U.S. patent application number 15/400581 was filed with the patent office on 2017-04-27 for systems and methods for the amplification of dna.
The applicant listed for this patent is Canon U.S. Life Sciences, Inc.. Invention is credited to Gregory A. Dale, Kenton C. Hasson, Shulin Zeng.
Application Number | 20170114380 15/400581 |
Document ID | / |
Family ID | 42007566 |
Filed Date | 2017-04-27 |
United States Patent
Application |
20170114380 |
Kind Code |
A1 |
Zeng; Shulin ; et
al. |
April 27, 2017 |
SYSTEMS AND METHODS FOR THE AMPLIFICATION OF DNA
Abstract
A system for amplifying nucleic acids is disclosed which, in one
embodiment, includes a fluidic device having a sample channel and a
heat exchange channel disposed sufficiently close to the sample
channel such that a heat exchange fluid in the heat exchange
channel can cause a sample in the sample channel to gain or lose
heat at desired levels. In one illustrative embodiment, the system
further includes three reservoirs coupled to the heat exchange
channel and a temperature control system configured to heat fluids
stored in the respective reservoirs at different temperatures. One
or more pumps and a controller are configured to cause fluid stored
in the reservoirs to enter and flow through the heat exchange
channel at different times.
Inventors: |
Zeng; Shulin; (Silver
Spring, MD) ; Hasson; Kenton C.; (Germantown, MD)
; Dale; Gregory A.; (Gaithersburg, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Canon U.S. Life Sciences, Inc. |
Rockville |
MD |
US |
|
|
Family ID: |
42007566 |
Appl. No.: |
15/400581 |
Filed: |
January 6, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
12233194 |
Sep 18, 2008 |
9540686 |
|
|
15400581 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/686 20130101;
C12P 19/34 20130101; C12Q 1/686 20130101; B01L 2300/1827 20130101;
B01L 2400/0487 20130101; B01L 2300/1838 20130101; B01L 2300/0816
20130101; B01L 3/502715 20130101; B01L 7/525 20130101; B01L
2200/147 20130101; B01L 2300/0883 20130101; C12Q 2527/101 20130101;
B01L 2300/1822 20130101 |
International
Class: |
C12P 19/34 20060101
C12P019/34; B01L 3/00 20060101 B01L003/00; B01L 7/00 20060101
B01L007/00; C12Q 1/68 20060101 C12Q001/68 |
Claims
1. A method of amplifying DNA using a system comprising a fluidic
device having a sample channel and a heat exchange channel that is
used to heat and/or cool a sample in the sample channel, the method
comprising: causing a sample of a test solution containing PCR
reagents to move through the sample channel of the fluidic device;
and while the sample is moving through at least a section of the
sample channel: (1) for a first period of time, causing a first
heat exchange fluid stored in a first container and regulated at a
first temperature while stored in the first container to: (1) exit
the first container and (2) move through the heat exchange channel
after exiting the first container; (2) for a second period of time,
causing a second heat exchange fluid stored in a second container
and regulated at a second temperature while stored in the second
container to: (1) exit the second container and (2) move through
the heat exchange channel after exiting the second container; and
(3) for a third period of time, causing a third heat exchange fluid
stored in a third container and regulated at a third temperature
while stored in the third container to: (1) exit the third
container and (2) move through the heat exchange channel after
exiting the third container, wherein the first period of time may
be different than the second period of time, which may be different
than the third period of time, and the first temperature is
different than the second temperature, which is different than the
third temperature.
2. The method of claim 1, further comprising causing the first heat
exchange fluid to enter the third container after exiting the heat
exchange channel.
3. The method of claim 2, further comprising causing the second
heat exchange fluid to enter the first container after exiting the
heat exchange channel.
4. The method of claim 3, further comprising causing the third heat
exchange fluid to enter the second container after exiting the heat
exchange channel.
5. The method of claim 1, further comprising causing the first heat
exchange fluid to enter the first container after exiting the heat
exchange channel.
6. The method of claim 5, further comprising causing the second
heat exchange fluid to enter the second container after exiting the
heat exchange channel.
7. The method of claim 6, further comprising causing the third heat
exchange fluid to enter the third container after exiting the heat
exchange channel.
8. The method of claim 1, wherein the first temperature is a
temperature such that when the first heat exchange fluid moves
through the heat exchange channel said fluid heats a sample in the
sample channel to a temperature over 80 degrees Celsius, the second
temperature is a temperature such that when the second heat
exchange fluid moves through the heat exchange channel said fluid
cools a sample in the sample channel to a temperature under about
60 degrees Celsius, and the third temperature is a temperature such
that when the third heat exchange fluid moves through the heat
exchange channel said fluid heats a sample in the sample channel to
a temperature between 60 and 80 degrees Celsius.
9. The method of claim 1, wherein at least a portion of the heat
exchange channel is beneath the sample channel and parallel with
the sample channel.
10. The method of claim 1, wherein at least one dimension of the
heat exchange channel and the sample channel is less than about
3000 micrometers.
11. The method of claim 10, wherein the heat exchange channel has a
width between about 20 and 2000 micrometers and a depth between
about 20 and 2000 micrometers.
12. The method of claim 1, wherein said first heat exchange fluid
is identical with the second heat exchange fluid, which is
identical with the third heat exchange fluid, and the first heat
exchange fluid comprises a gas and/or a liquid.
13. The method of claim 1, wherein said heat exchange fluids
comprise water and/or compressed air with pressure from 1 to 200
psia.
14. The method of claim 1, wherein said first heat exchange fluid
is different than the second heat exchange fluid, which can be the
same or different than the third heat exchange fluid.
15. A method of thermal exchange in a microfluidic chip comprising:
directing a first heat exchange fluid at a first temperature
through a heat exchange channel for a first period of time, wherein
the heat exchange channel is configured to exchange heat with a
portion of a sample channel, wherein at least one dimension of the
heat exchange channel and the sample channel are less than 1000
micrometers; directing a second heat exchange fluid at a second
temperature through the heat exchange channel for a second period
of time; and directing a third heat exchange fluid at a third
temperature through a heat exchange channel for a third period of
time.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application is a divisional of U.S. patent application
Ser. No. 12/233,194, filed on Sep. 18, 2008, the disclosure of
which is incorporated herein by reference in its entirety.
BACKGROUND
[0002] Field of the Invention
[0003] The present invention relates to systems and methods for
amplifying nucleic acids. In some embodiments, the invention
relates to microfluidic PCR analysis systems using microfluidic
temperature controlled channels.
[0004] Discussion of the Background
[0005] The amplification and detection of nucleic acids is central
to medicine, forensic science, industrial processing, crop and
animal breeding, and many other fields. The ability to detect
disease conditions (e.g., cancer), infectious organisms (e.g.,
HIV), genetic lineage, genetic markers, and the like, is ubiquitous
technology for disease diagnosis and prognosis, marker assisted
selection, correct identification of crime scene features, the
ability to propagate industrial organisms and many other
techniques. Determination of the integrity of a nucleic acid of
interest can be relevant to the pathology of an infection or
cancer. One of the most powerful and basic technologies to detect
small quantities of nucleic acids is to replicate some or all of a
nucleic acid sequence many times, and then analyze the
amplification products. PCR is perhaps the most well-known of a
number of different amplification techniques.
[0006] PCR is a powerful technique for amplifying short sections of
DNA. With PCR, one can quickly produce millions of copies of DNA
starting from a single template DNA molecule. PCR includes a three
phase temperature cycle of denaturation of DNA into single strands,
annealing of primers to the denatured strands, and extension of the
primers by a thermostable DNA polymerase enzyme. This cycle is
repeated so that there are enough copies to be detected and
analyzed. In principle, each cycle of PCR could double the number
of copies. In practice, the multiplication achieved after each
cycle is always less than 2. Furthermore, as PCR cycling continues,
the buildup of amplified DNA products eventually ceases as the
concentrations of required reactants diminish. For general details
concerning PCR, see Sambrook and Russell, Molecular Cloning--A
Laboratory Manual (3rd Ed.), Vols. 1-3, Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y. (2000); Current Protocols in
Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a
joint venture between Greene Publishing Associates, Inc. and John
Wiley & Sons, Inc., (supplemented through 2005) and PCR
Protocols A Guide to Methods and Applications, M. A. Innis et al.,
eds., Academic Press Inc. San Diego, Calif. (1990).
[0007] Real-time PCR refers to a growing set of techniques in which
one measures the buildup of amplified DNA products as the reaction
progresses, typically once per PCR cycle. Monitoring the
accumulation of products over time allows one to determine the
efficiency of the reaction, as well as to estimate the initial
concentration of DNA template molecules. For general details
concerning real-time PCR see Real-Time PCR: An Essential Guide, K.
Edwards et al., eds., Horizon Bioscience, Norwich, U.K. (2004).
[0008] Several different real-time detection chemistries now exist
to indicate the presence of amplified DNA. Most of these depend
upon fluorescence indicators that change properties as a result of
the PCR process. Among these detection chemistries are DNA binding
dyes (such as SYBR.RTM. Green) that increase fluorescence
efficiency upon binding to double stranded DNA. Other real-time
detection chemistries utilize Forster resonance energy transfer
(FRET), a phenomenon by which the fluorescence efficiency of a dye
is strongly dependent on its proximity to another light absorbing
moiety or quencher. These dyes and quenchers are typically attached
to a DNA sequence-specific probe or primer. Among the FRET-based
detection chemistries are hydrolysis probes and conformation
probes. Hydrolysis probes (such as the TaqMan.RTM. probe) use the
polymerase enzyme to cleave a reporter dye molecule from a quencher
dye molecule attached to an oligonucleotide probe. Conformation
probes (such as molecular beacons) utilize a dye attached to an
oligonucleotide, whose fluorescence emission changes upon the
conformational change of the oligonucleotide hybridizing to the
target DNA.
[0009] A number of commercial instruments exist that perform
real-time PCR. Examples of available instruments include the
Applied Biosystems PRISM 7500, the Bio-Rad iCylcer, and the Roche
Diagnostics LightCycler 2.0. The sample containers for these
instruments are closed tubes which typically require at least a 10
.mu.l volume of sample solution.
[0010] More recently, a number of high throughput approaches to
performing PCR and other amplification reactions have been
developed, e.g., involving amplification reactions in microfluidic
devices, as well as methods for detecting and analyzing amplified
nucleic acids in or on the devices. Thermal cycling of the sample
for amplification is usually accomplished in one of two methods. In
the first method, the sample solution is loaded into the device and
the temperature is cycled in time, much like a conventional PCR
instrument. In the second method, the sample solution is pumped
continuously through spatially varying temperature zones.
[0011] To have good yield of a target product, one has to control
the sample temperature at different levels very accurately. And to
reduce the process time, one has to heat up or cool down the sample
to desired temperature very quickly.
[0012] One specific approach for regulating temperature within the
devices is to employ external temperature control sources. Examples
of such sources include, but are not limited to, heating blocks and
water baths. Another option is to utilize a heating element such as
a resistive heater that can be adjusted to a particular
temperature. Another temperature controller includes Peltier
controllers (e.g., INB Products thermoelectric module model
INB-2-(11-4)1.5). This controller can be utilized to achieve
effective thermal cycling or to maintain isothermal incubations at
any particular temperature.
[0013] In some devices and applications, heat exchangers can also
be utilized in conjunction with one of the temperature control
sources to regulate temperature. Such heat exchangers typically are
made from various thermally conductive materials (e.g., various
metals and ceramic materials) and are designed to present a
relatively large external surface area to the adjacent region.
Often this is accomplished by incorporating fins, spines, ribs and
other related structures into the heat exchanger. Other structures
include coils and sintered structures. In certain devices, heat
exchangers such as these are incorporated into a holding space,
chamber or detection area.
[0014] Conventional heat exchangers that can be utilized in certain
applications are discussed, for example, in U.S. Pat. No. 6,171,850
which discloses a reaction receptacle that includes a plurality of
reservoirs disposed in the surface of a substrate. Additional
methods of temperature control for microfluidic systems are known
which include, for example: a thermal cycling system using the
circulation of temperature controlled water to the underside of a
microtiter plate (U.S. Pat. No. 5,508,197); a thermal cycling
system using infrared heating and air cooling (U.S. Pat. No.
6,413,766); a microfluidic chip where flow travels through several
static temperature zones (U.S. Pat. No. 6,960,437); the use of
exothermic and endothermic materials to heat up and cool down the
PCR samples (U.S. patent application publication
US2005/012982).
[0015] In conventional systems temperature accuracy and thermal
cycling speeds are issues to be resolved. For example, the accuracy
of the temperature of any bath used to heat a microchannel and the
bath's subsequent conduction of heat to the microchannel is
important in that certain stages of PCR processing take place at
well-defined temperatures. The thermal cycling speed refers to the
time between stabilization from one temperature to another in a
heating cycle. For example in the PCR process, the thermal cycling
speed refers to the time to shift from 95.degree. C. to 55.degree.
C. to 72.degree. C. The faster the thermal cycling speeds and the
more accurate the temperature stabilization, the more efficient PCR
processes can be performed.
[0016] There is a need for improved systems and methods for
amplifying nucleic acids and for systems and methods for
microfluidic thermal control.
SUMMARY
[0017] The present invention provides improved systems and methods
for amplifying nucleic acids and systems and methods for
microfluidic temperature control.
[0018] A method according to some embodiments of the invention
includes: causing a sample of a test solution containing PCR
reagents to move through a sample channel of a fluidic device and
while the sample is moving through at least a section of the sample
channel: (1) for a first period of time, causing a first heat
exchange fluid stored in a first container and regulated at a first
temperature while stored in the first container to exit the first
container and move through a heat exchange channel of the fluidic
device after exiting the first container; (2) for a second period
of time, causing a second heat exchange fluid stored in a second
container and regulated at a second temperature while stored in the
second container to exit the second container and move through the
heat exchange channel after exiting the second container; and (3)
for a third period of time, causing a third heat exchange fluid
stored in a third container and regulated at a third temperature
while stored in the third container to exit the third container and
move through the heat exchange channel after exiting the third
container. Steps (1)-(3) are preferably repeated at least several
times. Also, it is preferred that the first period of time is
different than the second period of time, which is different than
the third period of time, although there may be some overlap
between the time periods. It is also preferred that the first
temperature is different than the second temperature, which is
different than the third temperature.
[0019] In some embodiments, the method may further include causing
the first heat exchange fluid to enter the third container after
exiting the heat exchange channel, causing the second heat exchange
fluid to enter the first container after exiting the heat exchange
channel, and causing the third heat exchange fluid to enter the
second container after exiting the heat exchange channel. The heat
exchange fluids may be a gas, a liquid or a gas and liquid mixture.
For example, the heat exchange fluids may include water and/or
compressed air with pressure from 1 to 200 psia.
[0020] The heat exchange and sample channels may each have a
dimension less than 2000 micrometers. For example, the heat
exchange channel may have a width between about 20 and 2000
micrometers and a depth between about 20 and 2000 micrometers. The
containers may have a volume of less than 2000 ml. For example, the
containers may have a volume from 10 to 1000 ml.
[0021] A system according to an embodiment of the invention
includes: a fluidic device comprising a sample channel and a heat
exchange channel sufficiently close to the sample channel such that
a heat exchange fluid in the heat exchange channel can cause a
sample in the sample channel to appreciably gain or lose heat; a
first reservoir having an output port coupled to an input of the
heat exchange channel and having an input port coupled to an output
of the heat exchange channel through a first return valve, the
first reservoir storing a first heat exchange fluid; a second
reservoir having an output port coupled to the input of the heat
exchange channel and having an input port coupled to the output of
the heat exchange channel through a second return valve, the second
reservoir storing a second heat exchange fluid; a third reservoir
having an output port coupled to the input of the heat exchange
channel and having an input port coupled to the output of the heat
exchange channel through a third return valve, the third reservoir
storing a third heat exchange fluid; a temperature control system;
one or more pumps; and a controller.
[0022] The temperature control system may be configured to: (a)
regulate the heat exchange fluid stored in the first reservoir at a
first temperature, (b) regulate the heat exchange fluid stored in
the second reservoir at a second temperature, and (c) regulate the
heat exchange fluid stored in the third reservoir at a third
temperature.
[0023] The controller may be configured to operate the valves and
the one or more pumps such that: (a) for a first period of time,
the first heat exchange fluid stored in the first reservoir enters
the heat exchange channel, but the second and third heat exchange
fluids stored in the second and third reservoirs, respectively, do
not enter the heat exchange channel; (b) for a second period of
time, the second heat exchange fluid stored in the second reservoir
enters the heat exchange channel, but the first and third heat
exchange fluids stored in the first and third reservoirs,
respectively, do not enter the heat exchange channel; and (c) for a
third period of time, the third heat exchange fluid stored in the
third reservoir enters the heat exchange channel, but the first and
second heat exchange fluids stored in the first and second
reservoirs, respectively, do not enter the heat exchange
channel.
[0024] In other embodiments, a thermal exchange system for
microfluidic systems includes at least one heat exchange channel,
wherein the at least one heat exchange channel is configured to
carry a heat exchange fluid, wherein the heat exchange channel is
configured to exchange heat with a portion of a sample channel,
wherein the sample channel is configured to carry a genomic sample
in a buffer. The system further includes at least two reservoir
tanks, a first reservoir tank and a second reservoir tank, wherein
the first reservoir tank is configured to include a first heat
exchange fluid at a first temperature, and the second reservoir
tank is configured to include a second heat exchange fluid at a
second temperature, wherein either the first or the second heat
exchange fluids can be directed into the at least one heat exchange
channel. In other aspects of this system, three reservoirs are
included wherein the third reservoir includes a third heat exchange
fluid at a third temperature.
[0025] The thermal exchange system according to one embodiment is
further characterized in that the first heat exchange fluid is
flowing through the at least one heat exchange channel and the
portion of the sample channel is heated to about 95 degrees
Celsius, the second heat exchange fluid is flowing through the at
least one heat exchange channel and the portion of the sample
channel is heated to about 55 degrees Celsius, and the third heat
exchange fluid is flowing through the at least one heat exchange
channel and the portion of the sample channel is heated to about 72
degrees Celsius.
[0026] In some embodiments, the thermal exchange system has at
least one heat exchange channel that is substantially parallel to
the sample channel. In other embodiments, the thermal exchange
system has at least one heat exchange channel that is substantially
perpendicular to the sample channel. In still other embodiments,
the thermal exchange system has at least one heat exchange channel
that is configured to exchange heat with substantially one side of
the sample channel. In yet other embodiments, the thermal exchange
system has at least one heat exchange channel that is configured to
exchange heat with substantially two sides or three sides of the
sample channel.
[0027] The above and other embodiments of the present invention are
described below with reference to the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] The accompanying drawings, which are incorporated herein and
form part of the specification, illustrate various embodiments of
the present invention. In the drawings, like reference numbers
indicate identical or functionally similar elements.
[0029] FIG. 1 is a block diagram illustrating a system according to
embodiments of the invention.
[0030] FIGS. 2A-2B illustrate a temperature control system
according to some embodiments of the invention.
[0031] FIGS. 3A-3C illustrate various configurations of a heat
exchange channel according to embodiments of the invention.
[0032] FIG. 4 illustrates a process according to some embodiments
of the invention.
[0033] FIGS. 5A-5B illustrate portions of processes according to
some embodiments of the invention.
[0034] FIG. 5C illustrates a temperature profile that can result
from the use of a temperature control system according to at least
one exemplary embodiment.
[0035] FIG. 6 illustrates a temperature control system according to
other embodiments of the invention.
[0036] FIG. 7 illustrates a portion of a process according to some
embodiments of the invention.
[0037] FIG. 8 illustrates a temperature control system in
accordance with other embodiments of the invention.
[0038] FIG. 9 illustrates a temperature control system in
accordance with still other embodiments of the invention.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0039] FIG. 1 illustrates a functional block diagram of a system
100 for the amplification of DNA according to some embodiments of
the invention. As illustrated in FIG. 1, system 100 may include a
microfluidic device 102. Microfluidic device 102 may include one or
more microfluidic channels 104. In the example shown, device 102
includes two microfluidic channels, channel 104a and channel 104b.
Although only two channels are shown in the exemplary embodiment,
it is contemplated that device 102 may have fewer than two or more
than two channels. For example, in some embodiments, device 102
includes eight channels 104.
[0040] Device 102 may include two DNA processing zones, a DNA
amplification zone 131 (a.k.a., PCR zone 131) and a DNA melting
zone 132. A DNA sample traveling through the PCR zone 131 may
undergo PCR, and a DNA sample passing through melt zone 132 may
undergo high resolution thermal melting. As illustrated in FIG. 1,
PCR zone 131 includes a first portion of channels 104 and melt zone
132 includes a second portion of channels 104, which is down stream
from the first portion.
[0041] In order to achieve PCR for a DNA sample flowing through the
PCR zone 131, the temperature of the sample must be cycled, as is
well known in the art. Accordingly, in some embodiments, system 100
includes a temperature control apparatus 120. The temperature
control apparatus 120 may include a temperature sensor, a
heater/cooler, and a temperature controller. In some embodiments,
temperature controller 120 is interfaced with main controller 130
so that main controller 130 can control the temperature of the
samples flowing through the PCR zone and the melting zone.
[0042] To monitor the PCR process and the thermal melting process
that occur in PCR zone 131 and melt zone 132, respectively, system
100 may include an imaging system 118. Imaging system 118 may
include an excitation source, a detector, a controller, and an
image storage unit.
[0043] Further features of system 100 are described in U.S. patent
application Ser. No. 11/770,869, which is incorporated herein by
this reference.
[0044] FIGS. 2A and 2B further illustrate a temperature control
apparatus 120 in accordance with some embodiments of the invention.
FIG. 2A illustrates a single heat exchange channel 210 and a single
sample channel 104 (although, as discussed above, the invention is
not limited to a single heat exchange channel 210 and/or sample
channel 104). The heat exchange channel 210 is configured to carry
a heat exchange fluid and configured to exchange heat with a
portion 230 of a sample channel 220. The sample channel 220 can be
configured to carry a bolus 240 of genomic sample material, which
experiences temperature variation due to the heat exchange through
the portion 230. In the one exemplary embodiment, temperature
variations are performed temporally by varying the temperature of
the heat exchange fluid temporally or by switching to a different
heat exchange fluid at a particular time that has a different
temperature.
[0045] At least one exemplary embodiment includes multiple
reservoirs of various heat exchange fluids at various temperatures.
For example, FIG. 2B illustrates a reservoir system for storing and
directing heat exchange fluids through the heat exchange channel
210. FIG. 2B illustrates three fluid containers (a.k.a.,
reservoirs) T1, T2, and T3, each of which stores a fluid (e.g., a
gas, a liquid or a gas and liquid mixture) and pump 290 coupled to
each container for pumping fluid out of the containers and into a
heat exchange channel 210 formed in chip 102. In one embodiment,
the first heat exchange fluid is identical with the second heat
exchange fluid, which is identical with the third heat exchange
fluid, and the first heat exchange fluid comprises a gas and/or a
liquid. In other embodiments, the heat exchange fluids comprise
water and/or compressed air with pressure from 1 to 200 psia. In
still other embodiments, the heat exchanges fluids can be different
from one another. In one non-limiting example, the first heat
exchange fluid is a gas, the second heat exchange fluid is a liquid
and the third heat exchange fluid is gas and liquid mixture..
[0046] Each container T1-T3 includes an output port that is coupled
to an input of the heat exchange channel through a forward valve.
For example, the output port of T1 is coupled to the heat exchange
channel through forward valve V1F, the output port of T2 is coupled
to the heat exchange channel through forward valve V2F, and the
output port of T3 is coupled to the heat exchange channel through
forward valve V3F.
[0047] Each container T1-T3 also includes an input port that is
coupled to an output of the heat exchange channel through a return
valve. For example, the input port of T1 is coupled to the heat
exchange channel through return valve V1R, the input port of T2 is
coupled to the heat exchange channel through return valve V2R, and
the input port of T3 is coupled to the heat exchange channel
through return valve V3R.
[0048] As further illustrated, temperature control apparatus 120
may include a temperature control system that includes one or more
temperatures controllers. For example, in the illustrated
embodiment of FIG. 2B, temperature control apparatus 120 includes a
temperature controller C1 for regulating the temperature of the
fluid stored in T1 at a first temperature (e.g., C1 attempts to
maintain the temperature of the fluid in T1 at, or close to, a
predetermined temperature), a temperature controller C2 for
regulating the temperature of the fluid stored in T2 at a second
temperature, and a temperature controller C3 for regulating the
temperature of the fluid stored in T3 at a third temperature. Each
of C1, C2 and C3 may include, a sensor for sensing temperature,
heating/cooling elements, and computerized controllers for
controlling the heating/cooling elements based on output from a
sensor.
[0049] Referring now to FIGS. 3A-C, cross-sectional, end views of
chip 102 are shown and serve to illustrate various different
embodiments of heat exchange channel 304 and to illustrate the
relationship between a sample channel 104, which carries a sample
302, and heat exchange channel 304. Sample 302 may include a
solution that contains, among other things, a piece of DNA, DNA
polymerase, and a primer.
[0050] As illustrated in FIGS. 3A-C, heat exchange channel 304 may
only run along one side of channel 104 (see FIG. 3A), heat exchange
channel 304 may be generally L shaped and run along two sides of
channel 104 (see FIG. 3B), and heat exchange channel 304 may be
generally U shaped and run along three side of channel 104 (see
FIG. 3C). In some embodiments, channel 304 may have a width between
about 10 and 3000 micrometers (more preferably between about 20 and
2000 micrometers) and a depth between about 10 and 3000 micrometers
(more preferably between about 20 and 2000 micrometers).
[0051] Referring now to FIG. 4, a flow chart illustrates a process
400 according to some embodiments of the invention. Process 400 may
begin in step 402, where a fluid is stored in a first container
(e.g., container T1). In step 404, the temperature of the fluid in
the first container is regulated at a first temperature (e.g., at
least about 80 degrees Celsius). In step 406, a fluid is stored in
a second container (e.g., container T2). In step 408, the
temperature of the fluid in the second container is regulated at a
second temperature (e.g., a temperature not more than about 60
degrees Celsius). In step 410, a fluid is stored in a third
container (e.g., container T3). In step 412, the temperature of the
fluid in the third container is regulated at a third temperature
(e.g., a temperature between about 60 and 80 degrees Celsius). In
step 414, a sample (e.g., sample 302) is caused to flow though
sample channel 104. While the sample is flowing through channel
104, steps 416-420 can be performed.
[0052] In step 416, the fluid stored in the first container is
caused to flow through heat exchange channel 304 for a first amount
of time. Next, in step 418, the fluid stored in the second
container is caused to flow through heat exchange channel 304 for a
second amount of time. Next, in step 420, the fluid stored in the
third container is caused to flow through heat exchange channel 304
for a third amount of time. After step 420, steps 416-420 may be
repeated a number of times. The first amount of time may be
different than the second amount of time, which may be different
than the third amount of time.
[0053] In one exemplary, non-limiting embodiment, the fluid stored
in the first container (e.g. water) can be heated to a temperature
of approximately 97 degrees Celsius so that the sample material can
be heated to a temperature of approximately 95 degrees Celsius. The
fluid stored in the second container (e.g. water) can be maintained
at a temperature of approximately 53 degrees Celsius so that the
sample material can be cooled to a temperature of approximately 55
degrees Celsius. The fluid stored in the third container (e.g.
water) can be heated to a temperature of approximately 74 degrees
Celsius so that the sample material can be heated to a temperature
of approximately 72 degrees Celsius. Also in this exemplary
embodiment, the fluid stored in the first container is caused to
flow through heat exchange channel 304 for a first amount of time
that can be, for example, approximately 0.3 to 2 seconds and
preferably approximately 0.5 seconds. The fluid stored in the
second container is caused to flow through heat exchange channel
304 for a second amount of time that can be, for example,
approximately 1 to 5 seconds and preferably approximately 2
seconds. The fluid stored in the third container is caused to flow
through heat exchange channel 304 for a third amount of time that
can be, for example, approximately 1 to 10 seconds and preferably
approximately 5 seconds. Of course, the fluid stored in the
containers can be heated or cooled to different temperatures and
the time periods during which the fluid flows through the heat
exchange channel can be decreased or increased depending on the
requirements for a given amplification reaction.
[0054] Referring now to FIG. 5A, steps 416-420 are further
illustrated according to some embodiments where process 400 is
implemented using the apparatus shown in FIGS. 2A-B. As shown in
FIG. 5A, step 416 may include opening valve V1F, opening valve V3R
and closing the other valves (V2F, V3F, V1R, and V2R), step 418 may
include opening valve V2F, opening valve V1R and closing the other
valves (V1F, V3F, V2R, and V3R), step 420 may include opening valve
V3F, opening valve V2R and closing the other valves (V1F, V2F, V1R,
and V3R). Preferably, while all the steps 416-420 are being
performed, pump 290 is activated, thereby causing the fluids to
flow out of a container and back into a container. The container
from which the fluid flows and to which the fluid returns, of
course, depends on the valves that are open at the time. For
example, when step 416 is performed in accordance with the flow
shown in FIG. 5A, fluid will flow out of container T1 and into
container T3. Directing the fluid flow out of container T1 and into
container T3, in this particular embodiment, is one exemplary way
to allow more time for the fluid to reach the desired temperature
level, which can increase the temperature accuracy and efficiency
of the temperature cycling process.
[0055] Referring now to FIG. 5B, steps 416-420 are further
illustrated according to another embodiment where process 400 is
implemented using the apparatus shown in FIGS. 2A-B. As shown in
FIG. 5B, step 416 may include opening valve V1F, opening valve V1R
and closing the other valves (V2F, V3F, V2R, and V3R), step 418 may
include opening valve V2F, opening valve V2R and closing the other
valves (V1F, V3F, V1R, and V3R), step 420 may include opening valve
V3F, opening valve V3R and closing the other valves (V1F, V2F, V1R,
and V2R). Preferably, while all the steps 416-420 are being
performed, pump 290 is activated, thereby causing the fluids to
flow out of a container and back into a container. In this example,
when step 416 is performed in accordance with the flow shown in
FIG. 5B, fluid will flow, for example, out of container T1 and back
into container T1.
[0056] In another embodiment, one or more of the containers T1-T3
are constructed to have an internal bladder or baffle that
separates the internal portion of the container into a first
chamber and a second chamber, and wherein the first and second
chambers are in fluid communication with one another by, for
example, a controllable valve. In this embodiment, fluid can be
controllably released from one chamber of the container (e.g. T1)
through a forward valve (e.g. V1F) and can be controllably caused
to flow back into the other chamber of the container through the
return valve (e.g. V1R). As stated above, fluid also can
controllably flow between the first chamber and the second chamber
of a container through, for example, a controllable valve in the
bladder or baffle separating the chambers. This embodiment may be
useful, for example, in an embodiment where fluid flows out one
container and back into the same container before fluid flows out
of, or into, another container, as discussed in connection with the
process illustrated in FIG. 5B. This embodiment also may be useful
in connection with other embodiments where different fluids are
used in the containers such as, for example, when a gas is used in
container T1, a liquid is used in container T2 and a mixture of gas
and liquid is used in container T3.
[0057] FIG. 5C illustrates an example of a temperature versus time
plot of the temperature experienced by bolus 240 as it traverses
through the sample channel 220 as various heat exchange fluids flow
through the heat exchange channel 210 at various times, in
accordance with at least one exemplary embodiment of the present
invention.
[0058] Referring now to FIG. 6, an apparatus 120 is illustrated
according to another embodiment. The embodiment shown in FIG. 6 is
similar to that shown in FIGS. 2A-B, with the exception that the
forward valves V1F, V2F, and V3F are replaced with pumps 602, 604
and 606, respectively, and pump 290 is not present. The operation
of the apparatus 120 in accordance with this embodiment is
discussed below.
[0059] Referring now to FIG. 7, steps 416-420 are further
illustrated according to some embodiments where process 400 is
implemented using the apparatus shown in FIG. 6. As shown in FIG.
7, step 416 may include, activating only pump 602, opening valve
V3R and closing the other return valves (V1R and V2R), step 418 may
include activating only pump 604, opening valve V1R and closing the
other return valves (V3R and V2R), step 420 may include activating
only pump 606, opening valve V2R and closing the other return
valves (V1R and V3R). The container from which the fluid flows and
to which the fluid returns, of course, depends on the valves that
are open at the time and the pump that is activated. For example,
when step 418 is performed in accordance with the flow shown in
FIG. 7, fluid will flow out of container T2 and into container T1.
The apparatus of FIG. 6 also can be controlled such that fluid will
flow out of one container and back into the same container, as
discussed above.
[0060] FIG. 8 illustrates a thermal exchange system 800 in
accordance with another exemplary embodiment. The thermal exchange
system 800 is directed to a thermal exchange system that includes a
plurality of heat exchange channels (e.g., 810a-c), each configured
to carry a heat exchange fluid, where each heat exchange fluid
preferably is at a different temperature. The plurality of heat
exchange channels (810a-c) can be configured to lie substantially
perpendicular (orthogonal) (although the invention is not limited
to an orthogonal orientation) to a sample channel 820 and
configured to exchange heat with a portion (e.g., 830a-c) of the
sample channel. In this exemplary embodiment, a bolus 840 of
genomic material traveling along the sample channel 820 experiences
temperature change associated with heat exchanged in from portions
of heat exchange channels 830a-c, as discussed above in connection
with other embodiments.
[0061] In the embodiment of FIG. 8, heat exchange channel 810a is
in fluid communication with at least container T1, heat exchange
channel 810b is in fluid communication with at least container T2,
and heat exchange channel 810c is in fluid communication with at
least container T3. In one aspect of this embodiment, fluid is
caused to flow from containers T1-T3 and through heat exchange
channels 810a-c through one or more pumps and is caused to return
to the containers through one or more return valves, as disclosed
herein. The container from which the fluid flows and to which the
fluid returns, of course, depends on the valves that are open at
the time and the pump that is activated.
[0062] FIG. 9 illustrates a thermal exchange system 900 in
accordance with another exemplary embodiment, which includes a
curved sample channel 920, that directs a bolus 940 of genomic
material back and forth (905A-C) near a plurality of heat exchange
channels (910a-c), each configured to carry a heat exchange fluid,
where each heat exchange fluid preferably is at a different
temperature. In this exemplary embodiment, a bolus 940 of genomic
material traveling along the curved sample channel 920 experiences
temperature change associated with heat exchanged with portions of
heat exchange channels 910a-c, as discussed above in connection
with other embodiments. In this embodiment, the fluid flows from
the containers T1-T3 through heat exchange channels 910a-c, for
example, in the same manner described above in connection with the
FIG. 8 embodiments and other embodiments described herein.
[0063] While various embodiments/variations of the present
invention have been described above, it should be understood that
they have been presented by way of example only, and not
limitation. Thus, the breadth and scope of the present invention
should not be limited by any of the above-described exemplary
embodiments. Further, unless stated, none of the above embodiments
are mutually exclusive. Thus, the present invention may include any
combinations and/or integrations of the features of the various
embodiments.
[0064] Additionally, while the processes described above and
illustrated in the drawings are shown as a sequence of steps, this
was done solely for the sake of illustration. Accordingly, it is
contemplated that some steps may be added, some steps may be
omitted, and the order of the steps may be re-arranged.
* * * * *