U.S. patent application number 09/319441 was filed with the patent office on 2002-03-07 for reaction vessels.
Invention is credited to LEE, MARTIN A, LESLIE, DARIO.
Application Number | 20020028165 09/319441 |
Document ID | / |
Family ID | 26310574 |
Filed Date | 2002-03-07 |
United States Patent
Application |
20020028165 |
Kind Code |
A1 |
LEE, MARTIN A ; et
al. |
March 7, 2002 |
REACTION VESSELS
Abstract
A reaction vessel for holding reagents is described, which
vessel comprises an electrically conducting polymer capable of
emitting heat when an electric current is passed through it.
Suitably the reaction vessel comprises a reagent container, such as
a capillary tube, slide or chip, in close contact with the
electrically conducting polymer. For example, the polymer may be in
the form of a film which is wrapped around the tube to form a
sheath. This provides a readily controllable heating supply which
may be heated and cooled to desired temperatures rapidly. An
apparatus suitable for thermal cycling reactions, such as the
polymerase chain reaction (PCR) and comprising one or more reaction
vessels as described above, as well as methods for carrying out
such reactions are also described and claimed.
Inventors: |
LEE, MARTIN A; (SALISBURY,
GB) ; LESLIE, DARIO; (SALISBURY, GB) |
Correspondence
Address: |
NIXON & VANDERHYE
1100 NORTH GLEBE ROAD
8TH FLOOR
ARLINGTON
VA
222014714
|
Family ID: |
26310574 |
Appl. No.: |
09/319441 |
Filed: |
June 25, 1999 |
PCT Filed: |
November 20, 1997 |
PCT NO: |
PCT/GB97/03187 |
Current U.S.
Class: |
422/199 ;
422/109; 422/198; 422/400 |
Current CPC
Class: |
B01L 7/52 20130101; B01L
2300/1844 20130101; B01L 3/502707 20130101; B01L 3/50851 20130101;
B01L 2300/0822 20130101; B01L 2300/12 20130101; B01L 2300/1827
20130101; B01L 2300/0838 20130101; G05D 23/1904 20130101; G05D
23/22 20130101; G05D 23/2401 20130101 |
Class at
Publication: |
422/199 ;
422/102; 422/198; 422/109 |
International
Class: |
B01L 007/00; G05D
023/19 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 6, 1996 |
GB |
9625442.0 |
Jul 31, 1997 |
GB |
19716052.7 |
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 a 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 any one of claims 1 to 3 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 or claim 5 wherein the heater
polymer is in the form of a film.
7. Apparatus as claimed in claim 5 or claim 6 wherein the sheath is
integral with the container.
8. Apparatus as claimed in any one of claims 4 to 7 wherein the
heater polymer is perforated or reticulated.
9. Apparatus as claimed in any one of claims 1 to 3 wherein the
heater polymer forms a container for the reactants.
10. Apparatus as claimed in any one of claims 1 to 3 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 any one of the preceding claims wherein
each reaction vessel comprises a capillary tube.
12. Apparatus as claimed reaction vessel as claimed in any one of
claims 1 to 10 wherein the reaction vessel comprises a slide.
13. Apparatus as claimed in any one of claims 1 to 10 wherein the
reaction vessel comprises a chip.
14. Apparatus according to any one of the preceding claims wherein
the reaction vessels are provided in an array.
15. Apparatus as claimed in any one of the preceding claims,
wherein the control means is arranged to supply electric current so
as to conduct reactions requiring multiple temperature stages
within the reaction vessels.
16. Apparatus as claimed in claim 15 and wherein the control means
is programmed such that multiple cycles of the reaction can be
effected automatically.
17. Apparatus as claimed in claim 15 or claim 16 wherein the
control means is arranged to supply current according to a
predetermined time/temperature profile.
18. Apparatus as claimed in any one of claims 15 to claim 17 and
adapted for polymerase chain reaction processes.
19. Apparatus as claimed in any one the preceding claims further
comprising a means for detecting a signal from a sample in a
reaction vessel.
20. 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.
21. A method according to claim 20 wherein the reaction is a DNA
amplification method.
22. A method according to claim 21 wherein the amplification method
is a polymerase chain reaction (PCR).
23. A method according to any one of claims 19 to 22 wherein
reagents for a plurality of reactions are each placed in a reaction
vessel and heated simultaneously.
24. A method according to claim 23 wherein each reaction vessel is
heated individually to the temperature required for the reaction
taking place within that vessel.
25. A reaction vessel comprising a slide or a chip and an
electically 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.
26. A reaction vessel according to claim 25 wherein the said
polymer is integral with the slide or chip.
27. Apparatus for reactions requiring multiple temperature stages,
according to claim 1 and substantially as hereinbefore described
with reference to the accompanying drawings.
28. A reaction vessel comprising a container and an electrically
conducting polymer which is adapted for use in apparatus according
to anyone of claims 1 to 19.
29. A method according to claim 19 substantially as hereinbefore
described with reference to the Example.
Description
[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.
[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 multi-shot apparatus,
certainly not, mobile or portable such apparatus.
[0011] The applicants have developed an efficient system for rapid
heating and cooling of reactants which is particularly useful in
thermal cycling reactions.
[0012] Accordingly, the present invention provides a reaction
vessel comprising an electrically conducting polymer which emits
heat when ah electric current is passed through it.
[0013] 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.
[0014] 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.
[0015] 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.
[0016] Suitable cooling methods include forced air cooling, for
example by use of fans, immersion in ice or water baths etc.
[0017] 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.
[0018] The 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.
[0019] 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.
[0020] 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.
[0021] In another embodiment of the invention, the polymer is
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 wuith 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.
[0022] Vessels which comprise the polymer as an integral part may
provide particularly compact structures.
[0023] 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.
[0024] 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 operating temperature. 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.
[0025] 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.
[0026] 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.
[0027] 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.
[0028] In one 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.
[0029] Alternatively 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] Vessels of this type may be particularly useful for carrying
out in-situ PCR for example on tissue samples.
[0031] Other suitable reaction vessel are tubes and cuvettes, which
are known in the art.
[0032] The invention further provides apparatus for reactions
requiring multiple temperature stages, said apparatus comprising a
reaction vessel as described above, a means for generating an
electrical current within the polymer and a control means for
regulating the amount of electric current passing through the
polymer so as to control its temperature.
[0033] The control means is suitably an automatic control means
such as a computer controlled interface arrangement. By using a
programmable controller for the electrical circuit connected to the
polymer, a defined heating regime, for example a defined number of
cycles of predetermined temperature stages to be established over
predetermined time intervals and dwells can be pre-programmed using
the apparatus, including employing different temperature and time
profiles with different reaction vessels in the same apparatus at
the same time.
[0034] The control means may include a temperature monitoring
device such as a thermocouple, which monitors the temperature of
the reaction vessel and feeds this information into the control
system so that the desired regime of heating and/or cooling is
adhered to.
[0035] Alternatively, the temperature of the polymer may be
monitored directly by measuring its resistivity, for example by
arranging the polymer heating element as a resistor in a wheatstone
bridge circuit arrangement. This avoids the use of other
temperature measurement devices such as thermocouples.
[0036] Optionally, the apparatus further comprises artificial
cooling means such as one or more fans.
[0037] The apparatus may include a plurality of containers. The
polymer may be provided as an integral part of each container, as a
sheath around each container or arranged such that a layer of
polymer is interposed between adjacent containers. Any electrical
connection points on the polymer may be connected to a single
electrical supply, if a number of reactions requiring the same
temperature stages are being carried out.
[0038] However, in a preferred embodiment the apparatus is arranged
such that the polymer in contact with (or forming) a container or a
group of containers is connected to an individual supply, several
containers or groups of containers being connected to different,
independently controlled electrical supplies. With this
arrangement, a number of different reactions requiring different
temperature stages can be carried out at the same time as each
container or group of containers has its own heating element. This
arrangement allows users to carry out a number of small batch
reactions using a single apparatus which has not been possible
using existing equipment. The only apparatus previously available
for this type of use are certain designs of block heaters which
have between 2 and 4 segments which can be heated and cooled
independently. However, such apparatus is limited to use for 2 to 4
batches of reactions and has the disadvantage of slow cycle times
as previously described.
[0039] 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.
[0040] 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 a
reaction vessel as defined above.
[0041] 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.
[0042] 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.
[0043] Generic DNA intercollating 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.
[0044] Embodiments of the present invention will now be described,
by way of example, with reference to the accompanying drawings,
wherein
[0045] FIG. 1. Shows a reaction vessel heater comprising a sheath
of electrically conducting polymer arranged to fit around a
reaction tube;
[0046] FIG. 2. Shows a reaction slide having an electrically
conducting polymer coating over one of its surfaces;
[0047] FIG. 3. Shows a reaction slide having a layer of
electrically conducting polymer within a composite
construction;
[0048] 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;
[0049] FIG. 5 shows a diagram of apparatus according to the
invention for carrying out a PCR reaction;
[0050] FIG. 6 shows a thermocycling profile used with the apparatus
of FIG. 5;
[0051] FIG. 7 is a schematic diagram of a portable PCR
multidetector; and
[0052] FIG. 7a is a diagram of a detector element for use in the
apparatus of FIG. 7.
[0053] 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.
[0054] 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.
[0055] 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.
[0056] 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.
[0057] 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.
[0058] 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.
[0059] 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.
[0060] 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.
[0061] 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.
[0062] 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.
[0063] 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.
[0064] The apparatus shewn in FIG. 7 comprises a lidded box 70
having insulative partitioning defining a plurality of detector
element receptor bays 71. The box 71 is shewn 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
shewn) for monitoring the temperature therein.
[0065] The detector element shewn 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.
[0066] 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
shewn). The apparatus when fully connected is arranged to permit
displaying on the computer screen the connection status of each
tube 75.
[0067] Closure of the lid to the box 70 completes the insulation of
each bay and the retention of each tube 75 in its bay.
[0068] 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.
[0069] Of course the principle of the apparatus described in
relation to FIGS. 7 and 7a may be realized 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.
[0070] The following Example illustrates the invention.
EXAMPLE
[0071] Amplification of DNA
[0072] Using the apparatus of FIG. 5 with the K-type thermocouple
removed, the following PCR reaction was effected.
[0073] 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.
[0074] The reaction mixture placed in the tube 6 comprised the
following:
[0075] 50 mM Tris.HCl pH 8.3
[0076] 3 mM MgCl
[0077] 2.5 mg/ml Bovine Serum Albumen
[0078] 200 .mu.M each of DATP, dTTP, dCTP and dGTP
[0079] 10 .mu.g/ml each PCR primers
[0080] 25 Units/ml Taq Polymerase
[0081] 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..
[0082] 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.
[0083] 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.
* * * * *