U.S. patent application number 14/480512 was filed with the patent office on 2015-06-04 for systems and methods for fluorescence detection with a movable detection module.
This patent application is currently assigned to Bio-Rad Laboratories. The applicant listed for this patent is Bio-Rad Laboratories. Invention is credited to Michael J. Finney, Jeffrey A. Goldman, Igor Kordunsky.
Application Number | 20150152475 14/480512 |
Document ID | / |
Family ID | 33416498 |
Filed Date | 2015-06-04 |
United States Patent
Application |
20150152475 |
Kind Code |
A1 |
Kordunsky; Igor ; et
al. |
June 4, 2015 |
SYSTEMS AND METHODS FOR FLUORESCENCE DETECTION WITH A MOVABLE
DETECTION MODULE
Abstract
A fluorescence detection apparatus for analyzing samples located
in a plurality of wells in a thermal cycler and methods of use are
provided. In one embodiment, the apparatus includes a support
structure attachable to the thermal cycler and a detection module
movably mountable on the support structure. The detection module
includes one or more channels, each having an excitation light
generator and an emission light detector both disposed within the
detection module. When the support structure is attached to the
thermal cycler and the detection module is mounted on the support
structure, the detection module is movable so as to be positioned
in optical communication with different ones of the plurality of
wells. The detection module is removable from the support structure
to allow easy replacement.
Inventors: |
Kordunsky; Igor; (Newton,
MA) ; Goldman; Jeffrey A.; (Acton, MA) ;
Finney; Michael J.; (San Francisco, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Bio-Rad Laboratories |
Hercules |
CA |
US |
|
|
Assignee: |
Bio-Rad Laboratories
Hercules
CA
|
Family ID: |
33416498 |
Appl. No.: |
14/480512 |
Filed: |
September 8, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13542587 |
Jul 5, 2012 |
8835118 |
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14480512 |
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|
12827521 |
Jun 30, 2010 |
8236504 |
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13542587 |
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|
11555642 |
Nov 1, 2006 |
7749736 |
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12827521 |
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10431708 |
May 8, 2003 |
7148043 |
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11555642 |
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Current U.S.
Class: |
435/287.2 |
Current CPC
Class: |
B01L 7/52 20130101; B01L
2300/0654 20130101; G01N 2201/0446 20130101; B01L 2300/1822
20130101; G01N 21/276 20130101; B01L 2300/0829 20130101; G01N
21/6452 20130101; C12Q 1/686 20130101 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; B01L 7/00 20060101 B01L007/00; G01N 21/64 20060101
G01N021/64 |
Claims
1. (canceled)
2. A fluorescence detection module comprising: a detection module
housing having a plurality of openings at a first end; a mechanical
connector disposed on an outside surface of the detection module
housing at a second end opposite the first end, the mechanical
connector being configured to detachably attach the detection
module to a movable shuttle mounted inside a thermal cycler in an
orientation such that, by moving the movable shuttle, the openings
at the first end of the detection module housing are positionable
in optical communication with a plurality of sample wells inside
the thermal cycler; and a plurality of excitation/detection pairs
disposed within the detection module housing, each
excitation/detection pair being separated from each other
excitation detection pair by opaque walls, each
excitation/detection pair including: an excitation light generator
fixedly mounted inside the opaque walls; an emission light detector
fixedly mounted inside the opaque walls; and one or more optical
components fixedly mounted inside the opaque walls and arranged to
direct light from the excitation light generator toward a
respective one of the openings at the first end of the detection
module housing and to direct light from the respective one of the
openings toward the emission light detector.
3. The fluorescence detection module of claim 2 wherein each
excitation light generator comprises a light-emitting diode
(LED).
4. The fluorescence detection module of claim 2 wherein each
excitation light generator is configured to generate light at a
different range of wavelengths.
5. The fluorescence detection module of claim 2 wherein each
emission light detector is configured to detect light at a
different range of wavelengths.
6. The fluorescence detection module of claim 2 wherein the one or
more optical components of each excitation/detection pair include a
beam splitter oriented such that light from the excitation light
generator of the excitation/detection pair is directed toward the
respective one of the openings and light entering through the
respective one of the openings is directed toward the emission
light detector of the excitation/detection pair.
7. The fluorescence detection module of claim 6 wherein the beam
splitter is highly transparent to light of an excitation wavelength
generated by the excitation light generator and highly reflective
to light of a detection wavelength.
8. The fluorescence detection module of claim 6 wherein the one or
more optical components of each excitation/detection pair further
include a lens to focus light from the excitation light generator
onto the beam splitter.
9. The fluorescence detection module of claim 6 wherein the one or
more optical components of each excitation/detection pair further
include a lens to focus light from the beam splitter onto the
emission light detector.
10. The fluorescence detection module of claim 2 wherein the
plurality of openings are arranged such that each
excitation/detection pair is simultaneously positionable in optical
communication with a different one of the sample wells.
11. The fluorescence detection module of claim 2 wherein the
plurality of openings are arranged such that when a first one of
the excitation/detection pairs is positioned in optical
communication with one of the sample wells, a different one of the
excitation/detection pairs is not in optical communication with any
of the sample wells.
12. The fluorescence detection module of claim 2 wherein the
mechanical connector includes a receptacle for a ball plunger.
13. The fluorescence detection module of claim 2 further
comprising: an electrical connector disposed on the detection
module at the second end.
14. The fluorescence detection module of claim 13 wherein the
electrical connector includes electrical signal paths to pass
control signals to activate and deactivate the excitation light
generators and to pass data signals from the emission light
detectors.
15. A method of calibrating a movable detection module in a
fluorescence detection apparatus, the method comprising: providing
a lid heater having a plurality of openings to permit optical
communication with a plurality of sample wells, the lid heater
having a plurality of calibration locations arranged between the
openings, wherein each calibration location includes a material
that provides a known fluorescence response; attaching a detection
module to a movable shuttle inside a lid of the fluorescence
detection apparatus, wherein the detection module includes a
plurality of excitation/detection pairs, each excitation/detection
pair being separated from each other excitation detection pair by
opaque walls, each excitation/detection pair including an
excitation light generator, an emission light detector, and one or
more optical components arranged to direct light from the
excitation light generator toward a respective one of a plurality
of openings at a first end of the detection module housing and to
direct light from the respective one of the openings toward the
emission light detector; with the lid closed, operating the movable
shuttle to position the detection module such that a first one of
the excitation/detection pairs is in optical communication with a
first one of the calibration locations; obtaining a first
calibration measurement for the first one of the
excitation/detection pairs; operating the movable shuttle to
reposition the detection module such that a second one of the
excitation/detection pairs is in optical communication with a
second one of the calibration locations; obtaining a second
calibration measurement for the second one of the
excitation/detection pairs; operating the movable shuttle to
reposition the detection module such that the first one of the
excitation/detection pairs is in optical communication with a first
one of the sample wells; and interrogating the first one of the
sample wells using the first one of the excitation/detection
pairs.
16. The method of claim 15 wherein interrogating the first one of
the sample wells using the first one of the excitation/detection
pairs includes briefly flashing the excitation light source of the
first one of the excitation/detection pairs and reading a signal
from the emission light detector of the first one of the
excitation/detection pairs.
17. The method of claim 15 wherein, when the first one of the
excitation/detection pairs is in optical communication with the
first one of the sample wells, the second one of the
excitation/detection pairs is in optical communication with a
second one of the sample wells, the method further comprising:
while the first one of the excitation/detection pairs is in optical
communication with a first one of the sample wells, interrogating
the second one of the sample wells using the second one of the
excitation/detection pairs.
18. The method of claim 17 wherein interrogating the first one of
the sample wells using the first one of the excitation/detection
pairs and interrogating the second one of the sample wells using
the second one of the excitation/detection pairs are performed
sequentially.
19. The method of claim 17 wherein interrogating the first one of
the sample wells using the first one of the excitation/detection
pairs and interrogating the second one of the sample wells using
the second one of the excitation/detection pairs are performed
concurrently.
20. The method of claim 15 wherein, when the first one of the
excitation/detection pairs is in optical communication with the
first one of the sample wells, the second one of the
excitation/detection pairs is not in optical communication with any
of the sample wells, the method further comprising: subsequently to
interrogating the first one of the sample wells, operating the
movable shuttle to reposition the detection module such that a
second one of the excitation/detection pairs is in optical
communication with a second one of the sample wells; and
interrogating the second one of the sample wells using the second
one of the excitation/detection pairs.
21. The method of claim 15 further comprising: subsequently to
interrogating the first one of the sample wells using the first one
of the excitation/detection pairs, operating the movable shuttle to
reposition the detection module such that a second one of the
excitation/detection pairs is in optical communication with the
first one of the sample wells; and interrogating the first one of
the sample wells using the second one of the excitation/detection
pairs.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a continuation of application Ser. No.
13/542,587, filed Jul. 5, 2012, entitled "Systems and Methods For
Fluorescence Detection With A Movable Detection Module," which is a
continuation of application Ser. No. 12/827,521, filed Jun. 30,
2010, now U.S. Pat. No. 8,236,504, which is a continuation of
application Ser. No. 11/555,642, filed Nov. 1, 2006, now U.S. Pat.
No. 7,749,736, which is a continuation of application Ser. No.
10/431,708, filed May 8, 2003, now U.S. Pat. No. 7,148,043. The
respective disclosures of all applications are incorporated herein
by reference.
BACKGROUND OF THE INVENTION
[0002] The present invention relates in general to fluorescence
detection systems and in particular to a fluorescence detection
system having a movable excitation/detection module for use with a
thermal cycler.
[0003] Thermal cyclers are known in the art. Such devices are used
in a variety of processes for creation and detection of various
molecules of interest, e.g., nucleic acid sequences, in research,
medical, and industrial fields. Processes that can be performed
with conventional thermal cyclers include but are not limited to
amplification of nucleic acids using procedures such as the
polymerase chain reaction (PCR). Such amplification processes are
used to increase the amount of a target sequence present in a
nucleic acid sample.
[0004] Numerous techniques for detecting the presence and/or
concentration of a target molecule in a sample processed by a
thermal cycler are also known. For instance, fluorescent labeling
may be used. A fluorescent label (or fluorescent probe) is
generally a substance which, when stimulated by an appropriate
electromagnetic signal or radiation, absorbs the radiation and
emits a signal (usually radiation that is distinguishable, e.g., by
wavelength, from the stimulating radiation) that persists while the
stimulating radiation is continued, i.e. it fluoresces. Some types
of fluorescent probes are generally designed to be active only in
the presence of a target molecule (e.g., a specific nucleic acid
sequence), so that a fluorescent response from a sample signifies
the presence of the target molecule. Other types of fluorescent
probes increase their fluorescence in proportion to the quantity of
double-stranded DNA present in the reaction. These types of probes
are typically used where the amplification reaction is designed to
operate only on the target molecule.
[0005] Fluorometry involves exposing a sample containing the
fluorescent label or probe to stimulating (also called excitation)
radiation, such as a light source of appropriate wavelength,
thereby exciting the probe and causing fluorescence. The emitted
radiation is detected using an appropriate detector, such as a
photodiode, photomultiplier, charge-coupled device (CCD), or the
like.
[0006] Fluorometers for use with fluorescent-labeled samples are
known in the art. One type of fluorometer is an optical reader,
such as described by Andrews et al. in U.S. Pat. No. 6,043,880. A
sample plate containing an array of samples is inserted in the
optical reader, which exposes the samples to excitation light and
detects the emitted radiation. The usefulness of optical readers is
limited by the need to remove the sample plate from the thermal
cycler, making it difficult to monitor the progress of
amplification.
[0007] One improvement integrates the optical reader with a thermal
cycler, so that the sample plate may be analyzed without removing
it from the thermal cycler or interrupting the PCR process.
Examples of such combination devices are described in U.S. Pat. No.
5,928,907, U.S. Pat. No. 6,015,674, U.S. Pat. No. 6,043,880, U.S.
Pat. No. 6,144,448, U.S. Pat. No. 6,337,435, and U.S. Pat. No.
6,369,863. Such combination devices are useful in various
applications, as described, e.g., in U.S. Pat. No. 5,210,015, U.S.
Pat. No. 5,994,056, U.S. Pat. No. 6,140,054, and U.S. Pat. No.
6,174,670.
[0008] Existing fluorometers suffer from various drawbacks. For
instance, in some existing designs, different light sources and
detectors are provided for different sample wells in the array.
Variations among the light sources and/or detectors lead to
variations in the detected fluorescent response from one well to
the next. Alternatively, the light source and/or detector may be
arranged in optical communication with more than one of the wells,
with different optical paths to and/or from each well. Due to the
different optical paths, the detected fluorescent response varies
from one sample well to the next. To compensate for such
variations, the response for each sample well must be individually
calibrated. As the number of sample wells in an array increases,
this becomes an increasingly time-consuming task, and errors in
calibration may introduce significant errors in subsequent
measurements.
[0009] In addition, existing fluorometers generally are designed
such that the light sources and detectors are fixed parts of the
instrument. This limits an experimenter's ability to adapt a
fluorometer to a different application. For instance, detecting a
different fluorescent label generally requires using a different
light source and/or detector. Many existing fluorometers make it
difficult for an experimenter to reconfigure light sources or
detectors, thus limiting the variety of fluorescent labels that may
be used.
[0010] It is also difficult to perform concurrent measurements of a
number of different fluorescent labels that may be present in a
sample (or in different samples). As described above, to maximize
the data obtained in an assay, experimenters often include multiple
fluorescent labeling agents that have different excitation and/or
emission wavelengths. Each labeling agent is adapted to bind to a
different target sequence, in principle allowing multiple target
sequences to be detected in the same sample. Existing fluorometers,
however, do not facilitate such multiple-label experiments. Many
fluorometers are designed for a single combination of excitation
and emission wavelengths. Others provide multiple light sources and
detectors to allow detection of multiple labels; however, these
configurations often allow only one label to be probed at a time
because the excitation wavelength of one label may overlap the
emission wavelength of another label; excitation light entering the
detector would lead to incorrect results. Probing multiple labels
generally cannot be done in parallel, slowing the data collection
process.
[0011] Therefore, an improved fluorometer for a thermal cycler that
overcomes these disadvantages would be desirable.
BRIEF SUMMARY OF THE INVENTION
[0012] Embodiments of the present invention provide fluorescence
detection in a thermal cycling apparatus. According to one aspect
of the invention, a fluorescence detection apparatus for analyzing
samples located in a plurality of wells in a thermal cycler
includes a support structure attachable to the thermal cycler and a
detection module movably mountable on the support structure. The
detection module includes an excitation light generator and an
emission light detector, both disposed within the detection module.
When the support structure is attached to the thermal cycler and
the detection module is mounted on the support structure, the
detection module is movable so as to be positioned in optical
communication with different ones of the plurality of wells.
[0013] According to another aspect of the invention, the detection
module may include two or more excitation light generators and two
or more emission light detectors arranged to form two or more
excitation/detection pairs. In one embodiment, the
excitation/detection pairs are arranged such that each
excitation/detection pair is simultaneously positionable in optical
contact with a different one of the plurality of wells. In an
alternative embodiment, excitation/detection pairs are arranged
such that when a first one of the excitation/detection pairs is
positioned in optical contact with any one of the plurality of
wells, a different one of the excitation/detection pairs is not in
optical contact with any one of the plurality of wells. In some
embodiments, the detection module is detachably mounted on the
support structure, thereby enabling a user to replace the detection
module with a different detection module.
[0014] According to yet another aspect of the invention, a method
for detecting the presence of a target molecule in a solution is
provided. A plurality of samples is prepared, each sample
containing a fluorescent probe adapted to bind to a target
molecule. Each sample is placed in a respective one of a number of
sample wells of a thermal cycler instrument, the thermal cycler
instrument having a detection module movably mounted therein, the
detection module including an excitation/detection channel, the
excitation/detection channel including an excitation light
generator disposed within the detection module and an emission
light detector disposed within the detection module. The thermal
cycler instrument is used to stimulate a reaction, and the sample
wells are scanned to detect a fluorescent response by moving the
detection module and activating the excitation/detection channel.
During the scanning, the detection module is moved such that the
excitation/detection channel is sequentially positioned in optical
communication with each of the plurality of sample wells. Where the
detection module includes multiple excitation/detection pairs or
channels, channels may be active in parallel or sequentially.
[0015] The following detailed description together with the
accompanying drawings will provide a better understanding of the
nature and advantages of the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 is a perspective view of a thermal cycling apparatus
according to an embodiment of the present invention;
[0017] FIG. 2 is an exploded view of a lid assembly for a thermal
cycling apparatus according to an embodiment of the present
invention;
[0018] FIG. 3 is a bottom view of a fluorometer assembly for a
thermal cycling apparatus according to an embodiment of the present
invention;
[0019] FIG. 4 is a top view of detection module according to an
embodiment of the present invention;
[0020] FIGS. 5A-B are bottom views of detection modules according
to alternative embodiments of the present invention;
[0021] FIG. 6 is a schematic diagram of an excitation/detection
pair for a detection module according to an embodiment of the
present invention;
[0022] FIG. 7 is a block diagram illustrating electrical
connections for a lid assembly for a thermal cycling apparatus
according to an embodiment of the present invention; and
[0023] FIG. 8 is a flow diagram of a process for using a thermal
cycler having a fluorescence detection system according to an
embodiment of the present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0024] An exemplary apparatus embodiment of the present invention
will be described with reference to the accompanying drawings, in
which like reference numerals indicate corresponding parts. Methods
of using the apparatus will also be described. It is to be
understood that embodiments shown and described herein are
illustrative and not limiting of the invention.
I. Exemplary Apparatus
[0025] FIG. 1 is a perspective view of a thermal cycling apparatus
100 according to an embodiment of the present invention. Apparatus
100 consists of a base unit 110 and a lid assembly 112. Base unit
110, which may be of conventional design, provides power and
control functions for a thermal cycling process via conventional
electronic components (not shown), such as programmable processors,
clocks, and the like. Base unit 110 also provides a user interface
116 that may include a keypad 118 and an LCD display screen 120,
enabling a user to control and monitor operation of the thermal
cycler. Base unit 110 connects to an external power source (e.g.,
standard 120 V ac power) via a power cable 121. Some examples of
base unit 110 include the DNA Engine.RTM., Dyad.TM., and Tetrad.TM.
thermal cyclers sold by MJ Research, Inc., assignee of the present
application.
[0026] Lid assembly 112 includes a sample unit and a fluorescence
detection apparatus, disposed within a lid 122; these components
will be described below. Lid 122 has a handle 124 to aid in its
placement on and removal from base unit 110, and ventilation holes
126. Lid 122 provides optical and thermal isolation for the
components inside lid assembly 112.
[0027] FIG. 2 is an exploded view of the inside of lid assembly
112. Shown are a sample unit 202, a lid heater 204, and a
fluorometer assembly 206. Sample unit 202 contains a number of
sample wells 210 arranged in a regular array (e.g., an 8.times.12
grid). In one embodiment, each sample well 210 holds a removable
reaction vessel (not shown), such as a tube, that contains a
nucleic acid sample to be tested, together with appropriate PCR
reactants (buffers, primers and probes, nucleotides, and the like)
including at least one fluorescent label or probe adapted to bind
to or otherwise respond to the presence of a target nucleic acid
sequence. The reaction vessels are advantageously provided with
transparent sample caps (not shown) that fit securely over the tops
of the vessels to prevent cross-contamination of samples or
spillage during handling. Reaction vessels may also be sealed in
other ways, including the use of films such as Microseal.RTM.B
(made by MJ Research, Inc.), wax products such as Chill-out.TM.
(made by MJ Research, Inc.), or mineral oil. In an alternative
configuration, a removable sample tray (not shown) that holds one
or more distinct samples at locations corresponding to sample wells
210 is used. The sample tray may also be sealed in any of the ways
described above.
[0028] Sample unit 202 also includes heating elements (e.g.,
Peltier-effect thermoelectric devices), heat exchange elements,
electrical connection elements for connecting the heating elements
to base unit 110, and mechanical connection elements. These
components (not shown) may be of conventional design. Sample unit
202 also provides electrical connections for lid heater 204 and
fluorometer assembly 206 via multiwire cables 212, which are
detachably connected to connectors 214.
[0029] Lid heater 204 has holes 220 therethrough, matching the size
and spacing of the sample wells 210, and electronically controlled
heating elements (not shown). Lid heater 204 is coupled to lid 122.
The coupling mechanism (not shown) is advantageously movable (e.g.,
lid heater 204 may be attached to lid 122 by a hinge) in order to
provide access to fluorometer assembly 206 when lid 122 is removed
from sample unit 202. When lid 122 is in place on sample unit 202,
supports 224 hold lid heater 204 in position. Lower portions 226 of
supports 224 are advantageously designed to compress lid heater 204
toward sample unit 202, thereby reducing the possibility of sample
evaporation during operation of apparatus 100. This compression
also allows reaction vessels of different sizes to be used. Lid
heater 204 is used to control the temperature of the sample caps
(or other sealants) of reaction vessels sample wells 210, in order
to prevent condensation from forming on the caps during thermal
cycling operation.
[0030] Lid heater 204 advantageously includes one or more
calibration elements 222 positioned between selected ones of holes
220 or in other locations away from the holes, such as near the
periphery of lid heater 204. Calibration elements 222 provide a
known fluorescence response and may be used to calibrate
fluorescence detectors in fluorometer assembly 206. Calibration
elements 222 may be made, e.g., of a fluorescent coating on a glass
or plastic substrate, or they may consist of a plastic with a dye
impregnated in it, fluorescent glass, or a fluorescent plastic such
as polyetherimide (PEI). Neutral-density or other types of filters
may be placed over the fluorescent material in order to avoid
saturating the fluorescence detectors. In general, any material may
be used, provided that its fluorescence characteristics are
sufficiently stable over time with the application of light
(photo-bleaching) and heat. To the extent practical, the effect of
temperature on the fluorescence response is advantageously
minimized. Where multiple calibration elements 222 are provided,
different materials may be used for different ones of the
calibration elements. In an alternative embodiment, lid heater 204
may be omitted, and calibration elements 222 may be disposed on the
surface of sample unit 202.
[0031] Sample unit 202 and lid heater 204 may be of conventional
design. Examples of suitable designs include sample unit and lid
heater components of the various Alpha.TM. modules sold by MJ
Research, Inc., assignee of the present application.
[0032] Fluorometer assembly 206 includes a support frame or
platform 230 fixedly mounted inside lid 122. Movably mounted on the
underside of support frame 230 is a shuttle 232, which holds a
detection module 234. Shuttle 232 is movable in two dimensions so
as to position detection module 234 in optical communication with
different ones of the sample wells 210 in sample unit 202 through
the corresponding holes 220 in lid heater 204. Support frame 230
and supports 224 are advantageously dimensioned such that when lid
122 is positioned in base unit 110 and closed, detection module 234
is held in close proximity to lid heater 204; one of skill in the
art will appreciate that this arrangement reduces light loss
between the sample wells and the detection module.
[0033] FIG. 3 is a bottom view of fluorometer assembly 206, showing
a movable mounting of shuttle 232 and detection module 234. In this
embodiment, translation stages driven by stepper motors are used to
move the shuttle 232, to which detection module 234 is detachably
coupled, to a desired position. Specifically, support platform 230
has an x-axis stepper motor 302 and a lead screw 304 attached
thereto. Stepper motor 302 operates to turn lead screw 304, thereby
moving a translation stage 306 along the x direction (indicated by
arrow). Limit switches 308 are advantageously provided to restrict
the motion of translation stage 306 to an appropriate range, large
enough to allow detection module 234 to be placed in optical
contact with any of the wells while preventing translation stage
306 from contacting other system components, such as stepper motor
302.
[0034] Translation stage 306 has a y-axis stepper motor 316 and a
lead screw 318 mounted thereon. Stepper motor 316 operates to turn
lead screw 318, thereby moving shuttle 232 along the y direction
(indicated by arrow). Limit switches 320 are advantageously
provided to restrict the motion of shuttle 232 to an appropriate
range, large enough to allow detection module 234 to be placed in
optical contact with any of the wells, while preventing shuttle 232
from contacting other system components, such as stepper motor
316.
[0035] Stepper motors 302, 316, lead screws 304, 318, and limit
switches 308, 320 may be of generally conventional design. It will
be appreciated that other movable mountings may be substituted. For
example, instead of directly coupling the motors to the lead
screws, indirect couplings such as chain drives or belt drives may
be used. Chain drives, belt drives, or other drive mechanisms may
also be used to position the detection module without lead screws,
e.g., by attaching a translation stage to the chain, belt, or other
drive mechanism. Other types of motors, such as servo motors or
linear motors, may also be used. Different drive mechanisms may be
used for different degrees of freedom.
[0036] Shuttle 232 holds detection module 234 via connectors 330,
331. Connectors 330, 331 which may vary in design, are configured
to support and align detection module 234 on the underside of
shuttle 232. The connectors are advantageously adapted to allow
easy insertion and removal of detection module 234, to facilitate
replacement of the detection module. In one embodiment, connectors
330 provide mounting for a cylindrical member (not shown) that
pivotably holds an edge of detection module 234, while connectors
331 include ball plungers mounted on shuttle 232 that are
insertable into corresponding receptacles on detection module 234.
Electrical connections (not shown) between shuttle 232 and
detection module 234 may also be provided, as will be described
below.
[0037] FIG. 4 is a top view of detection module 234. Detection
module 234 includes fittings 420 that couple to corresponding
connectors 330 on the underside of shuttle 232, thereby securing
detection module 234 in place so that it moves as a unit with
shuttle 232. Detection module 234 also includes an electrical
connector 424 that couples to a corresponding electrical connector
on the underside of shuttle 232, thereby allowing control and
readout signals to be provided to and obtained from detection
module 234.
[0038] FIG. 5A is a bottom view of one embodiment of detection
module 234, showing four openings 502, 504, 506, 508 for four
independently controlled fluorescent excitation/detection channels
(also referred to as "excitation/detection pairs") arranged inside
the body of detection module 234. Examples of excitation/detection
channels will be described below. The spacing of openings 502, 504,
506, 508 corresponds to the spacing of sample wells 210. Thus, when
opening 502 is placed in optical communication with one of the
sample wells 210, openings 504, 506, and 508 are each in optical
communication with a different one of the sample wells 210.
Openings 502, 504, 506, 508 may simply be holes through the bottom
surface of detection module 234, or they may be made of any
substance that has a high degree of transparency to the excitation
and detection light wavelengths of their respective channels.
[0039] FIG. 5B is a bottom view of a detection module 234'
according to an alternative embodiment of the invention. In this
embodiment, four openings 512, 514, 516, 518 are provided, but they
are arranged in a staggered fashion so that only one opening at a
time may be in optical communication with any of the sample wells.
This configuration is useful for reducing cross-talk between the
excitation/detection pairs.
[0040] FIG. 6 is a schematic diagram illustrating a configuration
of optical elements for an excitation/detection channel (or
excitation/detection pair) 600 according to an embodiment of the
invention. Detection module 234 may include one or more instances
of excitation/detection pair 600, each of which provides an
independent fluorescence detection channel. Excitation/detection
pair 600 is arranged inside opaque walls 602, which provide optical
isolation from other excitation/detection pairs that may be
included in detection module 234, as well as from external light
sources. An excitation light path 604 includes a light-emitting
diode (LED) or other light source 606, a filter 608, a lens 610,
and a beam splitter 612. A detection light path 620 includes beam
splitter 612, a filter 624, a lens 626, and a photodiode or other
photodetector 628. Beam splitter 612 is advantageously selected to
be highly transparent to light of the excitation wavelength and
highly reflective of light at the detection (fluorescent response)
wavelength.
[0041] The components of excitation light path 604 are arranged to
direct excitation light of a desired wavelength into a reaction
vessel 616 held in a sample well 210 of sample block 202. The
desired wavelength depends on the particular fluorescent labeling
agents included in reaction vessel 616 and is controlled by
selection of an appropriate LED 606 and filter 608. Optical
communication between the excitation/detection pair 600 and
reaction vessel 616 is provided by opening 502 in opaque walls 602
and a hole 220 through lid heater 204, as described above. To
maximize light transmission to and from excitation/detection pair
600, the space between opening 502 and lid heater 204 is
advantageously made small during operation.
[0042] Excitation light that enters reaction vessel 616 excites the
fluorescent label or probe therein, which fluoresces, thereby
generating light of a different wavelength. Some of this light
exits reaction vessel 616 on detection light path 620 and passes
through opening 502. Beam splitter 612 directs a substantial
portion of the fluorescent light through filter 624, which filters
out the excitation frequency, and lens 626, which focuses the light
onto the active surface of photodiode 628. Photodiode 628 generates
an electrical signal in response to the incident light. This
electrical signal is transmitted by a readout signal path 630 to
circuit board 634, which routes the signal to electrical connector
424 for readout. Circuit board 634 and/or signal path 630 may also
include other components, such as pre-amplifiers, for shaping and
refining the electrical signal from photodiode 628.
[0043] LED 606 and photodiode 628 may be controlled by signals
received via connector 424, as indicated by respective control
signal paths 636, 638. Control signals for LED 606 may operate to
activate and deactivate LED 606 at desired times; control signals
for photodiode 628 may operate to activate and deactivate
photodiode 628 at desired times, adjust a gain parameter, and so
on.
[0044] While FIG. 6 shows one excitation/detection pair 600, it is
to be understood that an embodiment of detection module 234 may
contain any number of such pairs, each of which is advantageously
in optical isolation from the others and has its own opening for
optical communication with the sample wells (e.g., openings 504,
506, 508 of FIG. 5). The various excitation/detection pairs are
independently controlled and independently read out, but their
respective control and readout paths may all be coupled to circuit
board 634.
[0045] The configuration of excitation/detection pairs may be
varied from that shown, and the excitation and detection light
paths may include additional components, fewer components, or any
combination of desired components. The optics may be modified as
appropriate for a particular application (e.g., the optical path
may be shorter in embodiments where lid heater 204 is not included)
and use any number and combination of components including but not
limited to lenses, beam splitters, mirrors, and filters. While LEDs
provide a compact and reliable light source, use of other types of
coherent or incoherent light sources, such as laser diodes, flash
lamps, and so on, is not precluded. Similarly, the detectors are
not limited to photodiodes; any type of photodetector may be
substituted, including photomultipliers and charge-coupled devices
(CCDs). Each excitation/detection pair is advantageously configured
as a self-contained assembly, requiring only external electrical
connections to make it operational. Because the length of the
excitation and detection optical paths do not vary from one
experiment to the next, it is desirable to fixedly mount and
optimize the various optical components of each
excitation/detection pair 600 inside detection module 234 during
manufacture so that further adjustments during operation are not
required.
[0046] FIG. 7 is a block diagram illustrating electrical
connections for lid assembly 112. A main processing board 702 is
mounted in lid assembly 112. Main processing board 702 includes a
primary signal processor 704, a stepper motor driver unit 706, a
connection 708 for electrical power, and a connection 710 for an
external computer (e.g., a personal computer, or PC). Main
processing board 702 also provides connectors 214 for cables 212
that provide transmission of electrical signals to and from lid
122.
[0047] Lid 122 includes a secondary processing board 720 that
facilitates communication between main processing board 702 and
stepper motors 302, 316, as well as shuttle 232. Secondary
processing board 720 includes connectors 722 for cables 212, a
connector 724 that connects a cable 726 to shuttle 232, and
connectors 732 and 734 for cables 736, 738 that provide control
signals to the x and y stepper motors 302, 316. Routing paths (not
shown) in secondary processing board 720 establish appropriate
signal connections between the various connectors.
[0048] Cable 726 is used to communicate control signals for
detection module 234, such as activating and deactivating
individual light sources, and to receive signals from the
photodetectors included in detection module 234. Electrical
connector 730 is provided on shuttle 232 for passing signals to and
from detection module 234. Electrical connector 730 accepts the
mating connector 424 on the top surface of detection module 234
when detection module 234 is mounted on shuttle 232. In an
alternative embodiment, cable 726 may attach directly to detection
module 234.
[0049] As mentioned above, main processing board 702 provides a
connection 710 to an external computer (not shown). The external
computer may be used to control the motion of shuttle 232 and the
operation of detection module 234, as well as for readout and
analysis of fluorometry data obtained from detection module
234.
[0050] As described above, detection module 234 is designed to be
self-contained and detachable from shuttle 232. This allows for a
reconfigurable fluorometry system, in which an experimenter is able
to change detection modules as desired to perform different
measurements. For instance, different detection modules may be
optimized for different fluorescent labeling agents (or
combinations of agents). If the experimenter wishes to study a
different agent, she simply installs the appropriate detection
module. Installation is a matter of attaching electrical connector
424 and mechanical connectors 420 on the top of the desired
detection module 234 to corresponding connectors on the underside
of shuttle 234. In some embodiments, the connectors are designed
such that the electrical connection is made automatically as the
mechanical connection is engaged. As noted above, lid heater 204 is
advantageously movably mounted so as to allow access to
fluorescence assembly 230, thereby allowing experimenters to change
detection modules.
[0051] It will be appreciated that the apparatus described herein
is illustrative and that variations and modifications are possible.
For instance, the base and sample unit may be designed as an
integrated system or separated further into smaller modular
components. The fluorometer assembly need not be attached or
otherwise integrated into the lid, so long as it is mountable in a
fixed position relative to the sample wells. Any mechanism may be
used to make the detection module movable so as to position it in
optical communication with different ones of the sample wells, not
limited to translation stages or stepper motors. The detection
module may include any number (one or more) of excitation/detection
pairs operable as independent detection channels, and different
pairs may be designed to detect the same fluorescent probe or
different fluorescent probes. In one alternative embodiment, the
detection module includes a row of excitation/detection pairs with
optical windows arranged to correspond to a row of the sample
array, and the detection module is made movable in one direction to
interrogate different columns of the array.
[0052] The external computer is also optional, and any of its
functions may be integrated into the thermal cycler device;
conversely, control functions for the thermal cycler may be
implemented to operate on the external computer, thereby providing
a single control device for the entire apparatus. In one
embodiment, the external computer is used to control the position
of detection module 234 with respect to the sample wells and
operations of the light source(s) and detector(s). In addition, any
external computer may be special purpose control and
signal-processing devices as well as a general-purpose computer
such as a PC.
II. Methods of Use
[0053] The apparatus described herein can be used to detect the
amount of amplification product generated in an amplification
reaction by detecting the amount of fluorescence. Various
amplification techniques can be used to quantify target sequences
present in DNA or RNA samples. Such techniques, which involve
enzymatic synthesis of nucleic acid amplicons (copies) that contain
a sequence that is complementary to the sequence being amplified,
are well known in the art and widely used. These include, but are
not limited to the polymerase chain reaction (PCR), RT-PCR, strand
displacement amplification (SDA), transcription based amplification
reactions, ligase chain reaction (LCR), and others (see, e.g.
Dieffenfach & Dveksler, PCR Primer: A Laboratory Manual, 1995;
U.S. Pat. Nos. 4,683,195 and 4,683,202; PCR Protocols: A Guide to
Methods and Applications, Innis et al., eds, 1990; Walker, et al.,
Nucleic Acids Res. 20(7):1691-6, 1992; Walker, PCR Methods Appl
3(1):1-6, 1993; Phyffer, et al., J. Clin. Microbiol. 34:834-841,
1996; Vuorinen, et al., J. Clin. Microbiol. 33:1856-1859, 1995;
Compton, Nature 350(6313):91-2, 1991; Lisby, Mol. Biotechnol.
12(1):75-991999; Hatch et al., Genet. Anal. 15(2):35-40, 1999; and
Iqbal et al., Mol. Cell Probes 13(4):315-320; 1999). Nucleic acid
amplification is especially beneficial when the amount of target
sequence present in a sample is very low. By amplifying the target
sequence and detecting the amplicon synthesized, the sensitivity of
an assay may be vastly improved, since fewer copies of the target
sequence are needed at the beginning of the assay to better ensure
detection of nucleic acid in the sample belonging to the organism
or virus of interest.
[0054] Measurement of amplification products can be performed after
the reaction has been completed or in real time (i.e.,
substantially continuously). If measurement of accumulated
amplified product is performed after amplification is complete,
then detection reagents (e.g., fluorescent probes) can be added
after the amplification reaction. Alternatively, probes can be
added to the reaction prior to or during the amplification
reaction, thus allowing for measurement of the amplified products
either after completion of amplification or in real time. If
amplified products are measured in real time, initial copy number
can be estimated by determining the cycle number at which the
signal crosses a threshold and projecting back to initial copy
number, assuming exponential amplification.
A. Fluorescent Probes
[0055] A number of formats are available that make use of
fluorescent probes. These formats are often based on fluorescence
resonance energy transfer (FRET) and include molecular beacon, and
TaqMan.RTM. probes. FRET is a distance-dependent interaction
between a donor and acceptor molecule. The donor and acceptor
molecules are fluorophores. If the fluorophores have excitation and
emission spectra that overlap, then in close proximity (typically
around 10-100 angstroms) the excitation of the donor fluorophore is
transferred to the acceptor fluorophore. As a result, the lifetime
of the donor molecule is decreased and its fluorescence is
quenched, while the fluorescence intensity of the acceptor molecule
is enhanced and depolarized. When the excited-state energy of the
donor is transferred to a non-fluorophore acceptor, the
fluorescence of the donor is quenched without subsequent emission
of fluorescence by the acceptor. In this case, the acceptor
functions as a quenching reagent.
[0056] One FRET-based format for real-time PCR uses DNA probes
known as "molecular beacons" (see, e.g., Tyagi et al., Nat.
Biotech. 16:49-53, 1998; U.S. Pat. No. 5,925,517). Molecular
beacons have a hairpin structure wherein the quencher dye and
reporter dye are in intimate contact with each other at the end of
the stem of the hairpin. Upon hybridization with a complementary
sequence, the loop of the hairpin structure becomes double stranded
and forces the quencher and reporter dye apart, thus generating a
fluorescent signal. A related detection method uses hairpin primers
as the fluorogenic probe (Nazarenko et al., Nucl. Acid Res.
25:2516-2521, 1997; U.S. Pat. No. 5,866,336; U.S. Pat. No.
5,958,700). The PCR primers can be designed in such a manner that
only when the primer adopts a linear structure, i.e., is
incorporated into a PCR product, is a fluorescent signal
generated.
[0057] Amplification products can also be detected in solution
using a fluorogenic 5' nuclease assay, a TaqMan assay. See Holland
et al., Proc. Natl. Acad. Sci. U.S.A. 88: 7276-7280, 1991; U.S.
Pat. Nos. 5,538,848, 5,723,591, and 5,876,930. The TaqMan probe is
designed to hybridize to a sequence within the desired PCR product.
The 5' end of the TaqMan probe contains a fluorescent reporter dye.
The 3' end of the probe is blocked to prevent probe extension and
contains a dye that will quench the fluorescence of the 5'
fluorophore. During subsequent amplification, the 5' fluorescent
label is cleaved off if a polymerase with 5' exonuclease activity
is present in the reaction. The excising of the 5' fluorophore
results in an increase in fluorescence which can be detected.
[0058] In addition to the hairpin and 5'-nuclease PCR assay, other
formats have been developed that use the FRET mechanism. For
example, single-stranded signal primers have been modified by
linkage to two dyes to form a donor/acceptor dye pair in such a way
that fluorescence of the first dye is quenched by the second dye.
This signal primer contains a restriction site (U.S. Pat. No.
5,846,726) that allows the appropriate restriction enzyme to nick
the primer when hybridized to a target. This cleavage separates the
two dyes and a change in fluorescence is observed due to a decrease
in quenching. Non-nucleotide linking reagents to couple
oligonucleotides to ligands have also been described (U.S. Pat. No.
5,696,251).
[0059] Other amplification reactions that can be monitored using a
fluorescent reading include those that are quantified by measuring
the amount of DNA-binding dye bound to the amplification product.
Such assays use fluorescent dyes, e.g., ethidium bromide or SYBR
Green I (Molecular Probes, Inc., Eugene, Oreg.; U.S. Pat. Nos.
5,436,134 and 5,658,751) that exhibit increased fluorescence when
intercalated into DNA (see, e.g., U.S. Pat. Nos. 5,994,056 and
6,171,785). Use of SYBR Green I for this purpose is also described
in Morrison et al. (Biotechniques 24, 954-962, 1998). An increase
in fluorescence reflects an increase in the amount of
double-stranded DNA generated by the amplification reaction.
[0060] Other fluorescent probes include inorganic molecules,
multi-molecular mixtures of organic and/or inorganic molecules,
crystals, heteropolymers, and the like. For example, CdSe--CdS
core-shell nanocrystals enclosed in a silica shell may be easily
derivatized for coupling to a biological molecule (Bruchez et al.
(1998) Science, 281: 2013-2016). Similarly, highly fluorescent
quantum dots (zinc sulfide-capped cadmium selenide) have been
covalently coupled to biomolecules for use in ultrasensitive
biological detection (Warren and Nie (1998) Science, 281:
2016-2018).
[0061] Multiplex assays may also be performed using apparatus 100.
Multiplex PCR results in the amplification of multiple
polynucleotide fragments in the same reaction. See, e.g., PCR
PRIMER, A LABORATORY MANUAL (Dieffenbach, ed. 1995) Cold Spring
Harbor Press, pages 157-171. For instance, different target
templates can be added and amplified in parallel in the same
reaction vessel. Multiplex assays typically involve the use of
different fluorescent labels to detect the different target
sequences that are amplified.
B. PCR Conditions and Components
[0062] Exemplary PCR reaction conditions typically comprise either
two or three step cycles. Two step cycles have a denaturation step
followed by a hybridization/elongation step. Three step cycles
comprise a denaturation step followed by a hybridization step
followed by a separate elongation step. The polymerase reactions
are incubated under conditions in which the primers hybridize to
the target sequences and are extended by a polymerase. The
amplification reaction cycle conditions are selected so that the
primers hybridize specifically to the target sequence and are
extended.
[0063] Successful PCR amplification requires high yield, high
selectivity, and a controlled reaction rate at each step. Yield,
selectivity, and reaction rate generally depend on the temperature,
and optimal temperatures depend on the composition and length of
the polynucleotide, enzymes and other components in the reaction
system. In addition, different temperatures may be optimal for
different steps. Optimal reaction conditions may vary, depending on
the target sequence and the composition of the primer. Thermal
cyclers such as apparatus 100 provide the necessary control of
reaction conditions to optimize the PCR process for a particular
assay. For instance, apparatus 100 may be programmed by selecting
temperatures to be maintained, time durations for each cycle,
number of cycles, and the like. In some embodiments, temperature
gradients may be programmed so that different sample wells may be
maintained at different temperatures, and so on.
[0064] Fluorescent oligonucleotides (primers or probes) containing
base-linked or terminally-linked fluors and quenchers are
well-known in the art. They can be obtained, for example, from Life
Technologies (Gaithersburg, Md.), Sigma-Genosys (The Woodlands,
Tex.), Genset Corp. (La Jolla, Calif.), or Synthetic Genetics (San
Diego, Calif.). Base-linked fluors are incorporated into the
oligonucleotides by post-synthesis modification of oligonucleotides
that are synthesized with reactive groups linked to bases. One of
skill in the art will recognize that a large number of different
fluorophores are available, including from commercial sources such
as Molecular Probes, Eugene, Oreg. and other fluorophores are known
to those of skill in the art. Useful fluorophores include:
fluorescein, fluorescein isothiocyanate (FITC), carboxy tetrachloro
fluorescein (TET), NHS-fluorescein, 5 and/or 6-carboxy fluorescein
(FAM), 5- (or 6-) iodoacetamidofluorescein, 5-{[2(and
3)-5-(Acetylmercapto)-succinyl]amino} fluorescein
(SAMSA-fluorescein), and other fluorescein derivatives, rhodamine,
Lissamine rhodamine B sulfonyl chloride, Texas red sulfonyl
chloride, 5 and/or 6 carboxy rhodamine (ROX) and other rhodamine
derivatives, coumarin, 7-amino-methyl-coumarin,
7-Amino-4-methylcoumarin-3-acetic acid (AMCA), and other coumarin
derivatives, BODIPY.TM. fluorophores, Cascade Blue.TM. fluorophores
such as 8-methoxypyrene-1,3,6-trisulfonic acid trisodium salt,
Lucifer yellow fluorophores such as
3,6-Disulfonate-4-amino-naphthalimide, phycobiliproteins
derivatives, Alexa fluor dyes (available from Molecular Probes,
Eugene, Oreg.) and other fluorophores known to those of skill in
the art. For a general listing of useful fluorophores, see also
Hermanson, G. T., BIOCONJUGATE TECHNIQUES (Academic Press, San
Diego, 1996).
[0065] The primers for the amplification reactions are designed
according to known algorithms. For example, algorithms implemented
in commercially available or custom software can be used to design
primers for amplifying the target sequences. Typically, the primers
are at least 12 bases, more often 15, 18, or 20 bases in length.
Primers are typically designed so that all primers participating in
a particular reaction have melting temperatures that are within
5.degree. C., and most preferably within 2.degree. C. of each
other. Primers are further designed to avoid priming on themselves
or each other. Primer concentration should be sufficient to bind to
the amount of target sequences that are amplified so as to provide
an accurate assessment of the quantity of amplified sequence. Those
of skill in the art will recognize that the amount of concentration
of primer will vary according to the binding affinity of the
primers as well as the quantity of sequence to be bound. Typical
primer concentrations will range from 0.01 .mu.M to 0.5 .mu.M.
[0066] One of skill in the art will further recognize that it is
desirable to design buffer conditions to allow for the function of
all reactions of interest. Thus, buffer conditions can be designed
to support the amplification reaction as well as any enzymatic
reactions associated with producing signals from probes. A
particular reaction buffer can be tested for its ability to support
various reactions by testing the reactions both individually and in
combination. The concentration of components of the reaction such
as salt, or magnesium can also affect the ability of primers or
detection probes to anneal to the target nucleic acid. These can be
adjusted in accordance with guidance well known in the art, e.g.,
Innis et al., supra.
C. Exemplary PCR Process
[0067] FIG. 8 is a flow chart of a nucleic acid amplification and
measurement process 800 using apparatus 100. In this example,
apparatus 100 controls a PCR amplification process and detects the
presence of multiple target sequences in the nucleic acid
samples.
[0068] At step 802, reaction vessels 616 are prepared. Preparation
includes placing reaction components into the vessels and sealing
the vessels to prevent spillage or cross-contamination. The
reaction components include buffer, target nucleic acid,
appropriate primers and probes, nucleotides, polymerases, as well
as optional additional components. In one embodiment, four
fluorescent probes are included, each adapted to detect a different
target sequence, and a particular reaction vessel may include any
one or more of the fluorescent probes. Each probe advantageously
responds to light of a different incident wavelength and emits
light of a different wavelength.
[0069] At step 806, a detection module 234 is mounted on shuttle
232. As described above, detection module 234 may include any
number of detection channels (i.e., excitation/detection pairs). In
one embodiment, detection module 234 includes four detection
channels. Each channel is optimized for a different one of the
fluorescent probes included in reaction vessels 616.
[0070] At step 808, reaction vessels 616 are placed into sample
wells 210 of sample unit 202. At step 810, lid assembly 112 is
closed and positioned in base unit 110.
[0071] At step 812 each channel of detection module 234 is
calibrated. Calibration is performed by operating stepper motors
302, 316 to position detection module 234 such that at least one of
its channels is in optical communication with a calibration
location 222. As described above, each calibration location
provides a known fluorescent response. Accordingly, calibration
measurements can be used to correct subsequent sample measurements
for variations or fluctuations in detector response. Numerous
calibration techniques are known in the art. Where detection module
234 has multiple channels, each channel may be independently
calibrated.
[0072] At step 814, a PCR cycle is performed. In general, step 814
involves operation of base unit 110 to regulate the temperature of
sample unit 202, thereby holding the reaction vessels at desired
temperatures for desired lengths of time to complete a two-step or
three-step PCR cycle. Base unit 110 may be controlled via user
interface 116 or by an external computer.
[0073] At step 816, fluorometer assembly 206 scans and interrogates
the reaction vessels 616. The operation of fluorometer assembly 206
is advantageously controlled by an external computer and
synchronized with the operation of base unit 110, so that
measurements are identifiable as corresponding to particular times
in the PCR process.
[0074] More specifically, at step 816a, stepper motors 302, 316 or
other motion devices are operated to position detection module 234
such that each of the four detector channels is in optical
communication with a different one of sample wells 210 via
respective optical windows 502, 504, 506, 508. At step 816b, the
LED or other light source for each channel is activated (flashed on
for a brief period) to stimulate fluorescence. In one embodiment,
the LEDs of different channels are operated in parallel; in an
alternative embodiment, they are operated sequentially so as to
avoid reflected LED light from one channel causing false signals in
the photodetector of another channel.
[0075] At step 816c, resulting fluorescence is detected by the
corresponding photodiode or other detector of the channel, which is
read out to the external computer. The detectors may be read out in
various ways. For instance, a peak signal may be detected, the
signal may be integrated over a time interval, or the decay of the
fluorescent signal after the LED has been deactivated may be
measured.
[0076] Steps 816a-c are advantageously repeated, with the position
of the detection module being changed each time so that each
channel of detection module 234 eventually interrogates each of the
sample wells 210. In one embodiment, scanning and interrogating
four channels for each of 96 sample wells takes about 15 seconds.
The external computer advantageously executes a program (e.g., the
Opticon Monitor program sold by MJ Research, Inc.) that enables a
user to view measurement data as they are collected, in graphical
and/or tabular form. Such programs are well known in the art. An
example includes the Opticon Monitor.TM. program sold by MJ
Research, Inc.
[0077] Steps 814 and 816 may be repeated for any number of reaction
cycles. Persons of ordinary skill in the art will recognize that
real-time fluorescence measurements from process 800 may be used to
detect and quantify the presence of each target sequence. Such
measurements may also be used for purposes such as determining
reaction rates and adjusting reaction parameters for improved
efficiency, as well as determining when additional reaction cycles
are no longer needed in a particular experiment (e.g., when a
sufficient quantity of a target sequence has been produced).
[0078] It will be appreciated that process 800 is illustrative and
that variations and modifications are possible. Steps described as
sequential may be executed in parallel, order of steps may be
varied, and steps may be modified or combined. For example,
fluorescence measurements may be performed at any point during a
PCR cycle, performed multiple times during each PCR cycle
(including substantially continuous scanning of the sample wells),
or not performed until after some number of PCR cycles. Any number
of distinguishable fluorescent probes may be used in a single
reaction vessel, and the detection module may be adapted to include
at least as many channels as the number of probes in use. In some
embodiments, the detection module includes multiple channels
optimized for the same probe. This may reduce the scanning time
since only one of these channels needs to be used to interrogate a
particular sample well.
[0079] In addition, as mentioned above, in one alternative
embodiment, the various channels of detection module 234 are
arranged such that when one of its channels is in optical
communication with a sample well 210, other channels are not. This
arrangement allows for a "flyover" mode of operation, in which
detection module 234 is substantially continuously in motion during
a scanning pass over the wells. Cross-talk between the channels is
reduced because only one sample well at a time receives any
excitation light.
CONCLUSION
[0080] While the invention has been described with respect to
specific embodiments, one skilled in the art will recognize that
numerous modifications are possible. For instance, the fluorescence
detection assembly described herein may be adapted for use with a
wide variety of thermal cycler systems and may interrogate sample
wells from any direction (e.g., above or below) in accordance with
the design of a particular instrument. In addition, the system may
be adapted to detect a wide range of molecules of biological
interest that are identifiable by a fluorescent label or probe; it
is not limited to nucleic acids or to any particular amplification
process.
[0081] Thus, although the invention has been described with respect
to specific embodiments, it will be appreciated that the invention
is intended to cover all modifications and equivalents within the
scope of the following claims.
* * * * *