Description
Title of Invention: A METHOD OF MANIPULATING SOLID
CARRIERS AND AN APPARATUS OF MANIPULATING SOLID
CARRIERS
Technical Field
[0001]
[0002] The present invention relates to a method of manipulating solid carriers and an
apparatus of manipulating solid carriers.
Background Art
[0003] Medical treatments using genes, such as genetic diagnosis or gene therapies, have
received more attentions because of the advances in gene technologies in recent years.
Besides, many approaches have been developed which use genes for breeding and
breed identification in the agriculture and livestock fields. PCR (polymerase chain
reaction) has widely been used as a technique for making use of the genes. In recent
years, PCR is an essential tool for revealing information of biological substances. In
PCR, by subjecting a solution (reaction solution) containing a nucleic acid to be
amplified (target nucleic acid) and a reagent to thermal cycles, the target nucleic acid is
amplified. Typical thermal cycles for PCR are performed at two or three different tem
peratures.
[0004] On the other hand, kits for a simple and rapid test such as immunochromatography
have been the mainstream for the current diagnosis of an infectious disease including
influenza in medical practices. Such simple and rapid tests, however, often suffer from
poor accuracy and it has thus been desired to apply PCR, which can be expected to
provide diagnosis with higher accuracy, to the diagnosis of the infectious diseases. Fur
thermore, only short period of time can be used for the medical check in general
outpatient practices in a medical facility because of the limited time available for
clinical examinations. Therefore, current examinations of, for example, influenza
viruses are performed rapidly using an easy approach such as immunochromatography
at the expense of the accuracy of the examinations.
[0005] Under such circumstances, in the field of medical practices, it is necessary to reduce
the time required for reactions in order to achieve PCR examinations which can be
expected to provide a result with higher accuracy. As a device for achieving PCR
reactions in a short period of time, for example, some methods are disclosed as a
technique of PCR, such as a method using magnetic beads (Japanese Patent Laid-open
No. 2009-207459), a method of performing thermal cycles of PCR using magnetic
beads as a tool for moving liquid droplets which are moved in a region on a substrate
where the temperature is changed (Japanese Patent Laid-open No. 2008-012490), and a
method of extracting nucleic acids, by pouring magnetic particles and reagents into a
vessel having a shape of a funnel and externally forming a magnetic field using two or
more magnets to manipulate magnetic particles in the vessel,and performing different
reactions by replacing the reagents in the vessel with a pipette at the same time as ma
nipulating the magnetic particles (Japanese Patent Publication No. 2008-507705T).
[0006] According to the Japanese Patent Laid-open No. 2009-207459, biological samples
that travel in a flow passage along with a fluid are adsorbed onto magnetic particles.
The magnetic particles are used for breaking up the biological samples. In this case, a
magnetic force is applied to the magnetic particles from the outside of the flow passage
to manipulate or hold the magnetic particles at a predetermined position. In addition,
this publication also describes a method of adsorbing nucleic acids onto the magnetic
particles held in the flow passage. In these methods, the magnetic particles are used as
obstacles that can be manipulated. Biological substances are carried by a solution
flowing through the flow passage. This causes problems of disposal of wasted liquid
and difficult mixing of the reagents.
[0007] Japanese Patent Laid-open No. 2008-012490 describes a method of externally
applying a magnetic force to liquid droplets containing magnetic particles encapsulated
therein to move the liquid droplets across points on a substrate that are different in
temperature, by using a physical force exerted between the magnetic particles and their
surrounding liquid, to perform PCR. The publication also describes a method of
adsorbing nucleic acids onto the magnetic particles and moving them between different
reagents to adsorb and wash the nucleic acids in a similar way. In this publication, the
magnetic particles are encapsulated in the liquid droplets having a free interface, and
they are moved in a direction horizontal to the surface of the substrate. However, the
amount of the liquid that can be present around the magnetic particles is limited and
the motion of the magnetic particles and the liquid is completely synchronous with
each other. It seems that the magnetic particles are washed with the reagents only in a
limited region for washing, and it is thus hard to improve the efficiency of the washing
operation.
[0008] Japanese Patent Publication No. 2008-507705T uses more than one magnets for the
magnetic particles placed in a vessel to change, over a period of time, the position near
the vessel receiving the largest magnetic force to manipulate the magnetic particles in
the vessel. Efficiency in the steps of washing and elution required for the extraction of
nucleic acids is thus improved. In addition, the magnetic particles in this publication
are shaken in a predetermined region in the vessel. The surrounding reagent is replaced
using a suction/injection device such as a pipette to achieve several washing op
erations. This method requires troublesome operations to replace the reagent, separate
the magnetic particles and the reagent for the replacement, or hold them appropriately,
which could have an adverse effect on improving the efficiency of the operation.
Summary of Invention
Technical Problem
[0009] The present invention was made in order to overcome at least some of the afore
mentioned problems and can be implemented as the following modes or embodiments.
[0010] [Embodiment 1] A method of manipulating a solid carrier according to this em
bodiment is a method of manipulating a solid carrier in a liquid contained in a vessel,
the solid carrier being magnetic and capable of carrying a substance, the method
comprising: manipulating the solid carrier using a magnetic force application unit
adapted to applying a magnetic force to the solid carrier in the vessel so that the
relative position between the magnetic force application unit and the vessel is altered
over a period of time by means of directing the solid carrier in a direction along either
one or a combination of the vectors of the longitudinal direction of the vessel and a
cross-sectional direction crossing the longitudinal direction of the vessel.
[001 1] According to this embodiment, the solid carrier in the vessel is manipulated by
changing the relative position between the magnetic force application unit and the
vessel over a period of time, and changing the magnetic field near the solid carrier and
the vessel over a period of time. The solid carrier is moved in the vessel in the lon
gitudinal and horizontal directions of the vessel to thereby allowing the solid carrier in
the liquid in the vessel to pass through more regions.
[0012] [Embodiment 2] In the method of manipulating a solid carrier as described in the
aforementioned embodiment, the magnetic force application unit comprises at least
one permanent magnet.
[0013] According to this embodiment, heat is less likely to be generated, so that the solid
carrier can be directed in an efficient manner.
[0014] [Embodiment 3] In the method of manipulating a solid carrier as described in the
aforementioned embodiments, the position of the solid carrier is changed in the vessel
by moving the permanent magnet to a position of the peripheral surface of the vessel
and then moving the permanent magnet away from the position to move the permanent
magnet closer to another position of the peripheral surface of the vessel.
[0015] According to this embodiment, the solid carrier can be directed in an efficient
manner.
[0016] [Embodiment 4] In the method of manipulating a solid carrier as described in the
aforementioned embodiments, the solid carrier is directed using two permanent
magnets that are opposed to each other at a certain distance.
[0017] According to this embodiment, the solid carrier can be directed in a more efficient
manner.
[0018] [Embodiment 5] In the method of manipulating a solid carrier as described in the
aforementioned embodiments, the solid carrier is directed within the vessel as a result
of the relative rotation between the magnetic force application unet and the vessel.
[0019] According to this embodiment, the solid carrier is moved along the inner surface of
the vessel. By changing the distance between the permanent magnet and the vessel, the
magnitude of the magnetic force near the vessel is altered. The partices of the solid
carrier are repeatedly gathered and dispersed in the vessel. As a result, the solid carrier
is floated in many regions in the liquid, which improves the efficiency of the washing
operation.
[0020] [Embodiment 6] In the method of manipulating a solid carrier as described in the
aforementioned embodiments, at least three permanent magnets are provided, and the
solid carrier is directed in at least two directions in a plane perpendicular to the lon
gitudinal direction of the vessel as a result of the relative displacement of the position
between the vessel and the permanent magnets.
[0021] According to this embodiment, the rocking motion of a permanent magnet for use in
externally applying a magnetic force is performed in two- and three-dimensional
manners. The displacement can thus be performed efficiently.
[0022] [Embodiment 7] An apparatus of manipulating a solid carrier according to this em
bodiment is an apparatus of manipulating a solid carrier in a liquid contained in a
vessel, the solid carrier being magnetic and capable of carrying a substance, the
apparatus comprising: a magnetic force application unit adapted to applying a
magnetic force to the solid carrier in the vessel, the solid carrier being manipulated so
that the relative position between the magnetic force application unit and the vessel is
altered over a period of time by means of directing the solid carrier in a direction along
either one or a combination of the vectors of the longitudinal direction of the vessel
and a cross-sectional direction crossing the longitudinal direction of the vessel.
[0023] According to this embodiment, the solid carrier is manipulated in the vessel by
changing the relative position between the magnetic force application unit and the
vessel, and changing the magnetic field near the solid carrier and the vessel over a
period of time. In this way, it is possible to provide an apparatus of manipulating a
solid carrier in which the relative position of the magnetic force application unit and
the vessel is changed over a period of time, and the solid carrier is moved in the vessel
in the longitudinal and horizontal directions of the vessel, thereby to allow the solid
carrier to pass through more regions in the liquid in the vessel.
[Embodiment 8] A method of manipulating a solid carrier according to this embodiment
is a method of manipulating a magnetic solid carrier in a vessel comprising
liquid, the vessel having a longitudinal direction, the method comprising the step of:
altering a relative position between the magnetic force application unit adapted to
applying a agnetic force to the solid carrier from outside of the vessel and the vessel
over a period of time, to direct the solid carrier, in one of a planar direction crossing
the longitudinal direction of the vessel, the longitudinal direction of the vessel, and a
direction determined by combining the vectors of the longitudinal direction of the
vessel and the planar direction crossing the longitudinal direction of the vessel.
According to this embodiment, the solid carrier is manipulated in the vessel by
changing the relative position between the magnetic force application unit and the
vessel over a period of time, and changing the magnetic field near the solid carrier and
the vessel over a period of time. The solid carrier can be passed through more regions
in the liquid in the vessel by moving the solid carrier in the vessel in one of the planar
direction crossing the longitudinal direction of the vessel, the longitudinal direction of
the vessel, and the direction determined by combining the vectors of the longitudinal
direction of the vessel and the planar direction crossing the longitudinal direction of
the vessel.
[Embodiment 9] A method of manipulating a solid carrier according to this em
bodiment is the method of manipulating a solid carrier according to the Embodiment 8,
wherein: the magnetic force application unit comprises at least one permanent magnet.
According to this embodiment, heat is less likely to be generated, so that the solid
carrier can be directed in an efficient manner.
[Embodiment 10] A method of manipulating a solid carrier according to this em
bodiment is the method of manipulating a solid carrier according to the Embodiment 8
or 9, comprising: moving the magnet closer to a first position of the vessel; moving the
magnet away from the first position; moving the magnet closer to a second position of
the vessel, the second position being different from the first position; and moving the
magnet away from the second position, wherein the solid carrier is thereby directed in
the vessel.
According to this embodiment, the solid carrier can be directed in an efficient manner.
[Embodiment 11A method of manipulating a solid carrier according to this em
bodiment is the method of manipulating a solid carrier according to any one of the Em
bodiments 8 to 10, wherein: the magnet is two permanent magnets that are opposed to
each other at a certain distance.
According to this embodiment, the solid carrier can be directed in a more efficient
manner.
[Embodiment 12] A method of manipulating a solid carrier according to this em
bodiment is the method of manipulating a solid carrier according to the Embodiment
11, comprising: decreasing a relative distance between a first magnet and the vessel
while increasing a relative distance between a second magnet and the vessel, the
second magnet being opposed to the first magnet; and increasing the relative distance
between the first magnet and the vessel while decreasing the relative distance between
the second magnet and the vessel, wherein the solid carrier is directed in the vessel
thereby.
According to this embodiment, the solid carrier can be directed in a more efficient
manner.
[Embodiment 13] A method of manipulating a solid carrier according to this em
bodiment is the method of manipulating a solid carrier according to the Embodiment 8
or 9, wherein the solid carrier is directed in the vessel by means of rotating the magnet
around the vessel.
According to this embodiment, the solid carrier is moved along the inner surface of the
vessel. By changing the distance between the permanent magnet and the vessel, the
magnitude of the magnetic force near the vessel is altered. The particles of the solid
carrier are repeatedly gathered and dispersed in the vessel. As a result, the solid carrier
is floated in many regions in the liquid, which improves the efficiency of the washing
operation.
[Embodiment 14] A method of manipulating a solid carrier according to this em
bodiment is the method of manipulating a solid carrier according to the Embodiment 8
or 9, wherein at least three permanent magnets are provided, and one of a first distance,
second distance, and a third distance being determined to be smaller than the remaining
two distances, the first distance representing a distance between the vessel and a first
magnet, the second distance representing a distance between the vessel and a second
magnet, and the third distance representing a distance between the vessel and a third
magnet; and the solid carrier is thereby directed in a plane perpendicular to the lon
gitudinal direction of the vessel.
According to this embodiment, the rocking motion of a permanent magnet for use in
externally applying a magnetic force is performed in two- and three-dimensional
manners. The displacement can thus be performed efficiently.
[Embodiment 15] An apparatus of manipulating a solid carrier according to this em
bodiment is an apparatus of manipulating a magnetic solid carrier in a vessel
comprising liquid, the vessel having a longitudinal direction, the apparatus comprising:
magnetic force application unit adapted to applying a magnetic force to the solid
carrier from outside the vessel, wherein the magnetic force application unit is ma
nipulated so that the relative position between the magnetic force application unit and
the vessel is altered over a period of time, to direct the solid carrier, by using the
magnetic force application unit, in one of a planar direction crossing the longitudinal
direction of the vessel, the longitudinal direction of the vessel, and a direction de
termined by combining the vectors of the longitudinal direction of the vessel and the
planar direction crossing the longitudinal direction of the vessel.
According to this embodiment, the solid carrier is manipulated in the vessel by
changing the relative position between the magnetic force application unit and the
vessel, and changing the magnetic field near the solid carrier and the vessel over a
period of time. In this way, it is possible to provide an apparatus of manipulating a
solid carrier in which the relative position of the magnetic force application unit and
the vessel is changed over a period of time, and the solid carrier is moved in the vessel
in the longitudinal and horizontal directions of the vessel, thereby to allow the solid
carrier to pass through more regions in the liquid in the vessel.
[Embodiment 16] An apparatus of manipulating a solid carrier according to this em
bodiment is the apparatus of manipulating a solid carrier according to the Embodiment
15, wherein the vessel is a tube having a first oil plug, a washing solution plug, a
second oil plug, an elution solution plug, and a third oil plug in the longitudinal
direction thereof.
According to this embodiment, the nucleic acids can easily be purified from a sample.
Brief Description of Drawings
[fig. 1]FIG. 1 is a perspective view showing an example of an apparatus for the ex
traction of nucleic acids according to the Embodiment 1.
[fig.2]FIG. 2 is a view diagrammatically showing essential parts of a device for the ex
traction of nucleic acids according to the Embodiment 1.
[fig.3]FIG. 3 is a view diagrammatically showing essential parts of the device for the
extraction of nucleic acids according to a modified version.
[fig.4]FIG. 4 is a view diagrammatically showing essential parts of the device for the
extraction of nucleic acids according to a modified version.
[fig.5]FIG. 5 is a view diagrammatically showing a device for the extraction of nucleic
acids according to a modified version.
[fig.6]FIG. 6 a view diagrammatically showing essential parts of the device for the ex
traction of nucleic acids according to a modified version.
[fig.7]FIG. 7 is a view diagrammatically showing an example of a kit for the extraction
of nucleic acids according to the Embodiment 1.
[fig.8]FIG. 8 is a view diagrammatically showing an example of a kit for the extraction
of nucleic acids according to the Embodiment 1.
[fig.9]FIG. 9 is a view showing a method of externally applying a magnetic force to
move magnetic particles according to the Embodiment 1.
[fig. 10]FIG. 10 is a view showing a method of externally applying a magnetic force to
move magnetic particles according to the Embodiment 1.
[fig. 11]FIG. 11 is a view showing a method of externally applying a magnetic force to
move magnetic particles according to the Embodiment 1.
[fig. 12]FIG. 12 is a graph showing the results obtained in the Experiment 1.
[fig.l3]FIG. 13 is a graph showing the relationship between the elusion temperature
and the yield of DNAs according to the Experiment 4.
[fig. 14]FIG. 14 is a view showing an exemplified configuration of a permanent magnet
and a tube according to the Embodiment 2.
[fig.l5]FIG. 15 is a view showing an exemplified configuration of the permanent
magnet and the tube according to the Embodiment 2.
[fig. 16]FIG. 16 is a view showing an exemplified configuration of permanent magnets
and a tube according to the Embodiment 3.
[fig.l7]FIG. 17 is a perspective view showing an example of an apparatus for the ex
traction of nucleic acids according to the modified version 3.
[fig.l8]FIG. 18 is a view diagrammatically showing a device for the extraction of
nucleic acids according to a modified version 4.
[fig. 19]FIG. 19 is a diagrammatic representation for use in illustrating a method for the
extraction of nucleic acids according to a modified version 5.
Description of Embodiments
[0025] Now, some embodiments of the present invention are described. The embodiments
described below are for illustrating examples of the present invention. The present
invention is not limited to the following embodiments, and includes various modi
fications that can be achieved without changing the scope of the present invention. The
components and parts described below are not necessarily essential ones for the present
invention.
An apparatus for the extraction of nucleic acids is described as an embodiment of an
apparatus of manipulating a solid carrier for use in extracting nucleic acids with a kit
for the extraction of nucleic acids being mounted thereon.
[0026] (Embodiment 1)
1. Apparatus for the extraction of nucleic acids
Fig. 1 is a perspective view showing an example of an apparatus for the extraction of
nucleic acids according to the present embodiment. An apparatus for the extraction of
nucleic acids 3000 of this embodiment can suitably be applied to a kit for the ex
traction of nucleic acids, a device for the extraction of nucleic acids, and a method for
the extraction of nucleic acids, which are described later.
[0027] The apparatus for the extraction of nucleic acids 3000 of this embodiment comprises
a tube mount 300, a magnetic force application unit 400, and a moving mechanism
500. The tube mount 300 receives a tube (vessel) 100 that forms a device for the ex
traction of nucleic acids 1000. The magnetic force application unit 400 externally
applies a magnetic force to the tube 100, in particular, from the side of the tube 100
after the tube 100 is mounted on the tube mount 300. The moving mechanism 500
changes the relative position of the tube mount 300 and the magnetic force application
unit 400 in the longitudinal direction of the tube 100.
[0028] The tube 100 mounted on the tube mount 300 of the apparatus for the extraction of
nucleic acids 3000 is described later.
[0029] The tube mount 300 is a place where the tube 100 is mounted. An adsorption
chamber 120 connected to the tube 100 may also be mounted on the tube mount 300
along with the tube 100. A structure and a mechanism for receiving the tube 100 in the
tube mount 300 may appropriately be designed as long as a magnetic force can be
applied to the tube 100 and, if necessary, to the adsorption chamber 120 by the
magnetic force application unit 400. The tube mount 300 may be configured so that the
tube 100 is stretched straight and supported by the tube mount 300 when the tube 100
is flexible and has a curved or bent posture. The tube mount 300 in the illustrated em
bodiment has a support plate 310. The tube 100 is disposed against the support plate
310. Although the support plate 310 is not an essential element, it can suppress the
vibration of the tube 100. In addition, the tube mount 300 in the illustrated embodiment
has clip mechanisms 320 for securing the tube 100 at two positions.
[0030] The tube mount 300 and the magnetic force application unit 400 are configured so
that their relative position is changed in the longitudinal direction of the tube 100.
Therefore, when the tube mount 300 is designed so as to move relative to the magnetic
force application unit 400 without any movement of the latter, the apparatus for the ex
traction of nucleic acids 3000 has a moving mechanism 360 as the moving mechanism
500 to move the tube mount 300, as illustrated. Alternatively, when the magnetic force
application unit 400 has a moving mechanism, the moving mechanism 360 of the tube
mount 300 may be omitted. In the illustrated embodiment, the tube mount 300
comprises a hinge 330, a guiding rail 340, a drive belt 350, and a motor which is not
shown.
[0031] Although the apparatus for the extraction of nucleic acids 3000 in this embodiment
has one tube mount 300, it may have two or more tube mounts. In such a case, two or
more magnetic force application units 400 may be provided. The two or more tube
mounts 300 may be configured so that they function independently or in cooperation
with each other.
[0032] The magnetic force application unit 400 is configured to apply a magnetic force to
the tube 100 and, if necessary, to the adsorption chamber 120 after the tube 100 is
mounted on the tube mount 300. The magnetic force application unit 400 comprises,
for example, a permanent magnet, an electromagnet, or a combination thereof.
Although the magnetic force application unit 400 has at least one magnet, two or more
magnets may be provided. It is preferable that a permanent magnet is used for the
magnetic force application unit because this configuration is less likely to produce heat
in comparison to a case where an electromagnet is used. The permanent magnet used
may be, for example, a nickel-based, iron-based, cobalt-based, samarium-based, or
neodymium-based permanent magnet.
[0033] The magnetic force application unit 400 can apply a magnetic force to magnetic
particles (solid carriers) M provided in the adsorption chamber 120 and the tube 100.
By changing the relative position between the tube mount 300 and the magnetic force
application unit 400, the magnetic particles M can be moved in the adsorption chamber
120 and the tube 100.
[0034] In the illustrated embodiment, the magnetic force application unit 400 has a pair of
permanent magnets 410. The permanent magnets 410 are opposed to each other
relative to the adsorption chamber 120 and the tube 100. The permanent magnets 410
are separated from each other at a distance that is larger than the diameter of the tube
100. The direction of the polarity of the permanent magnets 410 is not specifically
limited. The magnetic force application unit 400 is configured so that the relative
position of the magnetic force application unit 400 and the tube mount 300 is changed
in the longitudinal direction of the tube 100. When the magnetic force application unit
400 is designed to be moved relative to the tube mount 300 without any movement of
the latter, the apparatus for the extraction of nucleic acids 3000 has a moving
mechanism as the moving mechanism 500 to move the magnetic force application unit
400.
[0035] In the illustrated embodiment, the magnetic force application unit 400 is configured
so that, when one of the permanent magnets 410 approaches the tube 100, the other
leaves from the tube 100. A motor 420 is used to move the pair of permanent magnets
410 laterally relative to the tube 100. Operation of the motor 420 causes the magnetic
particles M to rock laterally in the tube 100 in the direction crossing the longitudinal
direction of the tube 100.
[0036] The motor 420 can be operated at any appropriate time regardless of which part of
the adsorption chamber 120 or the tube 100 a magnetic force is applied to. It is,
however, noted that the efficiencies of washing and eluting the magnetic particles M in
the tube 100 can be increased by operating the motor 420 when the permanent magnets
410 are positioned at the level of the second plug 20 or the fourth plug 40 in the tube
100.
[0037] Each magnetic particle M has magnetism and can carry a substance. The magnetic
particles M are manipulated in a solution contained in the tube 100. More specifically,
the magnetic particles M are manipulated so that the relative position of the magnetic
force application unit 400 and the magnetic particles M is altered over a period of time
by directing the magnetic particles M in the tube 100 in one of (1) the longitudinal
direction of the tube 100, (2) a cross-sectional direction crossing the longitudinal
direction of the tube 100, and (3) a direction determined by combining the vectors of
the longitudinal direction of the tube 100 and the cross-sectional direction crossing the
longitudinal direction of the tube 100, or in one of (1) a lateral direction crossing the
longitudinal direction of the tube 100, (2) the longitudinal direction of the tube 100,
and (3) a direction determined by combining the vectors of the longitudinal direction
of the tube 100 and the lateral direction crossing the longitudinal direction of the tube
100, by using the magnetic force application unit 400 for applying a magnetic force to
the magnetic particles M.
[0038] What is required for manipulating the magnetic particles M in the tube 100 is to
change the relative position of the magnetic force application unit 400 and the tube
100. This can be achieved by keeping either of the tube 100 or the magnetic force ap
plication unit 400 stationary and moving the other, or alternatively, by moving both of
the tube 100 and the magnetic force application unit 400. This embodiment describes a
case where the tube 100 is fixed after being mounted on the tube mount 300 and the
magnetic force application unit 400 is moved.
[0039] In this embodiment, a "plug" of a liquid refers to a portion of an inner space of the
vessel (tube 100) that is substantially filled with the subject liquid alone. The inner
space of the vessel is partitioned longitudinally by the plugs. As used herein, the term
"substantially" indicates that a few amount (e.g., as a thin film or foam) of another
substance (e.g., a liquid or a gas) may be present around the plug, that is, on the inner
wall of the vessel or on the interface of the plug. In addition, the vessel refers to a
hollow component that may be deformed. The vessel has a section with such a size that
allows the liquid to keep the shape as the plug within the capillary.
[0040] The apparatus for the extraction of nucleic acids 3000 of this embodiment allows the
automation of a pre-treatment of PCR, which significantly reduces the time and labor
required for the pre-treatment. In addition, the magnetic force application unit 400 can
be rockd in the apparatus for the extraction of nucleic acids 3000 of this embodiment.
This provides more efficient washing (purification) of the magnetic particles M
carrying the nucleic acids adsorbed thereon, further improving the accuracy of PCR.
[0041] 2. Device for the extraction of nucleic acids
Fig. 2 is a view diagrammatically showing essential parts of the device for the ex
traction of nucleic acids 1000 according to this embodiment.
The device for the extraction of nucleic acids 1000 has the tube 100, a first plug 10, a
second plug 20, a third plug 30, a fourth plug 40, and a fifth plug 50. The device for
the extraction of nucleic acids 1000 has the longitudinal direction and has the tube
(vessel) 100 in which the first plug 10 made of an oil (liquid), the second plug 20 made
of a first washing solution (liquid) that is phase separated from the oil, the third plug
30 made of an oil, the fourth plug 40 made of an elution solution (liquid) that is phase
separated from the oil, and the fifth plug 50 is made of an oil are provided in this order.
The tube 100 in this embodiment has the first through fifth plugs 10 to 50, but a sixth
plug and a seventh plug, which are described later, may also be provided.
[0042] 2.1. Tube
The tube 100 forms an essential part of the device for the extraction of nucleic acids
1000. The device for the extraction of nucleic acids 1000 may comprise various
components and parts other than the tube 100. The device for the extraction of nucleic
acids 1000 may comprise, for example, a tubing connected to the tube 100, an stopper,
a joint, a pump, or a controller, which are not shown herein.
[0043] The tube 100 is a hollow component having a cavity therein through which a liquid
can be passed in its longitudinal direction. Although the tube 100 is straight in its lon
gitudinal direction, it may be curved or bent. The shape and size of the cavity in the
tube 100 are not specifically limited as long as the liquid can keep the shape of the
plug in the tube 100. The size of the cavity in the tube 100 and the cross-sectional
shape of the cavity perpendicular to the longitudinal direction of the tube 100 may be
varied along the longitudinal direction of the tube 100. Whether the liquid can keep the
shape of the plug in the tube 100 depends on factors including the material of the tube
100 and the type of the liquid. The cross-sectional shape of the tube 100 perpendicular
to its longitudinal direction is appropriately designed to the extent that the liquid can
keep the shape of the plug in the tube 100.
[0044] The shape of the outer periphery of the tube 100 as viewed in the cross section per
pendicular to the longitudinal direction of the tube 100 is not specifically limited. The
thickness (the length from the side of the inner cavity to the outer surface) of the tube
100 is not limited as well. When the cavity in the tube 100 has a circular cross section
in the direction perpendicular to the longitudinal direction of the cavity, the inner
diameter of the tube 100 (diameter of a circle in the cross section of the cavity per
pendicular to the longitudinal direction thereof) may be, for example, equal to or larger
than 0.5 mm but not larger than 3 mm. The tube 100 having an inner diameter within
this range is preferable because a plug of liquid can be formed easily for a wide variety
of materials of the tube 100 and for various liquids.
[0045] The material of the tube 100 is not specifically limited. Examples include glass,
polymers, or metals. It is, however, preferable that a material such as glass or a
polymer transparent to visible light is used for the tube 100 because the inside (within
the cavity) can be observed from the outside of the tube 100. It is also preferable that a
material that transmits the magnetic force or a non-magnetic material is used for the
tube 100 because the application of the magnetic force from the outside of the tube 100
facilitates the migration of the magnetic particles M through the tube 100.
[0046] The first plug 10 made of a first oil, the second plug 20 made of the first washing
solution that is phase separated from the oil, the third plug 30 made of a second oil that
is phase separated from the first washing solution, the fourth plug 40 made of the
elution solution that is phase separated from the oil, and the fifth plug 50 made of a
third oil that is phase separated from the elution solution are provided in this order in
the tube 100.
[0047] 2.2. First plug, third plug, and fifth plug
Each of the first plug 10, the third plug 30, and the fifth plug 50 is made of an oil.
The oil of the first plug 10, the third plug 30, and the fifth plug 50 may be the same or
different from each other. The oil may be selected from, for example, silicone oil such
as dimethyl silicone, paraffin oil, mineral oil and a mixture thereof. The liquid of the
first, second, third, fourth, and fifth plugs 10, 20, 30, 40, and 50 is selected so that the
adjacent plugs are phase separated from each other.
[0048] The second plug 20 is provided between the first plug 10 and the third plug 30. A
plug of another liquid may be provided adjacent to the first plug 10 opposite to the
second plug 20. Although the first plug 10 preferably contains neither air bubbles nor
any other liquid, air bubbles and another liquid may be present as long as the magnetic
particles M carrying the nucleic acids adsorbed thereon can pass through the first plug
10. In addition, it is preferable that no air or liquid is present between the first plug 10
and the second plug 20. The air or another liquid may, however, be present as long as
the magnetic particles M carrying the nucleic acids adsorbed thereon can pass from the
first plug 10 to the second plug 20. Likewise, it is preferable that no air or liquid is
present between the second plug 20 and the third plug 30. The air or another liquid
may, however, be present as long as the magnetic particles M carrying the nucleic
acids adsorbed thereon can pass from the second plug 20 to the third plug 30.
[0049] The fourth plug 40 is provided between the third plug 30 and the fifth plug 50. A
plug of another liquid may be provided adjacent to the fifth plug 50 opposite to the
fourth plug 40. Although the third plug 30 preferably contains neither air bubbles nor
any other liquid, air bubbles and another liquid may be present as long as the magnetic
particles M carrying the nucleic acids adsorbed thereon can pass through the third plug
30. In addition, it is preferable that no air or liquid is present between the third plug 30
and the fourth plug 40. The air or another liquid may, however, be present as long as
the magnetic particles M carrying the nucleic acids adsorbed thereon can pass from the
third plug 30 to the fourth plug 40. Likewise, it is preferable that no air or liquid is
present between the fourth plug 40 and the fifth plug 50. The air or another liquid may,
however, be present as long as the magnetic particles M carrying the nucleic acids
adsorbed thereon can pass from the fourth plug 40 to the fifth plug 50. Furthermore, it
is preferable that no air or liquid is present in the fifth plug 50.
[0050] The length (height) of the liquid column in the longitudinal direction of the tube 100
is not specifically limited as long as the first plug 10, the third plug 30, and the fifth
plug 50 can be formed. The specific length of each of the first plug 10, the third plug
30, and the fifth plug 50 in the longitudinal direction of the tube 100 is equal to or
longer than 1mm but not longer than 50 mm. The length is preferably equal to or
longer than 1mm but not longer than 30 mm, and more preferably, equal to or longer
than 5 mm but not longer than 20 mm, in order to avoid that a travel distance of the
magnetic particles M becomes too long. Of these, when the length of the third plug 30
in the longitudinal direction of the tube 100 is long, the second plug 20 is less likely to
be discharged in an aspect where the fourth plug 40 is discharged from the tube 100 on
the side of the fifth plug 50. In this case, a specific length of the third plug 30 may be
equal to or longer than 10 mm but not longer than 50 mm.
[0051] The first plug 10 and the fifth plug 50 each has a function of preventing the first
washing solution forming the second plug 20 and the elution solution forming the
fourth plug 40 from being contact with an external material (e.g., evaporation) or from
being contaminated even when at least one end of the tube 100 is opened. The volume
of the first washing solution and the elution solution can thus be kept constant even
when at least one end of the tube 100 is exposed to the outside air. This contributes to
reducing any fluctuation of the concentration of each liquid as well as contamination of
each liquid. Accordingly, the nucleic acids and various other substances can be
extracted more precisely during the extraction of the nucleic acids.
[0052] The third plug 30 serves to prevent the first washing solution forming the second
plug 20 and the elution solution forming the fourth plug 40 from being mixed with
each other. The third plug 30 may be made of an oil of a higher viscosity, which
increases a "repellent effect" of the oil when the magnetic particles M are moved
across the interface between the oil and the first washing solution forming the second
plug 20. As a result, fewer water-soluble components on the surface of the magnetic
particles M are brought into the oil forming the third plug 30 when the particles are
moved from the plug of the first washing solution forming the second plug 20 to the oil
forming the third plug 30.
[0053] 2.3. Second plug
The second plug 20 is positioned between the first plug 10 and the third plug 30 in
the tube 100. The second plug 20 is made of the first washing solution. The first
washing solution is a liquid that is phase separated from both the oil forming the first
plug 10 and the oil forming the third plug 30. Examples of the first washing solution
include water and a buffer having a solute at a concentration of 10 mM or lower,
preferably 7 mM or lower, and more preferably, 5 mM or lower. The composition of
the buffer is not specifically limited, and a tris-HCl buffer is an example. The buffer
may contain EDTA (ethylenediaminetetraacetic acid). Such first washing solution
allows efficient washing of the magnetic particles M carrying the nucleic acids
adsorbed thereon.
[0054] The volume of the second plug 20 is not specifically limited and can appropriately be
determined based on, for example, the amount of the magnetic particles M carrying the
nucleic acids adsorbed thereon. For example, when the volume of the magnetic
particles M is equal to 0.5 uL, the volume of 10 uL or larger is enough for the second
plug 20. It is preferable that the volume is equal to or larger than 20 uL but not larger
than 50 uL, and more preferably, equal to or larger than 20 uL but not larger than 30
uL. With the volume of the second plug 20 within this range, the magnetic particles M
can be washed sufficiently when the volume of the magnetic particles M is equal to 0.5
uL. Although a larger volume of the second plug 20 is preferable for washing the
magnetic particles M, the volume can appropriately be determined in consideration of
the length and the width of the tube 100 as well as the length of the second plug 20
depending thereon in the longitudinal direction of the tube 100.
[0055] The second plug 20 may be made up of two or more smaller plugs separated from
each other by a plug of an oil. When the second plug 20 is made up of two or more
plugs separated from the oil plug(s), this means that two or more plugs of the first
washing solution are formed. With the second plug 20 separated by the oil plug(s), the
concentration of a water-soluble substance (in a case where such water-soluble
substance is subjected to washing) achieved in the plugs of the first washing solution is
smaller than the concentration of the water-soluble substance achieved in the single
plug of the first washing solution of the same volume. The second plug 20 may be
separated into any number of smaller plugs. For example, when the second plug 20 is
divided into two smaller plugs having an equal volume, the concentration of the watersoluble
substance (in a case where such water-soluble substance is subjected to
washing) can be reduced to 1/4 of the concentration obtained without separating the
second plug 20 by calculation. The number of the divisions of the second plug 20 is
determined appropriately in consideration of, for example, the length of the tube 100
and the substance to be washed.
[0056] 2.4. Fourth plug
The fourth plug 40 is positioned between the third plug 30 and the fifth plug 50 in the
tube 100. The fourth plug 40 is made of the elution solution.
[0057] The elution solution is a liquid for use in releasing the nucleic acids adsorbed on the
magnetic particles M from the magnetic particles M into the solution. Examples of the
elution solution include purified water such as sterilized water, distilled water, or ionexchanged
water, or a solution of such water containing at least one enzyme, dNTP,
probe, primer, or buffer. The elution solution is a liquid that is phase separated from
both the oil forming the third plug 30 and the oil forming the fifth plug 50.
[0058] When water or an aqueous solution is used as the elution solution, the nucleic acids
adsorbed on the magnetic particles M can be released (eluted) into the elution solution
while the magnetic particles M carrying the nucleic acids adsorbed thereon are
captured in the elution solution. When a solution in which at least one enzyme, dNTP,
probe, primer or buffer is dissolved is selected as the elution solution, the nucleic acids
adsorbed on the magnetic particles M can be released (eluted) and some or all of the
components required for a reaction solution for PCR can be contained in the elution
solution. This further reduces the time and labor in preparing a reaction solution for
PCR using the elution solution. The concentration of at least one enzyme, dNTP,
probe, primer or buffer dissolved in the elution solution forming the fourth plug 40 is
not specifically limited and can be determined according to the reaction solution for
PCR to be prepared.
[0059] It is noted that dNTP as used herein is a mixture of four kinds of deoxynucleotide
triphosphates (dATP (deoxy adenosine triphosphate), dCTP (deoxycytidine
triphosphate), dGTP (deoxyguanosine triphosphate), and dTTP (thymidine
triphosphate).
[0060] The volume of the fourth plug 40 is not specifically limited and can appropriately be
determined based on, for example, the amount of the magnetic particles M carrying the
nucleic acids adsorbed thereon. For example, when the volume of the magnetic
particles M is equal to 0.5 uL, the volume of 0.5 uL or smaller is enough for the fourth
plug 40. It is preferable that the volume is equal to or larger than 0.8 uL but not larger
than 5 uL, and more preferably equal to or larger than 1 uL but not larger than 3 uL.
With the volume of the fourth plug 40 within this range, the nucleic acids can be eluted
sufficiently from the magnetic particles M the when the volume of the magnetic
particles M is equal to 0.5 uL. The volume of the fourth plug 40 for the elution of the
nucleic acids from the magnetic particles M can appropriately be determined in con
sideration of the length and the width of the tube 100 as well as a rapid progress of a
thermal cycle of PCR so that a heat capacity of the reaction solution does not increase
excessively.
[0061] 2.5. Effects
The device for the extraction of nucleic acids 1000 according to this embodiment has
the tube 100 containing the oil, the first washing solution, and the elution solution
provided as the plugs. Nucleic acids can thus be extracted in a significantly short
period of time by introducing the magnetic particles M carrying the nucleic acids
adsorbed thereon into the tube 100 through the first plug 10 and moving them to the
fourth plug 40. More specifically, the magnetic particles M carrying the nucleic acids
adsorbed thereon are introduced into the tube 100 through the first plug 10. They are
passed through the oil forming the first plug 10, washed with the first washing solution
forming the second plug 20, and then passed through the oil forming the third plug 30.
The nucleic acids are released from the magnetic particles M in the elution solution
forming the fourth plug 40. This means that the device for the extraction of nucleic
acids 1000 of this embodiment can be used to obtain the elution solution containing the
nucleic acids in a highly pure form by moving the magnetic particles M carrying the
nucleic acids adsorbed thereon through the tube 100. Accordingly, the device for the
extraction of nucleic acids 1000 can significantly reduce the time and labor required
for the pre-treatment for PCR.
[0062] (Modified version)
(Configuration of the device for the extraction of nucleic acids)
The device for the extraction of nucleic acids 1000 of this embodiment includes the
tube 100, the first plug 10, the second plug 20, the third plug 30, the fourth plug 40,
and the fifth plug 50. The device may, however, have a configuration with the addition
of other function(s). The device for the extraction of nucleic acids of this embodiment
may include a combination of the structures described below or a modified version of
these structures.
[0063] (End of the tube)
Fig. 3 is a view diagrammatically showing essential parts of the device for the ex
traction of nucleic acids according to this modified version. A device for the extraction
of nucleic acids 1010 of this embodiment may have the tube 100 that is opened at the
end at the side of the fifth plug 50. More specifically, as shown in Fig. 3, the tube 100
of the device for the extraction of nucleic acids 1010 is opened at the end at the side of
the fifth plug 50. In this device for the extraction of nucleic acids 1010, the fifth plug
50 and the fourth plug 40 are discharged successively by applying a pressure to the
inside of the tube 100 on the side of the first plug 10 in the tube 100. This allows for
easy removal of the elution solution forming the fourth plug 40 containing the target
nucleic acids into, for example, a reaction chamber for PCR using the device for the
extraction of nucleic acids 1010.
[0064] (Stopper)
Fig. 4 is a view diagrammatically showing essential parts of the device for the ex
traction of nucleic acids according to this modified version. A device for the extraction
of nucleic acids 1020 of this embodiment may further include, for example, a stopper
110 that can seal the end of the tube 100 at the side of the fifth plug 50 as shown in the
figure. The stopper 110 can freely be fitted to and removed from the end of the tube
100. The stopper 110 may be made of, for example, a rubber, an elastomer, or a
polymer. When the tube 100 is sealed with the stopper 110, the stopper 110 may
contact with the fifth plug 50 or, alternatively, a gas such as air may be present
between the fifth plug 50 and the stopper 110. In addition, while the stopper 110 can
freely be fitted to and removed from the tube 100, a mechanism to achieve this is not
specifically limited. In the embodiment shown in Fig. 4, a part of the stopper 110 is
inserted into the tube 100 and secured thereto. The stopper 110 may, however, have a
shape of a cap.
[0065] When the stopper 110 is removed from the device for the extraction of nucleic acids
1020, the end of the tube 100 at the side of the fifth plug 50 is opened. This cor
responds to the device for the extraction of nucleic acids 1010 shown in Fig. 3. The
device for the extraction of nucleic acids 1020 can thus be used for easy removal of the
elution solution forming the fourth plug 40 containing the target nucleic acids into, for
example, a reaction chamber for PCR. On the other hand, with the end of the tube 100
at the side of the fifth plug 50 sealed with the stopper 110 (as shown in Fig. 4), an
effect of preventing each plug from being displaced in the tube 100 is provided. Ac
cordingly, the plugs do not follow the movement of the magnetic particles M when the
magnetic particles M are moved in the tube 100.
[0066] It is noted that the end of the tube 100 (at the side of the fifth plug 50) is sealed with
the stopper 110 or connected to another vessel after a reagent is filled in the tube 100
in order to avoid any displacement of the fluid due to the gravity within the tube 100.
[0067] (Adsorption chamber)
Fig. 5 is a view diagrammatically showing a device for the extraction of nucleic acids
according to this modified version. As shown in Fig. 5, a device for the extraction of
nucleic acids 1030 further includes an adsorption chamber (vessel) 120 that can freely
be connected to and removed from the end of the tube 100 at the side of the first plug
10 in communication with each other.
[0068] The adsorption chamber 120 may be an independent member. The adsorption
chamber 120 can contain a liquid. The adsorption chamber 120 has an opening 121
through which a liquid or a solid can be put into and removed from the adsorption
chamber 120. In the embodiment shown in Fig. 5, the opening 121 in the adsorption
chamber 120 is communicated with the end of the tube 100 at the side of the first plug
10. In addition, the adsorption chamber 120 may have two or more openings 121. One
of such openings 121 may be communicated with the end of the tube 100 at the side of
the first plug 10.
[0069] The inner volume of the adsorption chamber 120 is not specifically limited and, may
be, equal to or larger than 0.1 mL but not larger than 100 mL. The opening(s) 121 of
the adsorption chamber 120 may have a structure that can be sealed with a lid 122
when appropriate. The material of the adsorption chamber 120 is not specifically
limited and may be, for example, a metal or a polymer.
[0070] The opening 121 in the adsorption chamber 120 can be connected to the end of the
tube 100 at the side of the first plug 10. The type of the connection between the ad
sorption chamber 120 and the tube 100 is not specifically limited as long as the content
therein does not escape therefrom. With the adsorption chamber 120 and the tube 100
connected, the inner space in the adsorption chamber 120 is communicated with the
inner space in the tube 100. The adsorption chamber 120 may be removed from the
tube 100, if necessary.
[0071] By providing the adsorption chamber 120 as in the case of the device for the ex
traction of nucleic acids 1030, the magnetic particles M, an adsorption solution, and a
sample can be contained in the adsorption chamber 120 to allow the magnetic particles
M to adsorb the nucleic acids. Then, the adsorption chamber 120 is connected to the
end of the tube 100 at the side of the first plug 10. In this way, the magnetic particles
M can easily be introduced into the tube 100 through the first plug 10.
[0072] The adsorption solution refers to a liquid in which nucleic acids are adsorbed onto
the magnetic particles M. The adsorption solution is an aqueous solution containing,
for example, a chaotropic agent. The adsorption solution may contain a chelating agent
or a surfactant. More specifically, the adsorption solution may have disodium dihydrogen
ethylenediamine tetraacetate or a dihydrate thereof dissolved therein or may
contain, for example, polyoxyethylene sorbitan monolaurate.
[0073] The chaotropic agent as used herein refers to a substance that disrupts the intermolecular
forces between water molecules and weakens the structure of the water
molecules. Specific examples of the chaotropic agent include guanidinium ions, ureas,
and iodide ions. When exposed to water containing the chaotropic agent, it is thermodynamically
more favorable for the nucleic acids to be adsorbed onto a solid than
being surrounded by water molecules. The nucleic acids in the water are thus adsorbed
on the surface of the magnetic particles M. Examples of the substance having
chaotropic properties in the water include guanidine hydrochloride and sodium iodide.
[0074] The adsorption chamber 120 can be shaken without being connected to the tube 100
to vigorously mix the liquid in the adsorption chamber 120. This allows quick ad
sorption of the nucleic acids onto the magnetic particles M. The adsorption chamber
120 may have the lid 122 that seals the opening 121. Furthermore, it is also possible to
quantitatively concentrate the nucleic acids in the sample in the elution solution
forming the fourth plug 40 by appropriately altering the amount of the sample to be in
troduced into the adsorption chamber 120 and the volume of the liquid (in particular,
the fourth plug 40) in the tube 100.
[0075] By using an elastic material such as a rubber, an elastomer, or a polymer as a
material of the adsorption chamber 120, the adsorption chamber 120 can be deformed
and the inside of the tube 100 can be pressurized with the adsorption chamber 120
connected to the tube 100. In this way, a pressure can easily be applied to the tube 100
from the side of the first plug 10 in order to discharge the elution solution forming the
fourth plug 40 from the end of the tube 100 at the side of the fifth plug 50. This con
figuration makes it possible to remove the elution solution into, for example, a reaction
chamber for PCR.
[0076] The nucleic acids are adsorbed onto the surface of the magnetic particles M by
previously introducing the magnetic particles M into the adsorption chamber 120 filled
with the nucleic acids and the adsorption solution, placing the lid 122 on the adsorption
chamber 120, and shaking the adsorption chamber 120 by using an agitator or by
hand(s) to vigorously mix the adsorption solution and the magnetic particles M in the
adsorption chamber 120. The magnetic particle M herein is not specifically limited as
long as it is a magnetic solid carrier that attracts objects and has a hydrophilic surface
capable of attracting the nucleic acids, i.e., holding them via reversible physical ad
sorption in the presence of chaotropic ions. More specifically, the magnetic particle M
is preferably a substance containing silicon dioxide such as silica, glass, and diatomite
or chemically modified versions thereof. It is more preferable that the magnetic
particle M is a complex with a magnetic substance or a superparamagnetic metal oxide.
[0077] After the magnetic particles M and the adsorption solution are mixed vigorously, the
lid 122 is opened to connect the adsorption chamber 120 to the tube 100 or to pour the
adsorption solution containing the magnetic particles M in the adsorption chamber 120
into the tube 100. In this event, the tube 100 is parallel to the direction of the gravity.
Hereinafter, movement in the vertically downward direction of the magnetic force ap
plication unit 400 is referred to as falling, and movement in the vertically upward
direction thereof (toward the adsorption chamber 120) is referred to as rising.
[0078] (Sixth plug and seventh plug)
The device for the extraction of nucleic acids according to this embodiment may
include a sixth plug and a seventh plug in the tube 100.
[0079] Fig. 6 is a view diagrammatically showing essential parts of the device for the ex
traction of nucleic acids of this modified version. More specifically, it is a view dia
grammatically showing a device for the extraction of nucleic acids 1100 having a sixth
plug 60 and a seventh plug 70 in the tube 100.
[0080] The device for the extraction of nucleic acids 1100 has the sixth plug 60 made of a
second washing solution that is phase separated from the oil and the seventh plug 70
made of an oil, in this order from the third plug 30, that are added between the third
plug 30 and the fourth plug 40 in the tube 100 of the aforementioned device for the ex
traction of nucleic acids.
[0081] The sixth plug 60 is provided adjacent to the third plug 30 in the tube 100 opposite to
the second plug 20. The sixth plug 60 is made of the second washing solution. The
second washing solution is a liquid that is phase separated from both the oil forming
the third plug 30 and the oil forming the seventh plug 70. Examples of the second
washing solution include water and a buffer having a solute at a concentration of 10
mM or lower, preferably 7 mM or lower, and more preferably, 5 mM or lower. The
composition of the buffer is not specifically limited, and a tris-HCl buffer is an
example. The buffer may contain EDTA (ethylenediaminetetraacetic acid). The second
washing solution may be the same or different in composition from the first washing
solution.
[0082] The volume of the sixth plug 60 is not specifically limited and can appropriately be
determined based on, for example, the amount of the magnetic particles M carrying the
nucleic acids adsorbed thereon. For example, when the volume of the magnetic
particles M is equal to 0.5 uL, the volume of 10 uL or larger is enough for the sixth
plug 60. It is preferable that the volume is equal to or larger than 20 uL but not larger
than 50 uL, and more preferably, equal to or larger than 20 uL but not larger than 30
uL. With the volume of the sixth plug 60 within this range, the magnetic particles M
can be washed sufficiently when the volume of the magnetic particles M is equal to 0.5
uL. Although a larger volume of the sixth plug 60 is preferable for washing the
magnetic particles M, the volume can appropriately be determined in consideration of
the length and the width of the tube 100 as well as the length of the sixth plug 60
depending thereon in the longitudinal direction of the tube 100.
[0083] The sixth plug 60 may be made up of two or more smaller plugs separated from each
other by a plug of an oil. When the sixth plug 60 is made up of two or more plugs
separated from the oil plug(s), this means that two or more plugs of the second
washing solution are formed. With the sixth plug 60 separated by the oil plug(s), the
concentration of a water-soluble substance (in a case where such water-soluble
substance is subjected to washing) achieved in the plugs of the second washing
solution is smaller than the concentration of the water-soluble substance achieved in
the single plug of the first washing solution of the same volume. The sixth plug 60 may
be separated into any number of smaller plugs. For example, when the sixth plug 60 is
divided into two smaller plugs having an equal volume, the concentration of the watersoluble
substance (in a case where such water-soluble substance is subjected to
washing) can be reduced to 1/4 of the concentration obtained without separating the
sixth plug 60 by calculation. The number of the divisions of the sixth plug 60 is de
termined appropriately in consideration of, for example, the length of the tube 100 and
the substance to be washed. When the first washing solution forming the second plug
20 and the second washing solution forming the sixth plug 60 are the same, a similar
effect can be obtained to the one obtained by dividing the second plug 20 in the device
for the extraction of nucleic acids that does not have the aforementioned sixth and
seventh plugs 60 and 70.
[0084] The seventh plug 70 is made of an oil that is phase separated from both the sixth plug
60 and the elution solution forming the fourth plug 40 which are adjacent to the
seventh plug 70. The oil forming the seventh plug 70 may be an oil that is different
from the oil forming each of the first plug 10, the third plug 30, and the fifth plug 50.
The oil may be similar to the one described in conjunction with the first plug 10.
[0085] Although the seventh plug 70 preferably contains neither air bubbles nor any other
liquid, air bubbles and another liquid may be present as long as the magnetic particles
M carrying the nucleic acids adsorbed thereon can pass through the seventh plug 70. In
addition, it is preferable that no air or liquid is present between the seventh plug 70 and
the adjacent fourth plug 40 and between the seventh plug 70 and the adjacent sixth
plug 60. The air or another liquid may, however, be present as long as the magnetic
particles M carrying the nucleic acids adsorbed thereon can pass through the tube 100.
It is preferable that no air or liquid is present in the seventh plug 70.
[0086] The length of the seventh plug 70 in the longitudinal direction of the tube 100 is not
specifically limited as long as the seventh plug 70 can be formed. The specific length
of the seventh plug 70 in the longitudinal direction of the tube 100 is equal to or longer
than 1mm but not longer than 50 mm. The length is preferably equal to or longer than
1mm but not longer than 30 mm, and more preferably, equal to or longer than 5 mm
but not longer than 20 mm, in order to avoid that a travel distance of the magnetic
particles M becomes too long. In the device for the extraction of nucleic acids 1100,
when the length of the seventh plug 70 in the longitudinal direction of the tube 100 is
long, the sixth plug 60 is less likely to be discharged in an aspect where the fourth plug
40 is discharged from the tube 100 on the side of the fifth plug 50. In this case, a
specific length of the seventh plug 70 may be equal to or longer than 10 mm but not
longer than 50 mm.
[0087] The seventh plug 70 serves to prevent the second washing solution forming the sixth
plug 60 and the elution solution forming the fourth plug 40 from being mixed with
each other. The seventh plug 70 may be made of an oil of a higher viscosity, which
increases a "repellent effect" of the oil when the magnetic particles M are moved
across the interface between the oil and the second washing solution forming the sixth
plug 60. As a result, fewer water-soluble components on the surface of the magnetic
particles M are brought into the seventh plug 70 (oil) when the magnetic particles are
moved from the plug of the second washing solution forming the sixth plug 60 to the
oil forming the seventh plug 70.
[0088] In the device for the extraction of nucleic acids 1100, the magnetic particles M
carrying the nucleic acids adsorbed thereon can be washed in the second plug 20 and
the sixth plug 60. This further improves an efficiency of washing the magnetic
particles M.
[0089] In addition, in the device for the extraction of nucleic acids 1100, the first washing
solution forming the second plug 20 may contain a chaotropic agent. For example,
when guanidine hydrochloride is contained in the first washing solution forming the
second plug 20, the magnetic particles M can be washed while the adsorption of the
nucleic acids on the magnetic particles M is maintained or enhanced in the second plug
20. The concentration of the guanidine hydrochloride contained in the second plug 20
is, for example, equal to or higher than 3 mol/L but not higher than 10 mol/L, and
preferably equal to or higher than 5 mol/L but not higher than 8 mol/L. With the con
centration of the guanidine hydrochloride within this range, any foreign substances can
be washed out while keeping stable adsorption of the nucleic acids on the magnetic
particles M.
[0090] Furthermore, water or a buffer is used for the second washing solution forming the
sixth plug 60. This allows washing with more stable adsorption of the nucleic acids on
the magnetic particles M in the first washing solution forming the second plug 20. In
addition, the magnetic particles M can further be washed while diluting the chaotropic
agent in the second washing solution forming the sixth plug 60.
[0091] It will easily be appreciated that the device for the extraction of nucleic acids 1100
having the sixth plug 60 and the seventh plug 70 in the tube 100 can have the stopper
and the adsorption chamber as described above as well as a liquid reservoir which will
be described later, to achieve a similar effect to those described above.
[0092] 3. Kit for the extraction of nucleic acids
Fig. 7 is a view diagrammatically showing an example of a kit for the extraction of
nucleic acids according to this embodiment. A kit for the extraction of nucleic acids
2000 shown in Fig. 7 includes the components that form an essential part of the afore
mentioned device for the extraction of nucleic acids 1000. Similar components that are
described in conjunction with the section entitled "2. Device for the extraction of
nucleic acids" are denoted by like reference numerals and detailed description thereof
will be omitted.
[0093] The kit for the extraction of nucleic acids 2000 of this embodiment has the tube 100
and the adsorption chamber 120. The tube 100 contains the first plug 10 made of an
oil, the second plug 20 made of the first washing solution that is phase separated from
the oil, the third plug 30 made of an oil, the fourth plug 40 made of the elution solution
that is phase separated from the oil, and the fifth plug 50 made of the oil, that are
provided in this order. The adsorption chamber 120 can be connected to and com
municated with the end of the tube 100 at the side of the first plug 10.
[0094] The tube 100 is similar to the tube 100 of the device for the extraction of nucleic
acids 1000 with the ends thereof opened. The tube 100 has a hollow shape including a
cavity formed therein through which a liquid can be passed in the longitudinal
direction thereof. The inner shape, outer shape, size, characteristic, and material of the
tube 100 are similar to those of the tube 100 of the device for the extraction of nucleic
acids 1000. The plugs provided within the tube 100 are similar to those provided in the
tube 100 of the device for the extraction of nucleic acids 1000. In addition, both ends
of the tube 100 may be sealed with stoppers 110 that can freely be fitted to and
removed from the tube 100. When the ends of the tube 100 are sealed with the stoppers
110, the kit for the extraction of nucleic acids 2000 can be stored and transferred more
easily. Furthermore, with the stopper 110 sealing the end of the tube 100 at the side of
the fifth plug 50, any displacement of the plugs in the tube 100 can be restricted while
moving the magnetic particles M in the tube 100 during the use of the tube 100, which
further facilitates the washing and the extraction. Moreover, the stopper 110 can freely
be fitted to and removed from the tube 100, so that the end of the tube 100 at the side
of the fifth plug 50 can be opened. Accordingly, it is easy to discharge the elution
solution forming the fourth plug 40 into which the nucleic acids are eluted from the
end of the tube 100 at the side of the fifth plug 50.
[0095] The adsorption chamber 120 is similar to the adsorption chamber 120 described in
conjunction with the device for the extraction of nucleic acids 1000.
[0096] In the embodiment shown in Fig. 7, both ends of the tube 100 are sealed with the
stoppers 110 that can freely be fitted to and removed from the tube 100. In addition,
the kit for the extraction of nucleic acids 2000 may include the lid 122 capable of
sealing the opening 121 in the adsorption chamber 120, which can freely be attached
thereto and removed therefrom. The opening 121 in the adsorption chamber 120 may
thus be sealed with the lid 122 that can freely be attached to and removed from the ad
sorption chamber 120. Furthermore, in the kit for the extraction of nucleic acids 2000,
some or all of the components of the adsorption solution (liquid) may be contained in
the adsorption chamber 120.
[0097] In the kit for the extraction of nucleic acids 2000, the adsorption chamber 120 may
hold the adsorption solution and the magnetic particles M. This makes it possible to
perform, in the adsorption chamber 120, a step of allowing the magnetic particles M to
adsorb the nucleic acids in a sample (substance) after the sample is introduced into the
adsorption chamber 120. No other vessel is thus required, so that a pre-treatment for
PCR can be performed more quickly. In this case, the opening 121 in the adsorption
chamber 120 may be sealed with the lid 122 that can freely be attached to and removed
from the adsorption chamber 120, if necessary. The magnetic particles M will be
described later.
[0098] By using an elastic material as a material of the adsorption chamber 120 as described
above, the adsorption chamber 120 can be deformed and the inside of the tube 100 can
be pressurized with the adsorption chamber 120 connected to the tube 100. In this way,
a pressure can easily be applied to the tube 100 from the side of the first plug 10 in
order to discharge the elution solution forming the fourth plug 40 containing the
nucleic acids eluted therein from the end of the tube 100 at the side of the fifth plug 50.
This configuration makes it possible to easily remove the elution solution into, for
example, a reaction chamber for PCR.
[0099] The kit for the extraction of nucleic acids 2000 may include other components such
as, for example, a stopper, a lid, an instruction manual, a reagent, and a case other than
the tube 100 and the adsorption chamber 120. The illustrated embodiment describes an
example where the tube 100 has five plugs therein. It is, however, readily understood
that the tube 100 may have the sixth plug 60, the seventh plug 70 or other plug(s), if
necessary, provided therein.
[0100] The kit for the extraction of nucleic acids 2000 of this embodiment has the ad
sorption chamber 120 that can be connected to and communicated with the end of the
tube 100 at the side of the first plug 10. Accordingly, it is possible to allow the
magnetic particles M to adsorb the nucleic acids by holding the magnetic particles M
and the sample in the adsorption chamber 120. By connecting the adsorption chamber
120 to the end of the tube 100 at the side of the first plug 10, the magnetic particles M
can easily be introduced into the tube 100 from the side of the first plug 10 in the tube
100. Furthermore, the kit for the extraction of nucleic acids 2000 of this embodiment
has the adsorption chamber 120, so that the adsorption chamber 120 can be shaken to
vigorously mix the liquid in the adsorption chamber 120. This allows the quick ad
sorption of the nucleic acids onto the magnetic particles M.
[0101] In addition, by connecting the adsorption chamber 120 to the tube 100, it is easy to
introduce the magnetic particles M carrying the nucleic acids adsorbed thereon into the
tube 100 through the first plug 10 and move them to the fourth plug 40. This allows the
extraction of the nucleic acids in a significantly short period of time. The kit for the ex
traction of nucleic acids 2000 can be used to obtain the elution solution containing the
nucleic acids in a highly pure form by moving the magnetic particles M carrying the
nucleic acids adsorbed thereon through the tube 100. Accordingly, the kit for the ex
traction of nucleic acids 2000 can significantly reduce the time and labor required for
the pre-treatment for PCR.
[0102] 4. Method for the extraction of nucleic acids
The apparatus for the extraction of nucleic acids 3000, the kit for the extraction of
nucleic acids 2000, and the device for the extraction of nucleic acids 1000, which are
described above, and their variations which will be described later can all suitably be
used for a method for the extraction of nucleic acids as a method of manipulating the
solid carriers of this embodiment.
A method using the aforementioned kit for the extraction of nucleic acids 2000 is
described as an example of the method for the extraction of nucleic acids of this em
bodiment.
[0103] The method for the extraction of nucleic acids of this embodiment comprises the
steps of: introducing a sample containing the nucleic acids into the flexible adsorption
chamber 120 in which the magnetic particles M and the adsorption solution are
contained; shaking the adsorption chamber 120 to allow the magnetic particles M to
adsorb the nucleic acids; connecting the adsorption chamber 120 to the end of the tube
100 at the side of the first plug 10 so that the adsorption chamber 120 is communicated
with the tube 100, the tube 100 having the first plug 10 made of an oil, the second plug
20 made of the first washing solution that is phase separated from the oil, the third plug
30 made of an oil, the fourth plug 40 made of the elution solution that is phase
separated from the oil, and the fifth plug 50 made of an oil provided therein in this
order; applying a magnetic force to move the magnetic particles M from the inside of
the adsorption chamber 120 to the position of the fifth plug 50 through the tube 100;
and eluting the nucleic acids from the magnetic particles M into the elution solution
forming the fourth plug.
[0104] In the method for the extraction of nucleic acids of this embodiment, various
particles (e.g., silica particles, polymer particles, and particles of a magnetic substance)
may be used as long as they are magnetic particles capable of adsorbing nucleic acids
in the adsorption solution and can be moved in the tube 100 using a magnetic force. In
an embodiment of the method for the extraction of nucleic acids described below, such
magnetic particles are used as the magnetic particles M that contain a magnetic
substance and can adsorb nucleic acids on the surface thereof. When particles other
than the magnetic particles are moved in the tube, the gravity or a potential difference
may also be used.
[0105] In the method for the extraction of nucleic acids of this embodiment, a material that
transmits the magnetic force is selected for the adsorption chamber 120 and the tube
100. The magnetic force is applied to the adsorption chamber 120 and the tube 100
from the outside to move the magnetic particles M within the adsorption chamber 120
and the tube 100.
[0106] The sample contains the target nucleic acids. Hereinafter, this may merely be referred
to as the target nucleic acids. The target nucleic acids may be, for example, DNAs or
RNAs (DNA is a deoxyribonucleic acid, and/or RNA is a ribonucleic acid). The target
nucleic acids are extracted from the sample and eluted into the elution solution using
the method for the extraction of nucleic acids of this embodiment, and then used as, for
example, a template for PCR. Examples of the sample include blood, nasal mucus, oral
mucus, and various other biological samples.
[0107] 4.1. Step of introducing the sample into the vessel
The step of introducing a sample into the adsorption chamber 120 may be performed
by, for example, taking the sample with a cotton swab, inserting the cotton swab into
the adsorption chamber 120 through the opening 121, and dipping it in the adsorption
solution. The sample may be introduced into the adsorption chamber 120 through the
opening 121 using, for example, a pipette. When the sample is a paste or a solid
material, it may be put into the adsorption chamber 120 or adhered to the inner surface
of the adsorption chamber 120 using, for example, a spoon or tweezers inserted
through the opening 121.
[0108] 4.2. Step of allowing the magnetic particles to adsorb the nucleic acids
The step of allowing the magnetic particles to adsorb the nucleic acids is performed
while shaking the adsorption chamber 120. This step can be performed more e f
ficiently by using the lid 122, if used for sealing the opening 121 in the adsorption
chamber 120, to seal the adsorption chamber 120. During this step, the target nucleic
acids are adsorbed onto the surface of the magnetic particles M due to the effect of the
chaotropic agent. Proteins or nucleic acids other than the target nucleic acids may also
be adsorbed onto the surface of the magnetic particles M in this step.
[0109] The shaking of the adsorption chamber 120 may be achieved by using an apparatus
such as a vortex shaker. Alternatively, the adsorption chamber 120 may manually be
shaken by an operator using his or her hand(s). In addition, the adsorption chamber 120
may be shaken while externally applying a magnetic force taking advantage of the
magnetism of the magnetic particles M. The time duration during which the adsorption
chamber 120 is shaken can appropriately be determined. For example, when an ap
proximate shape of the adsorption chamber 120 is a hollow cylindrical shape having a
diameter of about 20 mm and a height of about 30 mm, the content of the adsorption
chamber 120 is mixed vigorously by shaking the adsorption chamber 120 by hands for
10 seconds and the nucleic acids are adsorbed onto the surface of the magnetic
particles M.
[01 10] 4.3. Step of connecting the vessel to the tube
Fig. 8 is a view diagrammatically showing an example of a kit for the extraction of
nucleic acids according to this embodiment.
As shown in Fig. 8, the adsorption chamber 120 is connected to the end of the tube
100 at the side of the first plug 10. The plugs in the tube 100 are less likely to move in
the tube even after the stopper 110 is removed from the end at the side of the first plug
10 because the stopper 110 is left at the end at the side of the fifth plug 50. This step is
performed after the stopper 110 at the end of the tube 100 at the side of the first plug
10, if any, is removed. The adsorption chamber 120 and the tube 100 are connected so
that the content thereof does not escape therefrom. The adsorption chamber 120 is
communicated with the tube 100 so that the content can flow between them.
[0111] 4.4. Step of moving the magnetic particles
After the aforementioned step, the magnetic particles M carrying the nucleic acids
adsorbed thereon in the adsorption chamber 120 are ready to be introduced into the
tube 100. In the liquids contained in the tube 100, the magnetic particles M having the
magnetism that are capable of carrying the nucleic acids are manipulated. A method of
introducing the magnetic particles M carrying the nucleic acids adsorbed thereon into
the tube 100 is not specifically limited and, for example, the gravity or a centrifugal
force may be used. This embodiment, however, uses the external application of a
magnetic force to the adsorption chamber 120 and the tube 100. The magnetic force
may be applied by using, for example, a permanent magnet or an electromagnet. It is
preferable that a permanent magnet is used for this application purpose because less
heat is generated. When permanent magnets are used, the magnets may be moved by
an operator with his or her hand(s) or, alternatively, by a mechanical device. The
magnetic particles M are attracted by magnetic force, so that this property is used to
alter the relative position between the adsorption chamber 120 plus the tube 100 and
the permanent magnets to move the magnetic particles M from the adsorption chamber
120 to the tube 100. The magnetic particles M are manipulated using the magnetic
force application unit 300 adapted to applying a magnetic force to the magnetic
particles M in the tube 100 so that the relative position between the magnetic force ap
plication unit 400 and the magnetic particles M is altered over a period of time by
means of directing the magnetic particles M in a direction along either one or a com
bination of the vectors of the longitudinal direction of the tube 100 and the direction
crossing the longitudinal direction of the tube 100. As a result, the magnetic particles
M are moved sequentially from the first plug 10 to the fourth plug 40. The resident
time of the magnetic particles M in each plug when they pass through that plug is not
specifically limited. In addition, the magnetic particles M may be rockd within a single
plug in the longitudinal direction of the tube 100.
[0112] Now, an example of a method of externally applying a magnetic force is described in
which the magnetic particles M are moved by applying a magnetic force to the ad
sorption chamber 120 and the tube 100 of this embodiment from the outside.
Figs. 9 to 11 are views showing a method of externally applying a magnetic force to
move the magnetic particles M according to this embodiment. The magnetic force ap
plication unit 400 comprises, as shown in Fig. 9(A), a pair of permanent magnets
410A, 410B opposed to each other at a certain distance. The permanent magnets 410A,
4 10B are faced to each other with the tube 100 placed between them after the tube 100
is mounted on the tube mount 300 (see Fig. 1).
First, in order to introduce the magnetic particles M in the adsorption chamber 120 into
the tube 100, the magnetic force application unit 400 is lowered from the level of the
adsorption chamber 120 until the pair of permanent magnets 4 1OA, 4 10B of the
magnetic force application unit 400 reaches the level of the second plug 20 in the tube
100.
[0113] Next, in order to wash the magnetic particles M, the magnetic force application unit
400 is lowered in the longitudinal direction of the tube 100 while repeatedly moving
the permanent magnets in a horizontal direction, as shown in Fig. 9(B). More
specifically, the permanent magnets are moved so that one permanent magnet 4 1OA (or
the permanent magnet 410B) is moved closer to the side of the tube 100 while the
other permanent magnet 4 10B (or the permanent magnet 4 1OA) is moved away from
the side of the tube 100, and then vise versa so that the relative magnitudes of the
magnetic force applied to the tube 100 by the permanent magnets 4 1OA, 4 10B is a l
ternated. It is preferable that the washing of the magnetic particles M by the first
washing solution forming the second plug 20 is performed while passing the magnetic
particles M through the first washing solution forming the second plug 20 over a long
distance. With the present method, the magnetic particles M can travel in the liquid
over a distance that is enough to achieve the effect of washing the surface of the
magnetic particles M with the first washing solution forming the second plug 20 in the
tube 100 by falling the magnetic particles M in the longitudinal direction of the tube
100 while moving them laterally.
[0114] Next, in order to move the magnetic particles M from the second plug 20 to the third
plug 30, one permanent magnet 410A (or the permanent magnet 410B) of the magnetic
force application unit 400 is moved closer to the side of the tube 100, and then the
magnetic force application unit 400 is lowered to the level of the third plug 30, as
shown in Fig. 10(A). Since the magnetic force is larger on a portion of the side of the
tube 100 because of the permanent magnet closer to the tube, the magnetic particles M
are gathered or collected at that position on the side of the tube 100. These gathered
magnetic particles M can be moved to the third plug 30 without being captured at the
interface between the first washing solution forming the second plug 20 and the oil
forming the third plug 30.
[0115] Next, in order to move the magnetic particles M from the third plug 30 to the fourth
plug 40, the magnetic force application unit 400 is lowered until the permanent magnet
4 1OA (or the permanent magnet 4 10B) of the magnetic force application unit 400
reaches the level of the fourth plug 40 while keeping one permanent magnet 4 1OA (or
the permanent magnet 410B) closer to the side of the tube 100 as shown in Fig. 10(A).
The magnetic particles M can thus be moved from the third plug 30 to the fourth plug
40.
[01 16] Subsequently, as shown in Fig. 10(B), one permanent magnet 410A (or the
permanent magnet 410B) is moved closer to the side of the tube 100 while the other
permanent magnet 4 10B (or the permanent magnet 4 1OA) is moved away from the side
of the tube 100, and then vise versa, to alternate the relative magnitudes of the
magnetic force applied to the tube 100 by the permanent magnets 4 1OA, 4 10B when
the magnetic particles M are present in the fourth plug 40. In other words, the magnetic
particles M are displaced in the fourth plug 40 at least between two different points in
the direction perpendicular to the longitudinal direction of the tube 100. As a result, the
nucleic acids adsorbed on the magnetic particles M can be eluted in the elution solution
forming the fourth plug 40. In addition to the aforementioned lateral movement of the
magnetic force application unit 400, it may be moved vertically in the longitudinal
direction of the tube 100. This ensures the magnetic particles M to reside in the fourth
plug 40.
[01 17] After the magnetic particles M are rockd enough in the fourth plug 40, one
permanent magnet 410A (or the permanent magnet 410B) of the magnetic force ap
plication unit 400 is moved closer to the side of the tube 100, and the permanent
magnets 4 1OA and 4 10B are moved upward the permanent magnet 4 1OA (or the
permanent magnet 410B) of the magnetic force application unit 400 reaches the level
of the second plug 20, as shown in Fig. 11. The magnetic force is larger on a portion of
the side of the tube 100 and the magnetic particles M are thus gathered on the inner
surface of the tube 100 near the permanent magnet 4 1OA (or the permanent magnet
410B). Accordingly, the magnetic particles M can be moved to the second plug 20
without being captured at the interface between the elution solution forming the fourth
plug 40 and the oil forming the third plug and at the interface between the first washing
solution forming the second plug 20 and the oil forming the third plug 30.
[0118] Subsequently, the elution solution forming the fourth plug 40 containing the nucleic
acids eluted therein is dispensed from the tube 100 or directly introduced into a
reaction chamber for PCR. In this way, the intended nucleic acids are extracted from
the sample for a PCR reaction.
[01 19] 4.5. Step of eluting the nucleic acids
After the magnetic particles M reaches the fourth plug 40, the nucleic acids carried
on the magnetic particles M are eluted into the elution solution forming the fourth plug
40 due to the action of the elution solution. During this step, the nucleic acids are
eluted from the sample into the elution solution. The nucleic acids are thus extracted
from the sample.
[0120] 4.6. Step of discharging the fourth plug from the tube
The method for the extraction of nucleic acids of this embodiment may include a step
of discharging the fifth plug 50 and the fourth plug 40 from the end of the tube 100
opposite to the end connected to the adsorption chamber 120 by deforming the ad
sorption chamber.
[0121] This step can be performed by deforming the adsorption chamber 120 after the "4.5.
Step of eluting the nucleic acids." The fifth plug 50 is discharged first before the
extrusion of the fourth plug 40. It should be noted that the stopper 110 sealing the end
of the tube 100 at the side of the fifth plug 50 is removed prior to this step to open the
end of the tube 100 at the side of the fifth plug 50.
[0122] When an external force is applied to the adsorption chamber 120 to deform it by the
increase of the inner pressure, the plugs are pushed down and moved due to the
pressure in the direction from the first plug 10 to the fifth plug 50 in the tube 100. As a
result, the fifth plug 50 and the fourth plug 40 are discharged in this order through the
end of the tube 100 at the side of the fifth plug 50. The third plug 30 (or the seventh
plug 70) may also be discharged, but the second plug 20 (or the sixth plug 60) is
prevented from being discharged. In this case, for example, it is easy to prevent the
second plug 20 (or the sixth plug 60) from being discharged when the volume of the
third plug 30 (or the seventh plug 70) is larger than the volume of other plugs, and the
third plug 30 (or the seventh plug 70) is long enough in the longitudinal direction of
the tube 100.
[0123] The fourth plug 40 and the fifth plug 50 are discharged into, for example, a reaction
chamber for PCR. The elution solution and the oil are thus removed into the reaction
chamber for PCR, but the oil does not usually affect reactions of PCR. Accordingly, an
oil similar to the oil forming the fifth plug 50 may previously be contained in the
reaction chamber for PCR. In this case, the present step may be performed with the end
of the tube 100 dipped in the oil. This allows the introduction of the elution solution
containing the target nucleic acids into the reaction chamber for PCR without exposing
the solution to the outside air. When the method for the extraction of nucleic acids of
this embodiment includes the present step, the elution solution containing the target
nucleic acids can easily be removed into, for example, the reaction chamber for PCR.
[0124] 4.7. Effects
In the method for the extraction of nucleic acids of this embodiment, extraction of
the nucleic acids can easily be performed in a significantly short period of time. Using
the method for the extraction of nucleic acids of this embodiment, it is possible to
obtain the elution solution containing the nucleic acids in a highly pure form by
moving the magnetic particles M carrying the nucleic acids adsorbed thereon through
the tube 100. Accordingly, the method for the extraction of nucleic acids of this em
bodiment can significantly reduce the time and labor required for the pre-treatment for
PCR.
[0125] 5. Experiments
Experiments are now described and the present invention is described more in detail
below, but the present invention is not limited to these experiments.
[0126] 5.1. Experiment 1
In the Experiment 1, the aforementioned kit for the extraction of nucleic acids 2000
was used with the first plug 10 through the seventh plug 70 provided in the tube 100.
[0127] First, a suspension of 375 uL of the adsorption solution and 1 uL of the magnetic
beads (the magnetic particles M) was contained in a polyethylene vessel (the ad
sorption chamber 120) having a volume of 3 mL. The composition of the adsorption
solution was an aqueous solution of 76 wt. of guanidine hydrochloride, 1.7 wt. of
disodium dihydrogen ethylenediamine tetraacetate dihydrate, and 10 wt.% of polyoxyethylene
sorbitan monolaurate (TOYOBO, MagExtractor-Genome-, NPK-1). In
addition, a suspension containing 50 vol.% of magnetic silica particles and 20 wt.% of
lithium chloride was used as a suspension of magnetic beads.
[0128] Using a pipette, 50 uL of the blood collected from a human subject was added to the
adsorption chamber 120 through the opening therein. The adsorption chamber 120 was
fitted with the lid 122 and the content was mixed vigorously by hands for 30 seconds.
Thereafter, the lid 122 of the adsorption chamber 120 was removed and the adsorption
chamber 120 was connected to the tube 100. The tube 100 had the stoppers 110 at both
ends thereof, and the stopper 110 at the end closer to the first plug 10 was removed
before connecting the adsorption chamber 120 to the tube 100.
[0129] A silicone oil was used as the first, third, seventh, and fifth plugs 10, 30, 70, and 50.
An aqueous solution of 76 wt.% of guanidine hydrochloride was used as the first
washing solution of the second plug 20. In addition, a tris HC1 buffer (solute con
centration of 5 mM) of pH 8.0 was used as the second washing solution of the sixth
plug 60. Sterilized water was used as the elution solution of the fourth plug 40.
[0130] The permanent magnets 410 were moved by hands and the magnetic particles M in
the adsorption chamber 120 were introduced into the tube 100. Then, the magnetic
particles M were moved to the fourth plug 40. The magnetic particles M were present
in the plugs in the tube 100 for approximately the following time durations: 3 seconds
each in the first, third, and seventh plugs 10, 30, and 70, 20 seconds in the second plug
20, 20 seconds in the sixth plug 60, and 30 seconds in the fourth plug 40. In the second
plug 20 and the sixth plug 60, the magnetic particles M were not moved laterally. In
addition, the volumes of the second plug 20, the sixth plug 60, and the fourth plug 40
were 25 uL, 25 uL, and 1 uL, respectively.
[0131] Next, the stopper at the end of the tube 100 at the side of the fifth plug 50 was
removed, and the vessel was deformed by hands to discharge the fifth plug 50 and the
fourth plug 40 into a reaction chamber for PCR. This operation was performed after
the magnetic particles M were moved using the permanent magnets 410 back to the
second plug 20.
[0132] Subsequently, 19 uL of PCR reagent was added to the elution solution to perform
real-time PCR according to an ordinary method. The PCR reagent used was a mixture
of: 4 uL of LightCycler 480 Genotyping master (Roche Diagnostics 4 707 524), 0.4 uL
of SYBR Green I (S7563 available from Life Technologies) diluted to 1/1000 with
sterilized water, 0.06 uL of 100 uM beta-actine primers (F/R), and 14.48 uL of
sterilized water. A PCR amplification curve in the Experiment 1 is shown in Fig. 12.
Fig. 12 is a graph showing the results obtained in this experiment. The ordinate in
Fig. 12 represents the fluorescent intensity while the abscissa represents PCR cycles.
[0133] 5.2. Experiment 2
In the Experiment 2, the nucleic acids were extracted using an ordinary method of
extracting nucleic acids.
[0134] First, a suspension of 375 uL of the adsorption solution, and 20 uL of the magnetic
beads (the magnetic particles M) was contained in a polyethylene vessel (the ad
sorption chamber 120) having a volume of 1.5 mL. The composition of the suspension
of the adsorption solution and the magnetic beads was similar to the one described in
the aforementioned experiment.
[0135] Using a pipette, 50 uL of the blood collected from a human subject was added to the
adsorption chamber 120 through the opening therein. The adsorption chamber 120 was
fitted with the lid 122 and the content was mixed vigorously for 10 minutes using a
vortex mixer. B/F separation was then performed using the magnetic stand and a
pipette. In this state, the magnetic particles M and a small amount of the adsorption
solution were left in the adsorption chamber 120.
[0136] Next, 450 uL of the first washing solution having the same composition as in the Ex
periment 1 was introduced into the adsorption chamber 120, and the lid 122 was fitted
to the adsorption chamber 120. The content was mixed vigorously for 5 seconds using
a vortex mixer. The first washing solution was removed using the magnetic stand and
the pipette. This operation was repeated twice. In this state, the magnetic particles M
and a small amount of the first washing solution were left in the adsorption chamber
120.
[0137] Next, 450 uL of the second washing solution having the same composition as in the
Experiment 1 was introduced into the adsorption chamber 120, and the lid 122 was
fitted to the adsorption chamber 120. The content was mixed vigorously for 5 seconds
using a vortex mixer. The second washing solution was removed using the magnetic
stand and the pipette. This operation was repeated twice. In this state, the magnetic
particles M and a small amount of the second washing solution were left in the ad
sorption chamber 120.
[0138] Furthermore, 50 uL of sterilized water (the elution solution) was added to the ad
sorption chamber 120. The adsorption chamber 120 was fitted with the lid 122 and the
content was mixed vigorously for 10 minutes using a vortex mixer. A supernatant was
collected using the magnetic stand and the pipette. This supernatant contains the target
nucleic acids.
[0139] Subsequently, 1 uL of the elution solution was removed into a new tube and 19 uL of
PCR reagent was added to the elution solution to perform real-time PCR according to
an ordinary method. The PCR reagent used was a mixture of: 4 uL of LightCycler 480
Genotyping master (Roche Diagnostics 4 707 524), 0.4 uL of SYBR Green I (S7563
available from Life Technologies) diluted to 1/1000 with sterilized water, 0.06 uL of
100 uM beta-actine primers (F/R), and 14.48 uL of sterilized water. A PCR ampli
fication curve in this experiment is shown in Fig. 12.
[0140] 5.3. Experiment 3
In the Experiment 3, the aforementioned kit for the extraction of nucleic acids 2000
was used with the first plug 10 through the fifth plug 50 provided in the tube 100.
[0141] The composition of the suspension of the adsorption solution and the magnetic beads
was similar to those used in the Experiment 1, and a silicone oil was used as the first,
third, and fifth plugs 10, 30, and 50 as in the case of the Experiment 1.
[0142] A tris-HCl buffer (solute concentration of 5 mM) of pH 8.0 was used as the first
washing solution of the second plug 20. Sterilized water was used as the elution
solution of the fourth plug 40.
[0143] Using a pipette, 50 uL of the blood collected from a human subject was added to the
adsorption chamber 120 through the opening therein. The adsorption chamber 120 was
fitted with lid 122 and the content was mixed vigorously by hands for 30 seconds.
Thereafter, the lid 122 of the adsorption chamber 120 was removed and the adsorption
chamber 120 was connected to the tube 100. The tube 100 had the stoppers 110 at both
ends thereof, and the stopper 110 at the end closer to the first plug 10 was removed
before connecting the adsorption chamber 120 to the tube 100.
[0144] The permanent magnets 410 were moved by hands and the magnetic particles M in
the adsorption chamber 120 were introduced into the tube 100. Then, the magnetic
particles M were moved to the fourth plug 40. The magnetic particles M were present
in the plugs in the tube 100 for approximately the following time durations: 3 seconds
each in the first and third plugs 10 and 30, 20 seconds in the second plug 20, and 30
seconds in the fourth plug 40. In the second plug 20, the magnetic particles M were not
moved laterally. In addition, the volumes of the second plug 20 and the fourth plug 40
were 25 uL and 1 uL, respectively.
[0145] Next, the stopper 110 at the end of the tube 100 at the side of the fifth plug 50 was
removed, and the adsorption chamber 120 was deformed by hands to discharge the
fifth plug 50 and the fourth plug 40 into a reaction chamber for PCR. This operation
was performed after the magnetic particles M were moved using the permanent
magnets 410 back to the second plug 20.
[0146] Subsequently, 19 uL of PCR reagent was added to the elution solution to perform
real-time PCR according to an ordinary method. The PCR reagent used was a mixture
of: 4 uL of LightCycler 480 Genotyping master (Roche Diagnostics 4 707 524), 0.4 uL
of SYBR Green I (S7563 available from Life Technologies) diluted to 1/1000 with
sterilized water, 0.06 uL of 100 uM beta-actine primers (F/R), and 14.48 uL of
sterilized water.
[0147] The amplification curves were similar to those shown in Fig. 12. In this experiment,
when a similar experiment was performed using 76 wt. of guanidine hydrochloride
as the first washing solution of the second plug 20, a delay of the rising edge corre
sponding to 10 cycles or more was observed as compared to the amplification curve
obtained in the Experiment 1.
[0148] 5.4. Experiment 4
(Effect of elution temperature on the yield of DNAs)
In the Experiment 4, the nucleic acids were extracted using an ordinary method of
extracting nucleic acids.
[0149] First, a suspension of 375 uL of the adsorption solution, and 20 uL of the magnetic
beads (the magnetic particles M) was contained in a polyethylene vessel (the ad
sorption chamber 120) having a volume of 1.5 mL. The composition of the suspension
of the adsorption solution and the magnetic beads was similar to the one described in
the aforementioned experiment.
[0150] Next, using a pipette, 50 uL of the genomic DNA solution adjusted to 1 ng/uL was
added to the adsorption chamber 120 through the opening therein. The adsorption
chamber 120 was fitted with the lid 122 and the content was mixed vigorously for 10
minutes using a vortex mixer. B/F separation was then performed using the magnetic
stand and the pipette. In this state, the magnetic particles M and a small amount of the
adsorption solution were left in the adsorption chamber 120.
[0151] Next, 450 uL of the first washing solution having the same composition as in the Ex
periment 1 was introduced into the adsorption chamber 120, and the lid 122 was fitted
to the adsorption chamber 120. The content was mixed vigorously for 5 seconds using
a vortex mixer. The first washing solution was removed using the magnetic stand and
the pipette. This operation was repeated twice. In this state, the magnetic particles M
and a small amount of the first washing solution were left in the adsorption chamber
120.
[0152] Next, 450 uL of the second washing solution having the same composition as in the
Experiment 1 was introduced into the adsorption chamber 120, and the lid 122 was
fitted to the adsorption chamber 120. The content was mixed vigorously for 5 seconds
using a vortex mixer. The second washing solution was removed using the magnetic
stand and the pipette. This operation was repeated twice. In this state, the magnetic
particles M and a small amount of the second washing solution were left in the ad
sorption chamber 120.
[0153] Furthermore, 50 uL of sterilized water (the elution solution) was added to the ad
sorption chamber 120. The adsorption chamber 120 was fitted with the lid 122 and the
content was mixed vigorously for 5 seconds using a vortex mixer, which was heated
for 2 minutes using a tube heater. Thereafter, the content was mixed vigorously for
another 10 seconds using the vortex mixer. A supernatant was collected using the
magnetic stand and the pipette. Three different temperatures were used to heat the tube
heater: 23 °C (allowed to stand at a room temperature), 45°C, and 65°C.
[0154] Subsequently, 1 uL of the elution solution was removed into a new tube and 19 uL of
PCR reagent was added to the elution solution to perform real-time PCR according to
an ordinary method. As a comparative sample, a genomic DNA solution adjusted to 1
ng/uL was also added to the PCR sample. The PCR reagent used was a mixture of: 4
uL of LightCycler 480 Genotyping master (Roche Diagnostics 4 707 524), 0.4 uL of
SYBR Green I (S7563 available from Life Technologies) diluted to 1/1000 with
sterilized water, 0.06 uL of 100 uM beta-actine primers (F/R), and 14.48 uL of
sterilized water.
[0155] The relationship between the elution temperature and the yield of DNAs is shown in
Fig. 13.
Fig. 13 is a graph showing the relationship between the elution temperature and the
yield of DNAs in this embodiment. This result was calculated from the cycle of the
rising edge of the real-time PCR. The yield of DNAs can be given as a ratio relative to
the comparative sample (corresponding to 1) by the equation 2 (CtO - Ctl), in which
CtO represents cycle of the rising edges for the comparative sample and Ctl represents
a cycle of the rising edges for the extraction sample.
[0156] 5.5. Results of the experiments
The following things were found from the aforementioned experiments.
(1) When the time duration required for the extraction of the nucleic acid, that is a
pre-treatment of PCR, was compared, the time after the insertion of the sample into the
adsorption chamber 120 and before the introduction of the target nucleic acids into the
reaction chamber for PCR was approximately 2 minutes in the Experiment 1. It was
approximately 30 minutes in the Experiment 2. From these results, it was found that
the time required for the extraction of the nucleic acids was significantly shorter in the
method for the extraction of nucleic acids of the Experiment 1 than in the method for
the extraction of nucleic acids of the Experiment 2.
[0157] (2) In addition, the amount of the washing solution in the Experiment 1 was 1/18 of
the amount in the Experiment 2. Furthermore, the amount of the elution solution in the
Experiment 1 was about 1/50 of the amount in the Experiment 2. It was thus found that
the Experiment 1 was significantly smaller in the amounts of the washing solution and
the elution solution as compared to the Experiment 2.
[0158] (3) Moreover, when the concentration of the target nucleic acids in the elution
solution is compared under the assumption that the amounts of the adsorption solution
and the elution solution are the same, the concentration is ideally 50 times higher in the
Experiment 1 than in the Experiment 2. In the experiments described above, however,
the blood sample contains a large amount of the nucleic acids, which exceeds the
maximum amount that can be adsorbed onto 1 uL of the magnetic particles M. Ac
cordingly, not all the nucleic acids contained in the blood sample can be collected, so
the concentration in the Experiment 1 is not 50 times higher than that in the Ex
periment 2. For such samples that contain a small amount of the nucleic acids and
these nucleic acids can all be adsorbed onto 1 uL of the magnetic particles M, the con
centration in the Experiment 1 will be 50 times higher than that in the Experiment 2.
[0159] (4) From the graph shown in Fig. 12, it was found that the rising edge of the ampli
fication curve for the nucleic acids was observed about 0.6 cycle earlier in the Ex
periment 1 than in the Experiment 2 even in the whole blood sample containing a large
amount of the nucleic acids. In other words, it was found that the reaction solution for
PCR used in the Experiment 1 contained the target nucleic acids at a higher con
centration than in the reaction solution for PCR used in the Experiment 2. This
confirmed that the concentration of the target nucleic acids in the elution solution was
higher in the Experiment 1 than in the Experiment 2.
[0160] (5) From the result in the Experiment 3, it was found that nucleic acids could be
extracted enough even if the second plug 20 contains a buffer. When the guanidine
solution was used as the second plug 20, it was found that a significant delay of the
rising edge of the PCR amplification curve occurred due to the inhibition of the
enzyme reaction. In addition, it was also found that the effect of the inhibition of the
enzyme reaction in the guanidine solution could be reduced by diluting the elusion
solution by a factor of at least 1000.
[0161] (6) From the result in the Experiment 4, it was found that the yield of DNAs could be
large enough for use in PCR when the temperature of the fourth plug 40 was around 40
°C or higher.
[0162] (Embodiment 2)
A method of externally applying a magnetic force that is different from that
described in the Embodiment 1 is described according to the method of manipulation
of the present invention.
Figs. 14 and 15 are views showing an exemplified configuration of the permanent
magnet 410 and the tube 100 according to this embodiment. Fig. 14 shows a structure
and Fig. 15 shows a moving procedure.
[0163] The permanent magnet 410 is connected to the magnetic force application unit 400.
Either one or both of the permanent magnet 410 and the tube 100 are connected to a
moving mechanism that can change the relative position between them. As shown in
the Embodiment 1, as a method of introducing the magnetic particles M carrying the
nucleic acids adsorbed thereon into the tube 100 having the first washing solution
forming the second plug 20 and the elution solution forming the fourth plug 40 that are
filled as the plugs separated by the oil forming the third plug 30, and moving them in
the tube 100, the permanent magnet 410 of the magnetic force application unit 400
having this single permanent magnet 410 is moved closer to the side of the tube 100. In
this case, the tube 100 and the permanent magnet 410 are not necessarily contact to
each other, provided that the magnetic force generated by the permanent magnet 410
acts on the magnetic particles M within the tube 100. After the permanent magnet 410
is moved closer to the side of the tube 100, as shown in Fig. 15, the permanent magnet
410 is moved down in the longitudinal direction of the tube 100 while being rotated
around the side of the tube 100 (A to H in the figure). As a result, the magnetic
particles M pass through many regions of the reagents (the first washing solution
forming the second plug 20 or the elution solution forming the fourth plug 40)
provided as the plug surrounded by the inner wall of the tube 100. In this way, the
magnetic particles M are washed with the reagent in an efficient manner.
[0164] In this embodiment, in order to move the magnetic particles M in the tube 100, what
is required is to change the relative position between the permanent magnet 410 and
the side of the tube 100 close to it. As described in the embodiments, the magnetic
force application unit 400 having the permanent magnet(s) 410 may be moved around
the side of the tube 100, or alternatively, the tube 100 may rotate on the longitudinal
axis of the tube 100 rather than rotating the magnetic force application unit 400 around
the tube 100. Alternatively, the rotation of the magnetic force application unit 400 may
be combined with the rotation of the tube 100.
[0165] (Embodiment 3)
A method of externally applying a magnetic force that is different from that
described in the aforementioned embodiments is described according to the method of
manipulation of the present invention.
Fig. 16 is a view showing an exemplified configuration of the permanent magnets
410A, 410B, 410C and the tube 100 according to this embodiment. Fig. 16(A) shows a
structure and Fig. 16(B) shows a moving procedure.
[0166] The permanent magnets 4 1OA, 4 10B, 4IOC are connected to the magnetic force application
unit 400. Either the permanent magnets 4 1OA, 4 1OB, 4 IOC or the tube 100, or
alternatively, both of them is/are connected to a moving mechanism that can change
the relative position between them. In this embodiment, at least three permanent
magnets 4 1OA, 4 10B, 4 IOC are used to move the magnetic particles M introduced into
the tube 100 to at least three points in a cross-sectional plane perpendicular to the lon
gitudinal direction of the tube 100, using the following procedures. In this way, as
shown in Fig. 16(B), the magnetic particles M can be washed with the reagent more e f
ficiently by allowing the magnetic particles M to pass through the regions of the
reagent which otherwise they do not pass through when they merely travel in the liquid
in the tube 100 between the permanent magnets.
[0167] Following is a description of a process of a method of externally applying a magnetic
force for use in manipulating the magnetic particles M in the liquid in the tube 100
using at least three permanent magnets 410A, 410B, 410C. In this example, a distance
between the tube 100 and any one of the three or more permanent magnets 4 1OA,
4 10B, 4 IOC is smaller than the distances between the tube 100 and the remaining
permanent magnets.
[0168] 1. At the step 1, the permanent magnet 410A is moved closer to near a first position,
i.e., a point "a" on the side of the tube 100 and a relative distance with respect to the
tube 100 is thus decreased. The remaining permanent magnets 4 10B, 4 IOC are further
away from the tube 100 than the permanent magnet 4 1OA. The magnetic field is thus
strongest at and near the point "a" around the tube 100.
[0169] 2. At step 2, the permanent magnet 4 1OA is moved away from the position near the
point "a" and the relative distance with respect to the tube 100 is thus increased. On the
other hand, the permanent magnet 410B is moved closer to near a second position, i.e.,
a point "b" on the side of the tube 100. The point "b" is different from the point "a" and
is located at a position from which the magnetic field generated can reach the point "a"
sufficiently. The relative distance between the permanent magnet 4 10B and the tube
100 is thus decreased. As in the step 1, the remaining permanent magnets 4 1OA, 4 IOC
are away from the tube 100 at a longer distance than the permanent magnet 4 10B so
that the magnetic field is strongest at and near the point "b" in the space around the
tube 100.
[0170] 3. At step 3, the permanent magnet 4 10B is moved away from the position near the
point "b" and the relative distance with respect to the tube 100 is thus increased. On the
other hand, the permanent magnet 4 IOC is moved closer to near a third position, i.e., a
point "c" on the side of the tube 100. The point "c" is different from the points "a" and
"b" and is located at a position from which the magnetic field generated can reach the
point "b" sufficiently. The relative distance between the permanent magnet 4 IOC and
the tube 100 is thus decreased. As in the step 1, the remaining permanent magnets
4 1OA, 4 1OB are away from the tube 100 at a longer distance than the permanent
magnet 410C so that the magnetic field is strongest at and near the point "c" in the
space around the tube 100.
[0171] 4. The operation returns to the step 1. The point "a" on the side of the tube 100 to
which the permanent magnet 4 1OA approaches may be the same or different from the
point "a" in the step 1. It should be noted, however, that the point "a" is a different
position from the points "b" and "c" in the steps 2 and 3. In addition, it is preferable
that the point "a" is located at a position from which the magnetic field generated can
reach the point "c" sufficiently.
[0172] The magnetic particles M can be moved in the liquid in the tube 100 among at least
three points in the plane perpendicular to the longitudinal direction of the tube 100 by
moving the magnetic force application unit 400 having the permanent magnets 4 1OA,
4 10B, 4IOC using the process described above. In addition, in the aforementioned
steps, the magnetic force application unit 400 having the permanent magnets 410A,
4 10B, 4IOC is moved. However, either one or both of the magnetic force application
unit 400 and the tube 100 may be moved as long as the relative position between each
of the permanent magnet 4 1OA, 4 10B, 4IOC and the tube 100 is altered in a similar
manner. Furthermore, movement of the magnetic particles M in the longitudinal
direction of the tube 100 may be made in addition to the movement of the magnetic
particles M in the aforementioned cross-sectional plane of the tube 100. The magnetic
particles M are successively moved through the reagents having different functions
while passing through many regions in the liquid to do the intended washings.
[0173] While the embodiments have thus been described specifically, the present invention
is not limited to the aforementioned embodiments and various other modifications can
be made.
[0174] (Modified version 1)
In the aforementioned embodiments, a combined motion is used in which the ro
tational movement of the permanent magnet 410 around the periphery of the tube 100
is combined with the relative movement to alter the distance between the permanent
magnet 410 and the axis of the tube 100. As a result, the magnetic particles M travel
along the inner surface of the side of the tube 100. By changing the distance between
the permanent magnet 410 and the tube 100, the magnitude of the magnetic force
around the tube 100 is also varied. The magnetic particles M in the tube 100 are r e
peatedly gathered and dispersed. As a result, the magnetic particles M reside in many
regions in the reagents, which increases the efficiency of the washing.
[0175] (Modified version 2)
Although the aforementioned embodiments describe and illustrate how the magnetic
field is changed around the single tube 100, two or more tubes 100 may be arranged in
parallel or in circle. A similar method of changing the magnetic force may be used to
simultaneously manipulate the magnetic particles M in these tubes 100.
[0176] In this case, permanent magnets similar to the pair of permanent magnets 410A,
4 10B in the Embodiment 1 are arranged relative to the tubes 100 that are disposed in
parallel. These two or more pairs of permanent magnets 4 1OA, 4 10B are operated in a
similar manner to those described above to simultaneously move the magnetic particles
M in the tubes 100 in the back-and-force direction as well as in the up-and-down
direction.
[0177] In addition, a permanent magnet is placed near the central axis of the circle formed
by the tubes 100 that are arranged annually. A cylindrical permanent magnet is
provided at a position outside the circle formed by the tubes 100. The outer permanent
magnet moves in an eccentric path and the permanent magnet near the center moves in
an eccentric path in synchronism with the outer permanent magnet. In this way, the
magnitude of the magnetic field is changed over a period of time between the outer and
inner sides of the tubes 100 that are arranged in circle.
[0178] (Modified version 3)
(Apparatus for the extraction of nucleic acids)
Fig. 17 is a perspective view showing an example of an apparatus for the extraction
of nucleic acids according to this modified version. An apparatus for the extraction of
nucleic acids 3100 is similar to the aforementioned apparatus for the extraction of
nucleic acids 3000 except for a heating unit 600. The components and parts providing
similar functions and operations are thus represented by like reference numerals, and
description thereof will be omitted.
[0179] The heating unit 600 is for heating a part of the tube 100 after the tube 100 is
mounted on the tube mount 300. The heating unit 600 may be, for example, a heat
source and a heat block, a heater, and a coil for electromagnetic heating. The heating
unit 600 may have any shape adapted to receive the tube 100 inserted thereinto or to
contact with the side of the tube 100, as long as the heating unit 600 can heat the liquid
in the tube 100.
[0180] A portion of the tube 100 heated by the heating unit 600 include a region where the
fourth plug 40 is present in the longitudinal direction of the tube 100. The heating unit
600 may heat other portions of the tube 100. It is preferable, however, that the heating
unit 600 does not heat the portion corresponding to the second plug 20 in the lon
gitudinal direction of the tube 100.
[0181] In the apparatus for the extraction of nucleic acids 3100 shown in Fig. 17, a heater
610 is provided as the heating unit 600. The heater 610 is disposed in parallel to the
support plate 310 and is adapted to heat a position including the fourth plug 40 in the
tube 100. The heater 610 is in contact to an approximately half of the outer peripheral
surface of the tube 100.
[0182] The apparatus for the extraction of nucleic acids 3100 can elute a sufficient amount
of the nucleic acids into the elution solution forming the fourth plug 40 by means of
the washing with at least one of the first washing solution forming the second plug 20
and the second washing solution forming the sixth plug 60 even when the amount of
the nucleic acids adsorbed on the magnetic particles M is not so large. This contributes
to increasing the washing efficiency and eluting the nucleic acids into the elution
solution at a concentration sufficient for PCR.
[0183] (Modified version 4)
(Liquid reservoir)
Fig. 18 is a view diagrammatically showing a device for the extraction of nucleic
acids according to this modified version. As shown in Fig. 18, a device for the ex
traction of nucleic acids 1040 has a liquid reservoir (vessel) 130 formed at the end of
the tube 100 at the side of the first plug 10 in communication with the tube 100. The
liquid reservoir 130 is communicated with the tube 100.
[0184] The liquid reservoir 130 can hold liquid therein. The liquid reservoir 130 has an
opening 131 through which a substance can be introduced into the liquid reservoir 130
from outside. The position at which the opening 131 is formed in the liquid reservoir
130 is not specifically limited. The liquid reservoir 130 may have two or more
openings 131. The inner volume of the liquid reservoir 130 is not specifically limited,
and may be, for example, equal to or larger than 0.1 mL but not larger than 100 mL.
The material for use in making the liquid reservoir 130 is not specifically limited, and
may be, for example, a polymer or a metal. It may be the same as the material used to
make the tube 100.
[0185] By providing the liquid reservoir 130 as in the case of the device for the extraction of
nucleic acids 1040, the magnetic particles M, the adsorption solution, and the sample
can be contained in the liquid reservoir 130 to allow the magnetic particles M to adsorb
the nucleic acids. These magnetic particles M can easily be introduced into the tube
100 through the end of the tube 100 at the side of the first plug 10.
[0186] In addition, the liquid reservoir 130 can be shaken along with the tube 100 to mix the
liquid in the liquid reservoir 130 vigorously. This results in the rapid adsorption of the
nucleic acids onto the magnetic particles M. Furthermore, by appropriately changing
the amount of the sample to be introduced into the liquid reservoir 130 and the volume
of the liquid in the tube 100, the nucleic acids in the sample can be concentrated quan
titatively in the elution solution.
[0187] When the liquid reservoir 130 is provided as in the case of the device for the ex
traction of nucleic acids 1040, a lid 132 may be provided to seal the opening 131 in the
liquid reservoir 130. The lid 132 is freely attached to the liquid reservoir 130 and
removed therefrom. When a flexible and elastic material such as a rubber, an
elastomer, or a polymer is selected as a material of the liquid reservoir 130, the inside
of the tube 100 can be pressurized by deforming the liquid reservoir 130 with the lid
132 being fitted to the liquid reservoir 130.
[0188] This facilitates the application of a pressure to the tube 100 at the end thereof at the
side of the first plug 10 in order to discharge the elution solution forming the fourth
plug 40 having the nucleic acids eluted thereinto, from the tube 100 through the end at
the side of the fifth plug 50. As a result, it becomes possible to perform from the step
of introducing the sample into the liquid reservoir 130 to the step of easily removing
the elution solution into, for example, a reaction chamber for PCR. In addition, the lid
132 can serve to prevent the liquid in the liquid reservoir 130 from being leaked
therefrom when the liquid reservoir 130 is shaken along with the tube 100, which
improves the efficiency of adsorbing the nucleic acids onto the magnetic particles M.
[0 189] (Modified version 5)
(Variation of the step of moving the magnetic particles)
Fig. 19 is a diagrammatic representation for use in illustrating a method for the ex
traction of nucleic acids of this modified version.
In the aforementioned "4.4. Step of moving the magnetic particles," the magnetic
particles M are moved from the first plug 10 to the fourth plug 40 through the in
termediate plugs(s) by externally applying a magnetic force to the magnetic particles
M. The magnetic force that is externally applied may, however, be varied to shake,
collect, and disperse the magnetic particles M in the second plug 20 when the magnetic
particles M are moved to the second plug 20. This can improve the effect of washing
the magnetic particles M using the first washing solution forming the second plug 20.
[0190] More specifically, as shown in I and J in Fig. 19, when a pair of permanent magnets
410 is used as means for applying the magnetic force, the magnetic particles M are
moved from the adsorption chamber 120 and passed through the first plug 10 using the
permanent magnets 410. The magnetic particles M then reach the second plug 20. At
that time, one of the permanent magnets 410 is moved away from the tube 100 and the
other of the permanent magnets 410 is moved closer to the tube 100 from the opposite
side. This results in the shaking of the magnetic particles M in the second plug 20 in
the direction crossing the longitudinal direction of the tube 100 (repeated between I
and J in the figure). This can improves the effect of washing the magnetic particles M
using the first washing solution forming the second plug 20. This washing operation
using the magnetic particles M may be applied to the second plug 20 and/or the sixth
plug 60 in the case where the second plug 20 is divided into smaller plugs or in the
case where the sixth plug 60 is provided within the tube 100.
[0191] In addition, as shown in K in Fig. 19, the magnetic particles M can be distributed in
the second plug 20 merely by means of moving the permanent magnet 410 away from
the tube 100. The magnetic particles M is less likely to be entered into the first plug 10
or the oil forming the third plug 30 even when they are distributed in the second plug
20 with a smaller magnetic force because each of the magnetic particles M has a hydrophilic
surface. Therefore, such an aspect may also be used.
[0192] More specifically, when the magnetic particles M reach the second plug 20 after the
magnetic particles M are moved from the adsorption chamber 120 and passed through
the first plug 10 using the permanent magnets 410, the permanent magnets 410 are
moved away from the tube 100 to distribute the magnetic particles M in the second
plug 20. Then, the magnetic particles M are again moved and passed through the third
plug into the fourth plug 40 using the magnetic force generated by the permanent
magnets 410.
[0193] The aspect in which the magnetic force that is externally applied is changed to shake
and repeatedly collect and disperse the magnetic particles M may be applied to a case
where the magnetic particles M are present in the adsorption solution in the adsorption
chamber 120 or a case where the magnetic particles M are present in the fourth plug 40
(the elution solution).
[0194] (Modified version 6)
(Variation of the step of eluting the nucleic acids)
The aforementioned "4.5. Step of eluting the nucleic acids" may be performed while
heating the fourth plug 40. The fourth plug 40 can be heated using one of appropriate
methods including, for example, by contacting a thermal medium such as a heat block
to a position corresponding to the fourth plug 40 in the tube 100, by using a heat
source such as a heater, or using electromagnetic heating.
[0195] When the fourth plug 40 is heated, the plugs other than the fourth plug 40 may also
be heated. It is, however, preferable that the plug of the washing solution is not heated
if the magnetic particles M carrying the nucleic acids adsorbed thereon are present in
the plug of the washing solution. The temperature to be reached while heating the
fourth plug 40 is preferably equal to or higher than 35°C but not higher than 85°C,
more preferably, equal to or higher than 40°C but not higher than 80°C, and yet more
preferably, equal to or higher than 45°C but not higher than 75°C, in terms of the e f
ficiency of the elution as well as in terms of inhibiting deactivation of an enzyme for
PCR when the enzyme is contained in the elution solution.
[0196] In the step of eluting the nucleic acids, it is possible to elute the nucleic acids carried
by the magnetic particles M into the elution solution in a more efficient manner with
the fourth plug 40 being heated. In addition, it is possible to elute, into the elution
solution, the nucleic acids that are left on the magnetic particles M without having
been eluted in the washing solution even when the composition of the first washing
solution or the second washing solution is identical or similar to the composition of the
elution solution. In other words, the nucleic acids can further be eluted into the elution
solution after the magnetic particles M having the nucleic acids adsorbed thereon are
washed with the first washing solution or the second washing solution. It is thus
possible to achieve both the sufficient washing and the elution at a sufficient con
centration into the elution solution even when the composition of the washing solution
is identical or similar to the composition of the elution solution.
[0197] (Modified version 7)
(Variation of the step of discharging the fourth plug from the tube)
When the aforementioned "4.6. Step of discharging the fourth plug from the tube" is
used, the magnetic particles M after the elution of the adsorbed nucleic acids into the
elution solution in that step may be present in the fourth plug 40. Alternatively, the
magnetic particles M may be transferred to either one of the first plug 10, the second
plug 20, and the third plug 30 or to the adsorption chamber 120 by further applying a
magnetic force. This process makes it possible to discharge the fourth plug 40 from the
tube 100 without containing any magnetic particles M in the elution solution. When the
magnetic particles M is transferred to the second plug 20 or the adsorption chamber
120, the magnetic particles M are less likely to enter the oil forming the third plug 30
after the magnetic force is removed. Accordingly, the fourth plug 40 can be discharged
more easily from the tube 100.
[0198] The present invention is not limited to the aforementioned, embodiments, and
various other modifications can be made. For example, the present invention includes a
configuration that is substantially similar to the configurations described in the em
bodiments (such as a configuration that is identical in function, method, and result or a
configuration that is identical in object and effect). In addition, the present invention
includes a configuration in which a non-essential part of the configuration described in
one embodiment has been replaced with. Furthermore, the present invention includes a
configuration that has the same effect or that achieves the same object as the con
figuration described in one embodiment. Moreover, the present invention includes a
configuration that is a combination of the configuration described in the embodiments
with a known technique.
Reference Signs List
[0199] 10 ... first plug
20 ... second plug
30 ... third plug
40 ... fourth plug
50 ... fifth plug
60 ... sixth plug
70 ... seventh plug
100 ... tube (vessel)
110 ... stopper
120 ... adsorption chamber (vessel)
121 ... opening
122 ... lid
130 ... liquid reservoir (vessel)
131 ... opening
132 ... lid
300 ... tube mount
310 ... support plate
320 ... clip mechanism
330 ... hinge
340 ... guiding rail
350 ... drive belt
360 ... moving mechanism
400 ... magnetic force application unit
410, 4 1OA, 4 10B, 4IOC ... permanent magnet
420 ... motor
500 ... moving mechanism
600 ... heating unit
610 ... heater
1000, 1010, 1020, 1030, 1040, 1100 ... device for the extraction of nucleic acids
2000 ... kit for the extraction of nucleic acids
3000, 3100 ... apparatus for the extraction of nucleic acids (apparatus of manipulating a
solid carrier)
M ... magnetic particles (solid carrier)

PCT/JP2013/006657
Claims
[Claim 1] A method of manipulating a solid carrier in a liquid contained in a
vessel, the solid carrier being magnetic and capable of carrying a
substance, the method comprising:
manipulating the solid carrier using a magnetic force application unit
adapted to applying a magnetic force to the solid carrier in the vessel so
that the relative position between the magnetic force application unit
and the vessel is altered over a period of time by means of directing the
solid carrier in a direction along either one or a combination of the
vectors of the longitudinal direction of the vessel and a cross-sectional
direction crossing the longitudinal direction of the vessel.
[Claim 2] The method of manipulating a solid carrier according to Claim 1,
wherein:
the magnetic force application unit comprises at least one permanent
magnet.
[Claim 3] The method of manipulating a solid carrier according to Claim 2,
wherein:
the position of the solid carrier is changed in the vessel by moving the
permanent magnet to a position of the peripheral surface of the vessel
and then moving the permanent magnet away from the position to
move the permanent magnet closer to another position of the peripheral
surface of the vessel.
[Claim 4] The method of manipulating a solid carrier according to Claim 2 or 3,
wherein:
the solid carrier is directed using two permanent magnets that are
opposed to each other at a certain distance.
[Claim 5] The method of manipulating a solid carrier according to any one of
Claims 1 to 3, wherein:
the solid carrier is directed within the vessel as a result of the relative
rotation between the magnetic force application unit and the vessel.
[Claim 6] The method of manipulating a solid carrier according to Claim 2 or 3,
wherein:
at least three permanent magnets are provided, and the solid carrier is
directed in at least two directions in a plane perpendicular to the lon
gitudinal direction of the vessel as a result of the relative displacement
of the position between the vessel and the permanent magnets.
[Claim 7] An apparatus of manipulating a solid carrier in a liquid contained in a
WO 2014/073218 PCT/JP2013/006657
vessel, the solid carrier being magnetic and capable of carrying a
substance, the apparatus comprising:
a magnetic force application unit adapted to applying a magnetic force
to the solid carrier in the vessel, the solid carrier being manipulated so
that the relative position between the magnetic force application unit
and the vessel is altered over a period of time by means of directing the
solid carrier in a direction along either one or a combination of the
vectors of the longitudinal direction of the vessel and a cross-sectional
direction crossing the longitudinal direction of the vessel.
[Claim 8] A method of manipulating a magnetic solid carrier in a vessel
comprising liquid, the vessel having a longitudinal direction, the
method comprising the step of:
altering a relative position between the magnetic force application unit
adapted to applying a magenetic force to the solid carrier from outside
the vessel and the vessel is altered over a period of time, to direct the
solid carrier, in one of a planar direction crossing the longitudinal
direction of the vessel, the longitudinal direction of the vessel, and a
direction determined by combining the vectors of the longitudinal
direction of the vessel and the planar direction crossing the longitudinal
direction of the vessel.
[Claim 9] The method of manipulating a solid carrier according to Claim 8,
wherein:
the magnetic force application unit comprises at least one permanent
magnet.
[Claim 10] The method of manipulating a solid carrier according to Claim 8 or 9,
comprising:
moving the magnet closer to a first position of the vessel;
moving the magnet away from the first position;
moving the magnet closer to a second position of the vessel, the second
position being different from the first position; and
moving the magnet away from the second position; wherein the solid
carrier is directed in the vessel thereby.
[Claim 11] The method of manipulating a solid carrier according to Claims 9 or 10,
wherein:
the magnet is two permanent magnets that are opposed to each other at
a certain distance.
[Claim 12] The method of manipulating a solid carrier according to Claim 11,
comprising
WO 2014/073218 PCT/JP2013/006657
decreasing a relative distance between a first magnet and the vessel
while increasing a relative distance between a second magnet and the
vessel, the second magnet being opposed to the first magnet; and
increasing the relative distance between the first magnet and the vessel
while decreasing the relative distance between the second magnet and
the vessel; wherein the solid carrier is directed in the vessel thereby.
[Claim 13] The method of manipulating a solid carrier according to Claim 8 or 9,
wherein:
the solid carrier is directed in the vessel by means of rotating the
magnetic force application unit around the vessel.
[Claim 14] The method of manipulating a solid carrier according to Claim 9,
wherein:
at least three permanent magnets are provided, and one of a first
distance, second distance, and a third distance being determined to be
smaller than the remaining two distances, the first distance representing
a distance between the vessel and a first magnet, the second distance
representing a distance between the vessel and a second magnet, and
the third distance representing a distance between the vessel and a third
magnet; and the solid carrier is directed in a plane perpendicular to the
longitudinal direction of the vessel thereby.
[Claim 15] An apparatus of manipulating a magnetic solid carrier in a vessel
comprising liquid, the vessel having a longitudinal direction, the
apparatus comprising:
magnetic force application unit adapted to applying a magnetic force to
the solid carrier from outside the vessel, wherein the magnetic force ap
plication unit is manipulated so that the relative position between the
magnetic force application unit and the vessel is altered over a period
of time, to direct the solid carrier, by using the magnetic force ap
plication unit, in one of a planar direction crossing the longitudinal
direction of the vessel, the longitudinal direction of the vessel, and a
direction determined by combining the vectors of the longitudinal
direction of the vessel and the planar direction crossing the longitudinal
direction of the vessel.
[Claim 16] The apparatus of manipulating a solid carrier according to Claim 15,
wherein:
the vessel is a tube having a first oil plug, a washing solution plug, a
second oil plug, an elution solution plug, and a third oil plug in the lon
gitudinal direction thereof.