U.S. patent application number 15/436118 was filed with the patent office on 2017-06-08 for systems and methods for monitoring the amplification and dissociation behavior of dna molecules.
This patent application is currently assigned to CANON U.S. LIFE SCIENCES, INC.. The applicant listed for this patent is CANON U.S. LIFE SCIENCES, INC.. Invention is credited to Gregory A. DALE, Kenton C. HASSON.
Application Number | 20170157607 15/436118 |
Document ID | / |
Family ID | 38895127 |
Filed Date | 2017-06-08 |
United States Patent
Application |
20170157607 |
Kind Code |
A1 |
HASSON; Kenton C. ; et
al. |
June 8, 2017 |
SYSTEMS AND METHODS FOR MONITORING THE AMPLIFICATION AND
DISSOCIATION BEHAVIOR OF DNA MOLECULES
Abstract
The present invention relates to systems and methods for
monitoring the amplification of DNA molecules and the dissociation
behavior of the DNA molecules. A method according to one embodiment
of the invention may include the steps of: forcing a sample of a
solution containing real-time PCR reagents to move though a
channel; and while the sample is moving through an analysis region
of the channel, performing the steps of: (a) cycling the
temperature of the sample until the occurrence of a predetermined
event; (b) after performing step (a), causing the sample's
temperature to gradually increase from a first temperature to a
second temperature; and (c) while the step of gradually increasing
the sample's temperature is performed, using an image sensor to
monitor emissions from the sample.
Inventors: |
HASSON; Kenton C.;
(Gaithersburg, MD) ; DALE; Gregory A.;
(Gaithersburg, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CANON U.S. LIFE SCIENCES, INC. |
Rockville |
MD |
US |
|
|
Assignee: |
CANON U.S. LIFE SCIENCES,
INC.
Rockville
MD
|
Family ID: |
38895127 |
Appl. No.: |
15/436118 |
Filed: |
February 17, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11606006 |
Nov 30, 2006 |
9573132 |
|
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15436118 |
|
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60806440 |
Jun 30, 2006 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B01L 2200/143 20130101;
B01L 7/52 20130101; G06T 2207/10004 20130101; Y10T 436/117497
20150115; C12Q 1/686 20130101; B01L 2400/0487 20130101; B01L
2300/0654 20130101; G06T 2207/30072 20130101; G01N 35/08 20130101;
B01L 7/525 20130101; B01L 3/50273 20130101; B01L 2200/10 20130101;
B01L 2300/1811 20130101; C12Q 1/686 20130101; B01L 2300/0829
20130101; C12Q 2565/629 20130101; B01L 3/5027 20130101; B01L
2300/1822 20130101; B01L 3/502715 20130101; B01L 2300/1877
20130101; B01L 2300/1827 20130101; G06T 7/0012 20130101; B01L
2300/0627 20130101; G01N 21/71 20130101; B01L 2300/18 20130101;
G06T 7/11 20170101; Y10T 436/115831 20150115; C12Q 2561/113
20130101 |
International
Class: |
B01L 3/00 20060101
B01L003/00; G01N 21/71 20060101 G01N021/71; G06T 7/00 20060101
G06T007/00; B01L 7/00 20060101 B01L007/00 |
Claims
1. An optical analysis system, comprising: a substrate comprising a
microfluidic channel; a thermal generating unit operable to provide
heat to and absorb heat from at least a portion of the microfluidic
channel; an image sensor disposed in relation to the substrate such
that said portion of the channel is within the field of view of the
image sensor; and an image processing system coupled to the image
sensor and configured to: (i) receive image data from the image
sensor and (ii) use image data from the image sensor to determine
whether (a) the intensity of emissions from a nucleic acid sample
traveling through said portion of the channel equals or exceeds a
predetermined emission intensity threshold or (b) the rate of
change in the intensity of emissions from the nucleic acid sample
traveling through said portion of the channel equals or is less
than a predetermined threshold; and a temperature controller
configured to control the thermal generating unit and configured to
cause the thermal generating unit to gradually ramp the temperature
of the portion of the channel from a first temperature to a second
temperature in response to the image processing system determining
that (a) the intensity of emissions from a nucleic acid sample
traveling through said portion of the channel equals or exceeds a
predetermined emission intensity threshold or (b) the rate of
change in the intensity of emissions from the nucleic acid sample
equals or is less than the predetermined threshold.
2. The system of claim 1, wherein the temperature controller is
further configured to cause the thermal generating unit to cycle
the temperature of the portion of the channel in response to a
predetermined input.
3. The system of claim 2, further comprising an image sensor
controller, wherein the image sensor controller is operable to
cause the image sensor to capture images of at least a segment of
said portion of the channel that is within the field of view of the
image sensor while the thermal generating unit cycles the
temperature of the portion of the channel.
4. The system of claim 3, wherein the image sensor controller is
operable to cause the image sensor to capture images of at least a
segment of said portion of the channel that is within the field of
view of the image sensor while the thermal generating unit ramps
the temperature of the portion of the channel.
5. The system of claim 4, wherein the image sensor controller is
operable to cause the image sensor to capture at least 1 image
every 90 seconds of at least a segment of said portion of the
channel that is within the field of view of the image sensor while
the thermal generating unit cycles the temperature of the portion
of the channel.
6. The system of claim 5 wherein the image sensor controller is
operable to cause the image sensor to capture at least 5 images per
second of at least a segment of said portion of the channel that is
within the field of view of the image sensor while the thermal
generating unit ramps the temperature of the portion of the
channel.
7. The system of claim 6, wherein the thermal generating unit is
configured to ramp the temperature of the portion of the channel by
steadily increasing the amount of heat provided to the portion of
the channel at a thermal ramp rate of between about 0.1 to 2 degree
Celsius (C) per second.
8. The system of claim 1, further comprising an excitation source
for producing electromagnetic radiation directed at the portion of
the channel.
9. The system of claim 8, further comprising a second excitation
source for illuminating at least a segment of the portion of the
channel in response to the image processing system determining that
(a) the intensity of emissions from a nucleic acid sample traveling
through said portion of the channel equals or exceeds a
predetermined emission intensity threshold or (b) the rate of
change in the intensity of emissions from the nucleic acid sample
equals or is less than the predetermined threshold.
10. The system of claim 9, wherein the image sensor is a CMOS image
sensor and the second excitation source is a laser.
Description
[0001] The present application is a divisional of U.S. Ser. No.
11/606,006 filed Nov. 30, 2006 which claims the benefit to U.S.
Provisional Patent Application Ser. No. 60/806,440, filed on Jun.
30, 2006, which is incorporated herein by this reference.
BACKGROUND
[0002] Field of the Invention
[0003] The present invention relates to systems and methods for
monitoring the amplification of DNA molecules and the dissociation
behavior of the DNA molecules.
[0004] Discussion of the Background
[0005] The 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. Polymerase chain reaction
(PCR) is a well-known technique for amplifying DNA.
[0006] 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 the 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 a number of times so that at the end of the
process there are enough copies to be detected and analyzed. 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] In some applications, it is important to monitor the
accumulation of DNA products as the amplification process
progresses. 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
amplification process over time allows one to determine the
efficiency of the process, as well as 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] 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. See, for
example, Lagally et al. (Anal Chem 73:565-570 (2001)), Kopp et al.
(Science 280:1046-1048 (1998)), Park et al. (Anal Chem 75:6029-6033
(2003)), Hahn et al. (WO 2005/075683), Enzelberger et al. (U.S.
Pat. No. 6,960,437) and Knapp et al. (U.S. Patent Application
Publication No. 2005/0042639).
[0009] Once there are a sufficient number of copies of the original
DNA molecule, the DNA can be characterized. One method of
characterizing the DNA is to examine the DNA's dissociation
behavior as the DNA transitions from double stranded DNA (dsDNA) to
single stranded DNA (ssDNA) with increasing temperature. The
process of causing DNA to transition from dsDNA to ssDNA is
sometimes referred to as a "high-resolution temperature (thermal)
melt (HRTm)" process, or simply a "high-resolution melt"
process.
[0010] Accordingly, what is desired is a system for monitoring the
DNA amplification process and for determining the DNA's
dissociation behavior.
SUMMARY OF THE INVENTION
[0011] The present invention relates to systems and methods for
performing and monitoring real-time PCR and HRTm analysis.
[0012] In one aspect, the present invention provides a method that
includes the steps of: (a) introducing into a microchannel at least
one bolus containing nucleic acid; (b) forcing the bolus to move
through the microchannel; (c) while the bolus is moving through the
microchannel, amplifying nucleic acid contained in the bolus and
using an image sensor to determine whether the nucleic acid has
been sufficiently amplified; (e) if the nucleic acid has not been
sufficiently amplified, then repeating step (c), otherwise, while
the bolus is still moving through the microchannel, causing dsDNA
within the bolus to transition to ssDNA and using an image sensor
to capture images of the bolus. In some embodiments, the step of
using the image sensor to capture an image of the bolus comprises
determining a region of interest and then reading out only those
pixels of the image sensor that are within the region of interest
and capturing at least about 5 images of the bolus per second.
[0013] In some embodiments, the step of using the image sensor to
determine whether the nucleic acid has been sufficiently amplified
includes using the image sensor to capture an image of the bolus
and processing the captured image data to determine the intensity
of light emitted from the bolus. A value representing the
determined intensity may be compared to a threshold value to
determine whether the nucleic acid has been sufficiently
amplified.
[0014] In some embodiments, once it has been determined that the
nucleic acid has been sufficiently amplified, a process for causing
the dsDNA within the bolus to transition to ssDNA is initiated
(e.g., if the result of the comparison indicates that the value
representing the determined intensity is greater than the threshold
value, then the melting process is initiated at least shortly after
the determination is made).
[0015] In some embodiments, the step of initiating the process
comprises configuring a heating system to gradually increase the
temperature of the bolus. Preferably, the temperature is increased
at or about a constant rate (e.g., a constant rate of between about
0.1 and 1 degree Celsius per second).
[0016] In another aspect, the present invention provides a system
that includes the following elements: a substrate comprising a
microfluidic channel; a thermal generating unit operable to provide
heat to and absorb heat from at least a portion of the microfluidic
channel; an image sensor disposed in relation to the substrate such
that said portion of the channel is within the field of view of the
image sensor; and an image processing system coupled to the image
sensor and configured to: (i) receive image data from the image
sensor and (ii) use image data from the image sensor to determine
whether the intensity of emissions from a nucleic acid sample
traveling through said portion of the channel equals or exceeds a
predetermined emission intensity threshold; and a temperature
controller configured to control the thermal generating unit and
configured to cause the thermal generating unit to gradually ramp
the temperature of the portion of the channel from a first
temperature to a second temperature in response to the image
processing system determining that the intensity of emissions from
a nucleic acid sample traveling through said portion of the channel
equals or exceeds a predetermined emission intensity threshold.
[0017] In some embodiments, the thermal generating unit is
configured to ramp the temperature by steadily increasing the
amount of heat provided at a thermal ramp rate of between about 0.1
to 1 degree Celsius (C) per second. The temperature controller may
further be configured to cause the thermal generating unit to cycle
the temperature of the portion of the channel in response to a
predetermined input.
[0018] The system may further include an image sensor controller.
The image sensor controller may be operable to cause the image
sensor to capture images of at least a segment of said portion of
the channel that is within the field of view of the image sensor
while the thermal generating unit cycles the temperature of the
portion of the channel. The image sensor controller may also be
operable to cause the image sensor to capture images of at least a
segment of said portion of the channel that is within the field of
view of the image sensor while the thermal generating unit ramps
the temperature of the portion of the channel. In some embodiments,
the image sensor controller is operable to cause the image sensor
to capture at least 1 image every 90 seconds while the thermal
generating unit cycles the temperature and to capture at least 5
images per second while the thermal generating unit ramps the
temperature.
[0019] The system may also include an excitation source for
producing electromagnetic radiation directed at the portion of the
channel. In some embodiments, the system may further include a
second excitation source for illuminating at least a segment of the
portion of the channel in response to the image processing system
determining that the intensity of emissions from a nucleic acid
sample traveling through said portion of the channel equals or
exceeds a predetermined emission intensity threshold.
[0020] In another aspect, the present invention provides a method
that includes the steps of: (i) forcing a sample of a solution
containing real-time PCR reagents to move though a channel; and
(ii) while the sample is moving through an analysis region of the
channel, performing the steps of: (a) cycling the temperature of
the sample until the occurrence of a predetermined event; (b) after
performing step (a), causing the sample's temperature to gradually
increase from a first temperature to a second temperature; and (c)
while the step of gradually increasing the sample's temperature is
performed, using an image sensor to monitor emissions from the
sample. In some embodiments, the step of gradually increasing the
temperature of the sample from the first temperature to the second
temperature comprises increasing the temperature at or about a
constant rate (e.g., between about 0.1 and 1 degrees Celsius per
second).
[0021] In some embodiment, the method may further include the step
of determining whether the predetermined event has occurred. This
step may include determining whether nucleic acid in the sample has
been sufficiently amplified by, for example, determining the
intensity of light emitted from the sample and comparing the
determined intensity to an intensity threshold value. The step of
determining the intensity of light emitted from the sample may
include the step of using an image sensor to capture an image of
the sample and the image capturing step may include determining a
region of interest and then reading out only those pixels of the
image sensor that are within the region of interest.
[0022] The above and other embodiments of the present invention are
described below with reference to the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] 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. Additionally,
the left-most digit(s) of a reference number identifies the drawing
in which the reference number first appears.
[0024] FIG. 1 is a functional block diagram of a genomic analysis
system according to one embodiment.
[0025] FIG. 2 is a top view of a biochip according to one
embodiment.
[0026] FIG. 3 is a view of an image sensor according to one
embodiment.
[0027] FIG. 4 is a flow chart illustrating a process according to
one embodiment.
[0028] FIG. 5 is functional block diagram of a genomic analysis
system according to one embodiment.
[0029] FIG. 6 illustrates a first temperature profile according to
one embodiment.
[0030] FIG. 7 illustrates a second temperature profile according to
one embodiment.
[0031] FIG. 8 is a functional block diagram illustrating an
embodiment of image processing system.
[0032] FIG. 9 is a functional block diagram of a nucleic acid
analysis system according to one embodiment.
[0033] FIG. 10 is a flow chart illustrating a process according to
an embodiment.
[0034] FIG. 11 is an example temperature profile.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0035] Referring to the drawings, FIG. 1 illustrates a nucleic acid
analysis system 100 according to an embodiment. As shown in FIG. 1,
system 100 includes a microfluidic biochip 102 having a PCR
processing zone 104 (i.e., a zone in which DNA is amplified) and a
HRTm analysis zone 106 (i.e., a zone in which the dissociation
behavior of the amplified DNA is examined).
[0036] FIG. 2 is a top view of biochip 102 according to some
embodiments. As shown in FIG. 2, biochip 102 includes a number of
microcfluidic channels 202. In the example shown, there are 8
microfluidic channels, but it is contemplated that chip 102 may
have more or less than 8 channels. As shown, a first portion of
each microfluidic channel may be within the PCR processing zone 104
and a second portion of each microfluidic channel may be within the
HRTm analysis zone 106. As further shown in FIG. 1, zone 106 may
immediately follow zone 104 and the length of zone 104 may be
significantly greater than the length of zone 106 (e.g., the length
of zone 104 may be 5 times the length of zone 106).
[0037] Although FIG. 1 shows that there is a small gap between
zones 104 and 106, it is contemplated that there no gap exists
between the zones (i.e., it is contemplated that zone 106 not only
immediately follows zone 104, but also shares a common boundary
with zone 104).
[0038] In some embodiments, when system 100 is in use, at least one
channel 202 receives a sample (or "bolus") of a solution containing
real-time PCR reagents. A force may be used to cause the bolus to
travel through the channel such that the bolus traverses PCR zone
104 prior to entering HRTm zone 106. One system and method for
performing PCR in a microfluidic device is disclosed in U.S. patent
application Ser. No. 11/505,358, filed on Aug. 17, 2006,
incorporated herein by reference.
[0039] Referring back to FIG. 1, genomic analysis system 100
further includes an image sensor 108, a controller 110 for
controlling image sensor 108, and an image processing system 112
for processing the image data produced by image sensor 108. Image
sensor 108 may be implemented using a CMOS image sensor, a CCD
image sensor, or other image sensor. For example, in one
embodiment, sensor 108 is a CMOS sensor with an effective 12.7 mega
pixel resolution and having a size of 36.times.24 mm, which is
available from Canon Inc.
[0040] Image sensor 108 has a first image sensor region 121 and a
second image sensor region 122. Image sensor region 121 has a
different field of view than image sensor region 122. In preferred
embodiments, image sensor 108 is positioned with respect to chip
102 such that at least a portion of PCR processing zone 104 is
within the field of view of sensor region 121 and at least a
portion of HRTm zone 106 is within the field of view of sensor
region 122.
[0041] Referring now to FIG. 3, FIG. 3 is a view of the light
sensitive surface of image sensor 108. This view better illustrates
the two image sensor regions 121, 122. As shown, the area of image
sensor region 122 may be significantly smaller than the area of
image sensor region 121 (e.g., 1/5 the area or less). In some
embodiments the widths of the two regions 121, 122 are the same,
but the lengths are different.
[0042] Referring now to FIG. 8, FIG. 8 is a functional block
diagram illustrating an embodiment of image processing system 112.
As shown in FIG. 8, system 112 receives data output from image
sensor 108. System 112 may include an amplifier 802 to amplify the
data from image sensor 108. In one non-limiting embodiment,
amplifier 802 may amplify the data for greater than 3200 ISO
sensitivity. The amplified data may be converted to a digital
signal by, for example, a 16 bit analog-to-digital (A/D) converter
804. In one embodiment, utilization of a 16 bit A/D converter
provides a high level of dynamic range and low end bit resolution.
The digital signal output from A/D converter 804 may be processed
by a framing circuit 806, which may be configured to store data
produced from image sensor region 121 in a zone 1 data buffer 808
and store data produced from image sensor region 122 in a zone 2
data buffer 810. A programmable data processor 812 may be
programmed to process data in buffers 810 and 812 to, among other
things, determine and record the intensity of the fluorescence from
zones 104 and 106.
[0043] By configuring image sensor 108 and chip 102 as described
above, a single image sensor is able to (i) produce data
corresponding to the intensity of emissions from PCR zone 104 and
(ii) produce data corresponding to the intensity of emissions from
HRTm zone 106. Thus, while utilizing only a single image sensor,
system 100 can simultaneously monitor (1) the amplification of a
sample of DNA and (2) the dissociation behavior of a different DNA
sample.
[0044] As further illustrated in FIG. 1, system 100 may include one
or more thermal generating apparatuses. In the embodiment shown,
system 100 includes a first thermal generating apparatus 114 and a
second thermal generating apparatus 116 and a controller 118 for
controlling apparatuses 114, 116. In one embodiment, first thermal
generating apparatus creates a first thermal zone in PCR processing
zone 104 and second thermal generating apparatus creates a second
thermal zone in the HRTm analysis zone 106.
[0045] Each thermal generating apparatus 114, 116 is configured to
provide heat to and/or absorb heat from chip 102, and, thus, may
include one or more heat sources and/or heat sinks (e.g., each
thermal generating apparatus 114, 116 may include a peltier device
or other heat source or sink). More specifically, in the embodiment
shown, thermal apparatus 114 is configured to provide heat to
and/or absorb heat from PCR zone 104, and thermal apparatus 116 is
configured to provide heat to and/or absorb heat from HRTm zone
106.
[0046] While only one temperature controller is shown, it is
contemplated that each thermal generating apparatus may have its
own controller. Additionally, although system 100 may have a single
temperature controller, the thermal generating apparatuses may be
operated independently so apparatus 116 can be used to perform HRTm
analysis in zone 106 while at the same time apparatus 114 is used
to cause PCR to occur in zone 104.
[0047] That is, in some embodiments, the first thermal generating
apparatus 114 is configured such that while a bolus is within zone
104, thermal generating apparatus 114 cycles the temperature in
zone 104 to achieve PCR, and thermal generating apparatus 116 is
configured such that, when a bolus enters zone 106, thermal
generating apparatus 116 provides a substantially steadily
increasing amount of heat to zone 106 to cause the bolus to undergo
HRTm analysis (i.e., to cause the dsDNA in the bolus to transition
to ssDNA). In one example, thermal generating apparatus 116 may
provide a thermal ramp rate of typically 0.1 to 2 degree Celsius
(C) per second, with the preferred ramp rate being between 0.5 and
1 degree Celsius (C) per second.
[0048] Referring now to sensor controller 110, sensor controller
110 may be configured so that, for each bolus that undergoes HTRm
analysis in the HRTm analysis zone, image sensor controller 110
causes sensor 108 to capture preferably at least about 10 images
per second from image sensor region 122 for at least about 1 minute
while the bolus undergoes the HRTm analysis (typically it captures
the images for an uninterrupted duration of about 5 minutes). In
embodiments where the ramp rate is faster, the image sensor
controller 110 may cause sensor 108 to capture the images at a rate
of about 20 images per second. In many embodiments, the goal is to
achieve a temperature resolution of 0.1 degree Celsius or
better.
[0049] In some embodiments, to achieve the high 10 images/second
frame rate, the sensor may be implemented using a CMOS sensor and
the controller may be configured to window the CMOS sensor to read
out only the pixels of interest (e.g., some or all of the pixels
within image sensor region 122).
[0050] In some embodiments, system 100 may further include an
excitation source 130 (e.g., a laser or other excitation source)
for illuminating zones 104 and/or 106. Additional excitation
sources (e.g., source 131) may also be employed. System 100 may
further include a lens 140 that is disposed between chip 102 and
image sensor 108. In such embodiments, lens 140 may be configured
to focus onto the first image sensor region 121 light 145 coming
from the PCR processing zone 104 and to focus onto the second image
sensor region 122 light 146 coming from the HRTm analysis zone
106.
[0051] Referring now to FIG. 4, FIG. 4 is a flow chart illustrating
a process 400 according to an embodiment. Process 400 may begin in
step 402, where a microfluidic device 102 having a mircochannel
having a PCR processing zone 104 (the first thermal zone) and a
HRTm analysis zone 106 (the second thermal zone) is obtained (see
FIG. 5). In step 403, an image sensor (e.g., image sensor 108) is
obtained and positioned such that both the first and second thermal
zones are simultaneously within the image sensor's field of
view.
[0052] In step 404, a series of boluses of a test solution are
introduced into the microchannel (the test solution may be stored
in a test solution reservoir 150 (see FIG. 1)). In step 406, each
bolus is forced to move along the channel such that each bolus
passes through the PCR processing zone and then enter and moves
through the HRTm analysis zone. This step is pictorially
illustrated in FIG. 5. Arrow 501 shows the direction in which the
series of boluses move. In some embodiments, the boluses
continuously move at a constant speed.
[0053] In step 408, while one or more boluses are moving through
the PCR processing zone, the temperature of the PCR processing zone
is cycled to amplify the DNA in each bolus. FIG. 6 illustrates the
temperature profile of the PCR processing zone, according to some
embodiments, as a result of step 408 being performed. As shown in
FIG. 6, and as is well known in the art, one temperature cycle
consists of: (1) holding the temperature of the PCR processing zone
at a first temperature (t1) (e.g., 52 degrees C.) for a first
period of time (p1) (e.g., 5 seconds), (2) then rapidly increasing
the temperature from t1 to t2 (e.g., 72 degrees C.) and holding the
temperature at t2 for a second period of time (p2) (e.g., 10
seconds), (3) then rapidly increasing the temperature from t2 to t3
(e.g., 94 degrees C.) and holding the temperature at t3 for a third
period of time (p3) (e.g., 5 seconds), and (4) then rapidly
dropping the temperature back to t1 so that the cycle may repeat.
The above described temperature cycle may be referred to as a "PCR
cycle." In some embodiments, the PCR cycles repeat for period of
time (e.g., as long as necessary to produce a sufficient amount of
DNA--typically about 20-40 PCR cycles).
[0054] In step 410, image sensor 108 is used to capture images of
at least one bolus within the PCR processing zone as the bolus
moves through the zone and as the temperature of the zone is cycled
as described above. In some embodiments, the images are captured
only during the "middle" of the PCR cycle (i.e., the time during
which the temperature is held at t2), as shown in FIG. 6. In some
embodiments, sensor controller 110 controls the image capturing and
windows images sensor 108 so that the step of capturing an image of
the bolus includes reading only the pixels of the image sensor 108
that are within image sensor region 121, or a subset of those
pixels, such as, for example, the pixels that receive light from
the bolus and one or more immediately surrounding pixels.
[0055] In step 412, the images captured in step 410 are processed
by, for example, image processing system 112. Image processing
system 412 may include one or more processors programmed by
software to determine the intensity of fluorescence emitted from
the bolus as a function of time.
[0056] In step 418, when (or shortly after) a bolus enters the HRTm
analysis zone and while it moves though the zone, the temperature
of the HRTm analysis zone is increased to cause dsDNA within the
bolus to transition to ssDNA. FIG. 7 illustrates the temperature
profile of the HRTm analysis zone, according to some embodiments.
In the example shown in FIG. 7, a first bolus enters the HRTm
analysis zone at (or shortly after) time t1 and remains within the
zone until (or shortly after) time t2 and a second bolus enters the
HRTm analysis zone at (or shortly after) time t3 and remains within
the zone until (or shortly after) time t4. As shown in FIG. 7,
while the first and second samples are within the HRTm analysis
zone, the temperature of the zone may increase, at a substantially
constant rate (e.g., 0.1 to 1 degrees C. per second), from
temperature t1 (e.g., about 65 degrees C.) to temperature t2 (e.g.,
about 95 degrees C.), which temperature increase should cause dsDNA
within the samples to transition to ssDNA.
[0057] In one embodiment, amplification by PCR is performed in the
presence of a dsDNA binding fluorescent dye. The dye does not
interact with ssDNA but actively binds with dsDNA and fluoresces
brightly in this state. This shift in fluorescence can be used
firstly to measure the increase in DNA concentration in the PCR
processing zone and then to directly measure thermally-induced DNA
dissociation by HRTm. Initially, fluorescence is high in a melt
analysis because the sample starts as dsDNA, but fluorescence
diminishes as the temperature is raised and DNA dissociates into
single strands. The observed "melting" behavior is characteristic
of a particular DNA sample. A melt curve is typically made and
plots the transition from high fluorescence of the initial pre-melt
phase through the sharp fluorescence decrease of the melt phase to
basal fluorescence at the post-melt phase. Fluorescence decreases
as DNA binding dye is released from double-stranded DNA as it
dissociates (melts) into single strands. The midpoint of the melt
phase, at which the rate of change in fluorescence is greatest,
defines the temperature of melting (TM) of the particular DNA
fragment under analysis.
[0058] Suitable dsDNA binding dyes included SYBR.RTM. Green 1
(Invitrogen Corp., Carlsbad, Calif.), SYTO.RTM. 9 (Invitrogen
Corp., Carlsbad, Calif.), LC Green.RTM. (Idaho Technologies, Salt
Lake City, Utah) and Eva Green.TM. (Biotium Inc, Hayward, Calif.).
Of these dyes, SYTO.RTM. 9, LC Green.RTM. and Eva Green' have low
toxicity in an amplification reaction and can therefore be used at
higher concentrations for greater saturation of the dsDNA sample.
Greater dye saturation means measured fluorescent signals have
higher fidelity, apparently because there is less dynamic dye
redistribution to non-denatured regions of the nucleic strand
during melting and because dyes do not favor higher melting
temperature products (Wittwer et al., Clinical Chemistry 49:853-860
(2003)). The combination of these characteristics provides greater
melt sensitivity and higher resolution melt profiles.
[0059] In step 420, image sensor 108 is used to capture images of
the bolus within the HRTm analysis zone as the bolus moves through
the zone and while the temperature of the zone is increased as
described above. In some embodiments, when the image sensor is used
to capture images of the bolus within the HRTm analysis zone the
image sensor is in the same position and orientation it was in when
it was used in step 410 to capture images of the bolus within the
PCR processing zone.
[0060] In some embodiments, in step 420, the images are captured at
a high frame rate (e.g., more than 5 images per second and
preferably at least about 10 images per second). In some
embodiments, sensor controller 110 controls the image capturing and
windows images sensor 108 so that the step of capturing an image of
the bolus includes reading only the pixels of the image sensor 108
that are within image sensor region 122, or a subset of those
pixels, such as, for example, the pixels that receive light from
the bolus and one or more immediately surrounding pixels.
[0061] In step 422, the images captured in step 420 are processed
by, for example, image processing system 112. Image processing
system 412 may include one or more processors programmed by
software to determine the intensity of fluorescence emitted from
the bolus as a function of time.
[0062] As illustrated in FIG. 4, steps 418-420 may occur
simultaneously with steps 408-410.
[0063] Referring now to FIG. 9, FIG. 9 is a functional block
diagram illustrating a nucleic acid analysis system 900 according
to another embodiment of the invention.
[0064] As illustrated in FIG. 9, system 900 may include many of the
same components as system 100. For example, system 900 includes a
substrate 102 having at least one microfluidic channel; at least
one thermal generating unit 114, which is operable to provide heat
to and/or absorb heat from at least a portion of the microfluidic
channel, such as for example, a thermal generating unit disclosed
in U.S. patent application Ser. No. 11/505,358, incorporated herein
by reference. The system 900 may also include a temperature
controller 118 for controlling apparatus 114; an image sensor 108
disposed in relation to the substrate such that at least a portion
of the microfluidic channel is within the field of view of image
sensor 108; an image sensor controller 110; and an image processing
system 112 coupled to image sensor 108.
[0065] Like system 100, system 900 may be used to analyze a sample
of nucleic acid (e.g., bolus 902). More specifically, system 900
may be used to amplify DNA and then melt the amplified DNA. In one
embodiment, to amplify the DNA, a sample of a solution containing
real-time PCR reagents may be introduced into the microfluidic
channel and then forced to move through the channel in the
direction of arrow 901 using techniques well known in the art.
[0066] While the sample is moving through the channel, temperature
controller 118 may be configured to cause thermal generating
apparatus 114 to cycle the temperature of the sample as described
above. For example, in response to receiving a predetermined input,
temperature controller 118 may cause thermal generating apparatus
114 to cycle the temperature of the sample.
[0067] While the sample is undergoing amplification, image sensor
controller 110 may cause the image sensor to capture images of a
segment of the channel in which the sample is located, thereby
capturing image data corresponding to emissions from the sample. In
some embodiments, image sensor controller may cause image sensor
108 to capture at least one image each cycle and, to improve
throughput, may window the image sensor to read out only a
predetermined subset of the sensor's pixel array.
[0068] Image processing system 112 may be configured to receive
this image data from image sensor 108 and use the image data to
determine whether the intensity of emissions from the sample equals
or exceeds a predetermined emission intensity threshold.
Additionally or alternatively, image processing system 112 may be
configured to use the image data to determine the rate of change in
the intensity of emissions from the sample.
[0069] Further, temperature controller 118 may be configured to
cause thermal generating apparatus 114 to gradually ramp the
temperature of the sample from a first temperature (e.g., about 65
degrees Celsius) to a second temperature (e.g., about 95 degrees
Celsius) in response to the occurrence of a particular event.
[0070] For example, temperature controller 118 may be configured to
cause thermal generating apparatus 114 to gradually ramp the
temperature of the sample from the first temperature to the second
temperature in response to image processing system 112 determining
that (a) the intensity of emissions from the sample equals or
exceeds a predetermined emission intensity threshold or (b) the
rate of change in the intensity of emissions from the sample is
less than or equal to a predetermined threshold. In other
embodiments, temperature controller 118 may be configured to cause
thermal generating apparatus 114 to gradually ramp the temperature
of the sample from the first temperature to the second temperature
in response to: a determination that (a) the sample has been
exposed to at least a certain number of temperature cycles, (b) a
predetermined amount of time has elapsed from a specific earlier
point in time (e.g., the point in time when the sample entered the
PCR processing zone 104 or the point in time when the sample was
exposed to its first temperature cycle), or (c) the sample is
within a predefined region of the channel (e.g., within HRTm zone
106).
[0071] In some embodiments, in response to the particular event,
temperature controller 118 may cause thermal generating apparatus
114 to ramp the temperature by increasing the amount of provided
heat at a thermal ramp rate of between about 0.1 to 2 degree
Celsius (C) per second.
[0072] While the temperature of the sample is being ramped, image
sensor controller 110 may cause image sensor 108 to capture images
of the segment of the channel in which the sample is located,
thereby capturing image data corresponding to emissions from the
sample. More specifically, in some embodiments, image sensor
controller 110 causes image sensor 108 to capture at least 5 images
per second of the segment of the channel in which the sample is
located (and preferably 10 images or more per second).
[0073] In some embodiments, in response to image processing system
112 determining that the intensity of emissions from the sample
equals or exceeds the predetermined emission intensity threshold,
excitation source 131 is employed to illuminate the segment of the
channel in which the sample is located, thereby illuminating the
sample while the sample undergoes thermal melting.
[0074] Referring now to FIG. 10, FIG. 10 is a flow chart
illustrating at least some of the steps of the process described
above. The process shown in FIG. 10 may start in step 1002 where a
device having a microfluidic channel is obtained. In step 1003, an
image sensor is positioned so that at least a portion of the
channel is within the image sensor's field of view. In step 1004, a
sample of solution is introduced into the channel. In step 1006,
the sample is forced to move along the channel. In step 1008, while
the sample is moving through the channel, the temperature of the
sample is cycled to amplify DNA contained in the sample.
[0075] In step 1010, while step 1008 is being performed, the image
sensor is used to produce image data corresponding to emissions
from the sample. In step 1012, the image data is processed (e.g.,
the image data may be processed to determine the intensity of the
emissions). In step 1013, a determination is made as to whether the
DNA melting process (e.g., HRTm) should be initiated. If not, the
process may return to step 1008, otherwise it may proceed to step
1018.
[0076] As described above, there are several ways this
determination can be made. For example, in one embodiment, a
processor (e.g., a processor of temperature controller 118) keeps
track of the number of temperature cycles the sample has been
exposed to and if the number meets or exceeds a threshold, then the
melting process should be initiated (i.e., the process should
proceed to step 1018). In another embodiment, if it is determined
that the emission intensity from the sample is greater than or
equal to a predetermined threshold, then the process should proceed
to step 1018. In yet another embodiment, the step of determining
whether to begin the melting process includes examining the rate of
change of the emission intensity from the sample and initiating the
melting process if the rate of change is less than or equal to a
predetermined threshold. In still another embodiment, the step of
determining whether to begin the melting process includes
determining whether (i) a predetermined amount of time has elapsed
from a specific earlier point in time (e.g., the point in time when
the sample entered the PCR processing zone 104 or the point in time
when the sample was exposed to its first temperature cycle) or (ii)
the sample is within a predefined region of the channel (e.g.,
within HRTm zone 106).
[0077] In step 1018, heat may be provided or removed from the
sample so that the sample reaches a predetermined temperature
(e.g., about 65 degrees Celsius) and then heat is provided to the
sample to melt DNA within the sample. As described above, the heat
may be provided so that the temperature of the sample increases
substantially at a constant rate to a predetermined temperature
(e.g., at least about 95 degrees Celsius). In step 1020, while step
1018 is being performed, an image sensor is used to produce image
data corresponding to emissions from the sample. Preferably, in
step 1020 the images are captured at a higher frame rate than the
images captured in step 1010 (for example, in step 1020 the image
sensor may be configured to capture at least 5, and preferably
about 10, images per second). To achieve the higher frame rate, the
image sensor may be windowed such that fewer than all of the pixels
of the sensor's pixel array are readout. In step 1022, the image
data collected in step 1020 may be processed.
[0078] Referring now to FIG. 11, FIG. 11 illustrates the
temperature profile of a sample that undergoes the processing
described above. As shown in FIG. 11, the temperature of the sample
is cycled for a period of time so that the sample is exposed to
some number of temperature cycles. In the illustrated example, at
time t=Ti, the image processing system determines that (a) the
intensity of the emissions from the sample meets or exceeds a
threshold or (b) the rate of change of the intensity of the
emissions from the sample is less than or equal to a threshold,
either of which may be an indication that the DNA in the sample has
been sufficiently amplified. Accordingly, as shown in FIG. 11, at
about time t=Ti, the temperature of the sample is brought to about
65 degrees Celsius and then the temperature of the sample increases
at substantially a constant rate so that the temperature reaches a
predetermined temperature (e.g., about 95 degrees Celsius).
[0079] While various embodiments 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.
[0080] Additionally, while the processes described above 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.
[0081] For the claims below the words "a" and "an" should be
construed as "one or more."
* * * * *