U.S. patent application number 13/023958 was filed with the patent office on 2011-12-15 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.. Invention is credited to Gregory A. Dale, Kenton C. HASSON.
Application Number | 20110306119 13/023958 |
Document ID | / |
Family ID | 38895127 |
Filed Date | 2011-12-15 |
United States Patent
Application |
20110306119 |
Kind Code |
A1 |
HASSON; Kenton C. ; et
al. |
December 15, 2011 |
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. The present invention in one
embodiment provides a system that includes a microfluidic channel
comprising a PCR processing zone and an HRTm analysis zone; and an
image sensor having a first image sensor region having a first
field of view and a second image sensor region having a second
field of view, wherein the second field of view is different than
the first field of view, wherein at least a portion of the PCR
processing zone is within the first field of view; and at least a
portion of the HRTm analysis zone is within the second field of
view.
Inventors: |
HASSON; Kenton C.;
(Germantown, MD) ; Dale; Gregory A.;
(Gaithersburg, MD) |
Assignee: |
Canon U.S. Life Sciences,
Inc.
Rockville
MD
|
Family ID: |
38895127 |
Appl. No.: |
13/023958 |
Filed: |
February 9, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11606204 |
Nov 30, 2006 |
7906319 |
|
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13023958 |
|
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60806440 |
Jun 30, 2006 |
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Current U.S.
Class: |
435/287.2 |
Current CPC
Class: |
B01L 3/502715 20130101;
B01L 2200/10 20130101; B01L 2300/1877 20130101; G01N 35/08
20130101; G06T 2207/30072 20130101; B01L 7/525 20130101; B01L
2300/0654 20130101; B01L 3/5027 20130101; B01L 2300/0627 20130101;
G01N 21/71 20130101; C12Q 1/686 20130101; G06T 2207/10004 20130101;
B01L 2300/18 20130101; B01L 2300/1811 20130101; B01L 2300/1827
20130101; B01L 2400/0487 20130101; Y10T 436/115831 20150115; Y10T
436/117497 20150115; G06T 7/0012 20130101; B01L 7/52 20130101; G06T
7/11 20170101; B01L 2300/1822 20130101; B01L 2300/0829 20130101;
C12Q 2565/629 20130101; C12Q 2561/113 20130101; B01L 2200/143
20130101; B01L 3/50273 20130101; C12Q 1/686 20130101 |
Class at
Publication: |
435/287.2 |
International
Class: |
C12M 1/34 20060101
C12M001/34 |
Claims
1. A system, comprising: a microfluidic channel comprising a PCR
processing zone and an HRTm analysis zone; and an image sensor
having a first image sensor region having a first field of view and
a second image sensor region having a second field of view, wherein
the second field of view is different than the first field of view,
wherein at least a portion of the PCR processing zone is within the
first field of view, and at least a portion of the HRTm analysis
zone is within the second field of view.
2-42. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of application Ser. No.
11/606,204, filed Nov. 30, 2006, which claims the benefit of
Provisional Application Ser. No. 60/806,440, filed Jun. 30, 2006,
which is incorporated herein by reference in its entirety.
BACKGROUND
[0002] 1. 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] 2. 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 system that
includes the following elements: a microfluidic channel comprising
a PCR processing zone and an HRTm analysis zone; and an image
sensor having a first image sensor region having a first field of
view and a second image sensor region having a second field of
view, wherein the second field of view is different than the first
field of view, wherein at least a portion of the PCR processing
zone is within the first field of view; and at least a portion of
the HRTm analysis zone is within the second field of view. In one
embodiment, the system may further include: a first thermal
generating apparatus configured to provide heat to and/or absorb
heat from the PCR processing zone; and a second thermal generating
apparatus configured to provide heat to and/or absorb heat from the
HRTm analysis zone. The two temperature generating apparatuses may
be operated independently so that HRTm analysis can occur in the
HRTm analysis zone while at the same time PCR is occurring in the
PCR processing zone. The system may also include an image sensor
controller configured such that, for each bolus that undergoes HTRm
analysis in the HRTm analysis zone, the image sensor controller
preferably captures at least about 10 images per second from the
second image sensor region for at least about 1 minute while the
bolus undergoes the HRTm analysis. In some embodiments, to achieve
the 10 images/second capture rate, the controller may window the
image sensor. In some embodiments, the system may further include a
lens that is disposed between the channel and the image sensor. In
such embodiments, the lens may be configured to focus onto the
first image sensor region only the light coming from the PCR
processing zone and to focus onto the second image sensor region
only the light coming from the HRTm analysis zone.
[0013] In another aspect, the present invention provides a system
that includes (i) a channel for receiving a bolus of solution
containing real-time PCR reagents, which channel includes a DNA
amplification zone and a DNA melting zone adjacent to the DNA
amplification zone, and (ii) an image sensor disposed in relation
to the channel such that both the DNA melting zone and the DNA
amplification zone are within the field of view of the sensor at
the same time. In some embodiments, the system may further include:
a first thermal generating apparatus configured to provide heat to
and/or absorb heat from the DNA amplification zone; and a second
thermal generating apparatus configured to provide heat to and/or
absorb heat from the DNA melting zone. The two temperature
generating apparatuses may be operated independently so that DNA
melting can occur in the DNA melting zone while at the same time
DNA amplification is occurring in the DNA amplification zone. In
some embodiments, the first thermal generating apparatus is
configured such that while a bolus is within the DNA amplification
zone, the first thermal generating apparatus cycles the temperature
in the DNA amplification zone in order to achieve PCR, and the
second thermal generating apparatus is configured such that, when a
bolus enters the DNA melting zone, the second thermal generating
apparatus provides a substantially steadily increasing amount of
heat to the DNA melting zone at a thermal ramp rate of typically
0.1 to 1 degree Celsius (C) per second. In some embodiments, the
length of the DNA melting zone is significantly smaller than the
length of the DNA amplification zone (e.g., the length of the DNA
melting zone may be 1/5 the length of the DNA amplification zone.
In some embodiments, the system includes an image controller. The
image controller may be configured such that, for each bolus that
enters the melting zone, the controller preferably captures at
least about 10 images per second from the second image sensor
region for at least about 1 minute. In some embodiments, to achieve
the 10 images/second capture rate, the controller may window the
image sensor.
[0014] In another aspect, the present invention provides a method
that includes the steps of: (a) using a PCR processing zone of a
channel to perform a PCR process; (b) using an HRTm analysis zone
of the channel to perform an HRTm process; (c) while performing
step (b) using an image sensor to obtain images of the HRTm
analysis zone; and (d) after performing step (c) and without moving
the image sensor from the position it was in relative to the
channel when step (c) was performed, using the image sensor to
obtain images of and the PCR processing zone while performing step
(a). In some embodiments, the step of using the PCR processing zone
to achieve PCR includes the steps of: moving a bolus of test
solution containing real-time PCR reagents through the PCR
processing zone; and cycling the temperature of the bolus while the
bolus is in the PCR processing zone, and the step of using the HRTm
analysis zone to perform an HRTm process includes the steps of:
moving a bolus of test solution containing real-time PCR reagents
through the HRTm analysis zone; and steadily increasing the
temperature of the bolus while the bolus is in the HRTm analysis
zone. In some embodiments, the step of cycling the temperature of
the bolus in order to achieve PCR comprises using a first thermal
generating apparatus to cycle the temperature, and the step of
steadily increasing the temperature of the bolus comprises using a
second thermal generating apparatus to steadily increase the
temperature. In some embodiments, the method may further include
the steps of causing a bolus of solution containing real-time PCR
reagents to move through the PCR processing zone and then through
the HRTm analysis zone, and using the image sensor to capture at
least about 10 images of the bolus per second for at least about 1
minute while the bolus is in the HRTm analysis zone.
[0015] In another aspect, the present invention provides a method
that includes the steps of: (a) performing a PCR process in a first
zone of a microfluidic channel; (b) performing an HRTm process in a
second zone of the microfluidic channel; (c) while performing step
(a), focusing radiation from the first zone onto a first region of
an image sensor, but not onto a second region of the image sensor;
and (d) while performing step (c), focusing radiation from the
second zone onto the second region of the image sensor, but not
onto the first region of the image sensor.
[0016] In some embodiments, the step of performing the PCR process
in the first zone comprises: moving a bolus of test solution
containing real-time PCR reagents through the first zone; and,
while the bolus is in the first zone, cycling the temperature of
the bolus in order to achieve PCR; and the step of performing an
HRTm process in the second zone comprises: moving a bolus of test
solution containing real-time PCR reagents through the second zone
after moving the bolus through the first zone; and while the bolus
is in the second zone, steadily increasing the temperature of the
bolus. In some embodiments, the step of cycling the temperature of
the bolus in order to achieve PCR comprises using a first thermal
generating apparatus to cycle the temperature, and the step of
steadily increasing the temperature of the bolus comprises using a
second thermal generating apparatus to steadily increase the
temperature. In some embodiments, the method further includes:
causing a bolus of solution containing real-time PCR reagents to
move through the first zone and then through the second zone, and
using the image sensor to capture preferably at least about 10
images of the bolus per second for at least about 1 minute while
the bolus is in the second zone.
[0017] The above and other embodiments of the present invention are
described below with reference to the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] 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.
[0019] FIG. 1 is a functional block diagram of a genomic analysis
system according to one embodiment.
[0020] FIG. 2 is a top view of a biochip according to one
embodiment.
[0021] FIG. 3 is a view of an image sensor according to one
embodiment.
[0022] FIG. 4 is a flow chart illustrating a process according to
one embodiment.
[0023] FIG. 5 is functional block diagram of a genomic analysis
system according to one embodiment.
[0024] FIG. 6 illustrates a first temperature profile according to
one embodiment.
[0025] FIG. 7 illustrates a second temperature profile according to
one embodiment.
[0026] FIG. 8 is a functional block diagram illustrating an
embodiment of image processing system.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0027] 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).
[0028] 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).
[0029] 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).
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.
[0030] 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.
[0031] 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.
[0032] 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.
[0033] 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.
[0034] 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.
[0035] 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.
[0036] 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.
[0037] 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.
[0038] 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.
[0039] 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.
[0040] 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).
[0041] 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.
[0042] 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 microchannel
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.
[0043] 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.
[0044] 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).
[0045] 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.
[0046] 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.
[0047] 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.
[0048] 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.
[0049] 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.TM. 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.
[0050] 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.
[0051] 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.
[0052] 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.
[0053] As illustrated in FIG. 4, steps 418-420 may occur
simultaneously with steps 408-410.
[0054] 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.
[0055] Additionally, while the process described above and
illustrated in FIG. 4 is 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.
[0056] For the claims below the words "a" and "an" should be
construed as "one or more."
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