer
Incubate for 8 min.
Add 20m I (20mg/ml) proteinase K. Incubate for another 10 min.
Collect the mixture with a 2ml plastic syringe
ii
Once the mixture has been drawn inside the syringe, attach a Zymo-Spin V column
and push the entire mixture out of the syringe through the column
Discard the flow through and detach the column from the syringe
Aspirate 1ml of wash buffer with a clean 2ml plastic syringe
Attach the column to the syringe, and push the wash buffer out of the syringe through
the column
Discard the flow through and detach the column from the syringe
Place the column in a clean 1.5ml micro-centrifuge tube
Pipette 100 m I of elution buffer to the column
Pipette 100 m I of mineral oil to the column
Place the column on heating block at 75-80°C for 5-1 0 min
Aspirate 2ml of air with a clean 2ml plastic syringe
Attach the column to the syringe, and push the eiuate out of the column into the
micro-centrifuge tube
Example 6
Nucleic acid yield using a method of the invention is improved in uncapped columns
In this example, RNA was extracted from human plasma spiked with HIV-1 RNA by
carrying out the procedure described in Example 5 using an uncapped column. For
comparison, extractions were also performed with: (i) a capped column; or (ii) an
uncapped column; without adding mineral oil after the addition of elution buffer.
HIV-1 RNA in the eiuate was amplified, and specifically detected by dipstick assay
using a method as described in Dineva et a/ (Journal of Clinical Microbiology, 2005,
Vol. 43(8): 401 5-402 1) . The assay signals were scored from 0.5 to 5 using a
scorecard, with 5 being strongest and 0.5 weakest. The results, shown in Figure 6,
demonstrate that the best dipstick signal was obtained when mineral oil was used,
indicating that the yield of RNA was greatest from the uncapped column with mineral
oil.
Example 7
Nucleic acid yield using a method of the invention is comparable with a centrifugation
method
Plasma was spiked with HIV-1 at 5000 copies/ml. RNA was extracted from the
plasma and bound to a porous solid phase in two separate columns. Elution buffer
was added to each column, followed by addition of a layer of mineral oil to form a
liguid seal at the upper end of the solid phase of one of the columns. RNA was then
eluted from the solid phase with the mineral oil by applying air pressure to the top of
the solid phase. RNA was eluted from the other column by centrifugation. HIV-1 RNA
in the eluted samples was quantified by reverse transcription-polymerase chain
reaction (RT-PCR). The results are shown in Figure 7 . The black horizontal bars
indicate average extraction yield. It is concluded that the yield of RNA using a method
of the invention was comparable with the centrifugation method.
Claims
1 A method for obtaining a liquid from a porous solid phase, which comprises:
forming a liquid seal at a first end of a porous solid phase to which a liquid is
bound, wherein liquid of the liquid seal is immiscible with the liquid bound to the solid
phase; and
applying a pressure differential across the porous solid phase to cause the
immiscible liquid to move through the porous solid phase towards a second end of
the porous solid phase, thereby displacing the liquid bound to the porous solid phase
towards the second end and releasing this liquid from the second end.
2 . A method according to claim 1, wherein the immiscible liquid is less dense
than the liquid bound to the solid phase.
3 . A method according to claim 1 or 2 , wherein the immiscible liquid comprises a
hydrophobic liquid.
4. A method according to claim 3 , wherein the hydrophobic liquid is a mineral
oil.
5 . A method according to any preceding claim, wherein the porous solid phase
is within a column.
6 . A method according to any preceding claim, wherein the liquid bound to the
porous solid phase comprises a component of a biological sample, which it is desired
to release from the solid phase.
7 . A method according to claim 6, wherein the component comprises a nucleic
acid.
8. A method according to claim 7, wherein the porous solid phase comprises
material to which nucleic acid binds at a lower pH and from which nucleic acid is
eluted at a higher pH.
9 . A method according to any preceding claim, wherein the porous solid phase
comprises an inorganic oxide, preferably silica.
10. A method according to claim 6 , wherein the component comprises a protein,
and the solid phase preferably comprises an ion-exchange material for protein
purification.
1. A method according to any preceding claim, wherein the liquid bound to the
solid phase comprises an elution buffer.
12. A method according to any preceding claim, wherein the pressure differential
is applied for a sufficient time that at least some of the immiscible liquid is released
from the second end of the solid phase.
13. A method according to claim 12, wherein the liquid and the immiscible liquid
released from the solid phase are collected in an uncapped collection tube.
14. A method according to any preceding claim, which further comprises heating
the porous solid phase.
15. A method according to any preceding claim for isolation of a biological
component, wherein the method further comprises binding the component to the
porous solid phase and releasing the component from the porous solid phase into the
liquid bound to the porous solid phase prior to forming the liquid seal at the first end
of the porous solid phase.
16. A method according to any preceding claim, which is an automated method.
17. A kit for obtaining a liquid from a porous solid phase, which comprises:
a porous solid phase; and
a liquid which is immiscible with the liquid to be obtained from the porous
solid phase, and which can form a liquid seal at an end of the porous solid
phase.
18 . A kit for obtaining a liquid from a porous solid phase, which comprises:
a porous solid phase; and
instructions for applying a liquid, which is immiscible with the liquid to be
obtained from the porous solid phase, to an end of the porous solid phase to
form a liquid seal at the end of the porous solid phase.
19 . A kit according to claim 18, which further comprises the immiscible liquid.
20. A kit according to any of claims 7 to 19, wherein the immiscible liquid is less
dense than the liquid to be obtained from the porous solid phase.
2 1. A kit according to any of claims 17 to 20, wherein the immiscible liquid is a
hydrophobic liquid, preferably a mineral oil.
22. Apparatus for obtaining a liquid from a porous solid phase, which comprises:
a porous solid phase;
a liquid which is immiscible with the liquid to be obtained from the porous
solid phase, and which can form a liquid seal at an end of the porous solid
phase; and
a means for applying a pressure differential across the porous solid phase.
23. Apparatus according to claim 22, wherein the means for applying a pressure
differential across the porous solid phase comprises a pump, a syringe, or a vacuum
pump.
24. Apparatus according to claim 22 or 23, wherein the immiscible liquid is less
dense than the liquid to be obtained from the porous solid phase.
25. Apparatus according to any of claims 22 to 24, wherein the immiscible liquid
is a hydrophobic liquid, preferably a mineral oil.