U.S. patent application number 09/903671 was filed with the patent office on 2001-12-06 for reaction vessels.
This patent application is currently assigned to The Secretary of State for Defence.. Invention is credited to Bird, Hilary, Lee, Martin A., Leslie, Dario Lyall.
Application Number | 20010049134 09/903671 |
Document ID | / |
Family ID | 27268625 |
Filed Date | 2001-12-06 |
United States Patent
Application |
20010049134 |
Kind Code |
A1 |
Lee, Martin A. ; et
al. |
December 6, 2001 |
Reaction vessels
Abstract
Apparatus for effecting reactions, said apparatus comprising a
plurality of reaction vessels for holding reagents, an electrically
conducting polymer which emits heat when an electric current is
passed through it, and control means for controlling supply of
current to the polymer, the polymer being connectable to an
electrical supply via the control means. The control means may be
arranged such that different currents and therefore different
temperatures can be achieved in each reaction vessel. Certain novel
reaction vessels are described and claimed. The apparatus are
reaction vessels may be used in carrying out reactions which
require multiple temperature stages such as amplification reactions
such as the polymerase chain reaction.
Inventors: |
Lee, Martin A.; (Salisbury,
GB) ; Bird, Hilary; (Salisbury, GB) ; Leslie,
Dario Lyall; (Salisbury, GB) |
Correspondence
Address: |
NIXON & VANDERHYE P.C.
1100 North Glebe Rd., 8th Floor
Arlington
VA
22201-4714
US
|
Assignee: |
The Secretary of State for
Defence.
|
Family ID: |
27268625 |
Appl. No.: |
09/903671 |
Filed: |
July 13, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09903671 |
Jul 13, 2001 |
|
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|
09229748 |
Jan 14, 1999 |
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Current U.S.
Class: |
435/286.1 ;
435/287.2; 435/303.1 |
Current CPC
Class: |
B01L 7/52 20130101; B01L
3/502707 20130101; B01L 2300/0854 20130101; B01L 2300/1827
20130101; B01L 2300/04 20130101; B01L 2300/0838 20130101; B01L
2300/0822 20130101; B01L 2300/1844 20130101; B01L 3/50851 20130101;
B01L 2300/12 20130101; B01L 2200/147 20130101 |
Class at
Publication: |
435/286.1 ;
435/287.2; 435/303.1 |
International
Class: |
C12M 001/38 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 6, 1996 |
GB |
9625442.0 |
Jul 31, 1997 |
GB |
9716052.7 |
Jan 16, 1998 |
GB |
9800810.5 |
Nov 20, 1997 |
GB |
PCT/GB97/03187 |
Claims
1. Apparatus for effecting reactions, said apparatus comprising a
plurality of reaction vessels for holding reagents, an electrically
conducting polymer which emits heat when an electric current is
passed through it, and control means for controlling supply of
current to the polymer, the polymer being connectable to an
electrical supply via the control means.
2. Apparatus according to claim 1 wherein different currents can be
supplied to heat each vessel or group of vessels.
3. Apparatus according to claim 2 wherein the control means is
arranged for the supply of current for a different temperature
and/or time profile for each of the reaction vessels.
4. Apparatus as claimed in claim 1 wherein each reaction vessel
comprises a container for reactants and the heater polymer is
contiguous with said container.
5. Apparatus as claimed in claim 4 wherein the heater polymer forms
a sheath around the container.
6. Apparatus as claimed in claim 4 wherein the heater polymer is in
the form of a film.
7. Apparatus as claimed in claim 5 wherein the sheath is integral
with the container.
8. Apparatus as claimed in claim 4 wherein the heater polymer is
perforated or reticulated.
9. Apparatus as claimed in any claim 1 wherein the heater polymer
forms a container for the reactants.
10. Apparatus as claimed in any one of claim 1 wherein the reaction
vessel comprises a container for reactants, wherein one of the
surfaces of the container is coated with the said heater
polymer.
11. Apparatus as claimed in claim 1 wherein each reaction vessel
comprises a capillary tube.
12. Apparatus as claimed in claim 1 wherein the reaction vessel
comprises a slide.
13. Apparatus as claimed in claim 1 wherein the reaction vessel
comprises a chip.
14. Apparatus as claimed in claim 1 wherein the reaction vessels
are provided in an array.
15. Apparatus as claimed in claim 1 wherein the reaction vessel
comprises a container and a cap member, the cap member being formed
so as to project into the container to reduce the capacity thereof
and to create a space therebetween of substantially consistent
proportions.
16. Apparatus as claimed in claim 1 wherein the control means is
arranged to supply electric current so as to conduct reactions
requiring multiple temperature stages within the reaction
vessels.
17. Apparatus as claimed in claim 1 and wherein the control means
is programmed such that multiple cycles of the reaction can be
effected automatically.
18. Apparatus as claimed in claim 1 wherein the control means is
arranged to supply current according to a predetermined
time/temperature profile.
19. Apparatus as claimed in any one of claims 1 and adapted for
polymerase chain reaction processes.
20. Apparatus as claimed in claim 1 further comprising a means for
detecting a signal from a sample in a reaction vessel.
21. A method of carrying out a chemical or biochemical reaction
which requires multiple temperature stages; said method comprising
placing reagents required for said reaction in a reaction vessel
which comprises an electrically conducting polymer which emits heat
when an electric current is passed through it, supplying current to
said polymer so as to heat reagents to a first desired temperature;
and thereafter adjusting the current so as to produce the
subsequent temperatures stages required for the reaction.
22. A method according to claim 21 wherein the reaction is a DNA
amplification method.
23. A method according to claim 22 wherein the amplification method
is a polymerase chain reaction (PCR).
24. A method according to claim 20 wherein reagents for a plurality
of reactions are each placed in a reaction vessel and heated
simultaneously.
25. A method according to claim 24 wherein each reaction vessel is
heated individually to the temperature required for the reaction
taking place within that vessel.
26. A reaction vessel comprising a slide or a chip and an
electrically conducting polymer which emits heat when an electric
current is passed through it, said polymer being arranged to heat
reactants on said slide or chip.
27. A reaction vessel according to claim 26 wherein the said
polymer is integral with the slide or chip.
28. A reaction vessel comprising a container, a cap member, and an
electrically conducting polymer which is arranged so as to heat
reagents in the reaction vessel when current is supplied to said
polymer, the cap member being formed so as to project into the
container to reduce the capacity thereof and to create a space
therebetween of substantially consistent proportions.
29. A reaction vessel according to claim 28 wherein the cap member
is adapted to seal the container.
30. A reaction vessel according to claim 29 wherein the cap member
is adapted to snap fit onto the container.
31. A reaction vessel according to claim 28 wherein the space
created between the cap member and the container when the cap
member is in place is of substantially similar proportions to a
capillary tube.
32. A reaction vessel according to claim 28 wherein the space
defined between the container and the lid member is from 0.4 to 1.2
mm at any point.
33. A reaction vessel according to claim 28 wherein the container
is of right cylindrical form and the cap member is arranged to
impinge upon the base so that the space created when the cap member
is in place has the form of an open cylinder.
34. Apparatus for carrying out reactions at controlled
temperatures, which apparatus comprises a reaction vessel as
claimed in claim 28 and a means for controlling the supply of
current to the electrically conducting polymer so as to controller
temperature thereof.
35. Apparatus according to claim 35 which further comprises means
for observing a signal generated in the space between the container
and the cap member of the reaction vessel.
36. Apparatus for carrying out reaction at controlled temperatures,
which apparatus comprises a reaction vessel as claimed in claim 26
and a means for controlling the supply of electric current to the
electricity conducting polymer so as to control the temperature
thereof.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to vessels and apparatus for
controlled heating of reagents for example those used in
biochemical reactions and to methods for using these.
BACKGROUND OF THE INVENTION
[0002] The controlled heating of reaction vessels is often carried
out using solid block heaters which are heated and cooled by
various methods. Current solid block heaters are heated by
electrical elements or thermoelectric devices inter alia. Other
reaction vessels may be heated by halogen bulb/turbulent air
arrangements. The vessels may be cooled by thermoelectric devices,
compressor refrigerator technologies, forced air or cooling fluids.
The reaction vessels fit into the block heater with a variety of
levels of snugness. Thus, the thermal contact between the block
heater and the reaction vessel varies from one design of heater to
another. In reactions requiring multiple temperature stages, the
temperature of the block heater can be adjusted using a
programmable controller for example to allow thermal cycling to be
carried out using the heaters.
[0003] This type of heater arrangement is particularly useful for
reactions requiring thermal cycling, such as DNA amplification
methods like the Polymerase Chain Reaction (PCR). PCR is a
procedure for generating large quantities of a particular DNA
sequence and is based upon DNA's characteristics of base pairing
and precise copying of complementary DNA strands. Typical PCR
involves a cycling process of three basic steps.
[0004] Denaturation: A mixture containing the PCR reagents
(including the DNA to be copied, the individual nucleotide bases
(A,T,G,C), suitable primers and polymerase enzyme) are heated to a
predetermined temperature to separate the two strands of the target
DNA.
[0005] Annealing: The mixture is then cooled to another
predetermined temperature and the primers locate their
complementary sequences on the DNA strands and bind to them.
[0006] Extension: The mixture is heated again to a further
predetermined temperature. The polymerase enzyme (acting as a
catalyst) joins the individual nucleotide bases to the end of the
primer to form a new strand of DNA which is complementary to the
sequence of the target DNA, the two strands being bound
together.
[0007] A disadvantage of the known block heaters arises from the
lag time required to allow the heating block to heat and cool to
the temperatures required by the reaction. Thus, the time to
complete each reaction cycle is partially determined by the thermal
dynamics of the heater in addition to the rate of the reaction. For
reactions involving numerous cycles and multiple temperature
stages, this lag time significantly affects the time taken to
complete the reaction. Thermal cyclers based on such block heaters
typically take around 2 hours to complete 30 reaction cycles.
[0008] For many applications of the PCR technique it is desirable
to complete the sequence of cycles in the minimum possible time. In
particular for example where respiratory air or fluids or foods for
human and animal stock consumption are suspected of contamination
rapid diagnostic methods may save considerable money if not health,
even lives.
[0009] An alternative thermal cycler contains a number of capillary
reaction tubes which are suspended in air. The heating and cooling
of the reaction tubes is effected using a halogen lamp and
turbulent air from a fan. The thermal dynamics of this system
represent a considerable improvement over the traditional block
heater design because heated and cooled air is passed across the
reaction tubes and the required temperatures are achieved quite
rapidly, the fan providing a homogeneous thermal environment and
forced cooling. Using this apparatus 30 reaction cycles can be
completed in about 15 minutes.
[0010] A disadvantage of this thermal cycler is that air cooling
and heating are not readily suitable in apparatus which is required
to provide different thermal cycling conditions to multiple
reactions at the same time, and is certainly not mobile or
portable.
SUMMARY OF THE INVENTION
[0011] The applicants have developed an efficient system for rapid
heating and cooling of reactants which is particularly useful in
thermal cycling reactions.
[0012] The present invention relates to a reaction vessel
comprising an electrically conducting polymer which emits heat when
an electric current is passed through it.
[0013] Thus in a first aspect, there is provided apparatus for
effecting reactions, said apparatus comprising a plurality of
reaction vessels for holding reagents, an electrically conducting
polymer which emits heat when an electric current is passed through
it, and control means for controlling supply of current to the
polymer, the polymer being connectable to an electrical supply via
the control means.
[0014] Further aspects of the invention include specific reaction
vessels used in the apparatus as detailed hereinafter as well as
methods for carrying out chemical or biochemical reactions.
[0015] Electrically conducting polymers are known in the art and
may be obtained from Caliente Systems Inc. of Newark, USA. Other
examples of such polymers are disclosed for instance in U.S. Pat.
No. 5,106,540 and U.S. Pat. No. 5,106,538. Suitable conducting
polymers can provide temperatures up to 300.degree. C. and so are
well able to be used in PCR processes where the typical range of
temperatures is between 30.degree. and 100.degree. C.
[0016] An advantage of the invention over a conventional block
heater is derived from the fact that polymers which conduct
electricity are able to heat rapidly. The heating rate depends upon
the precise nature of the polymer, the dimensions of polymer used
and the amount of current applied. Preferably the polymer has a
high resistivity for example in excess of 1000 ohm.cm. The
temperature of the polymer can be readily controlled by controlling
the amount of electric current passing through the polymer,
allowing it to be held at a desired temperature for the desired
amount of time. Furthermore, the rate of transition between
temperatures can be readily controlled after calibration, by
delivering an appropriate electrical current, for example under the
control of a computer programme.
[0017] Furthermore as compared to a block heater, rapid cooling can
also be assured because of the low thermal mass of the polymer. If
desired however, the reaction vessel may be subjected to artificial
cooling to further increase the speed of cooling. Suitable cooling
methods include forced air cooling, for example by use of fans,
immersion in ice or water baths etc.
[0018] In addition, the use of polymer as the heating element in a
reaction vessel will generally allow the apparatus to take a more
compact form than existing block heaters, which is useful when
carrying out chemical reactions in field conditions such as in the
open air, on a river, on a factory floor or even in a small
shop.
[0019] Each reaction vessel may take the form of a reagent
container such as a glass, plastics or silicon container, with
electrically conducting polymer arranged in close proximity to the
container. In one embodiment of the vessel, the polymer is provided
as a sheath which fits around the reaction vessel, in thermal
contact with the vessel. The sheath can either be provided as a
shaped cover which is designed to fit snugly around a reaction
vessel or it can be provided as a strip of film which can be
wrapped around the reaction vessel and secured.
[0020] The polymer sheath arrangement means that close thermal
contact is achievable between the sheath and the reaction vessel.
This ensures that the vessel quickly reaches the desired
temperature without the usual lag time arising from the insulating
effect of the air layer between the reaction vessel and the heater.
Furthermore, a polymer sheath can be used to adapt apparatus using
pre-existing reaction vessels. In particular, a strip of flexible
polymer film can be wrapped around a reaction vessel of various
different sizes and shapes.
[0021] Where a sheath is employed it may be advantageous for it to
be perforated or in some way reticulated. This may increase the
flexibility of the polymer and can permit even readier access by a
cooling medium if the polymer is not itself used to effect the
cooling.
[0022] Alternatively the polymer maybe provided as an integral part
of the reaction vessel. The reaction vessel may be made from the
polymer by extrusion, injection moulding or similar techniques.
Alternatively, the reaction vessel may be manufactured using a
composite construction in which a layer of the conducting polymer
is interposed between layers of the material from which the vessel
is made or in which the internal or external surfaces of the
reaction vessel is coated with the polymer, or again in which the
vessel is basically made of the polymer coated with a thin laminate
of a PCR compatible material. Such vessels may be produced using
lamination and/or deposition such as chemical or electrochemical
deposition techniques as is conventional in the art
[0023] Vessels which comprise the polymer as an integral part may
provide particularly compact structures.
[0024] If several reaction vessels are required for a particular
reaction, any electrical connection points can be positioned so
that a single supply can be connected to all the reaction vessels
or tubes. The reaction vessels may be provided in an array.
[0025] Alternatively, each of or each group of reaction vessels may
have its own heating profile set by adjusting the applied current
to that vessel or group of vessels. This provides a further and
particularly important advantage of reaction vessels with polymer
in accordance with the invention over solid block heaters or
turbulent air heaters, in that individual vessels can be controlled
independently of one another with their own thermal profile. It
means that a relatively small apparatus can be employed to carry
out a plurality of PCR assays at the same time notwithstanding that
each assay requires a different thermal profile i.e. a varying
operating temperature and/or dwell times in each stage of a cycle.
For example, PCR tests for detecting a fair plurality of organisms
in a sample can be carried out simultaneously, notwithstanding that
the nucleotide sequence which is characteristic of each organism is
amplified at different PCR operating temperatures.
[0026] The polymer may suitably be provided in the form of a sheet
material or film, for example of from 0.01 mm to 10 mm, such as
from 1 to 10 mm, and preferably 0.1 to 0.3 mm thick. By using thin
films, the volume of polymer required to cover a particular
reaction vessel or surface is minimised. This reduces the time
taken for the polymer to heat to the required temperature as the
heat produced by passing the current through the polymer does not
have to be distributed throughout a large volume of polymer
material.
[0027] In use, the polymer component of the reaction vessel is
arranged such that an electric current can be generated within the
polymer. This can either be achieved by providing the polymer with
connection points for connection to an electrical supply or by
inducing an electric current within the polymer, for example by
exposing the polymer to suitable electrical or magnetic fields.
[0028] The close thermal contact between the polymer and the
reagents or reagent container which may be established in the
reaction vessels of the invention reduces or eliminates the
insulating effect of the air layer between the heating element and
the reaction vessel.
[0029] The vessel may comprise a flat support plate such as a
two-dimensional array in particular a chip such as a silicon wafer
chip; or a slide, in particular a microscope slide, on which
reagents may be supported. The plate may be made from the polymer
or the polymer may be provided as an integral part of the plate,
either as a coating on one side of the plate or as a polymer layer
within a composite construction as previously described. Where
appropriate, and particularly when the plate is a chip, the polymer
may be deposited and/or etched in the preferred format on the chip
using for example printed circuit board (PCB) technology.
[0030] Where the reaction vessel comprises a slide or chip, the
apparatus may comprise the slide or chip, an electrical supply,
means for connecting the electrical supply to the slide or chip or
for inducing an electrical current in the polymer and a means for
controlling the current passing through the polymer layer in the
slide or chip.
[0031] Vessels of this type may be particularly useful for carrying
out in-situ PCR for example on tissue samples.
[0032] These vessels are novel. Thus in a further aspect the
invention provided a reaction vessel comprising a slide or a chip
and an electrically conducting polymer which emits heat when an
electric current is passed through it, said polymer being arranged
to heat reactants on said slide or chip.
[0033] Other suitable reaction vessels are tubes and cuvettes,
which are known in the art.
[0034] In a preferred embodiment of the invention, the vessel
comprises a capillary tube. The heat transfer from a capillary tube
to reagents contained within it is more rapid than that achieved
using conventional reagent vessels as the surface area to volume
ratio of the reagents in the capillary tube is larger than in a
conventional reagent vessel. Furthermore, the volume of samples
used in these reactions is frequently very small, of the order of
microliters or less and small volume vessels are thus
essential.
[0035] Also, the invention provides apparatus for carrying out
reaction at controlled temperatures, which apparatus comprises a
reaction vessel comprising a slide or chip and a means for
controlling the supply of electric current to the electricity
conducting polymer so as to control the temperature thereof.
[0036] Capillary tube reaction vessels are usually filled by
allowing the sample to be drawn into the tube under capillary
action. The ends of the tube are then sealed. In the case of a
glass tube, which is the usual form, sealing is typically effected
thermally.
[0037] This thermal sealing method has a major disadvantage in
being liable to degrade the sample. Also however, a glass tube of
what might well be less than 2 mm outside diameter and about 4 cm
length, is very fragile. There are capillary reaction vessels which
have one end presealed. These may be filled by employing centrifuge
or vacuum techniques. These are however time consuming and besides
entail a risk of retained air and contamination from air.
[0038] It is not uncommon for a newly opened box to contain four or
five broken tubes in a bank of 96 such vessels, which is one
popular quantity for use in biochemical thermocycling apparatus.
Further breakages are very likely to occur during filling and
mounting and even in use, not least because heating and cooling is
typically effected using turbulent hot and cold air.
[0039] There exist also reaction vessels formed from plastics
material and vessels which are not capillary in form. Such vessels
typically have a maximum internal diameter of 5 to 10 mm and are
conical or paraboloid tapering down to the base. These are
relatively easily filled and are provided with caps which seal
thereto. They are relatively unbreakable but have the disadvantage
that the required temperatures may not be accurately attained or
consistently attained throughout the sample or with each cycle.
Because of the low surface area to volume ratio, heat transfer is
poor in conventional tubes.
[0040] Reaction vessels in which a cap for a reaction vessel
projects into the vessel in order to reduce the volume thereof are
described for example in EF-A-245994 and U.S. Pat. No.
4,578,588.
[0041] In a particularly preferred embodiment, the reaction vessel
used in the present invention comprises a container, a cap member,
and an electrically conducting polymer which is arranged so as to
heat reagents in the reaction vessel when current is supplied to
said polymer, the cap member being formed so as to project into the
container to reduce the capacity thereof and to create a space
therebetween of substantially consistent proportions.
[0042] Thus in a further aspect the invention provides a reaction
vessel comprising a container, a cap member, and an electrically
conducting polymer which is arranged so as to heat reagents in the
reaction vessel when current is supplied to said polymer, the cap
member being formed so as to project into the container to reduce
the capacity thereof and to create a space therebetween of
substantially consistent proportions.
[0043] In this way, the insertion of the cap member into the
vicinity of the sample results in an increase in the surface area
to volume ratio of the sample, so that the required temperature can
be consistently and rapidly attained throughout the reagent mass.
In addition the reaction vessel which is easy to fill.
[0044] The expression "substantially consistent proportions" used
herein means that the space, which will form the reagent volume is
of substantially similar cross section throughout. This means that
externally applied factors such as heating or cooling means, will
be effective throughout the entire volume of the reagent in a
substantially consistent manner.
[0045] As before, the reaction vessel of this embodiment may take
the form of a reagent container such as a glass or plastics
container, with electrically conducting polymer arranged in close
proximity to the container. In one embodiment of the vessel, the
polymer is provided as a sheath which fits around the reaction
vessel, in thermal contact with the vessel. The sheath can either
be provided as a shaped cover which is designed to fit snugly
around a reaction vessel or it can be provided as a strip of film
which can be wrapped around the reaction vessel and secured.
[0046] In a preferred arrangement, the polymer is provided as an
integral part of the reaction vessel, and in this case, it may
either be as part of the container or the cap member. The container
and/or cap member may be made from the polymer by extrusion,
injection moulding or similar techniques Alternatively, the
container or cap member may be manufactured using a composite
construction in which a layer of the conducting polymer is
interposed between layers of the material, such as plastics or
glass, from which the container or cap member is made. In a further
alternative, the internal or external surfaces of the container
and/or cap member are coated with the polymer. Alternatively, the
container or cap member is basically made of the polymer coated
with a thin laminate of a PCR compatible material. Such vessels may
be produced using lamination and/or deposition such as chemical or
electrochemical deposition techniques as is conventional in the
art.
[0047] Reaction vessels and apparatus of the invention can be used
in a variety of situations where chemical or biochemical reactions
are required to be carried out. Thus the invention further provides
a method of carrying out a reaction such as a chemical or
biochemical reaction which method comprises heating reagents in
apparatus or in a reaction vessel as described above.
[0048] In particular the invention provides a method of carrying
out a chemical or biochemical reaction which requires multiple
temperature stages; said method comprising placing reagents
required for said reaction in a reaction vessel which comprises an
electrically conducting polymer which emits heat when an electric
current is passed through it, supplying current to said polymer so
as to heat reagents to a first desired temperature; and thereafter
adjusting the current so as to produce the subsequent temperatures
stages required for the reaction.
[0049] As well as amplification reactions such as PCR reactions
already mentioned above, the vessels and apparatus of the invention
can be used for the purposes of nucleic acid sequencing and in
enzyme kinetic studies wherein are studied the activity of enzymes
at various temperatures, likewise other reactions, especially those
involving enzymic activity, where precise temperatures need to be
maintained. The reaction vessels of the invention allow precise
temperatures to be reached and maintained for suitable time
periods, and then changed rapidly as desired, even in mobile or
portable apparatus in accordance with some embodiments of the
invention.
[0050] For PCR reactions, the temperature conditions required to
achieve denaturation, annealing and extension respectively and the
time required to effect these stages will vary depending upon
various factors as is understood in the art. Examples of such
factors include the nature and length of the nucleotide being
amplified, the nature of the primers used and the enzymes employed.
The optimum conditions may be determined in each case by the person
skilled in the art. Typical denaturation temperatures are of the
order of 95.degree. C., typical annealing temperatures are of the
order of 55.degree. C. and extension temperatures of 72.degree. C.
are generally of the correct order. When utilising the reaction
vessels and apparatus of the invention, these temperatures can
rapidly be attained and the rate of transition between temperatures
readily controlled.
[0051] Generic DNA intercalating dyes and strand specific gene
probe assays, eg Taqman.RTM. assays as described in U.S. Pat. No.
5,538,848 and Total Internal Reflection Fluorescence (TIRF) assays
such as those described in WO93/06241 can of course be employed
with many embodiments of the invention. In such assays, a signal
from the sample such as a fluorescent signal or an evanescent
signal is detected using a fluorescence monitoring device. When
this type of process is undertaken, the fluorescence monitoring
device must be arranged such that it is able to detect signal
emanating from the sample. In some instances, it may be helpful if
at least a part of the vessel, for example an end where the vessel
is a tube of the invention may be optically clear so that
measurements can be made through it. Alternatively the vessel can
be provided with means of conveying a signal from the sample to the
monitoring device, for example, an optic fibre or an evanescent
wave guide.
[0052] The fluorescence monitoring device may be set to read a
fluorescent signal at one or more wavelengths depending upon the
nature of the signally system being used.
DETAILED DESCRIPTION OF THE INVENTION
[0053] Embodiments of the present invention will now be described,
by way of example, with reference to the accompanying drawings,
wherein
[0054] FIG. 1. Shows a reaction vessel heater comprising a sheath
of electrically conducting polymer arranged to fit around a
reaction tube;
[0055] FIG. 2. Shows a reaction slide having an electrically
conducting polymer coating over one of its surfaces;
[0056] FIG. 3. Shows a reaction slide having a layer of
electrically conducting polymer within a composite
construction;
[0057] FIG. 4. Shows an apparatus for carrying out reactions
involving multiple temperature stages and which utilises a strip of
electrically conducting polymer to heat a capillary tube reaction
vessel;
[0058] FIG. 5 shows a diagram of apparatus according to the
invention for carrying out a PCR reaction;
[0059] FIG. 6 shows a thermocycling profile used with the apparatus
of FIG. 5;
[0060] FIG. 7 is a schematic diagram of a portable PCR
multidetector;
[0061] FIG. 7a is a diagram of a detector element for use in the
apparatus of FIG. 7;
[0062] FIG. 8 shows a section through a first embodiment of a
reaction vessel of the invention;
[0063] FIG. 9 shows a section through a different embodiment of a
reaction vessel of the invention;
[0064] FIG. 10 shows a section through yet a further embodiment of
a reaction vessel of the invention;
[0065] FIG. 11 shows a section through a modified embodiment of the
reaction vessel of FIG. 10; and
[0066] FIG. 12 Shows a section through an embodiment of a reaction
vessel of the invention which allows reaction monitoring to be
effected readily.
[0067] Referring to FIG. 1, a sheath of electrically conducting
polymer 2 is provided with electrical connection points 3 for
connection to an electrical supply. The size and shape of the
sheath 2 is determined by the dimensions and shape of a reaction
vessel 1 around which the sheath fits.
[0068] In use, the sheath 2 is placed around and in close thermal
contact with the reaction vessel 1. The connection points 3 are
then connected to an electrical supply (not shown) and current is
passed through the polymer sheath 2, thereby heating it and any
reagents inside the reaction vessel 1.
[0069] Referring to FIG. 2, a slide 1 is coated on one side with
electrically conducting polymer 2. Electrical connection points 3
are provided at either end of the slide 1, in electrical connection
with the polymer layer 2.
[0070] In FIG. 3, the vessel comprises a slide 1 having a composite
construction such that a layer of electrically conducting polymer 2
is interposed between layers of the usual material used to produce
such slides such as glass. Electrical connection points 3 are
provided at either and of the slide 1, in electrical connection
with the polymer layer 2.
[0071] In use, an electrical supply (not shown) is connected to the
electrical connection points 3 on the slide shown in FIGS. 2 and 3
and current is passed through the polymer layer 2, thereby heating
the slide 1 and any reagents placed on the slide 1.
[0072] Referring to FIG. 4, a strip of electrically conducting
polymer film 2 is wrapped around a capillary tube 1 and secured.
The strip of polymer film 2 is provided with electrical connection
points 3 to which an electrical supply 5 is connected via
connection clips 4.
[0073] In use, current is passed through the polymer film 2,
thereby heating the capillary tube 1 and any reagents placed inside
the capillary tube 1.
[0074] The device of FIG. 5 was constructed in order to conduct PCR
detections. A capillary tube 6 with a 1.12 mm internal diameter and
1.47 mm outer diameter was used as the reaction vessel. A strip of
electrically conducting polymer 7 was wrapped around the tube and
fastened so that it was held quite tightly to the external surface
of the tube. Heating is therefore from all sides of the tube 6
minimising the temperature gradient across a sample in the tube
6.
[0075] Heating was provided by an electrical power supply 8 which
was connected via an interface 9 to a computer 10 to allow the
heating cycles to be controlled automatically. A fan cooler 11 was
arranged to direct air onto the polymer 7. An infra-red
thermocouple 12 was provided on the outside of the polymer 7 in
order to monitor the temperature.
[0076] For the purposes of assessing the performance of the
apparatus prior to use, a K-type thermocouple was used to monitor
the temperature inside the tube 6. The internal and external
temperatures were then used to linearise the external temperature
readings to the predicted sample temperature.
[0077] The heating polymer is connected to the power supply 8 and
the circuit closed using the interface 9 and software. A switch 14
arranged to close the circuit was a fast optical relay which can
switch every 10 ms. A second circuit was used to control two small
electric fans 11 which provided forced air cooling of the reaction
sample and which are run continuously. The control software was
LabView which provides a user friendly graphical interface for both
programming and operation. Current was applied initially with
relatively high frequency in order the more rapidly to arrive at
the required temperature. When the designated operating temperature
was achieved the current was applied less frequently as required to
maintain the designated operating temperature for the predetermined
duration.
[0078] The apparatus shown in FIG. 7 comprises a lidded box 70
having insulative partitioning defining a plurality of detector
element receptor bays 71. The box 71 is shown electrically
connected via an interface unit 72 to a power source 73 and a
computer 74. The connection is such as to permit different supplies
to each of the bays 71. Each bay contains a thermocouple (not
shown) for monitoring the temperature therein.
[0079] The detector element shown in FIG. 7a comprises a reaction
tube 75 surrounded by a sheath 76. The sheath 76 is formed of a
heating polymer and is connected to supply terminals 77 and 78.
[0080] After a tube 75 has been filled and stopped it can be
offered to the appropriate bay 71 until the terminals 77 and 78
have clipped onto matching receptor terminals in the bays (not
shown). The apparatus when fully connected is arranged to permit
displaying on the computer screen the connection status of each
tube 75.
[0081] Closure of the lid to the box 70 completes the insulation of
each bay and the retention of each tube 75 in its bay.
[0082] The computer programme is arranged for the separate
identification of the molecule being searched for in each tube 75,
which done it is arranged for the control of the appropriate
temperature cycle for PCR to amplify that molecule if present. When
the cycles are complete the tube contents can be exposed to
appropriate gene probe detectors to determine whether the molecule
searched for was indeed present.
[0083] Alternatively it would be possible to utilise the apparatus
to effect "real time quantitation" where the reaction is monitored
throughout and not just at the end point.
[0084] Of course the principle of the apparatus described in
relation to FIGS. 7 and 7a may be realised in a variety of ways. It
can be mobile rather than portable and arranged for the reception
of detector elements in a form other than that of a tube, including
a slide. Typically it is arranged to deal with 96 or 192 detector
elements.
[0085] A preferred form of the reaction vessels of the invention
are illustrated in FIGS. 8 to 12. The embodiment of FIG. 8
comprises a conical container (13) and a cap member (14) which
projects into the container (13) so as to define a thin space (15)
therebetween. A sealing ring (16) ensures that the cap member (14)
effectively closes the container (13). A base portion (17) of the
container (13) is flattened and made of an optically clear material
so that contents of the space (15) may be observed. A sheath of
electrically conducting polymer (18) is provided around the
container (13) This is provided with electrical connections which
may be connected to a power supply.
[0086] In use, reagents are introduced into the container (13)
before application of the cap member (14). When the cap member (14)
is applied, the reagents become distributed through the space (15).
Current is then applied to the electrically conducting polymer
sheath in order to heat the reaction vessel at it contents to the
desired temperature.
[0087] The alternative embodiment of FIG. 9 shows a container (19)
of generally circular cross section but with a flattened base. In
this case, the lid (20) is provided with an upper portion (21),
which snap fits onto the container (19). Once again a consistent
thin space (22) is formed between the container (19) and the lid
(20). If desired the upper portion (21) may comprise a lens which
allows enhanced observation of contents of the container.
Additionally or alternatively, the projecting portion the lid (20)
may comprise an optical waveguide such as a fibre optic, which
forms an integral part of the reaction monitoring system.
[0088] One of the container (19) or lid (21) may comprise an
electrically conducting polymer which is connectable to a power
supply (not shown). Alternatively, the container may be provided
with a sheath of electrically conducting polymer (not shown).
[0089] This embodiment may be employed in a similar manner to the
embodiment of FIG. 8 above.
[0090] The modification shown in FIG. 10 includes a differently
shaped container (22) with a correspondingly differently shaped lid
(23) which snap fits onto the container (22). In this case however,
the lid (23) includes a channel (24) which can accommodate a
temperature monitoring device (25) such as a thermocouple or
resistive temperature device (RTD), in order to allow the
temperature of the reaction being effected in the container (22) to
be monitored.
[0091] Again, the container (22) and/or the lid (23) may comprise
an electrically conducting polymer, or a sheath of electrically
conducting polymer may be provided around the container (22).
[0092] Although the lid (23) is solid, it may be hollowed out in an
alternative embodiment (FIG. 11), in order to reduce the thermal
mass. In this case, a sealing ring (16) is provided in order to
enclose the space between the container (22) and the lid (23).
[0093] The embodiment of FIG. 12 illustrates a modification whereby
the reaction effected in the vessel may be monitored readily. In
this instance the container (26) is generally cylindrical in shape
but has an annular projection (27) extending from the base surface
thereof. A lid (28) is adapted to sit directly on the base of the
container (26) such that the space defined therebetween is
generally cylindrical (29). The container (26) may then be
surrounded by a sheath of electrically conducting polymer for
heating, and the vessel may optionally be placed in a cooling
apparatus (not shown).
[0094] If the container is illuminated in the direction of the
broad arrows, for example using a fluorescent excitation source,
any sample in the container will be illuminated. Signal generated
by the source may be monitored by an appropriate fluorescence
monitoring device which is arranged in line with the projection
(27) in the direction of the line arrows.
[0095] Various signals can be monitored simultaneously from
different points around the annular projection (27). Alternatively,
one or more capillary-like projections may be provided in place of
the annular projection (27) so that different signals can be
monitored from each. For instance, fluorescence at different
wavelengths can be monitored. This may be the wavelengths of for
example a reporter and a quencher molecule when these are used
together in a reaction such as a TAQMAN.TM. reaction.
[0096] The following Example illustrates the invention.
EXAMPLE
[0097] Amplification of DNA
[0098] Using the apparatus of FIG. 5 with the K-type thermocouple
removed, the following PCR reaction was effected.
[0099] A 100 base pair amplicon from a cloned Yersinia pestis
fragment was amplified. Reaction conditions had previously been
optimised using the Idaho RapidCycler.TM. and samples of the same
reaction mixture were amplified in the Idaho RapidCycler.TM. as
control reactions.
[0100] The reaction mixture placed in the tube 6 comprised the
following:
[0101] 50 mM Tris.HCl pH 8.3.
[0102] 3 mM MgCl.sub.2
[0103] 2.5 mg/ml Bovine Serum Albumen
[0104] 200 .mu.M each of dATP, dTTP, dCTP and dGTP
[0105] 10 .mu.g/ml each PCR primers
[0106] 25 Units/ml Taq Polymerase
[0107] The thermocycling profile was programmed as 95.degree. C.
for zero seconds, 55.degree. C. for zero seconds, 72.degree. C. for
zero seconds as illustrated in FIG. 6. By way of comparison, a
similar thermocycling profile was programmed into an Idaho
RapidCycler.TM.. Reaction volumes of 50 .mu.l were used in both the
polymer covered capillary vessel 6 and the Idaho RapidCycler.TM..
In this context, "zero seconds" means that as soon as the target
temperature is reached, the program instructs the subsequent
temperature to be induced. The precise time at which the reaction
is held at the target temperature is therefore dependent upon the
parameters and properties of the device used. In general however,
it will be less than one second.
[0108] After 40 cycles in the capillary vessel, a 50 .mu.l sample
of the PCR product from each of the reactions were size
fractionated by agarose gel electrophoresis in a 2% gel in 1.times.
TAE buffer. DNA was visualised using ethidium bromide staining. The
sample was run adjacent a sample from the Idaho RapidCycler.TM. (25
cycles) and a similar correctly sized amplicon was detected.
* * * * *