U.S. patent application number 11/768605 was filed with the patent office on 2008-03-06 for system for rapid nucleic acid amplification and detection.
This patent application is currently assigned to SPARTAN BIOSCIENCE INC.. Invention is credited to Nicole A. Arbour, Christopher J.E. Harder, John Lem, Paul Lem, Alan Shayanpour, Jamie Spiegelman.
Application Number | 20080057544 11/768605 |
Document ID | / |
Family ID | 35149993 |
Filed Date | 2008-03-06 |
United States Patent
Application |
20080057544 |
Kind Code |
A1 |
Lem; Paul ; et al. |
March 6, 2008 |
SYSTEM FOR RAPID NUCLEIC ACID AMPLIFICATION AND DETECTION
Abstract
A system is provided for carrying out rapid nucleic acid
amplification or other biological reactions requiring thermal
cycling. The system of this invention incorporates at least two
heating blocks, each having a groove for receiving a reaction
vessel such that only a portion of the outer surface of the walls
of the vessel are in direct contact with the heating block. The
remaining portion of the outer surface of the walls of the reaction
vessel remains exposed to ambient conditions. The reaction vessel
comes into contact with only one heating block at a time either by
movement of the vessel between the heating blocks or by movement of
the heating blocks in relation to the vessel. The system of this
invention provides rapid temperature cycling without the need for
extended ramping times generally associated with single block
designs, which include a single temperature block that is forced to
heat and cool. Heating and cooling of the reaction using the system
of the present invention is accomplished by the reaction vessel and
heating blocks coming into thermal contact and reaching thermal
equilibrium. The entire vessel need not be surrounded by the
heating block, with at least one side of the vessel partially open
to ambient conditions. As a result of the configuration of this
system it is readily combined with detection systems, for example,
fluorescence detection systems.
Inventors: |
Lem; Paul; (Toronto, CA)
; Lem; John; (Toronto, CA) ; Spiegelman;
Jamie; (Toronto, CA) ; Harder; Christopher J.E.;
(Gatineau, CA) ; Arbour; Nicole A.; (Ottawa,
CA) ; Shayanpour; Alan; (Stittsville, CA) |
Correspondence
Address: |
LEYDIG VOIT & MAYER, LTD
TWO PRUDENTIAL PLAZA, SUITE 4900, 180 NORTH STETSON AVENUE
CHICAGO
IL
60601-6731
US
|
Assignee: |
SPARTAN BIOSCIENCE INC.
Ottawa
CA
|
Family ID: |
35149993 |
Appl. No.: |
11/768605 |
Filed: |
June 26, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11578440 |
Oct 13, 2006 |
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PCT/CA05/00576 |
Apr 15, 2005 |
|
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11768605 |
|
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60563061 |
Apr 16, 2004 |
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Current U.S.
Class: |
435/91.2 ;
435/303.1 |
Current CPC
Class: |
B01L 7/525 20130101;
B01L 2300/1827 20130101; B01L 9/06 20130101; G01N 21/6452 20130101;
G01N 21/272 20130101; G01N 21/6428 20130101 |
Class at
Publication: |
435/91.2 ;
435/303.1 |
International
Class: |
C12P 19/34 20060101
C12P019/34; C12M 1/00 20060101 C12M001/00 |
Claims
1. In a thermal cycling process for polymerase chain reaction (PCR)
wherein a reaction vessel (RV) having a vessel wall--containing a
reaction mixture comprising a target nucleic acid (NA) and reagents
selected to achieve amplification of said target NA by means of the
PCR--is thermally cycled between at least two predetermined
temperatures, the improvement characterized by maintaining a
temperature gradient within said RV during said thermal cycling
process.
2. The process defined in claim 1, wherein said temperature
gradient is obtained by applying heat unequally to the wall of said
RV.
3. The process defined in claim 2 wherein said heat is applied
unequally to said vessel wall by contacting only a portion of said
wall with a heating element while exposing the remainder of said
wall to ambient air.
4. The process as defined in claim 1, further characterized by
rapid thermal cycling wherein a temperature spectrum of said NA and
reagents is maintained within said RV during the thermal cycling
process.
5. The process as defined in claim 1, further characterized by
annealing and extension of said NA occurring in a single step
during the PCR process.
6. The process as defined in claim 1, further characterized by RV
temperature ramp-time in excess of 2.degree. C./sec.
7. The process as defined in claim 1, further characterized by real
time fluorescence-based measurement of said NA amplification, if
present in said reaction mixture, during the PCR process.
8. The process as defined in claim 1 wherein said temperature
gradient consists of a large gradient.
9. A polymerase chain reaction (PCR) device for thermal cycling of
a reaction mixture contained in a reaction vessel (RV) having an
outer wall, comprising at least two heating sources, means for
maintaining said heating sources at different predetermined
temperatures, and means for causing said RV to be in successive
physical contact with each said heating sources for predetermined
times and for a number of successive contacts sufficient to promote
the PCR in said reaction mixture contained in said RV,
characterized by said heating sources being configured for
successively contacting only part of said outer wall of the RV
while the remainder of said surface is exposed to ambient air.
10. The PCR device as defined in claim 8, wherein said at least two
heating sources comprise spaced apart heating blocks each having a
groove contoured to receive and provide thermal contact with only a
portion of the outer wall of the RV that contains the reaction
mixture.
11. The PCR device as defined in claim 8, further comprising means
for real-time florescence-based measurement of a preselected
nucleic acid (NA), if present in said reaction mixture, during
operation of the PCR device.
12. The PCR device defined in claim 8 further comprising a
controlling means for operating said device so as to achieve a time
versus temperature profile which maintains a large temperature
gradient within said RV during said thermal cycling process.
12. The PCR device defined in claim 11, wherein said controlling
means controls the device to provide annealing and extension of
said NA within said RV in a single step during the PCR process by
maintaining essentially no holding time at the temperature required
for said annealing and extension step.
13. The PCR device defined in claim 9, wherein said at least two
heating blocks are in spaced apart opposing relation to each other
and said grooves oppose each other.
14. The PCR device defined in claim 13, further comprising a holder
for retaining said RV such that when engaged within said groove,
and drive means for sequentially moving said RV between said blocks
for successive contact with said opposing grooves.
15. The device defined in claim 9, wherein said grooves are
dimensioned to contact between about 40% and 50% of the wall of
said RV.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This is a continuation-in-part of co-pending U.S. national
phase application Ser. No. 11/578,440 entered nationally on Oct.
13, 2006, and filed internationally on Apr. 15, 2005 as PCT
International Application No. PCT/CA2005/000576, which claim the
benefit of U.S. provisional application Ser. No. 60/563,061 filed
on Apr. 16, 2004, and both of which are incorporated herein by
reference in their entirety.
TECHNICAL FIELD
[0002] The present invention relates to a temperature cycling
apparatus useful for performing nucleic acid amplification and
detection. More specifically, the present invention relates to a
thermal cycling apparatus for rapidly cycling the temperature of a
sample through a predetermined temperature cycle.
BACKGROUND OF THE INVENTION
[0003] In numerous areas of industry, technology, and research
there is a need to reliably and reproducibly subject relatively
small samples to thermal cycling. The need to subject a sample to
repeated temperature cycles is particularly acute in biotechnology
applications. In the biotechnology field, it is often desirable to
repeatedly heat and cool small samples of materials over a short
period of time. One such biological process that is regularly
carried out is cyclical DNA amplification.
[0004] Cyclic DNA amplification, using a thermostable DNA
polymerase, allows automated amplification of primer specific DNA,
widely known as PCR, or the polymerase chain reaction. It is well
accepted that automation of this process requires controlled and
precise thermal cycling of reaction mixtures.
[0005] PCR is a technique involving multiple cycles that results in
the geometric amplification of certain polynucleotide sequence each
time a cycle is completed. The technique of PCR is well known to
the person of average skill in the art of molecular biology.
[0006] Commercial programmable metal heat blocks have been used in
the past to affect the temperature cycling of samples in microfuge
tubes through the desired temperature versus time profile. Peltier
heating and cooling are usually utilized in changing temperatures
at a rate of approximately 0.5-4 degrees Celsius per second.
However, the inability to quickly and accurately adjust the
temperature of the heat blocks through a large temperature range
over a short time period, has rendered the use of heat block type
devices undesirable as a temperature control system when carrying
out the polymerase chain reaction in a rapid fashion.
[0007] A wide variety of instrumentation has been developed for
carrying out nucleic acid amplifications. Important design goals
fundamental to PCR instrument development have included fine
temperature control, minimization of sample-to-sample variability
in multi-sample thermal cycling, automation of pre- and post-PCR
processing steps, high speed cycling, minimization of sample
volumes, real-time measurement of amplification products,
minimization of cross-contamination, or sample carryover, and the
like.
[0008] A prior art system is represented by a temperature cycler in
which multiple temperature controlled blocks with vertical reaction
vessel wells are maintained at different desired temperatures (U.S.
Pat. Nos. 5,525,300, 5,779,981 and 6,054,263). A robotic arm is
utilized to move reaction mixtures from block to block. The
reaction vessels are lifted vertically from out of the heat block,
transported to another heating block, and placed vertically down
into said heating block. However, this system requires precision
movement, pressurized thermal contact and expensive microprocessor
controlled robotics. This robotic movement system also impedes a
real-time fluorescent detection system of the nucleic acid
amplification product during and after temperature cycling has
completed.
[0009] Rapid cycling has been described before (e.g. U.S. Pat. No.
6,174,670 and U.S. Pat. No. 5,455,175). According to this prior
art, rapid cycling techniques are made possible by the rapid
temperature response and temperature homogeneity of samples in high
surface area-to-volume sample containers such as capillary tubes.
For further information, see also: C. T. Wittwer, G. B. Reed, and
K. M Ririe, Rapid cycle DNA amplification, in K. B. Mullis, F.
Ferre, and R. A. Gibbs. The polymerase chain reaction, Birkhauser,
Boston, 174-181, (1994). According to this prior art, improved
temperature homogeneity allows the time and temperature
requirements of PCR to be better defined and understood, while
improved temperature homogeneity also increases the precision of
any analytical technique used to monitor PCR during
amplification.
[0010] The design of instruments that permit PCR to be carried out
in closed reaction chambers and monitored in real-time is highly
desirable. Closed reaction chambers are desirable for preventing
cross-contamination, e.g. Higuchi et al, Biotechnology, 10: 413-417
(1992) and 11: 1026-1030 (1993); and Holland et al, Proc. Natl.
Acad. Sci., 88: 7276-7280 (1991). Clearly, the successful
realization of such a design goal would be especially desirable in
the analysis of diagnostic samples, where a high frequency of false
positives and false negatives would severely reduce the value of
the PCR-based procedure. Real-time monitoring allows the coupling
of amplification and detection, thus decreasing contamination risks
and labour time. As well, real-time monitoring of a PCR permits far
more accurate measurement of starting target DNA concentrations in
multiple-target amplifications, as the relative values of close
concentrations can be resolved by taking into account the history
of the relative concentration values during the PCR. Real-time
monitoring also permits the efficiency of the PCR to be evaluated,
which can indicate whether PCR inhibitors are present in a
sample.
[0011] Holland et al (cited above) and others have proposed
fluorescence-based approaches to provide real-time measurements of
amplification products during a PCR. Such approaches have either
employed intercalating dyes (such as ethidium bromide) to indicate
the amount of double stranded DNA present, others have employed
probes containing fluorescent-quencher pairs (the so-called
"Taq-Man.TM." approach) that are cleaved during amplification to
release a fluorescent product whose concentration is proportional
to the amount of double stranded DNA present. Other fluorescent
probe technologies have also been used in real-time PCR, including
Fluorescent Resonance Energy Transfer (FRET) probes (U.S. Pat. Nos.
6,174,670 and 6,569,627), linear probes in which one probe
stimulates and adjacent probes to fluoresce, and molecular beacons
in which a hairpin loop is formed within the probe to quench the
florescence when the probe is not hybridized to the target nucleic
acid.
[0012] The accepted state of the art is that nucleic acid
reactions, such as PCR, require a uniform temperature in order to
be successful. This was expressed in 1998 (by Neumaier et al. and
Wagener et al. Fundamentals of quality assessment of molecular
amplification methods in clinical diagnostics. Clinical Chemistry.
44(1): 12-26) as follows: [0013] "Uniform temperature transition is
an important aspect of successful amplification": and [0014] "The
homogeneity of heat conduction in the reaction block is of crucial
importance. The heat performance of the cycler and the uniformity
of heat conduction in the heating block must be controlled
regularly to avoid false negative results".
[0015] Consequently, manufacturers of PCR machines have engineered
their instruments to generate uniform thermal gradients. For
example, in 1992, Stratagene introduced the RoboCycler temperature
cycler, a unique four-block instrument that claimed to achieve
unparalleled temperature uniformity (Renzi, P., et al. (1992)
Strategies 5: 41-42). More recently, Corbett Research developed the
Rotor-Gene instrument that heats and cools PCR reaction tubes via
air jets. The rotor containing the reaction tubes spins at very
high speeds, and the stated intention is to increase temperature
uniformity. Additionally, there are products such as the DRIFTCON
system (Appropriate Technical Resources, Inc.) that enable
researchers to test the temperature uniformity of the thermal block
in their PCR machines.
SUMMARY OF THE INVENTION
[0016] The present invention provides an apparatus for thermal
cycling of a reaction sample between two or more temperatures in
which heat is applied to a reaction vessel, unequally to the vessel
wall. It has been found that unequal application of heat from
heating means such as a heating block, to the reaction vessel,
permits a more rapid cycling of the reaction vessel and its
contents between different reaction temperatures, while still
permitting reactions such as PCR to effectively occur.
[0017] The prototype device of the present invention was able to
successfully perform PCR reactions using an apparatus where the
thermal gradient across the reaction tube was highly
non-uniform.
[0018] Specifically, the prototype apparatus consisted of fixed
temperature heat blocks, with an engraved groove that fit one-half
of a standard PCR reaction tube. During a temperature cycle, only
one side of the PCR reaction tube was in contact with the heat
source, while the opposite side was exposed to ambient air
temperature. This unequal heating resulted in a large thermal
gradient across the reaction tube. Contrary to conventional
thinking the PCR reaction was successful, and no non-specific
amplicons were seen in post-PCR gel electrophoresis. In fact, gel
electrophoresis analysis showed that the amplicon generated by the
prototype apparatus using a highly non-uniform thermal gradient was
the same as the amplicon generated with the GeneAmp 2700 machine
(Applied Biosystems), using a thermal block with a highly uniform
temperature gradient.
[0019] With the new knowledge that nucleic acid amplification
reactions can be successfully performed when the, reaction vessel
is subject to a non-uniform temperature gradient, it is possible to
design set-ups such as the prototype device, where a reaction tube
is moved in a single degree of freedom of motion between two heat
blocks, set at fixed temperatures. This makes it possible to do
away with the robotics and electronics of the Stratagene
RoboCycler, where the tube must be moved in three dimensions so
that it can fully (and uniformly) enclosed in the temperature
block.
[0020] Another advantage is that an entire side of the tube may be
conveniently exposed for real-time detection by an optical
detection system. This avoids the need for complicated optics that
monitor the progress of a real-time PCR reaction by directing
excitation light down through the top of the reaction tube, and
then collecting emitted light that is reflected back up through the
top of the tube, (for example, this is the system used in some of
the real-time PCR machines from Applied Biosystems). For example,
it is contemplated that a possible design would be to embed the
reaction tube partially in a heat block and perform PCR by ramping
the temperature of the heat block. At the same, real-time optical
detection could be performed by detecting emitted fluorescence from
the exposed portion of the reaction tube.
[0021] According to one aspect, it is an object of the present
invention to provide a thermal cycling apparatus for quickly and
accurately varying the temperature of biological samples according
to a predetermined temperature versus time profile.
[0022] According to another aspect, it is also an object of the
present invention to provide a thermal cycling apparatus that can
effectively subject samples to a large temperature gradient over a
very short period of time.
[0023] According to another aspect, it is a further object of the
present invention to provide an apparatus that can subject a
biological sample to rapid thermal cycling by rapidly alternating
the sample between two heating blocks, which may be in opposing
relation to one another.
[0024] According to another aspect, it is another object of the
present invention to provide a thermal cycling apparatus that will
heat samples located in a fluid chamber placed in an engraved
groove in the side of the heating blocks.
[0025] According to another aspect, the two heating blocks are
positioned in ambient air, and a portion of the reaction vessel
while in the heating block is exposed to air without compromising a
thermal gradient within the reaction vessel.
[0026] According to another aspect, it is also an object of the
invention to provide a real-time nucleic amplification product
detection mechanism between the two heating blocks by means of
fluorescence monitoring of the product labeled with a fluorescent
dye.
[0027] Thus according to the present invention there is provided a
thermal cycling process for polymerase chain reaction (PCR) wherein
a reaction vessel (RV) having a vessel wall--containing a reaction
mixture comprising a target nucleic acid (NA) and reagents selected
to achieve amplification of said target NA by means of the PCR--is
thermally cycled between two predetermined temperatures, the
improvement characterized by subjecting the RV to a temperature
gradient during said thermal cycling process, and preferably a
large temperature gradient which is maintained during all or
substantially all of the duration of the cycling step.
[0028] In the above process, the heat of each of the two
predetermined temperatures to obtain the temperature gradient is
applied unequally to the wall of the RV.
[0029] Preferably, the heat is applied unequally to the vessel wall
by contacting only a portion of the wall with a heating, element
while exposing the remainder of the wall to ambient air.
[0030] In the process rapid thermal cycling is provided wherein a
temperature spectrum of the NA and reagents is maintained within
the RV during the thermal cycling process.
[0031] Further, preferably annealing and extension of NA occur in a
single step during the PCR process.
[0032] A PCR device, according to the present invention, for
thermal cycling of a reaction mixture contained in a reaction
vessel (RV) having an outer surface, comprising at least two
heating sources, characterized by each of the at least two heating
sources having a different predetermined temperature, for
successively heating only part of the outer surface of the RV.
[0033] In a narrower aspect in the PCR device the two heating
sources are spaced apart heating blocks and each having a groove
contoured to receive and provide thermal contact with only a
portion of the outer surface of the RV; and the means for causing
the RV to be in successive physical thermal contact with each the
two heating blocks for predetermined times and for a number of
successive contacts sufficient to promote the PCR in the reaction
mixture contained in the RV.
[0034] The PCR device further comprises a means for maintaining the
heating sources at different predetermined temperatures and a means
for causing the RV to be in successive physical contact with each
of the heating sources for predetermined times and for a number of
successive contacts sufficient to promote PCR in the reaction
mixture contained in the RV, the heating sources being configured
for successively contacting only part of the outer wall of the RV
while the remainder of the surface is exposed to ambient air.
[0035] In the PCR device the two heating sources comprise spaced
apart heating blocks each having a groove contoured to receive and
provide thermal contact with only a portion of the outer wall of
the RV.
[0036] The PCR device further comprises, a controlling means for
operating the device so as to achieve a time versus temperature
profile which subjects the RV to a large temperature gradient
during the thermal cycling process.
[0037] In the PCR device the controlling means controls the device
to provide annealing and extension of the NA within the RV in a
single step during the PCR process by maintaining essentially no
holding time at the temperature required for the step.
[0038] In the PCR device the two heating blocks are in spaced apart
opposing relation to each other and the grooves oppose each
other.
[0039] The PCR device further comprises a holder for retaining the
RV such that when engaged within the groove, the drive means
sequentially causes relative movement between the RV and the blocks
for successive contact with the opposing grooves.
[0040] In the PCR device the grooves are dimensioned to contact
between about 40% and 50% of the wall of the RV.
[0041] The PCR device further comprises means for real-time
florescence-based measurement of a preselected nucleic acid (NA),
if present in the reaction mixture during operation of the PCR
device.
[0042] The apparatus of the present invention includes a
controlling means for operating the apparatus through the desired
time versus temperature profile. The present invention is
particularly well suited for carrying out automated and rapid
polymerase chain reactions.
[0043] The controller of the present invention allows the chamber,
and subsequently the samples located in the sample compartment
therein, to pass through a predetermined temperature cycle
corresponding to the denaturation and annealing/extension steps in
the polymerase chain reaction. In use, the apparatus of the present
invention allows rapid optimization of denaturation, and
annealing/extension steps in terms of time and temperature, and
shortened or minimal time periods (ramp times) between the
temperatures at each step.
[0044] The present invention particularly decreases the total time
required for completion of polymerase chain reaction cycling over
prior art thermal cycling devices by having high reaction vessel
liquid ramp time (>2.degree. C./sec), by decreasing the number
of steps per cycle from three to two (the annealing and extension
steps are done at one temperature), and by decreasing the amount of
time for each step of each cycle without compromising amplification
efficiency.
[0045] The invention relates to a system for carrying out rapid
nucleic acid amplification using PCR, which can be coupled with
real-time fluorescence-based measurements of nucleic acid
amplification products. In one preferred embodiment of the
invention, an excitation beam is focused into a reaction mixture
containing a nucleic acid intercalating dye or hybridization probe
which fluoresces proportionally to the amount of nucleic acid
amplification product, it is understood that the proportionality of
the fluorescent intensities is for a constant set of parameters
such as temperature, pH, salt concentration, and the like, that
independently influence the fluorescent emissions of organic
dyes.
[0046] Preferably, the excitation beam is focused into the reaction
mixture through a lens through a portion of a wall of a closed
reaction chamber containing the reaction mixture.
[0047] In one embodiment, the reaction chamber is a reaction vessel
with a closed end, referred to herein as the bottom of the vessel,
and an open end, referred to herein as the top of the vessel, which
can be closed with a cap such that a leak-proof seal is formed. In
other words, once a reaction mixture is placed in the vessel and
the cap is attached a closed reaction chamber is formed. In this
embodiment, (1) the reaction mixture fills a portion of the
reaction vessel, generally at the bottom of the vessel, such that a
void is left between the cap of the vessel and a top surface of the
reaction mixture, and (2) the reaction is completed in the
apparatus described above and (3) the lens, without contacting the
side of the vessel, allows the excitation beam through the vessel
wall into the reaction mixture through its side surface and (4) the
resulting fluorescence generated by the fluorescent indicator is
collected by a photosensor, such as a photodiode, at various time
points.
[0048] As discussed more fully below, an excitation beam generated
by a single light source, e.g. a Light Emitting Diode (LED), is
placed on one side of the vessel. The wavelength of the excitation
beam is restricted by appropriately selecting a colored type of LED
as well as using a color filter by which LED light travels through.
Likewise, a different color filter located on the opposite side of
the vessel restricts the resultant emission fluorescence
wavelength. A photodiode sensor collects the filtered emission
light and the amplitude of the emission is examined and compared by
an analysis system.
[0049] Preferably, the system is employed with the PCR
amplification of nucleic acids.
[0050] The system of the invention permits accurate real-time
monitoring of nucleic amplification reactions by providing
apparatus and fluorescent reagents for generating a stable
fluorescent signal proportional to the amount of amplification
product and independent of variations in the volume of reaction
mixture. The availability of data showing the progress of
amplification reactions leads to rapid assessment of the efficiency
of the amplification reactions, and opens the possibility of
reduced reagent usage and feedback reaction control.
BRIEF DESCRIPTION OF THE DRAWINGS
[0051] The invention will be better understood by reference to the
appended figures of which:
[0052] FIG. 1: is a perspective view of a portion of a thermal
alternator device according to one embodiment of the invention.
[0053] FIG. 2A is a perspective view of a thermal alternator
according to the first embodiment of the invention.
[0054] FIG. 2B is a perspective view of a second embodiment of the
invention.
[0055] FIG. 3: is a perspective view of the fluorescence detection
apparatus according to one embodiment of the present invention.
[0056] FIG. 4: is a top plan view of a reaction vessel holder for
carrying out a method according to one embodiment of the present
invention;
[0057] FIG. 5: is an isometric view of the reaction vessel holder
shown in FIG. 4;
[0058] FIG. 6: is a block diagram of the control and processing
electronics of the present invention;
[0059] FIG. 7: is a general flow-chart illustrating how the device
of the present invention is operated; and
[0060] FIGS. 8A & 8B: are flow-charts summarizing the control
software of the present device.
DETAILED DESCRIPTION
[0061] In describing and claiming the present invention, the
following definitions apply:
[0062] "Nucleic acid," "DNA," and similar terms also include
nucleic acid analogs, i.e. analogs having other than a
phosphodiester backbone. For example, the so-called "peptide
nucleic acids," which are known in the art and have peptide bonds
instead of phosphodiester bonds in the backbone, are considered
within the scope of the present invention.
[0063] "Amplification" of DNA denotes the use of polymerase chain
reaction (PCR) to increase the concentration of a particular DNA
sequence within a mixture of DNA sequences.
[0064] An "amplicon" is a product of the amplification of a target
genetic sequence.
[0065] A "PCR reaction mixture" denotes a mixture adaptable for
simultaneously amplifying multiple genetic targets under suitable
conditions for PCR.
[0066] A "genetic target" denotes a genetic sequence capable of
amplification by polymerase chain reaction (PCR). A genetic target
in accordance with the present invention includes any DNA sequence,
including bacterial, viral, fungal, human, plant, and animal DNA,
for example.
[0067] "Continuous monitoring" and similar terms refer to
monitoring multiple times during a cycle of PCR, preferably during
temperature transitions, and more preferably obtaining at least one
data point in each temperature transition.
[0068] "Fluorescence detection" and similar terms refer to labeling
nucleic acids with a fluorescence indicator. The fluorescence
indicator can be a nucleic acid intercalating dye such as Ethidium
Bromide, Thiazole orange, Pico.TM. Green or SyBr.TM. Green. As
well, labeled hybridization probes using FRET, Taq-Man.TM. or other
chemistries such as molecular beacons can also be used as
fluorescence detection tools.
[0069] "Effective amount" means an amount sufficient to produce a
selected effect. For example, an effective amount of PCR primers is
an amount sufficient to amplify a segment of nucleic acid by PCR
provided that a DNA polymerase, buffer, template, and other
conditions, including temperature conditions, known in the art to
be necessary for practicing PCR are also provided.
[0070] "Probe", refers to a nucleic acid oligomer that hybridizes
specifically to a target sequence in a nucleic acid, which, in the
context of the present invention, is an amplicon, under standard
conditions that promote hybridization. This allows detection of the
amplicon. Detection may either be direct (i.e., resulting from a
probe hybridizing directly to the amplicon sequence) or indirect
(i.e., resulting from a probe hybridizing to an intermediate
molecular structure that links the probe to the target amplicon). A
probe's "target" generally refers to a sequence within (i.e., a
subset of) an amplified nucleic acid sequence which hybridizes
specifically to at least a portion of a probe oligomer using
standard hydrogen bonding (i.e., base pairing). A probe may
comprise target-specific sequences and other sequences that
contribute to three-dimensional conformation of the probe (e.g., as
described in Lizardi et al., U.S. Pat. Nos. 5,118,801 and
5,312,728).
[0071] By "sufficiently complementary" is meant a contiguous
nucleic acid base sequence that is capable of hybridizing to
another base sequence by hydrogen bonding between a series of
complementary bases. By definition, this allows stable
hybridization of a probe oligomer to a target sequence in the
amplicon even though it is not completely complementary to the
probe's target-specific sequence. Complementary base sequences may
be complementary at each position in the base sequence of an
oligomer using standard base pairing or may contain one or more
residues that are not complementary using standard hydrogen bonding
(including a basic "nucleotides"), but in which the entire
complementary base sequence is capable of specifically hybridizing
with another base sequence in appropriate hybridization conditions.
Contiguous bases are preferably at least about 80%, more preferably
at least about 90%, and most preferably greater than 95%
complementary to a sequence to which an oligomer is intended to
specifically hybridize. To those skilled in the art, appropriate
hybridization conditions are well known, can be predicted based on
base composition, or can be determined empirically by using routine
testing (e.g., see Sambrook et al., Molecular Cloning, A Laboratory
Manual, 2.sup.nd ed. (Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, N.Y., 1989) at .sctn. 1.90-1.91, 7.37-7.57,
9.47-9.51 and 11.47-11.57 particularly at .sctn.9.50-9.51,
11.12-11.13, 11.45-11.47 and 11.55-11.57).
[0072] The terms "label" and "detectable label" refer to a
molecular moiety or compound that can be detected or can lead to a
detectable response. A label is joined, directly or indirectly, to
a nucleic acid probe. Direct labeling can occur through bonds or
interactions that link the label to the probe, including covalent
bonds or non-covalent interactions (e.g., hydrogen bonding,
hydrophobic and ionic interactions) or through formation of
chelates or co-ordination complexes. Indirect labeling can occur
through use of a bridging moiety or "linker," such as an antibody
or additional oligonucleotide(s), which is either directly or
indirectly labeled, and which can amplify a detectable signal. A
label can be any known detectable moiety, such as, for example, a
radionuclide, ligand (e.g., biotin, avidin), enzyme or enzyme
substrate, reactive group, chromophore, such as a dye or particle
that imparts a detectable color (e.g., latex or metal particles),
luminescent compound (e.g., bioluminescent, phosphorescent or
chemiluminescent labels) and fluorescent compound.
[0073] PCR techniques applicable to the present invention include
inter alia those described in "PCR Primer. A Laboratory Manual",
Dieffenback, C. W. and Dveksler, G. S., eds., Cold Spring Harbor
Laboratory Press (1995); "Enzymatic amplification of beta-globin
genomic sequences and restriction site analysis for diagnosis of
sickle cell anemia", Saiki R K, Scharf S, Faloona F, Mullis K B,
Horn G T, Erlich H A, Amheim N, Science (1985) December 20;
230(4732): 1350-4.
[0074] The PCR of the present invention is performed using a
modified 2-step cycling profile as compared to standard PCR, namely
successive cycles of denaturation of double stranded target nucleic
acid and annealing/extension of the primers to produce a large
number of copies of segments of the target DNA. Each cycle is a
thermal cycle in which the reaction temperature is raised to
denature the double stranded DNA and lowered to allow annealing and
extension.
[0075] In one embodiment of the present invention, the PCR makes
use of successive two-step cycles in which the temperature is
raised to a first temperature for denaturation of the double
stranded DNA and lowered to a second temperature to allow annealing
and extension of the primers.
[0076] Following amplification of a nucleic acid using the system
described herein, the amplicons may be detected using any method
known in the art.
[0077] Preferably, the label on a labeled probe is detectable in a
homogeneous assay system, i.e., where, in a mixture, bound labeled
probe exhibits a detectable change, such as stability or
differential degradation, compared to unbound labeled probe,
without physically removing hybridized from non-hybridized forms of
the label or labeled probe. A "homogeneous detectable label" refers
to a label whose presence can be detected in a homogeneous fashion,
for example, as previously described in detail in Arnold et al.,
U.S. Pat. No. 5,283,174; Woodhead et al., U.S. Pat. No. 5,656,207;
and Nelson et al., U.S. Pat. No. 5,658,737. Examples of labels that
can be used in a homogenous hybridization assay include, but are
not limited to, chemiluminescent compounds (e.g., see U.S. Pat.
Nos. 5,656,207, 5,658,737 and 5,639,604), such as acridinium ester
("AE") compounds, including standard AE or derivatives thereof.
Synthesis and methods of attaching labels to nucleic acids and
detecting labels are well known in the art (e.g., see Sambrook et
al., Molecular Cloning, A Laboratory Manual, 2nd ed. (Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), Chapter
10; U.S. Pat. Nos. 5,658,737, 5,656,207, 5,547,842, 5,283,174,
4,581,333 and European Patent Application No. 0 747 706).
[0078] In accordance with another embodiment of the present
invention, where the amplicons are detected using an assay without
prior separation of the amplicons, they are detected using
different detectable molecules to allow the amplicons from the
different primer pairs to be distinguishable. For example, probes
used to hybridize to the various amplicons can be labeled with
labels that are detectable at different wavelengths.
[0079] In accordance with another embodiment of the present
invention, the amplicon production is monitored in real-time using
procedures known in the art (e.g. see U.S. Pat. No. 6,569,627) and
using the detection apparatus of the present invention.
[0080] It has now been found that rapid thermal cycling within a
temperature spectrum can successfully achieve a PCR, and cycling
between exact temperatures is not required, In the past PCR has
been understood to be a combination of three sequential reactions
(i.e. denaturation, annealing, extension) occurring at three
different temperatures for three time periods. The present
invention makes use of the fact that each of the reactions within
PCR can occur over a range of temperatures and these temperatures
overlap. Denaturation and annealing each occur so rapidly that no
holding time at a particular temperature is necessary for these
reactions to occur. Extension occurs over a range of temperatures
at varying rates and can occur between the annealing temperature
and the denaturation temperature. As a result, the method of the
present invention makes use of a single temperature for both the
annealing and extension portions of PCR.
[0081] Some advantages of the techniques contained herein are based
on rapid cycling, with its advantages in speed and specificity.
Technical Description of the Rapid Thermal Alternator
[0082] The specification describes the apparatus positioned in a
convenient horizontal orientation as illustrated herein.
Accordingly, directional references such as "horizontal" refer to
the apparatus when in this orientation. The device may be operated
in other orientations, with suitable modifications. For convenience
of description, terms such as "horizontal" are used but understood
as being capable of suitable modification.
[0083] The present invention provides a thermal cycler device for
performing reactions, such as PCR, using at least two spaced apart
temperature blocks, each maintained at a different temperature.
Each temperature block is configured to receive one or more
reaction vessels such that only a portion of the outer surface of
the one or more reaction vessels is in direct contact with the
temperature block. As a result of this configuration, the reaction
vessel or vessels can be moved from one temperature block to the
other using a one-dimensional (e.g. lateral) movement of either the
reaction vessel(s) or the temperature blocks, or a combination
thereof.
[0084] In the past, thermal cyclers having more than one
temperature block have required that the reaction vessels be lifted
from a receptacle in one temperature block, moved over and lowered
into a receptacle in the second temperature block. In the past this
design has been used for performing PCR because it was previously
believed that the most effective way to achieve precise temperature
control of the reaction mixture was to maximize the surface area of
the reaction vessel in direct contact with the temperature source.
It has now been found that this is not required for efficient PCR.
In fact, only a portion of the outer surface of the reaction vessel
surface needs to be in direct contact with the temperature block.
It is only necessary that a limited amount of outer surface be in
contact with the temperature block.
[0085] The amount of surface area contact between the heating block
and the reaction vessel will influence the rate at which a
sufficient portion of the reaction mixture is heated or cooled
sufficiently to drive the reaction. The higher the amount of
surface area contact, the faster the speed at which the reaction is
driven, and vice versa. Faster thermal transfer can also be
influenced by other factors, including, but not limited to, the
thickness of the reaction vessel wall, the thermal conductivity of
the material of the reaction vessel, the thermal conductivity of
the heating block, the shape and size of the reaction vessel, the
shape of the reaction vessel receiving groove within each
temperature block and the volume of the reaction mixture.
[0086] The above factors can be altered depending on the intended
application of the device of the present invention. Similarly, the
amount of direct surface area contact between the outer surface of
the reaction vessel and the temperature contact can be varied
depending on the intended application. For example, if faster
thermal transfer is required then a larger direct surface area
contact is required than if a slow thermal transfer is
satisfactory.
[0087] A standard PCR tube may be used as the reaction vessel and
in one version at least about 40% of the outer surface of the
reaction vessel (wherein the outer surface does not include the
surface of any lid present on the reaction vessel) must be in
direct contact with the temperature block. As seen in the figures,
an effective region of contact is about 50% of the vessel wall.
[0088] In one embodiment, the system contains two temperature
blocks mounted in side-by-side spaced apart relation. Transfer of
the reaction vessel or vessels between the two reaction blocks is
achieved either by one-dimensional (lateral) movement of the vessel
or vessels or by one-dimensional movement of the temperature
blocks. The reaction vessel or vessels may be held by holding means
attached to a horizontal transfer means, such as a robotic arm,
that transfers the vessels or vessels via a substantially
horizontal movement between the two temperature blocks.
[0089] An alternative embodiment composes three or more temperature
blocks. As the two-block system, the reaction vessel(s) is moved,
or the blocks are moved, such that the reaction vessel is in direct
contact with only one temperature block at a time.
[0090] As shown in FIG. 1, each heating block 10,20 has a
vessel-shaped slot 22 cut into the upper side facing each other
that acts as receiving means for the reaction vessel. Each slot 22
may be coated with a low-friction, high heat transfer film such as
Teflon.TM., to ensure uniform and rapid heating of the vessel as it
is moved into the slot. The slot 22 is shaped as to completely
contact the reaction vessel on all sides except the facing side
opposite the other heating block.
[0091] The reaction vessel can be a standard conical PCR reaction
tube 23 (e.g. 200 .mu.l volume) or alternatively can be a
rectangular cuvette. The reaction vessel is preferably designed
with a thin-walled material that facilitates heat transfer between
the heating block and the reaction mixture within the reaction
vessel when a portion of the outer surface of the vessel is in
direct contact with the heating block. Preferably the vessel has a
rim about the periphery of its top, which provides a seating
surface for contact with the holding means attached to the
mechanical arm 24. A flat cap that sits partially inside the top of
the vessel seals the top of the tube, providing an air tight seal
to prevent evaporation. The walls of the vessel are formed to be
vertically rigid up to a temperature of about 110.degree. C. to
ensure a tight seal with the vessel slot of the heating block 10,20
during heating. The vessel is formed from a transparent material,
preferably a plastic such as a polypropylene derivative or glass.
Preferably, the vessel has high transmittance of visible light, low
vessel wall gas permeability and sterile inner surface.
[0092] As shown in FIG. 2, the arm mechanism 24 is designed as to
transport the reaction vessel horizontally between the vessel slots
of the heating blocks. The arm 24 includes a platform 25 having
openings 27 to hold the reaction tubes 23. To move the arm
mechanism, a IX" motor 30 with a threaded shaft 32 is attached to a
gear or set of gears 34. A lubricating substance may be placed
between the gears to increase smooth gear movement. The gearing of
the DC motor reduces the effective rotations per minute (RPM) of
the motor and provides frictional force to stop and start the arm
mechanism. As shown in FIGS. 2 A and B, the arm 24 which supports
the reaction vessel is mounted parallel to two circular metal rods
36. On each end of the platform is a semi-circular "U" shaped clip
38 which slideably engages a corresponding rod 36 to the arm 24 to
move freely, with minimal friction, along the two parallel circular
metal rods 36. Alternatively, as seen in FIG. 1, the clip 38 may
fully encircle the rod 36. A lubricating substance can be placed
between the "U" shaped attachments and metal rods to reduce
frictional force. On one end of the arm 24, a straight gear 40 with
teeth is attached to the DC motor gear set 34. As the motor 30
rotates and drives the gear set 34, the shaft 32, by method of
frictional movement between gears 34,40, drives the arm 24 along
the two parallel metal rods 36. The motor 30 is connected via wires
42 to a circuit and controller which will be described farther
below.
[0093] As shown in FIG. 2, the purpose of the platform 25 is to
hold in place the reaction vessel 23. Located near the middle of
the platform 25 is an opening through which the reaction vessel 23
is placed. The rim 44 of the vessel sits directly around the
opening to hold the vessel in place. Optionally, as seen in FIG. 3,
a flat metal lid 46 is attached and covers the area of the platform
which houses the top of the vessel. The metal lid 46 is attached to
a hinge 48 and has a clamping mechanism (not shown) to provide
pressure on the top of vessel. The pressure of the metal lid on the
top of the vessel ensures the vessel is held securely in place and
will remain perpendicular to the horizontal orientation of the
platform. The metal lid may also have a resistive heating element
50 mounted to its surface. The purpose of the resistive heating
element is to transfer heat from the metal lid to the top of the
vessel. If the heated metal lid is set to a temperature above the
maximum temperature of the vessel, it will minimize condensation of
the reaction solvent at the top of the cap of the vessel, which
would otherwise increase reactant concentration and potentially
adversely affect the formation of PCR products.
[0094] The heated metal lid 46 is connected via wires 52 to a
circuit and controller which will be described further below.
Alternatively, if a heated metal lid is not included, the reaction
can be overlaid with a mineral oil, or a similar substance, which
would also minimize or eliminate condensation of the reaction
solvent on the lid of the vessel. A substance, such as mineral oil,
with a boiling point much greater than the reaction solvent reduces
evaporation of the reaction solvent.
[0095] As shown in FIG. 2, the entirety of the arm mechanism 24 is
mounted so that it does not come in contact with the heating blocks
10,20 nor impede the movement of the vessel 23. One such method is
by securing the arm mechanism with posts attached to the base and
arm mechanism.
[0096] A circuit board 53 electrically attached by means of wires
54 to the resistive heating elements attached to each block 10,20,
the temperature sensors attached to each block, the motor 30
driving the arm movement, and the microcontroller 26. The circuit
board consists of: [0097] Circuit 1--a current or voltage regulator
circuit for the resistive heating elements [0098] Circuit 2--a
temperature sensor circuit for the block temperature sensors [0099]
Circuit 3--a motor driver circuit for the arm movement motor
[0100] The current or voltage regulator circuit, Circuit 1,
regulates the current and voltage passed to the resistive heating
elements. Circuit 1 can be any well-known current and voltage
regulator, such as a MOSFET circuit driver or relay driver. The
current and voltage passed to the resistive heating elements is
regulated by the microcontroller 26.
[0101] The temperature sensor circuit, Circuit 2, can be any well
known circuit that responds to a change in voltage, current or
resistance transmitted by the temperature sensor. In this
embodiment, the temperature sensor circuit consists of a linear
resistance input varying with temperature from the temperature
sensor, National Semiconductor.TM. model number LM335AZ, connected
to an analog to digital integrated circuit, National Semiconductor
model number ADC0831CCN. The digital number representation of the
temperature sensor is transmitted via wire to the
microcontroller.
[0102] The motor driver circuit, Circuit 3, can be any well known
motor driver circuit, such as Texas Instruments.TM. motor driver
integrated circuit model number L293D. The motor driver transmits
current to the arm motor and is capable of forward and reverse
current polarity to move the arm mechanism horizontally back and
forth. The motor driver is connected via wire to the
microcontroller 26.
[0103] The microcontroller 26, controls the overall operation of
all circuit components and mechanical parts. In the embodiment of
the invention shown in FIG. 2A and FIG. 2B, the microcontroller 26
used is a Parallax BS2-IC.TM. with the Microchip PIC16C57c.TM. and
a Parallax.TM. protoboard. The microcontroller 26 receives a
digital signal from the temperature sensor circuit which represents
the temperature of each block 10,20. The microcontroller is
programmed with a predetermined hold temperature of each block.
Until the temperature of each block reaches its hold temperature,
the microcontroller maintains the current flow through Circuit 1
which drives each resistive heating element. Once each individual
heating block 10,20 reaches its respective hold temperature, the
microcontroller stops current flow through Circuit 1. As the
temperature of each heating block drops below the hold temperature,
current flow through Circuit 1 is reactivated. The microcontroller
effectively regulates and monitors the temperature of each heating
block and maintains temperature uniformity of each heating
block.
[0104] The microcontroller 26 can be programmed to actuate the
motor driver circuit, Circuit 2, at predetermined time intervals,
directions and durations. This has the effect of activating the DC
motor 30, thereby driving the arm gearing system 34,40. This
translates into horizontal movement of the arm platform which
shuttles the vessel between the slots of the heating blocks
10,20.
[0105] An example operation of the thermal cycling device consists
of programming the microcontroller 26 via keypad (not shown) and
display (not shown) to hold an individual temperature of each
heating block 10,20. The microcontroller 26 is also programmed with
a set number of cycles of arm movement. The microcontroller is also
programmed with a set dwell time of the vessel as it is moved into
the slot of each heating block and as it is located between the two
heating blocks.
[0106] A sample programmed run of the microcontroller 26 could
consist of (A) waiting for heating blocks to reach set
temperatures, (B) movement of the vessel by the mechanical arm to
the slot opening of the first block, (C) holding the cuvette at the
first block for a set period of time (D) movement of the vessel by
the mechanical arm to the slot opening of the second block, (E)
holdings the vessel at the second block for a set period of time,
(F) repeating steps B to E for a set number of cycles, (G) moving
the vessel to a location between the two heating blocks for
removal.
[0107] The thermal cycling device component of the present
invention, due to the constant temperature heating blocks, is
capable of cycling reaction samples in a vessel through
significantly shortened temperature versus time profiles compared
to prior art. The device depicted FIGS. 1 and 2 can be used for a
two-step DNA amplification reaction. The length of each reaction
cycle is significantly reduced in comparison to that observed in
standard, single-block thermal cyclers, since there is no
temperature ramp-up and ramp-down of the temperature blocks
required. The same reaction cycle using prior art devices would
take approximately 5-10 times longer because of the ramping times.
Decreased cycle times can lead to better yield and specificity of
the polymerase chain reaction over prior art cycling. Specifically,
in the past it has been found that ultra fast ramping times
resulted in improved specificity and increased yield; in PCR
amplifications. Rapid cycling results in less time for primer
extension at nonspecific annealing sites, consequently, the amount
of non-specific product is directly related to the tune at the low
temperature, which is the annealing temperature in standard PCR
(Wittwer C. T. et al, Biotechniques, 10:76-83 (1991). A rapid
cool-down (>5.degree. C./sec) of the PCR mixture favors the
kinetics of primer annealing over the thermodynamic advantage of
product reannealing. This, in turn, results in an increased product
yield.
[0108] Furthermore, a shortened time (for example, less than 5
seconds) required to bring the temperature of the reaction mixture
from one temperature level to the next temperature level
corresponding to phases in the amplification process, is
facilitated in the system of the present invention. Specifically,
the time is shortened in comparison to ramp times in standard PCR,
especially standard PCR performed using a single-block device. The
decrease in time required to change the temperature of the
reaction, decreases the overall time required for to complete
nucleic acid amplification.
[0109] The simplicity of the horizontal movement of the mechanical
arm system between the two heating blocks, significantly decreases
the complexity of control and cost of the thermal cycling device
compared to those currently in use. Previous device require
complicated robotic arm construction and precise microprocessor
control to achieve a similar movement of a reaction sample between
heating blocks.
[0110] Amplification products obtained through the use of the
thermal cycling device of the present invention are qualitatively
and quantitatively similar to those obtained through the standard
Peltier heating block cycling method. However, advantages in
specificity and yield are possible with rapid thermal control of
the reaction mixture using the device of the present invention.
Such a rapid response is not possible with prior art systems.
[0111] By reducing the ramping time of the reaction sample, the
present invention can markedly decrease the total time required for
the polymerase chain reaction. In addition, the vessel can be
designed to hold small reaction samples which reduces the amounts
of expensive reagents which must be used thus further reducing the
cost of carrying out procedures using the present invention.
[0112] The thermal cycling apparatus component of the present
invention is useful for amplifying DNA from any source. Although
particular configurations and arrangements of the present invention
have been discussed in connection with the specific embodiments of
the thermal cycling device as constructed in accordance with the
teachings of the present invention, other arrangements and
configurations may be utilized. For example, various cuvette or
heating block configurations may alternatively be used in the
thermal cycling device.
[0113] As will be appreciated by a worker skilled in the art the
thermal cycling device of the present invention provides even
greater improvement over the prior art in the speed at which
thermal cycling can be carried out, e.g., 30 cycles of DNA
amplification in 10-30, or fewer, minutes.
[0114] It will be appreciated that the apparatus described herein
can readily be used for many different applications including;
polymerase chain reaction processes; cycle sequencing; and, other
amplification protocols such as the ligase chain reaction, The
present invention also advantageously provides an apparatus for
accurately controlling the temperature of samples located in the
reaction vessel and quickly and accurately varies the temperature
of samples located in a vessel according to a predetermined
temperature versus time profile.
[0115] The configuration of the thermal alternator device of the
present invention allows it to be readily combined with detection
systems, such as the fluorescence detection system described in
more detail below. It should be readily appreciated, however, that
the device is not limited to combination with a fluorescence
detection system. For example, it can be easily adapted for use
with systems, including but not limited to, a visible light
detection system, a luminescence detection system or a magnetic
detection or separation system. The configuration of the present
device permits such adaptation to be well within the abilities of
the skilled worker.
Technical Description of the Fluorescence Detection System
[0116] As shown in FIG. 3, the device of the present invention
optionally includes a fluorescence detection system, which can be
located directly between the two temperature heating blocks 10,20
and beneath the horizontal translation means. The fluorescence
detection system consists primarily of an excitation source 60 and
a detector 62. The excitation source is a light source, which will
be described further below, which generates emissions from the
sample inside the cuvette 23. The sample fluoresces when
illuminated by the excitation source 60. The detector 62 consists
of a photosensitive sensor, capable of quantifying the intensity of
light produced by the emission source.
[0117] The excitation source 60 is located on one side of the
vessel 23 at an optimal distance to focus light into the chamber,
thereby illuminating the sample. Preferably, the excitation source
has a peak wavelength compatible with fluorescent dyes; for example
480 nm. In this embodiment, the excitation source is a blue Light
Emitting Diode (LED) with a 3000 mcd at 30 mA and 15-degree
focusing angle. The excitation source 60 is enclosed within an
opaque tube to prevent excess leakage of light from the source. The
excitation light source wavelength is restricted with an optical
low pass filter 64 placed directly in the path of light. An optical
filter is needed to differentiate the emission from excitation
wavelength. In this embodiment, a 500 nm low pass blue dichronic
filter is placed in the path of light from the excitation
source.
[0118] The emission source consists of a fluorescent entity and a
nucleic acid amplification product. When illuminated by the
excitation source, the entity (for example, a double-stranded DNA
specific dye or a fluorescently labeled probe) and nucleic acid
amplification product emits light at a different peak wavelength
than the excitation source. Examples of suitable fluorescent dyes
include, but are not limited to, thiazole orange, SYBR.TM. GREEN I,
ethidium bromide, pico green, acridine orange, YO-PRO-1, and
chromomycin A3. Alternatively the fluorescent entity is a nucleic
acid probe that is specific for the amplification product and that
is labeled with a fluorescent tag.
[0119] The detector 62 is located on one side of the vessel 23,
directly opposite the excitation source 60, at an optimal distance
to collect light from the emission source. Preferably, the detector
is a photosensitive sensor capable of differentiation of visible
light at a chosen peak wavelength. In this embodiment, the detector
consists of a CDS photodiode. The detector is enclosed within an
opaque tube to prevent excess light from being detected by the
sensor. The wavelength of light detected by the sensor is
restricted with an optical filter placed direction in the path of
the emission source. An optical filter is needed to differentiate
the emission source from the excitation wavelength, hi this
embodiment, a 520 nm band pass green dichronic filter is placed in
the path of light from the emission source.
[0120] Both the excitation source and the detector are activated by
means of the microcontroller 26. In this embodiment, when a
fluorescence reading of the sample within the vessel is desired,
the microcontroller 26 activates the excitation source 60. The
excitation source then illuminates the emission source. The light
generated by the emission source strikes the detector. In this
embodiment, a relative amount of light from the emission translates
into a change in resistance of the photodiode detector. This
resistance is monitored via wire by the microcontroller. Any change
in fluorescence translates into a change detected by the
microcontroller.
[0121] In a sample run of the fluorescence detection system, a
sample within a reaction vessel is placed into the thermal cycling
device. A fluorescence measurement is taken at the ambient
temperature of the device. Following this measurement, the PCR
reaction takes place over a predetermined number of cycles.
Following the completion of the PCR reaction, the vessel is
positioned between the fluorescence detection system and another
measurement is taken. By comparing the initial and final
fluorescence of the sample in the vessel, a corresponding increase
in nucleic acid amplification product can be determined.
Fluorescence measurements may also be taken at the completion of
each PCR cycle, thereby quantifying per cycle the relative amount
of nucleic acid amplification product increase.
[0122] The fluorescence detection component of the present
invention, due to the simplicity of design, is capable of measuring
per cycle results of nucleic acid amplification product by means of
fluorescence. Compared to prior art fluorescence detection systems
implemented in heating block thermal cyclers, this device component
offers significant reduction in mechanical complexity and cost,
while maintaining similar performance capabilities. DNA
amplification can be measured by means of fluorescence at the
beginning and end of the PCR reaction, as well as during each step.
This same performance measurement using prior art system would be
approximately 100 times more expensive. The simplistic optics,
excitation source and detector of the present invention, have
proven also to produce comparable results to more expensive and
complex prior art systems.
[0123] Furthermore, the rapid cycling of the thermal cycling
component means a quantitative fluorescence measurement of nucleic
acid product can be accomplished much faster than prior art
systems. This greatly reduces the time required to quantify any
nucleic acid product generated by the PCR reaction.
[0124] Turning now to FIG. 4 and FIG. 5, a thermal cycling device
with two fixed temperature heat blocks was constructed based on the
principles described in U.S. Patent Application 60/563,061 (which
is incorporated herein by reference), but modified as follows as
shown in FIG. 4 and FIG. 5.
[0125] A holder 100 for the reaction vessels 120 was constructed by
drilling holes of the appropriate size into a chassis 140
comprising a flat sheet of metal. The reaction vessel holder 100
was bolted onto chassis 140. Underneath the reaction vessel holder
100 proximal heater block 160a, and distal heater block 160b were
affixed to a support board 180. Grooves (not seen) were machined
into the proximal heater block 160a, and distal heater block 160b,
the grooves being shaped precisely to fit the shape of the reaction
vessels 120. The proximal heater block 160a, and distal heater
block 160b also contained resistive heaters (not seen) which were
controllable to maintain a set temperature. The support board 180
was configured and arranged to be able to move in one dimension by
sliding along two metal shafts 120. Motion of the support board 180
was driven by a cam shaft 122 which was configured and structured
to be rotatable in one direction by a motor (not seen). The cam
shaft 122 was thus configured to rotate in between a pair of
plastic or metal leaf springs 124. The cam shaft 122 was configured
and structured to have three main positions, namely: (1) pointing
parallel away from the proximal heater block 160a, and distal
heater block 160b to result in a configuration where the distal
heater block 160b came into contact with the reaction vessels 120;
(2) pointing perpendicular to the proximal heater block 160a, and
distal heater block 160b to result in a configuration where neither
of the proximal heater block 160a, and distal heater block 160b
were in contact with the reaction vessels 120; and (3) pointing
parallel towards the proximal heater block 160a, and distal heater
block 160b to result in a configuration where the proximal heater
block 160a came into contact with the reaction vessels 120.
[0126] Thus, when the cam shaft 122 were in the 2.sup.nd position,
the reaction vessels 120 are not in contact with either the
proximal heater block 160a, nor the distal heater block 160b. If
the middle section 126 were to be cut out of the support board 180,
then this middle position 126 would be convenient for imaging the
bottom of the reaction vessels 120. Specifically, a blue LED light
source may be shone at the bottom of the reaction vessel to excite
the contents of the vessel, e.g. SYBR.RTM. Green Dye (Molecular
Probes, Inc.). Emitted light from the vessel may be detected by
means of a CCD camera. To filter out blue light from the LED
source, a bandpass filter may be placed in front of the CCD camera
so that only higher wavelengths e.g., green and red are allowed to
pass through. This helps improve the signal-to-noise ratio.
[0127] The use of a cam shaft 122 with the leaf springs 124
attached to the support board 180 helps ensure good contact between
the proximal heater block 160a, and distal heater block 160b and
the reaction vessels 120. The reason is because the cam shaft 122
is able to deflect the leaf springs 124 when the cam shaft 122 is
parallel to, and facing either towards or away from the proximal
heater block 160a, and distal heater block 160b. This enables the
cam shaft 122 to exert extra force, thereby to drive the proximal
heater block 160a, and distal heater block 160b into contact with
the reaction vessels 120, and to correct for dimensional
tolerances.
[0128] It is important to note that the according to certain
aspects of the present invention the reaction vessels 120 only come
into partial contact with the proximal heater block 160a, and
distal heater block 160b. This means that there is a non-uniform
(i.e. nonzero) temperature gradient across the reaction vessel 120.
The reason is because, although the proximal heater block 160a, and
distal heater block 160b are set at a certain temperature, the top
of the reaction vessels 120 experience a different temperature
because it is held in place by a material which serves as a passive
insulator, and the side walls of the reaction vessels 120 which are
not in contact with the proximal heater block 160a, and distal
heater block 160b are exposed to the temperature of the ambient
air.
[0129] FIG. 6 shows the control and processing electronic block
diagram of the apparatus of FIGS. 4 and 5 (depicted pictorially in
FIG. 6)
[0130] The steps through which the microprocessor (.mu.P) shown in
FIG. 6 goes are shown in FIG. 7. These steps are accomplished as
shown in more detail in FIGS. 8A and 8B. Once the samples have been
prepared and loaded into the holder 100 (FIG. 4) and the operator
gave the "start" signal (e.g. by pressing a button or key), the
.mu.P is in control of the entire session as shown in FIGS. 8A and
8B. At the end, each of the four reaction vessels or "cuvettes" 12
is tested for sufficient fluorescence, which indicates presence of
the target DNA, and the result is acquired and stored/displayed
accordingly, and test completion is indicated.
[0131] It will be understood by those skilled in the art that the
embodiments described herein are merely exemplary and that the
person skilled in the art may make many modifications and
variations without departing from the scope of the invention. The
various embodiments may be practiced in the alternative or in
combination as appropriate. All such variations and modifications
are intended to be included within the scope of the invention.
* * * * *