Note: Descriptions are shown in the official language in which they were submitted.
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DIRECT ASPIRATION-REACTION AND INJECTION DEVICE AND
METHODS OF USE
Field of the Invention:
The present invention relates to a device which is suitable for
aspiration and analysis of biological samples, and for injection of samples
into biological materials. The present invention also relates to the use of
this
device in methods for detecting target molecules in samples.
1o Background of the Invention:
Clinical diagnostic assays typically involve the collection of biological
samples and subsequent analysis of those samples by assay procedures. In
many cases, the sample collection and subsequent analysis are conducted in
different areas of the laboratory, or outside the laboratory, thereby
15 necessitating transport and/or transfer of biological samples and assay
reagents. The handling of these samples gives rise to increased risk of
contamination by other biological materials.
The risk of contamination is of particular concern when the process
includes nucleic acid amplification reactions such as the polymerase chain
20 reaction (PCR), which is capable of multiplying a single strand of nucleic
acid (target nucleic acid) into millions of copies (amplicons). Although PCR
technology is of enormous potential utility in the clinical diagnostic
laboratory, nucleic acid amplification reactions can easily become
contaminated with foreign nucleic acid molecules. For example,
25 contamination may occur during the sampling procedure. In addition, the
sample may become contaminated with the amplification products
(amplicons) of previous amplification reactions, where opening of the
reaction chamber, and the utilization of standard pipetting techniques,
creates aerosols. Such contaminants may be amplified in the PCR reaction,
30 leading to a false positive indication of the substrate to be detected and
ultimately to incorrect diagnosis.
Due the problems discussed above, many reactions, particularly highly
sensitive PCR assays, are slow and require considerable operator skill in
order to ensure that reliable results are routinely obtained.
PCT/AU00/00931
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2
Accordingly, products and procedures which simplify sample
collection and analysis and which reduce the risk of contamination are
highly desirable.
Summary of the Invention:
The present inventors have now developed a device which reduces
problems associated with the multiple steps involved in clinical diagnostic
assays, allowing accurate results to be obtained more reliably. In particular,
the device enables the sample collection and analysis to be performed in a
single chamber, thereby reducing the risk of contamination.
In a first aspect, the present invention provides a device comprising a
chamber having a first open end and a second closed end and an inner and
an outer surface, an elongate member having first and second open ends and
having an inner and an outer surface, and sealing means providing a seal
z5 between the elongate member and the chamber, the second end of the
elongate member being slidably movable from a first position within the
chamber to a second position within the chamber, the movement of the
second end of the elongate member from the first position to the second
position causing a change in the pressure within the chamber sufficient to
2o enable uptake of fluid through the elongate member into the chamber.
In a preferred embodiment of the first aspect, the chamber is elongate.
In a further preferred embodiment of the present invention, the
chamber allows transmission of electromagnetic radiation. The second end
of the elongate chamber may comprises a lens for transmitting
25 electromagnetic radiation. Preferably, the electromagnetic radiation is
light
of a wave length between 400 to 800 nm. More preferably, the
electromagnetic radiation is fluorescence.
In a further preferred embodiment, the chamber is cylindrical.
Preferably, the chamber has an outer diameter of up to 2.8 mm, and/or an
3o inner diameter of up to 2.5 mm. Preferably, the chamber has a length of
between 20 to 40 mm.
Preferably, the elongate member has an outer diameter of up to 2.5 mm
and an inner diameter of between 0.2 mm and 1.2 mm.
In a further preferred embodiment, the first end of the elongate
35 member comprises a formed tip. Preferably, the tip is capable of piercing
biological tissue. In a preferred embodiment, the tip is in the order of l~.m,
AMEtv~Et~ SHEL-1'
~~E~t,IAE~
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3
but can extend up to 200~m or more in diameter. Preferably the diameter of
the tip is between 20 and40 ~.m.
In yet another embodiment, the chamber and elongate member are
formed of the same material. Preferably, this material is glass or plastic. In
a
preferred embodiment, however, the elongate chamber is made of plastic and
the elongated member is made of glass. Suitable plastics include, but are not
limited to, polyethylene terephthalate, polypropylene and polycarbonate.
In a preferred embodiment, the sealing means comprises a sleeve
located on the outer surface of the elongate member proximate the second
end. Preferably, the sleeve co-operates with the inner surface of the chamber
to form a seal. The sleeve may be of any suitable length. For example, the
sleeve may be between 10 and 80 mm in length. It will be appreciated,
however, that a longer sleeve may be desirable as it will impart additional
rigidity to the elongate member. The sleeve may co-operate with the inner
surface of the chamber along the entire length of the sleeve. It is preferred,
however, that the sleeve comprises a flared region of about 0.2 to 2.0 mm in
length which co-operates with the inner surface of the chamber to form the
seal.
In a p:eferred embodiment, the chamber further comprises a collar
2o proximate the first open end of the chamber, the collar comprising an inner
and an outer surface.
The device may further comprises a first guide located within the
collar. The first guide preferably comprises an annular outer surface which
co-operates with the inner surface of the collar thereby forming a seal
between the first guide and the inner surface of the collar. Preferably, the
first guide further comprises an aperture foi receiving the elongate member.
The elongate member may be received by the first guide through the aperture
such that a seal is formed between the external surface of the elongate
member and the guide.
3o Preferably, the device further comprises a second guide located on the
external surface of and proximate the first end of the elongate member. This
second guide may be used to hold the elongate member in place during
aspiration.
Since many biological reactions provide a quantitative result, it is
preferable to regulate the volume of sample used in the reaction.
Accordingly, in a further preferred embodiment, the device is adapted for use
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in conjunction with a means for regulating the volume of the sample
aspirated. The means for regulating the volume of the sample may be, for
example, a manual or an electronic pipettor. It will be appreciated that
currently available electronic pipettors may be used with the device of the
present invention. Examples of such electronic pipettors include the
UtlraMicroPump-Ih''~ and the Nanoliter 2000TM, both produced by World
Precision Instruments.
In yet another preferred embodiment, the interior surface of the
chamber and/or the elongate member is treated with a blocking agent in order
1o to prevent the sample from adhering to the chamber or elongate member.
The blocking agent may be selected from the group consisting of bovine
serum albumin, fetal calf serum, human serum albumin, polyvinyl alcohol,
polyvinyl pyrrolidone, a silicone compound and other suitable protein
sources. Most preferably, the solution comprises a silicone compound sold
under the trade name "Sigmacote" (Sigma).
In a further preferred embodiment, the elongate member is filled with
oil in order to facilitate the uptake of the sample.
The present invention also provides a device which allows a series of
separate reactions to be performed sequentially in the chamber.
2o Accordingly, in a second aspect, the present invention provides a
device according to the first aspect, wherein the chamber comprises
a first reagent composition;
a first wax layer providing a seal over the first reagent composition;
a second reagent composition positioned adjacent the first wax layer
and separated from the first reagent composition by the first wax layer; and
a second wax layer substantially covering the second reagent
composition;
wherein the second wax layer has a melting temperature which is
lower than the melting temperature of the first wax layer such that the first
wax layer is solid at the melting temperature of the second wax layer.
In a preferred embodiment of the second aspect at least one of, more
preferably each of, the first and second reagent compositions are in a liquid
form at room temperature. However, it is also envisaged that the reagent
compositions) may be solid at room temperature. For example, the reagent
compositions) may be lyophilized.
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In a third aspect, the present invention provides a device according to
the first aspect, wherein the chamber comprises
a first reagent composition in admixture with a first wax carrier; and
a second reagent composition in admixture with a second wax carrier;
5 wherein the second reagent composition is at least partially coated,
covered or layered upon the first reagent composition and wherein the
second wax carrier has a melting temperature which is lower than the first
wax carrier such that the first wax carrier is solid at the melting
temperature
of the second wax carrier.
1o In a fourth aspect the present invention provides a method for
detecting a substance in a sample, the method comprising
(i) introducing a sample and a reagent composition into the chamber
of a device according to the first aspect such that the sample and reagent
composition form a reaction mix, wherein the reagent composition comprises
at least one reagent which interacts with the substance to be detected such
that the level of electromagnetic radiation emitted by the reaction mix is
altered when compared to the level of electromagnetic radiation emitted by
the reagent composition alone;
(ii) optionally exciting the reaction mix with electromagnetic
2o radiation;
(iii) subjecting the chamber to conditions which allow the reaction to
occur, and
(iv) detecting the electromagnetic radiation emitted by the reaction
1111X.
In a fifth aspect the present invention provides a method for
amplifying at least one target nucleic acid molecule in a sample, the method
comprising
(i) introducing a sample and a reagent composition into the chamber
of a device according to the first aspect such that the sample and reagent
composition form a reaction mix, wherein the reagent composition comprises
reagents for amplification of the target sequence; and
(ii) subjecting the chamber to conditions which allow amplification
of the target sequence.
In a sixth aspect the present invention provides a method for
amplifying at least one target nucleic acid molecule in a sample, the method
comprising
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(i) introducing a sample and a reagent composition into the chamber
of a device according to the first aspect such that the sample and reagent
composition form a reaction mix,
wherein the reagent composition comprises reagents for amplification
of the target sequence and at least one reagent which interacts with the
amplified sequence such that the level of electromagnetic radiation emitted
by the reaction mix is altered when compared to the level of electromagnetic
radiation emitted by the reagent composition alone;
(ii) optionally exciting the reaction mix with electromagnetic
radiation;
(iii) subjecting the chamber to conditions which allow amplification
of the target sequence, and
(iv) detecting the electromagnetic radiation emitted by the reaction
mix.
In a seventh aspect, the present invention provides a method for
performing a series of reactions in a single vessel, the method comprising
(i) loading a sample into the chamber of a device according to the
second aspect such that the sample is positioned adjacent the second wax
layer;
(ii) heating the chamber to a temperature at which the second wax
layer melts but the first wax layer remains solid;
(iii) allowing the sample to interact with the second reagent
composition to form a reaction mix;
(iv) heating the chamber to a temperature at which the first wax layer
melts; and
(v) allowing the reaction mix to interact with the first reagent
composition.
In an eighth aspect the present invention provides a method for
performing a series of reactions in a single vessel, the method comprising
(i) loading a sample into the chamber of a device according to the third
aspect such that the sample is positioned adjacent the second reagent
composition in admixture with the second wax carrier;
(ii) heating the chamber to a temperature at which the second wax
carrier melts but the first wax carrier remains solid;
(iii) allowing the sample to interact with the second reagent
composition to form a reaction mix;
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(iv) heating the chamber to a temperature at which the first wax carrier
melts; and
(v) allowing the reaction mix to interact with the first reagent
composition.
hi a preferred embodiment of the fourth to sixth aspects, the sample is
introduced into the chamber through the elongate member.
In a preferred embodiment of the fourth and sixth aspects,
electromagnetic radiation is transmitted from an external source through the
chamber to the reaction mix. Furthermore, it is preferred that any alteration
1o in the electromagnetic radiation of the reaction mix is detected through
the
chamber.
In a preferred embodiment of the sixth aspect the reagent composition
comprises components which allow for the fluorescent based detection of an
amplified nucleic acid molecule product by oligonucleotide hybridization.
In a preferred embodiment of the second and third aspects, the first
wax layer has a melting temperature of about 70°C. Any wax which has a
melting temperature of about 70°C may be suitable. Preferably, the wax
is T
Wax (EnerGeneO) or F wax (20% paraflint C80N6 from Schumann Sasol,
80% T wax).
2o In a further preferred embodiment of the second and third aspects, the
second wax layer has a melting temperature of about 55°C. Any wax which
has a melting temperature of about 55°C may be suitable. Preferably,
the wax
is AmpliWax~ (PE Applied Biosystems).
In yet a further preferred embodiment of the second and third aspects,
an oil layer covers the second wax layer. The presence of the oil layer may
perform one or more of a number functions, including maintaining a sample
as a bolus adjacent the second wax layer, assisting transfer of the sample
fTOIIl the elongated member to the chamber, and reducing contamination.
In yet a further preferred embodiment of the second and third aspects,
the first reagent composition comprises reagents required to amplify a target
nucleic acid molecule. More preferably, the first reagent composition
comprises components which allow for fluorescent based detection of an
amplified nucleic acid product by oligonucleotide hybridization.
In yet a further preferred embodiment of the second and third aspects,
the second reagent composition comprises components which result in cell
lysis, protein digestion and/or modification, and/or DNA digestion and/or
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8
modification. Preferably, the second reagent composition comprises an
alkaline buffer in order to enhance cell lysis. More preferably, the second
reagent composition comprises proteinase K or protease in a buffer
compatible with proteinase K or protease activity.
In a preferred embodiment of the seventh and eighth aspects, step (iii)
and/or step (v) include the step of centrifuging the elongate chamber.
Preferably, the centrifugal force is between 5g and 200g, more preferably
between 5g and 70g. However, as would be appreciated by those skilled in
the art, the centrifugal force required will vary depending upon parameters
1o such as the temperature of the chamber, the wax melting temperature, the
viscosity of the compositions etc.
It will be appreciated by those skilled in the art that the chamber of a
device of the second and third aspects may comprise additional reagent
compositions and wax layers enabling more than two different reactions to
i5 occur in the same chamber.
In a preferred embodiment of the present invention, the sample
comprises biological material. Preferably, the biological material comprises
cells or viral particles.
In a further preferred embodiment of the present invention, the sample
2o is selected from the group consisting of: red and white blood cells,
embryonic
cells, bacteria, sperm, pollen, cell cultures, pap smears, single cells, blood
cell parasites, single cells infected with a virus, and other fluids
containing
DNA, RNA or protein.
It will be appreciated that the device of the first aspect of the present
25 invention may also be used for injecting a sample into biological tissue or
transferring a sample from one source to another.
Accordingly, in a ninth aspect, the present invention provides the use
of a device of the first aspect for injecting a fluid sample into a cell.
Preferably, the fluid sample comprises genetic material such as DNA or
3o sperm.
In a preferred embodiment of the ninth aspect, the device is adapted
for use in conjunction with a micromanipulator in order to control the
introduction of the sample into the cell.
Throughout this specification, the word "comprise", or variations such
35 as "comprises" or "comprising", will be understood to imply the inclusion
of a
stated element, integer or step, or group of elements, integers or steps, but
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not the exclusion of any other element, integer or step, or group of elements,
integers or steps.
The invention will hereinafter be described by way of the following
non-limiting Figures and Examples.
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Brief Description of the Accompanyin~ Drawing
Figure 1 is a perspective view of a device in accordance with a preferred
embodiment of the present invention in an assembled state.
5 Figure 2 is a cross-sectional view of a preferred device showing the
elongate
member in a first position.
Figure 3 is a cross-sectional view of a preferred device showing the elongate
member in a second position.
Figure 4 is an exploded cross sectional view of a device of Figure 2.
1o Figure 5 is a cross-sectional view of a preferred device of the present
invention showing (a) an assembled device; and (b) an elongate member and
sealing means.
Figure 6 shows the results of a sex determination assay performed on human
white blood cells by direct aspiration and single step real time amplification
and detection using a device in accordance with a preferred embodiment of
the present invention.
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Detailed Description of Preferred Embodiments:
With reference to Figures 1 to 4, an aspiration device (1) constructed in
accordance with a preferred embodiment of the present invention will now
be described. The device comprises a chamber (2) having a first open end (3)
and a second closed end (4), and an inner (5) and outer (6) surface. The
chamber is cylindrical and has an outer diameter of up to 2.8 mm, an inner
diameter of up to 2.5 mm and a length of between 20 and 40 mm. Preferably,
the chamber can hold sample volumes of up to 201.
1o It is preferred that the chamber (2) is made of a material that is
chemically inert, biocompatible and stable. Chemically inert means that the
material is stable to deformation, disclororation, cracking, splitting etc.
upon
exposure to heat, autoclaving, extraction reagents, diluents or other chemical
solutions. Biocompatible means that the material does not bind biological
materials from a solution, affect the stability, functionality, or
conformation
of a biological material upon contact with that material, or in any way
contaminate the biological solution with components that leach from the
material in the biological solution. Stable means that the material retains
all
of the above characteristics for years at room temperature.
2o Preferred polymers include polypropylene, polyethylene terephthalate
and polycarbonates, from which the entire device can be molded. Many
grades of polypropylene are commercially available. A resin like Himont
PD701 natural (Himont USA, Inc., Wilmington, Del.) is preferred as it
exhibits sufficient inertness and can be autoclaved. Examples of suitable
polycarbonates include, makrolon (Bayer), calibre (Dow Chemicals), Texan ,
(GE) and acrifix 192 (Rohm Chemical Fabrik). The entire device may be
injection molded under high injection pressures. Alternatively, the elongate
chamber may be made of other materials such as glass.
It is preferred that the material allows transmission of electromagnetic
3o radiation. Alternatively or in addition, the second closed end (4) of the
chamber comprises a lens (7) for transmitting electromagnetic radiation.
T'he device further comprises an elongate member (8) having first (9)
and second (10) open ends and an inner (11) and outer (12) surface. The
elongate member is slidably movable from a first position (13) to a second
position (14) in the elongate chamber (2). The first end (9) comprises a
needle tip (15). Preferably, the needle tip has a diameter of between 1gm and
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12
200~.m. More preferably, the tip has a diameter of between l~,m to 50~,m.
Preferably, the needle tip is capable of piercing biological tissue, such as
embryo tissue. Accordingly, it is preferred that at least the needle tip
portion
of the elongate member is made of glass. In one preferred embodiment, the
entire elongate member (8) is made of glass.
A sealing means (16) provides a seal between the outer surface (12) of
the elongate member and the inner surface (5) of the chamber (2). The
sealing means comprises a sleeve (17) located on the outer surface (12) of the
elongate member (8) proximate the second end (10). Teflon is preferably
used as the material for the sleeve (17) although other materials such as
rubber, plastic or glass may be used. The sleeve (17) co-operates with the
inner surface (5) of the chamber (2) so as to provide a seal between the
sleeve
(17) and the inner surface (5) of the chamber. The sleeve may co-operate
with the inner surface of the chamber along the entire length of the sleeve or
along a substantial portion of the length of the sleeve. Figure 5 shows an
alternative embodiment lIl WhlCh the sleeve (29) comprises a flared portion
(30) which co-operates with the inner surface (31) of the chamber (32) to
provide a seal.
In use, a fluid sample may be introduced into the chamber (2) via the
2o elongate member (8) by movement of the elongate member (8) from a first
position (13) to a second position (14). This movement may be achieved by
sliding the chamber (2) away from the first end (9) of the elongate member
(8). It will be appreciated that this movement of the chamber (2) relative to
the elongate member (8) causes a decrease in pressure within the chamber (2)
which draws the sample through the elongate member (8) into the chamber
(2).
The chamber further comprises a raised collar (18) located on the
chamber (2) proximate the first open end (3). The raised collar has an inner
(19) and an outer (20) surface . The outer surface (20) may be held by the
3o user in order to slide the chamber (2) away from or toward the first end
(9) of
the elongate member (8).
The device further comprises a first guide (21) located within the
raised collar (18) . The first guide comprises an annular outer surface (22)
which engages the inner surface (19) of the collar (18) thereby forming a seal
between the annular outer surface (22) and the inner surface (19). The first
guide further comprises an aperture (23) for receiving the elongate member
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(8). The elongate member (8) is received by the first guide (21) through the
aperture (23) such that the elongate member (8) is positioned co-axially with
respect to the chamber (2). The external surface (12) of the elongate member
(8) and the surface defining the aperture co-operate to form a seal. It is
preferred that the first guide is made of silicon.
The device further comprises a second guide (24) located on the
external surface (12) of and proximate the first end (9) of the elongate
member (8). This second guide (24) may be used to hold the elongate
member (8) in place whilst the chamber (2) is slidably moved away from or
1o toward the first end (9) of the elongate member (8).
A preferred device (1) of the present invention will now be described
generally in use.
The device of the present invention may be used to inject a fluid
sample into a cell. It will be appreciated that sliding the chamber (2) toward
the first end (9) of the elongate member (8) will increase the pressure within
the chamber (2) and cause expulsion of any fluid in the chamber (2) out
through the elongate member (8). In this manner, the device may be used to
inject foreign genetic material, such as DNA or sperm, into a cell.
It is preferred, however, that the device of the present invention is
2o used for aspiration of a sample into the chamber (2) and subsequent
analysis
of the sample. This analysis may be achieved by performing a desired
reaction on the sample within the chamber. In the context of this
embodiment, the elongate chamber may be loaded with a unit dose reagent
composition necessary to perform a reaction. "Unit dose" refers to a reagent
ccmposition comprising all or nearly all of the reagents needed to accomplish
a reaction except for the sample to be analysed. Preferably, the user need
only add the sample in order to start the reaction. The reaction composition
may be pre-loaded into the chamber (2) before the device is assembled.
Alternatively, the reaction composition may be drawn into the chamber (2)
3o through the elongate member (8). Preferably, the sample is drawn separately
into the chamber (2) through the elongate member after the chamber has
been loaded with the reagent composition. The reagent composition and
sample then react within the chamber (2) to form a reaction mix.
The inner surface (5) of the chamber (2) and/or the inner surface (11) of
the elongate member (8) may be treated with a blocking agent in order to
facilitate the loading of a sample into the chamber (2). By "blocking agent"
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we mean an agent which reduces or prevents the binding of biological
materials in a solution to the inner surface (11) of the elongate member (8)
or
inner surface (5) elongate chamber (2). The inner surface (11) of the elongate
member (8) or inner surface (5) of the chamber (2) may be contacted with the
blocking agent in order to inactivate any binding sites prior to the
aspiration
of the sample. Alternatively or in addition, the blocking agent may be added
directly to the sample prior to aspiration. The presence of the blocking agent
in the sample may facilitate the progression of the sample along the elongate
member during aspiration. The blocking agent may be a surfactant.
1o Examples of suitable blocking agents include, but are not limited to,
bovine
serum albumin, fetal calf serum, human serum albumin, polyvinyl alcohol,
polyvinyl pyrrolidone, and other suitable protein sources. Commercially
available products suitable for use as blocking agents in the context of the
present invention would be known by those skilled in the art. Suitable
examples include, "Sigmacote" (Sigma) and "Vigro Retrieval".
The elongate member (8) may also be filled with oil in order to
facilitate the passage of the sample through the elongate member into the
chamber.
Although a device (1) according to the present invention may be used
to perform a variety of reactions, in a preferred embodiment the device (1) is
used for collecting a biological sample and analysing that sample for the
presence of a target molecule. Preferably, the nature of the reagent
composition is such that the presence of the target molecule in the biological
substance may be detected by a change in the level of electromagnetic
radiation emitted by the reaction mix.
In a further preferred embodiment, the target molecule is a nucleic
acid sequence. Preferably, the reagent composition comprises reagents
which allow amplification of the target nucleic acid molecule. Preferably,
the reagent composition comprises at least one reagent which interacts with
3o the amplified sequence to cause a change in the level of electromagnetic
radiation emitted by the reaction mix.
An "amplification" reaction is a reaction in which multiple copies of an
original nucleic acid sequence are generated, typically by repeating an
enzymatic duplication process for a number of cycles. When additional
copies can be made from each of the duplicate copies made in an earlier
cycle, the amplification process is said to be exponential with respect to the
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number of cycles, While exponential amplification is desirable to improve
assay sensitivity, this heightened degree of sensitivity is also a drawback if
the amplification products are not carefully contained, resulting in
contamination.
5 Techniques for amplifying nucleic acid molecules are well known to
those skilled in the art. Examples of these techniques are the polymerase
chain reaction (PCR), disclosed in U.S. Patent Nos. 4,683,202 and 4,683,195
(Mullis); the ligase chin reaction (LCR) disclosed in EP-A-320 308 (Backman
et al); and gap filling LCR (GLCR) or variations thereof, which are disclosed
10 in WO 90/01069 (Segev), EP-A-439-182 (Backman, et al), GB 2,225,112A
(Newton, et al) and WO 93/00447 (Birkenmeyer et al). Other amplification
techniques include Q-Beta Replicase, as described in the literature; Strand
Displacement Amplification (SDA) as described in EP-A-497 272 (Walker),
EP-A-500 224 (Walker et al) and in Walker, et al., in Proc. Nat. Acad. Sci.
15 U.S.A., 89:392(1992); Self-Sustained Sequence Replication (3SR) as
described
in Fahy, et al., in PCR Methods and Applications 1:25 (1991); and Nucleic
Acid Sequence-Based Amplification (NASBA) as described in the literature.
Some amplification reactions, for example PCR and LCR, involve
cycles of alternately high and low set temperatures, a process known as
"thermal cycling". PCR or "Polymerase Chain Reaction" is an amplification
reaction in which a polymerase enzyme, usually thermostable, generates
multiple copies of the original sequence by extension of a primer using the
original nucleic acid as a template. PCR is described in more detail in U.S.
Pat. Nos. 4,683,202 and 4,683,195. LCR or "Ligase Chain Reaction" is a
nucleic acid amplification reaction in which a ligase enzyme, usually
thernostable, generates multiple copies of the original sequence by ligating
two or more oligonucleotide probes while they are hybridized to the target.
LCR, and its variation, Gap LCR, are described in more detail in EP-A-320-
308 (Baclanan et al), EP-A-439-182 (Backman, et al) and WO 90/100447
3o (Birkenmeyer et al.) and elsewhere.
In addition, amplification techniques which include assays which
involve fluorescent based detection are known to those skilled in the art.
Examples of these include the Molecular Beacons assay (Tyagi and Kramer,
1996), TaqMan~~"~ fluorescent energy transfer assay (Livak et al., 1995) and
hybridization probes which result in Forster Resonance Energy Transfer
(Deniz A.A. et al., Proc. Natl. Acad. Sci., USA 96:3670 (1999).
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Once the reagent composition and sample have been loaded into the
chamber (2), the elongate member (8) can be sealed, preferably by cutting the
elongate member (8) at a point proximal to the chamber (2) followed by heat
sealing. For example, the elongate member (8) may be simultaneously cut
and sealed by the application of heat. Alternatively, the elongate member (8)
may be removed from the chamber (2) and the open end (3) of the chamber
(2) may be heat sealed or sealed by the application of a cap.
The sealed chamber may then be placed in a thermal cycler so that the
target nucleic acid molecule in the sample undergoes amplification. At the
1o same time, emitted electromagnetic radiation may be detected through the
walls of the chamber or the lens in the base of the chamber.
The device and method of the present invention is suitable for use
with a number of direct reaction detection technologies/chemistries such as
Taqman (Perkin-Elmer), molecular beacons and the LightCycler~ fluorescent
hybridization probe analysis (Roche Molecular Systems).
A preferred system for real time DNA amplification and detection is
the LightCyclerz~~'I fluorescent hybridization probe analysis. This system
involves the use of three essential components: two different
oligonucleotides (labelled) and the amplification product. Oligonucleotide 1
carries a fluorescein label at its 3' end whereas oligonucleotide 2 carries
another label, LC Red 640 or LC Red 705, at its 5' end. The sequences of the
two oligonucleotides are selected such that they hybridize to the amplified
DNA fragment in a head to tail arrangement. When the oligonucleotides
hybridize in this orientation, the two fluorescence dyes are positioned in
close proximity to each other. The first dye (fluorescein) is excited by the
LightCycler's LED (Light Emitting Diode) filtered light source, and emits
green fluorescent light at a slightly longer wavelength. When the two dyes
are in close proximity, the emitted energy excites the LC Red 640 or LC Red
705 attached to the second hybridization probe that subsequently emits red
fluorescent light at an even longer wavelength. This energy transfer, referred
to as FRET (Forster Resonance Energy Transfer, or Fluorescence Resonance
Energy Transfer) is highly dependent on the spacing between the two dye
molecules. Only if the molecules are in close proximity (a distance between
1-5 nucleotides) the energy is transferred at high efficiency. Choosing the
appropriate detection channel, the intensity of the light emitted by the LC
Red 640 or LC Red 705 is filtered and measured by optics in the
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thermocycler. The increasing amount of measured fluorescence proportional
to the increasing amount of DNA generated during the ongoing PCR process.
Since LC Red 640 and LC Red 705 only emits a signal when both
oligonucleotides are hybridized, the fluorescence measurement is performed
after the annealing step. Using hybridization probes can also be beneficial if
samples containing very few template molecules are to be examined. DNA-
quantification with hybridization probes is not only sensitive but also highly
specific. It can be compared with agarose gel electrophoresis combined with
Southern blot analysis but without all the time consuming steps which are
required for the conventional analysis.
The 'TaqMan' fluorescence energy transfer assay uses a nucleic acid
probe complementary to an internal segment of the target DNA. The probe is
labelled with two fluorescent moieties with the property that the emission
spectrum of one overlaps the excitation spectrum of the other; as a result the
emission of the first fluorophore is largely quenched by the second. The
probe is present during PCR and if PCR product is made, the probe becomes
susceptible to degradation via a 5'-nuclease activity of Taq polyrnerase that
is
specific for DNA hybridized to template. Nucleolytic degradation of the
probe allows the two fluorophores to separate in solution, which reduces the
2o quenching and increases intensity of emitted light.
Probes used as molecular beacons are based on the principle of single-
stranded nucleic acid molecules that possess a stem-and-loop structure. The
loop portion of the molecule is a probe sequence that is complementary to a
predetermined sequence in a target nucleic acid. The stem is formed by the
annealing of two complementary arm sequences that are on either side of the
probe sequence. The arm sequences are unrelated to the target sequence. A
fluorescent moiety is attached to the end of one arm and a non-fluorescent
quenching moiety is attached to the end of the other arm. The stem keeps
these two moieties in close proximity to each other, causing the fluorescence
of the fluorophore to be quenched by fluorescence resonance energy transfer.
The nature of the fluorophore-quencher pair that we prefer to use is such that
energy received by the fluorophore is transferred to the quencher and
dissipated as heat, rather than being emitted as light. As a result, the
fluorophore is unable to fluoresce. When the probe encounters a target
molecule, it forms a hybrid that is no longer and more stable than the hybrid
formed by the arm sequences. Since nucleic acid double helices are
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18
relatively rigid, formation of a probe-target hybrid precludes the
simultaneous existence of a hybrid formed by the arm sequences. Thus, the
probe undergoes a spontaneous conformational change that forces the arm
sequences apart and causes the fluorophore and quencher to move away from
each other. Since the fluoropho~e is no longer in close proximity to the
quencher, it fluoresces when illuminated by an appropriate light source. The
probes are termed "molecular beacons" because they emit a fluorescent signal
only when hybridized to target molecules.
A number of real time fluorescent detection thermocyclers are
1o currently available with the chemistries being interchangeable with those
discussed above as the final product is emitted fluorescence. Such
thermocyclers include the Perkin Elmer Biosystems 7700, Corbett Research's
Rotogene, and the Hoffman La Roche Light Cycler. It is envisaged that any of
the above thermocyclers could be adapted to accommodate the device, and
perform the method, of the present invention.
In one preferred embodiment, the device of the present invention may
be used to perform a series of sequential reactions in a single chamber. In
this embodiment, the elongate chamber (2) is preloaded with two or more
reagent compositions, the reagent compositions being separated by layers of
wax.
Figure 1 depicts a preloaded device suitable for performing sequential
reactions. A first reagent composition (25) is covered by a first wax layer
(Z6). A second reagent composition (27) is loaded on top of the first wax
layer (26) and is covered by a second wax layer (28). The first wax layer (26)
has a higher melting point temperature than the second wax layer (28). Oil
(29) may be overlayed on the second wax layer (28).
It vvill be appreciated that a chamber (2) preloaded with reagent
compositions as depicted iii Figure 1 may be stored prior to use in a device
(1) according to the present invention. The open end of the chamber (3) may
3o be sealed with a cap prior to storage. The wax layers, when solidified in
the
chamber, preferably remain attached to the inner surface of the chamber
during routine shipping and handling.
When used herein, "wax" refers to a wax-like organic substance, solid
but much harder than greases at temperatures below about 40°C, which
melts
at somewhat higher temperatures to form a liquid and which has a lower
density than water. Waxes may be composed of hydrocarbons, alcohols, fatty
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19
acids and esters. They can be from plant, animal or mineral origin or may be
synthetic. Examples of mineral waxes include Petroleum wax and Montan
wax. Examples of synthetic waxes include Fischer-Tropsch waxes and
Polyolefin waxes. Preferably, waxes contain principally esters of fatty acids
and higher alcohols, free fatty acids and alcohols, and saturated
hydrocarbons. Typical pure compounds which are useful waxes include
eicosane, octacosane, cetyl palmitate, and pentaerythritol tetrabehenate.
Wax-like substances include, for example, polyester gels. Non-silicone
thixotropic gel-like substances may be used as wax-like substances. For
1o example, a hydrocarbon gel-like material polybutene H-100, marketed by
Amoco Chemicals Corporation, Chicago, IIL, and described in that company's
bulletin as 12-H as butylene polymer composed predominantly of high
molecular weight mono-olefins (85-98/0), the balance being isoparaffins,
when mixed with AEROSIL OX50, a fumed silica powder marketed by
Degussa, Inc., Pigments Division, New York, N.Y., may be suitable. Another
example of a useful hydrocarbon gel-like material is Poiy bd~ R-45HT,
marketed by ARCO Chemical Company, New York, N.Y. and described in
that company's general bulletin of April, 1976 as a hydroxyl terminated
homopolymer of butadiene with the degree of polymerization being in the
2o range of 50. Another example of a suitable gel-like material is a mixture
of
silicon fluid and very fine hydrophobic silicon dioxide powder.
Typical useful wax mixtures include paraffin, Paraplast (tradename of
Sherwood Medical), Ultraflex (tradename of Petrolite Corporation), and
BeSquare 175 (tradename of Petrolite Corporation). Particularly useful waxes
for the present invention include; T waxT"~ (Energene, Regensburg, Germany),
AmpliWaxl~"~ (PE Applied Biosystems, and Polyester Wax (Electron
Microscopy Sciences). Waxes can be prepared by mixing pure or mixed
waxes with one another or with greases or oils in any ratios which preserve
the relative hardness and stickiness characteristic of a wax. Preferably,
waxes used in the present invention are sterilized before use, using any
method known effective for this purpose.
The amount of wax required for the present invention is that amount
sufficient to cover the surface of the reagent composition. The amount can
be determined by routine experimentation. The amount varies depending on
several parameters, including, for example, the size of the reaction chamber.
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For example, 0.015 to 0.025 g of wax in a 0.2 ml microtube, or 0.023 to 0.04 g
of wax in a 0.5 ml microtube are useful.
"Oil" refers to a water-immiscible organic substance, liquid at
temperatures below about 40°C, which has a lower density than water.
5 "Mineral oil," also known as liquid petrolatum and paraffin oil, is a
colourless, optically clear, mixture of high-molecular weight hydrocarbons
with a density near 0.84 g/ml, widely available commercially and commonly
used as a vapour barrier over PCR reactions. Melting Point Bath Oil by Sigma
(silicon fluid) (catalogue no. M6884, density 0.96 g/ml or catalogue no.
10 M9389, density 1.05 g/ml) may also be suitable.
A biological sample may be drawn into the chamber (2) through the
elongate member (8) by movement of the chamber (2) relative to the elongate
member (8) as described above. It will be understood that a sample
introduced into the chamber (2) in this manner will be loaded adjacent the
15 second wax layer (28). The chamber (2) may then be sealed and heated to a
temperature at which the second wax layer (28) melts but the first wax layer
(26) remains solid. This melting of the second wax layer (28) preferably
brings the sample into fluid communication with the second reagent
composition (27). The chamber (2) may be centrifuged at this stage in order
20 to ensure admixing of the sample with the second reagent composition (27)
to form a reaction mix. The centrifugal force may be between 5 to 200g,
more preferably between 5 and 70g. The chamber (2) may then be subject to
conditions which allow the desired reaction to occur.
The chamber is then heated to a temperature at which the first wax
layer (26) melts. This melting of the first wax layer (26) preferably brings
the
reaction mix into fluid communication with the first reagent composition
(25). The chamber (2) may be centrifuged at this stage as described above in
order to ensure admixing of the reaction mix with the first reagent
composition (25). The chamber (2) may then be subject to conditions which
allow the desired reaction to occur.
It is also envisaged that the loaded chamber may be sealed and
inverted prior to melting of the wax layers and mixing of the sample and
reagents. For example, after the sample has been aspirated into the chamber,
the open end of the chamber may be sealed before inverting the chamber and
placing the inverted chamber in a thermal cycler. In this case, it is
preferred
that the collar is removed from the chamber. The same sequence of events as
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21
described above will occur in this embodiment. The second and first reagent
compositions will descend toward the sample in a temperature dependent
manner, however, rather than the sample descending towards the reagent
compositions.
As discussed above, in a preferred embodiment the device of the
present invention is used for detecting a target nucleic acid molecule in a
biological sample. Accordingly, it is preferred that the first reagent
composition (25) comprises reagents which allow amplification of a target
nucleic acid molecule.
1o As will be understood by those skilled in the art, the presence of
proteins, particularly DNA degrading enzymes, in a biological sample can
significantly hinder nucleic; acid amplification reactions. Furthermore, it is
important to liberate the nucleic acid from a cell, as well as to liberate the
nucleic from binding proteins such as histones, in order to ensure that the
nucleic acid is accessible to the polymerase in the amplification reaction
composition.
Accordingly, it is preferred that the second reagent composition (27)
comprises reagents which result in protein digestion and/or modification and
which lead to the liberation of nucleic acid molecules from cellular
2o components. It will be appreciated that interaction of the sample with a
second reagent composition (27) such as this results in cellular lysis,
protein
digestion and release of nucleic acid molecules prior to performing an
amplification reaction. Moreover, the exposure of the chamber to heat in
order to melt the first wax layer (26) may inactivate any proteinase enzymes
present in the second reagent composition prior to performing the
amplification reaction. Accordingly, this embodiment allows the user to
perform efficient and reliable amplification reactions on biological samples.
Because the cellular digestion and amplification reactions occur sequentially
in the same chamber, the risk of contamination is significantly reduced.
3o It will be appreciated that the chamber (2) may comprise more than
two reagent compositions separated by wax layers. It is envisaged, for
example, that a plurality of wax layers, each wax layer having a different
melting point, rnay be used to separate a plurality of reagent compositions.
This will allow more than two separate reactions to be performed
sequentially within the chamber.
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It will be appreciated that the device and methods of the present
invention will be useful in a range of applications such as forensic analysis,
diagnostic genetic tests, examining gene expression in single cells and sex
determining tests. In particular, it is envisaged that the reaction chamber
and methods of the present invention will be useful for reactions performed
on small samples, such as single cells, embryonic cells, sperm, pollen, blood
cell parasites, single cells infected with a virus, and DNA containing fluids.
Example 1 : Preparation of aspiration device.
A PCR reaction mix (17.1 ) was added to a chamber with a diameter of
approximately 1.6m.m. The 17.1 PCR reaction mix contained DNA
polymerise, reaction buffer including nucleotides and MgCIZ (TaqMan
Universal PCR Master Mix - PE Applied Biosystems), and two sets of
oligonucleotide primers, one set specific for the target region (SRY HMG
Box) of the Hunran Y chromosome and the other set of primers specific for
the autosomal target region G6PD on the Human X chromosome. The PCR
primers were each present at a final concentration of 0.3~M. The reaction
mix also contained PCR product detection probes each at a concentration of
0.2~.M. One probe detects products amplified from the Human Y
chromosome, whereas the other probe detects products amplified from the
Human X chromosome.
F wax (20% paraflint C80N6 from Schumann Sasol, 80% T wax) with a
melting temperature of about 76°C was heated to 90°C and 3.3,1
aspirated
using a Drummond Capillary Positive Displacement Pipette, allowed to
solidify, and expelled into the capillary device. The device was then loaded
onto a Rotogene (Corbett Research), centrifuged for 30 seconds at 78°C,
removed from the machine and cooled to 4°C, thus providing a solid wax
layer covering the PCR reaction mix.
3U 2.5,1 of proteinase K mix (2x Gold PCR buffer (Perkin Elmer), 0.4mg/ml
proteinase K (Boehringer Mannheim), l8uM SDS and the balance water) was
then layered on the top of the solidified F wax. A wax (AmpliWax Gem 50,
PE Applied Biosystems) with a melting temperature of about 55°C
was then
added to the device in the same manner as the F wax, however, the A wax
was melted at 75°C and centrifuged for 30 seconds in the Rotogene at
60°C.
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23
Finally, 15 ~tl of mineral oil (Sigma Chemicals) was loaded on top of
the A wax.
This chamber was then assembled with a glass elongate member
comprising a needle tip to produce a device as depicted in Figure 1.
Examine 2: Detection of a target nucleic acid in Human White Blood Cells.
Human white blood cells (WBC) from either male or female Human
were collected in cell preparation tubes with sodium citrate(CPT) (Becton
Dickinson, Cat #362761) for separating WBC from red blood cells, and
1o washed 2 times in phosphate buffered saline (0.01M phosphate buffer,
0.0027M KCI, 0.137M NaCI). The final WBC pellet was resuspended in
"Vigro Retrieval" (A.B. Technology). The "Vigro Retrieval" functions to
prevent the WBC adhering to the walls of the tube. The WBC were then
placed in a petri dish, and overlayed with mineral oil for pickup.
The required number of WBC were in 2.5 ~,l of "Vigro Retrieval"
preparation visually counted and aspirated using a device which had been
prepared as described in Example 1. For controls, similar volumes of male or
female Human DNA or "Vigro Retrieval" solution only (no DNA) were loaded
in to separate devices.
The WBC were aspirated into the elongate chamber on top of the
second wax layer underneath the oil and the glass elongate member was then
removed. After capping the device was placed in a Rotogene thermocycler.
To allow the first protein digestion reaction to occur the device was heated
to
60°C for 10 minutes. In this step the A wax melted and the F wax
remained
solid. Following this reaction, the chamber was then heated to72°C for
10
rains in order to inactivate the proteinase K. The chamber was then exposed
to the following PCR conditions: 1 x 94°C for 10 min (melting of the F
wax
layer occurs); 50 x 93°C for 15s, 60°C for 40s, 60°C for
10s.
The final results of the PCR are shown in Figure 6. The x-axis clearly
3o indicates the type of sample- either isolated DNA or WBC, the source-either
male or female, and the number of genomes or WBC added to the reaction
chamber.
All control samples and 11 out of the 12 samples analysed gave the
expected result for sex and were positive for the X internal control with the
exception of the no DNA controls (samples 3 & 16) which was correct for no
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24
sex or autosomal signal, and sample number 6 which failed to provide a sex
or autosomal signal.
Notably, for each of the male WBC and DNA samples analysed, a
signal was detected with no false positive signals detected in any of the
female deri«ed samples. Furthermore, as can be seen in samples 10 to 15,
the method was of sufficient sensitivity to detect a single copy of a target
sequence, namely a single Y chromosome sequence present in a single WBC.
Each of the patents, patent applications and literature documents
specifically cited above is incorporated herein in its entirety by reference.
It will be appreciated by persons skilled in the art that numerous
variations and/or modifications may be made to the invention as shown in
the specific embodiments without departing from the spirit or scope of the
invention as broadly described. The present embodiments are, therefore, to
be considered in all respects as illustrative and not restrictive.
