U.S. patent application number 12/142888 was filed with the patent office on 2008-10-16 for systems and methods for detecting and analyzing polymers.
This patent application is currently assigned to U.S. Genomics, Inc.. Invention is credited to Martin Fuchs, John Harris, Ray Meyer.
Application Number | 20080254549 12/142888 |
Document ID | / |
Family ID | 36119345 |
Filed Date | 2008-10-16 |
United States Patent
Application |
20080254549 |
Kind Code |
A1 |
Fuchs; Martin ; et
al. |
October 16, 2008 |
SYSTEMS AND METHODS FOR DETECTING AND ANALYZING POLYMERS
Abstract
A detection system and methods for improving the ability of the
detection system to recognize labels that are disposed on a
polymer. Embodiments of the invention include schemes for selecting
emitters and labels used within the system in a manner that allows
an increase in the number of distinct labels that can be used
together in a system. In other embodiments, the detection system
and methods are directed to identifying portions of a detection
signal that may be associated with extra labels residing within a
detection zone. In other embodiments, the detection system and
methods relate to using wide field imaging detectors while reducing
out of focus noise contributions to detection signals of the
system. Still, other embodiments relate to the use of linear array
detectors to detect labels
Inventors: |
Fuchs; Martin; (Uxbridge,
MA) ; Harris; John; (Foxboro, MA) ; Meyer;
Ray; (Wakefield, MA) |
Correspondence
Address: |
WOLF GREENFIELD & SACKS, P.C.
600 ATLANTIC AVENUE
BOSTON
MA
02210-2206
US
|
Assignee: |
U.S. Genomics, Inc.
Woburn
MA
|
Family ID: |
36119345 |
Appl. No.: |
12/142888 |
Filed: |
June 20, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11448411 |
Jun 7, 2006 |
7402422 |
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12142888 |
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11210155 |
Aug 23, 2005 |
7351538 |
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11448411 |
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60603760 |
Aug 23, 2004 |
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Current U.S.
Class: |
436/94 ;
702/19 |
Current CPC
Class: |
G01N 21/6428 20130101;
C12Q 2565/102 20130101; Y10T 436/143333 20150115; G01N 2021/6421
20130101; C12Q 1/6816 20130101; C12Q 1/6816 20130101; G01N
2021/6419 20130101; G01N 2021/6441 20130101 |
Class at
Publication: |
436/94 ;
702/19 |
International
Class: |
G01N 33/00 20060101
G01N033/00 |
Claims
1. A detection system for analyzing a polymer, the detection system
comprising: a first detection zone disposed in a first area of a
microchannel and adapted to detect a polymer having first and
second polymer portions to create a first detection signal when the
polymer is in the first zone; a second detection zone disposed in a
second area of the microchannel that is different from the first
area, the second detection zone adapted to detect the second
polymer portion to create a second detection signal when the second
portion is in the second area; a data processor adapted to
comparing the first and second detection signals and identify
components that are not common to both the first and second
detection signals.
2. The detection system of claim 1, wherein the second detection
zone is substantially overlapped with the first detection zone.
3. The detection system of claim 2, wherein the first detection
zone and the second detection zone are substantially circular, the
second detection zone being concentric with the first detection
zone and the second detection zone being disposed entirely within
the first detection zone.
4. The detection system of claim 3, wherein the first detection
zone has a diameter of approximately 1.00 microns and the second
detection zone has a diameter of approximately 0.50 microns.
5. The detection system of claim 1, further comprising: an emitter
for illuminating the first detection zone, the emitter adopted to
emit on excitation signal having low intensity components and high
intensity components.
6. The detection system of claim 5, wherein the first detection
zone covers areas having both low and high intensity components of
the excitation signal while the second detection zone covers areas
having substantially only high intensity components of the
excitation signal.
7. The detection system of claim 6, wherein the data processor is
adapted to remove at least some portions of emission signals
associated with the first detection zone of the low intensity
components of the excitation signal from the first detection signal
to improve the identification of the second polymer portion by the
first detection signal.
8. The detection system of any of claim 1, wherein the polymer is a
nucleic acid.
9. The detection system of claim 8, wherein the polymer is DNA or
RNA.
10. The detection system of claim 9, wherein the polymer is genomic
DNA.
11. The detection system of claim 9, wherein the RNA comprises
miRNA, siRNA, or RNAi.
12. The detection system of claim 1, wherein the first and second
detection zones are adapted to detect a polymer or polymer portion
having a label that fluoresces.
13. The detection system of claim 1, wherein the polymer comprises
a plurality of polymers, each of the first and second polymer
portions comprising separate polymers.
14. A method of analyzing a polymer in a detection system, the
method comprising: passing a plurality of polymers comprising a
first polymer portion and a second polymer portion through a first
detection zone to create a first detection signal, the first
detection zone disposed in a first area of the microchannel;
passing the second portion of polymers through a second detection
zone to create a second detection signal, the second detection zone
disposed in a second area of the microchannel; identifying
components of the first detection signal that are associated with
the second polymer portion by comparing the first detection signal
with the second detection signal; and detecting components of the
first detection signal associated with the second polymer portion
to analyze the polymers.
15. The method of claim 14, further comprising: modifying the first
detection signal by removing components of the first detection
signal that are not also associated only with the second detection
signal to improve the identification of the second polymer portion
by the first detection signal.
16. The method of claim 14, wherein passing the plurality of
polymers comprises moving both the first detection zone and the
second detection zone relative to the first and second portion of
polymers.
17. The method of claim 14, wherein passing the plurality of
polymers comprises flowing the plurality of polymers within a fluid
through the microchannel.
18. The method of claim 14, wherein the polymer is a nucleic
acid.
19. The method of claim 18, wherein the polymer is DNA or RNA.
20. The method of claim 19, wherein the polymer is genomic DNA.
21. The method of claim 19, wherein the RNA is miRNA, siRNA, or
RNAi.
22. The method of claim 14, wherein passing the second portion of
polymers through the second detection zone comprises passing the
second portion of polymers through the second detection zone that
is completely overlapped with the first detection zone.
23. The method of claim 14, wherein passing a plurality of polymers
through the first detection zone comprises passing a plurality of
polymers through the first detection zone defined by a high
intensity region and a low intensity region; and further wherein
passing the second portion of polymers through the second detection
zone comprises passing the second portion of the polymer through
the second detection zone that does not include the low intensity
region.
24. The method of claim 14, wherein identifying the components of
the first detection signal that are associated with the second
polymer portion comprises comparing a histogram of signal
intensities for the first detection signal with a histogram of
signal intensities for the second detection signal.
25. The method of claim 14, wherein modifying the first detection
signal comprises removing low intensity components from the first
detection signal that are not also present in second detection
signal.
26. The method of claim 14, wherein passing a plurality of polymers
through a first detection zone comprises passing a plurality of
polymers through a circular detection zone having a diameter of
approximately 1.00 microns; and further wherein, passing the second
portion of polymers through a second detection zone comprises
passing a plurality of polymers through a second circular detection
zone having a diameter of approximately 0.50 microns and being
substantially centered within the first detection zone.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 11/448,411, filed Jun. 7, 2006, which is a
divisional of U.S. patent application Ser. No. 11/210,155, filed
Aug. 23, 2005, which claims the benefit of U.S. Provisional
Application No. 60/603,760, filed Aug. 23, 2004. Each of which is
hereby incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates generally to systems and
methods for detecting and analyzing the structures of polymers, and
more particularly to systems and methods for improving the
detection of labels disposed on polymers, such as DNA or RNA.
BACKGROUND OF THE INVENTION
[0003] Polymer detection and analysis systems and their associated
methods have been used to detect and analyze polymers for many
years. Generally, such systems involve a sample, such as a polymer,
labeled with a known probe that will bind to the polymer in a
particular manner. The polymer is placed in a detection zone of the
system where an emitter, such as a laser, is used to excite the
label on the probe bound to the polymer. The label then emits an
emission signal that is seen by a detector in the system as a
portion of a detection signal. The characteristics of the emission
signal relative to the sample, the excitation signal, the
surroundings, and/or other characteristics are then used by the
system to analyze the polymer structure.
[0004] Detection systems often have difficulty distinguishing
emission signals from noise and/or disturbances within the system.
Such noise and/or disturbances may come from any number of sources.
By way of example, the excitation signal, the detection zone,
hardware of the system, the sample itself, impurities within the
sample, the solution in which the sample resides, and other
emission signals from the same or other polymers may be the source
of such noise or disturbances to a particular emission signal.
Noise in these systems may reduce the quality of the detection and
analysis that can be accomplished.
[0005] Labeling
[0006] Many technologies relating to genomic sequencing and
analysis require site-specific labeling of nucleic acids. Most
site-specific labeling is carried out using nucleic acid based
probes that hybridize to their complementary sequences within a
target molecule (e.g., a nucleic acid). The specificity of these
probes will vary, however, depending upon their length, their
sequence, the hybridization conditions, and the like. Moreover,
because these probes are usually labeled with a detectable label
such as a fluorophore or a radioactive label, they are expensive to
synthesize. The ability to increase the specificity of these
probes, and at the same time, use less of them would make labeling
reactions more efficient and less expensive to run.
SUMMARY OF THE INVENTION
[0007] The systems and methods of the present invention are
directed to improving the ability of a detection system to
recognize labels that are disposed on polymers. Frequently, these
polymers include DNA or RNA, that are being detected and analyzed
by a detection system.
[0008] In one embodiment a detection system for analyzing a polymer
having three or more distinct fluorophores is disclosed. Each
fluorophore has an excitation wavelength and a corresponding
emission bandwidth. The detection system comprises a polymer
interrogation zone constructed and arranged to accept the polymer
and at least three different emitters, each of the emitters
constructed and arranged to emit an excitation signal substantially
at an excitation wavelength of a corresponding one of the at least
three fluorophores such that each fluorophore emits a distinct
emission signal within its respective emission bandwidth and each
distinct emission signal has an emission maximum separated by at
least 60 nm from any other of the emission maximums. The detection
system also comprises a detector constructed and arranged to
distinctly detect the emission signal from each of the at least
three fluorophores.
[0009] In another embodiment, a detection system for analyzing a
polymer having three or more distinct fluorophores is disclosed.
Each fluorophore has an excitation wavelength and a corresponding
emission bandwidth. The detection system comprises a polymer
interrogation zone constructed and arranged to accept the polymer
and at least three different emitters, each of the emitters
constructed and arranged to emit an emission signal substantially
at an excitation wavelength of a corresponding one of the at least
three fluorophores such that each fluorophore emits a distinct
emission signal within its respective emission bandwidth that does
not overlap any other of the distinct emission signals at
normalized intensities above 70%. The detection system also
comprises a detector constructed and arranged to detect the
distinct emission signal from each of the at least three
fluorophores.
[0010] In a related embodiment, the detection system comprises each
of the emitters constructed and arranged to emit its excitation
signal within the excitation wavelength of one of the at least
three distinct fluorophores such that each fluorophore emits a
distinct emission signal within its respective emission bandwidth
that does not overlap any other of the distinct emission signal at
normalized intensities above 50%.
[0011] In another related embodiment, the detection system
comprises each of the emitters constructed and arranged to emit its
excitation signal within the excitation wavelength of one of the at
least three distinct fluorophores such that each fluorophore emits
a distinct emission signal within its respective emission bandwidth
that does not overlap any other of the distinct emission signal at
normalized intensities above 30%.
[0012] In another embodiment, a detection system for analyzing a
polymer having three or more distinct fluorophores is disclosed.
Each fluorophore has an excitation wavelength and a corresponding
emission bandwidth. The detection system comprises a polymer
interrogation zone constructed and arranged to accept the polymer.
The detection system also comprises at least three different
emitters, each of the emitters constructed and arranged to emit an
excitation signal substantially at an excitation wavelength of a
corresponding one of the at least three fluorophores such that each
fluorophore emits a distinct emission signal within its respective
emission bandwidth, each of the at least three emitters and
corresponding at least three fluorophores selected from a group
consisting of: an emitter emitting an excitation signal
substantially at a wavelength of 488 nm and a corresponding
fluorophore having an emission maximum substantially located at 512
nm, an emitter emitting an excitation signal substantially at a
wavelength of 532 nm and a corresponding fluorophore having an
emission maximum substantially located at 575 nm, an emitter
emitting an excitation signal substantially at a wavelength of 633
nm and a corresponding fluorophore having an emission maximum
substantially located at 665 nm, and an emitter emitting an
excitation signal substantially at a wavelength of 750 nm and a
corresponding fluorophore having an emission maximum substantially
located at 775 nm or 806 nm. The detection system further comprises
a detector constructed and arranged to detect the distinct emission
signal from each of the at least three fluorophores.
[0013] Another embodiment of the invention includes a method of
detecting emissions of three or more distinct fluorophores bound to
a polymer. The method comprises selecting three or more
fluorophores, each of the fluorophores characterized by an
excitation wavelength and a corresponding emission bandwidth, the
emission bandwidth of each of the fluorophores not overlapping the
emission bandwidth of any other of the fluorophores at normalized
intensities above 70%. The method also comprises attaching the
three or more fluorophores to the polymer in a sequence specific
manner and illuminating each of the fluorophores with an excitation
signal within the excitation wavelength of the corresponding
fluorophores, thereby causing each of the fluorophores to emit an
emission signal within the emission bandwidth of the fluorophore.
The method also includes detecting and analyzing the emission
signal of each of the fluorophores.
[0014] In a related method, the selecting three or more
fluorophores comprises selecting three or more fluorophores, each
of the fluorophores characterized by an excitation bandwidth and a
corresponding emission bandwidth, the emission bandwidth of each of
the fluorophores not overlapping the emission bandwidth of any
other of the fluorophores at normalized intensities above 50%.
[0015] In another related method, selecting three or more
fluorophores comprises selecting three or more fluorophores, each
of the fluorophores characterized by an excitation bandwidth and a
corresponding emission bandwidth, the emission bandwidth of each of
the fluorophores not overlapping the emission bandwidth of any
other of the fluorophores at normalized intensities above 30%.
[0016] Yet another method is disclosed for analyzing a polymer. The
method comprises selecting at least three distinct fluorophores and
at least three different excitation signals each emitted from a
corresponding emitter, each of the at least three fluorophores
having an excitation wavelength and a corresponding emission
bandwidth, each of the excitation signals having an excitation
wavelength of a corresponding one of the at least three
fluorophores such that each fluorophore emits a distinct emission
signal having an emission maximum within its respective emission
bandwidth, each of the at least three excitation signals and
corresponding at least three fluorophores selected from a group
consisting of: an excitation signal having a wavelength of 488 nm
and a corresponding fluorophore having an emission maximum
substantially located at 512 nm, an excitation signal having a
wavelength of 532 nm and a corresponding fluorophore having an
emission maximum substantially located at 575 nm, an excitation
signal having a wavelength of 633 nm and a corresponding
fluorophore having an emission maximum substantially located at 665
nm, and an excitation signal having a wavelength of 750 nm and a
corresponding fluorophore having an emission maximum substantially
located at 775 nm or 806 nm. The method also comprises binding the
at least three fluorophores to a polymer in a sequence specific
manner and illuminating each of the fluorophores with the
corresponding emitter. Detecting and analyzing the emission signal
of each of the fluorophores are also included in the method.
[0017] In some related embodiments the at least three fluorophores
are selected from the group consisting of: Bodipy FL fluorophore,
Tamra fluorophore, Alexa 647 fluorophore, Alexa 750 fluorophore,
and IR 38 fluorophore.
[0018] In some embodiments, the detector comprises a plurality of
detectors. In one embodiment, the plurality of detectors comprises
a plurality of avalanche photon detectors.
[0019] Some embodiments also comprise a plurality of dichroic
mirrors constructed and arranged to direct each of the distinct
emission signals into one of the plurality of detectors. Still,
some embodiments comprise a polychroic mirror adapted to prevent
excitation signals emitted from the at least three different
emitters from reaching the detector. Embodiments may also comprise
a bandpass filter for removing noise from each of the emission
signals.
[0020] Still, in some embodiments the at least three different
emitters comprise four different emitters and the at least three
different fluorophores comprise four different fluorophores.
[0021] The emitters comprise lasers in some of the disclosed
embodiments. Still in some embodiments, the excitation signals
comprise coherent light. The polymer, in some embodiments, is a
single polymer.
[0022] In an embodiment, a detection system for analyzing a polymer
is disclosed. The detection system comprises a first detection zone
disposed in a first area of a microchannel and adapted to detect a
polymer having first and second polymer portions to create a first
detection signal when the polymer is in the first zone and a second
detection zone disposed in a second area of the microchannel that
is different from the first area, the second detection zone adapted
to detect the second polymer portion to create a second detection
signal when the second portion is in the second area. The
embodiment also comprises a data processor adapted to comparing the
first and second detection signals and identify components that are
not common to both the first and second detection signals.
[0023] Also disclosed is a method of analyzing a polymer in a
detection system. The method comprises passing a plurality of
polymers comprising a first polymer portion and a second polymer
portion through a first detection zone to create a first detection
signal, the first detection zone disposed in a first area of the
microchannel and passing the second portion of polymers through a
second detection zone to create a second detection signal, the
second detection zone disposed in a second area of the
microchannel. The method also comprises identifying components of
the first detection signal that are associated with the second
polymer portion by comparing the first detection signal with the
second detection signal and detecting components of the first
detection signal associated with the second polymer portion to
analyze the polymers.
[0024] In some embodiments, the second detection zone is
substantially overlapped with the first detection zone. Still, in
some embodiments the first detection zone and the second detection
zone are substantially circular. The second detection zone is
concentric with the first detection zone and the second detection
zone is disposed entirely within the first detection zone.
[0025] In some embodiments, the first detection zone has a diameter
of approximately 1.00 microns and the second detection zone has a
diameter of approximately 0.50 microns.
[0026] In some illustrative embodiments the system further
comprises an emitter for illuminating the first detection zone, the
emitter adopted to emit on excitation signal having low intensity
components and high intensity components.
[0027] In some embodiments the first detection zone covers areas
having both low and high intensity components of the excitation
signal while the second detection zone covers areas having
substantially only high intensity components of the excitation
signal.
[0028] Still, in some embodiments the data processor is adapted to
remove at least some portions of emission signals associated with
the first detection zone of the low intensity components of the
excitation signal from the first detection signal to improve the
identification of the second polymer portion by the first detection
signal.
[0029] In some aspects of some embodiments, the first and second
detection zones are adapted to detect a polymer or polymer portion
having a label that fluoresces.
[0030] Still, in some embodiments the polymer comprises a plurality
of polymers, each of the first and second polymer portions
comprising separate polymers.
[0031] Related embodiments further comprise modifying the first
detection signal by removing components of the first detection
signal that are not also associated only with the second detection
signal to improve the identification of the second polymer portion
by the first detection signal.
[0032] In some of the embodiments, passing the plurality of
polymers comprises moving both the first detection zone and the
second detection zone relative to the first and second portion of
polymers. Still, in some embodiments, passing the plurality of
polymers comprises flowing the plurality of polymers within a fluid
through the microchannel. In some of the embodiments, passing the
second portion of polymers through the second detection zone
comprises passing the second portion of polymers through the second
detection zone that is completely overlapped with the first
detection zone.
[0033] In an illustrative embodiment, passing a plurality of
polymers through the first detection zone comprises passing a
plurality of polymers through the first detection zone defined by a
high intensity region and a low intensity region and passing the
second portion of polymers through the second detection zone
comprises passing the second portion of the polymer through the
second detection zone that does not include the low intensity
region.
[0034] In one illustrative embodiment, identifying the components
of the first detection signal that are associated with the second
polymer portion comprises comparing a histogram of signal
intensities for the first detection signal with a histogram of
signal intensities for the second detection signal.
[0035] In another illustrative embodiment modifying the first
detection signal comprises removing low intensity components from
the first detection signal that are not also present in second
detection signal.
[0036] In one illustrative embodiment, passing a plurality of
polymers through a first detection zone comprises passing a
plurality of polymers through a circular detection zone having a
diameter of approximately 1.00 microns and passing the second
portion of polymers through a second detection zone comprises
passing a plurality of polymers through a second circular detection
zone having a diameter of approximately 0.50 microns and that is
substantially centered within the first detection zone.
[0037] In one illustrative embodiment, a detection system is
disclosed to detect a label disposed on a polymer. The detection
system comprises a channel constructed and arranged to receive the
polymer carried in a carrier fluid. The channel has a sample area
defined by upper and lower channel surfaces separated from one
another by a channel height less than about 0.500 microns. Also
disclosed is an emitter constructed and arranged to illuminate the
sample area with an excitation signal to excite the label in the
sample area, thereby causing the label to emit an emission signal
and a detector constructed and arranged to detect a detection
signal from a detection zone disposed within the sample area, the
detection signal including the emission signal, the detection zone
being disposed at least partially within the sample area.
[0038] In one of the embodiments, channel height is less than about
0.100 microns and in some embodiments, channel height is less than
about 0.050 microns. In the detection system of some embodiments,
the sample area of the channel has a channel width less than about
250 microns. The detection system of some embodiments, includes the
sample area constructed and arranged to receive not more than 50
nanoliters/second of carrier fluid.
[0039] The emitter is a laser in some of these embodiments. In
particular, in some embodiments of the detection system, wherein
the laser is one or more lasers selected from the group consisting
of: a laser emitting light substantially at a wavelength of 488 nm,
a laser emitting light substantially at a wavelength of 532 nm, a
laser emitting light substantially at a wavelength of 633 nm, and a
laser emitting light substantially at a wavelength of 750 nm.
[0040] In some embodiments of the detection system, the label is a
fluorophore. Still, in some embodiments, the fluorophore is one or
more fluorophores selected from the group consisting of: a
fluorophore having an emission maximum substantially located at 512
nm, a fluorophore having an emission maximum substantially located
at 575 nm, a fluorophore having an emission maximum substantially
located at 665 nm, a fluorophore having an emission maximum
substantially located at 775 nm, and a fluorophore having an
emission maximum substantially located at 806 nm.
[0041] It is also disclosed that in some embodiments of the
detection system, the upper or lower surface of the channel are
adapted to transmit the excitation signal or detection signal
without contributing noise. The material is fused silica in some of
these embodiments.
[0042] Additionally, in some embodiments of the detection system,
the detector comprises a CCD array. In some of these embodiments,
the CCD array comprises a linear CCD array.
[0043] In another embodiment, a method to detect a label disposed
on a polymer is disclosed. The method comprises providing a carrier
fluid containing the polymer and providing a channel constructed
and arranged to receive the carrier fluid. The channel has a sample
area defined by upper and lower channel surfaces separated from one
another by a channel height less than about 0.500 microns. Also
disclosed is exciting the label with an excitation signal, causing
the label to emit an emission signal and detecting the emission
signal.
[0044] In yet another embodiment, a detection system is disclosed
to detect a label disposed on a polymer. The detection system
comprises a channel adapted to allow a carrier fluid containing a
polymer to pass in a flow direction through the channel and through
a detection zone within the channel. Also disclosed is an emitter
constructed and arranged to emit an excitation signal as a sheet of
light into the detection zone and a detector constructed and
arranged to detect a detection signal emanating from the detection
zone from a direction substantially orthogonal to the sheet of
light. The detection signal includes an emission signal from the
label when the label is present in the zone and excited by the
excitation signal.
[0045] In some embodiments of the detection system, the detector
has a focal plane lying substantially within the sheet of light.
Still, according to some embodiments, the detection system further
comprises a cylindrical lens adapted to form the sheet of
light.
[0046] Also disclosed are some embodiments of the detection system
where the sheet of light is disposed in the channel such that
substantially all of the carrier fluid passes through the sheet of
light when passing through the channel.
[0047] In still another embodiment, a method for detecting a label
on a polymer having the label. The method comprises providing a
carrier fluid containing the polymer and providing a channel having
a detection zone located within the channels. The method also
comprises flowing the carrier fluid through the channel in a flow
direction and through a detection zone in the channel, and emitting
an excitation signal as a sheet of light into the detection zone.
Also disclosed is detecting, with a detector, an emission signal
from the label when the label is present in the zone and excited by
the excitation signal. The detector is located in a direction
substantially orthogonal to the sheet of light.
[0048] In yet another embodiment, a detection system for detecting
a first and second distinct labels on a polymer is disclosed. The
detection system comprises a detection zone adapted to receive the
polymer for detection and an emitter for exciting each of the first
and second distinct labels on the polymer when in the detection
zone, causing each of the first and second distinct labels to emit
a first and second emission signal, respectively. The system also
comprises a mirror adapted to substantially separate the first and
second emission signals from one another and a wide field detector
adapted to receive the first and second emission signals on
spatially separate portions of a detection surface.
[0049] Also disclosed is a method of detecting a first and a second
distinct label on a polymer. The method comprises providing a
detection zone and placing the polymer and the label into the
detection zone. The method also comprises emitting an excitation
signal for exciting the first and second distinct labels, causing
the first and second labels to emit a first and second emission
signal, respectively. Also discloses is substantially separating
the first and second emission signals from one another and
detecting the first and second emission signals on a spatially
separated portions of a detector.
[0050] In another embodiment, a detection system is disclosed for
analyzing a polymer having a label. The detection system comprises
a channel adapted to provide a carrier fluid containing a polymer
through the channel in a flow direction and a detection zone lying
within the channel. The detection zone comprises a substantially
linear array, the array arranged in a direction substantially
orthogonal to the flow direction. The system also comprises an
emitter constructed and arranged to emit an excitation signal into
the detection zone. The excitation signal comprises a sheet of
light extending into the detection zone. Also discloses is a
detector constructed and arranged to detect an emission signal from
the label when the label is present in the detection zone and
excited by the excitation signal. In some embodiments of the
detection system, the detector is a linear CCD array.
[0051] In some of these embodiments, the polymer is a nucleic acid.
Also, in some embodiments, the polymer is DNA or RNA. Still,
sometimes the nucleic acid is genomic DNA. In other embodiments,
the RNA is miRNA, siRNA, or RNAi.
[0052] Each of the limitations of the invention can encompass
various embodiments of the invention. It is, therefore, anticipated
that each of the limitations of the invention involving any one
element or combinations of elements can be included in each aspect
of the invention. This invention is not limited in its application
to the details of construction and the arrangement of components
set forth in the following description or illustrated in the
drawings. The invention is capable of other embodiments and of
being practiced or of being carried out in various ways. Also, the
phraseology and terminology used herein is for the purpose of
description and should not be regarded as limiting. The use of
"including," "comprising", or "having", "containing", "involving",
and variations thereof as recited herein, is meant to encompass the
items listed thereafter and equivalents thereof as well as
additional items.
[0053] Further features and advantages of the present invention, as
well as the structure of various embodiments, are described in
detail below with reference to the accompanying drawings.
BRIEF DESCRIPTION OF DRAWINGS
[0054] The Figures are illustrative only and are not required for
enablement of the invention disclosed herein.
[0055] Various embodiments of the invention will now be described
by way of example, with references to the accompanying drawings, in
which:
[0056] FIG. 1 shows a representation of a detection system and
several of the components that comprise many embodiments of
detection systems;
[0057] FIG. 2 shows an intensity versus wavelength representation
of a detection signal having two emission signals sharing
overlapping wavelengths;
[0058] FIG. 3 shows a gain versus wavelength representation of a
response curve for a dichroic mirror;
[0059] FIG. 4 shows the detection signal shown in FIG. 2 being
reflected and transmitted by a dichroic mirror that has a response
curve like that shown in FIG. 3;
[0060] FIG. 5 shows an intensity versus wavelength plot of four
emission signals that comprise a particular detection signal;
[0061] FIG. 6 shows a set of emission signals and the excitation
signals used to cause the labels to emit each of the emission
signals;
[0062] FIG. 7 shows a response curve for a polychroic mirror used
to remove excitation signals from a detection signal, according to
one embodiment of the invention;
[0063] FIG. 8 shows the detection signal of FIG. 6 after having
been passed through a polychroic mirror like that represented by
FIG. 7;
[0064] FIG. 9 is a schematic diagram of a detection system, showing
dichroic mirrors extracting emission signals from a detection
signal according to an embodiment of the present invention;
[0065] FIG. 10 is a schematic diagram of a detection system,
showing dichroic mirrors extracting emission signals from a
detection signal according to another embodiment of the present
invention;
[0066] FIG. 11 shows a representation of signal intensity as it
varies across the cross section of a Guassian laser beam;
[0067] FIG. 12 shows an intensity versus time plot representing a
label passing through a high intensity portion of a detection zone
and a low intensity portion of a detection zone;
[0068] FIG. 13 shows a histogram of a peak intensity associated
with a label passing through a central portion of a detection zone,
according to one embodiment of the invention;
[0069] FIG. 14 shows a histogram of peak intensity associated with
a first label passing through a central portion of a detection zone
and a second label passing through a peripheral portion of a
detection zone, according to an embodiment of the present
invention;
[0070] FIG. 15 shows overlapped concentric detection zones,
according to an embodiment of the present invention;
[0071] FIG. 16 shows a detection signal plotted on a graph of
intensity versus time;
[0072] FIG. 17 shows an overlay of the histograms of both FIGS. 13
and 14;
[0073] FIG. 18 shows schematic representations of detection systems
using both point detectors and wide field imaging detectors;
[0074] FIG. 19 shows a microchannel defining a thin sample area
adapted to lie within the focal plane of a wide field imaging
detector, according to one embodiment of the invention;
[0075] FIG. 20 shows a sample area being illuminated by a thin
light sheet introduced into the sample area from a side
direction;
[0076] FIG. 21 shows spatial separation of emission signals in a
wide field imaging system and projection of the separated signals
onto separate portions of a wide field imaging detector; and
[0077] FIG. 22 shows a linear array detector and a corresponding
detection zone disposed within a channel of a detection system.
DETAILED DESCRIPTION
[0078] The present invention relates, in part, to a detection
system for accepting a polymer that has been labeled, an emitter
for emitting an excitation signal to excite the label causing it to
emit an emission signal, a detector adapted to detect the emission
signal as a part of an entire detection signal that is received
from a detection zone, and a data processor to analyze the
detection and/or emission signals received by the detector. Various
aspects of the present invention relate to improving the ability of
the detection system to recognize labels within the detection zone,
particularly in view of noise or disturbances that might be present
in the system.
[0079] As used herein, the term "emission signal" is used to denote
the emissions of a label, such as the fluorescent or radioactive
emissions of some labels. In this regard, an emission signal is
defined by the label that provides it. The term "detection signal"
is used to denote an entire signal received from the detection zone
of a system, regardless of how many emission signals it may contain
and/or the amount and type of noise or disturbances that it may
also contain. In this manner, the detection zone and its contents
define the detection signal.
[0080] It is to be understood that when emissions signals are said
to be "overlapped", it is meant that each of the emissions signals
comprise at least a portion of photons that share a common
wavelength in the electromagnetic spectrum.
[0081] As used herein, the term "sample area" is used to define a
portion of a detection system adapted to accept a sample for
analysis. The sample may include a polymer but is not limited to
the polymer alone. For example, the sample may include the polymer
and a buffer solution along with any other elements floating within
the buffer solution.
[0082] As used herein, the term "excitation signal" is used to
describe the emissions of an emitter that may be used to excite a
label. As used herein, the term "excitation zone" defines an area
illuminated by an excitation signal of an emitter.
[0083] As used herein, the term "detection signal" is used to
define all of the optical signal that is received by the optics of
a detection system. For instance, this may include reflected
excitation signals, noise, emission signals, and the like, that are
received by an objective lens or microscope of a detection
system.
[0084] As used herein, the term "detection zone" is used to denote
the zone within a sample area from which optical emissions are
received by detectors in the system. This may include all or a
portion of an excitation zone and/or all or a portion of a sample
area. In many systems, the detection zone may be defined by a
confocal aperture that restricts which portions of a detection
signal are passed to a detector. In other systems, particularly
those using wide field imaging detectors, the detection zone may be
defined by all that the wide field imaging detectors are adapted to
observe.
[0085] As used herein, the term "multi-channel" is used to denote a
detection system adapted to detect multiple labels. In particular,
it is used to denote a system adapted to detect labels each
associated with a particular portion of the electromagnetic
spectrum.
[0086] As used herein, the term "normalized intensity" particularly
when used with reference to an emissions signal, is used to
describe an emissions signal that has been divided by its maximum
value such that it may be compared with other emissions signals on
a normalized basis.
[0087] Some aspects of the present invention include one or more
particular lasers for exciting one or more particular labels that
are used within the system. In such embodiments, the lasers and
labels are chosen such that the excitation signals of the lasers do
not adversely impact any of the emission signals of the labels to a
degree that impedes detection of the labels. Alternatively, the
labels may be chosen such that each of their emission signals do
not adversely affect the analysis of any other labels within the
system. In many embodiments, the labels are fluorophores.
[0088] Other aspects are directed to reducing or eliminating
portions of detection signals that are associated with extraneous
labels residing within the detection zone. In this regard, these
aspects focus on reducing or eliminating the possibility of
emission signals, such as from additional polymers or unbound
labels within the detection zone, being associated with an emission
signal from a primary polymer that is being detected or
analyzed.
[0089] Still other aspects are directed to reducing noise emanating
from points out of the focal plane when detecting the labels with
wide field imaging devices. In some embodiments, the illumination
of the sample area is held substantially within the focal plane of
the wide field detector. Thus, only portions of the detection zone
lying within the focal plane of the wide field imaging device are
excited by the emitter, which reduces or eliminates contributions
to the detection signal from outside of the focal plane.
[0090] According to another aspect, instrumentation is included
within the system to assist wide field imaging devices in
differentiating between emission signals at different wavelengths.
In this regard, the number of detectors required by some
multi-channel systems may be reduced, thus potentially reducing the
cost and complexity of the detection system.
[0091] Still other aspects are related to using detectors that
optimize the benefits of point detection and wide field detection.
In such systems, an array of point detectors, such as in a linear
CCD array, may be disposed about a sample area to increase the size
of the detection zone and the probability that a polymer passing
through will be detected while only requiring single linear array
for detection.
[0092] Turn now to the figures, and particularly FIG. 1, which
shows basic components of many embodiments of detection systems.
Central to the detection system is a sample area where a labeled
sample, such as a polymer or multiple polymers, is directed for
detection. An emitter 52, such as a laser, emits an excitation
signal that is projected into the sample area 54 such that it may
strike a labeled polymer and cause the label to emit an emission
signal. If the label is within a detection zone, which may comprise
a portion of the sample area or all of the sample area, the
emission signal will be a portion of a detection signal that is
received by optics 56 of the detection system, such as a lens or
microscope. The optics of the system receive the detection signal
and directs it through various optical signal processing
instruments, such as objective lenses, filtering lenses or mirrors,
and the like. Such optical signal processing instruments may be
used to extract an emission signal from other portions of the
detection signal, including noise, disturbances, etc. After an
emission signal, or a portion thereof, has been extracted from the
detection signal, the emission signal may be directed to a one or
more downstream detectors 58 in the system. The downstream
detectors convert the optical signal into an electrical signal for
processing by a data processor 60, which is typically a digital
computer. The data processor receives the emission signal and
analyzes it along with other inputs, such as the spatial location
of the polymer relative to the detector, time when the emission
signal was detected, the spatial or temporal relationship between
the emission signal and other emission signals that are detected,
or other characteristics that may be used by a particular detection
analysis system.
[0093] Multi-Channel Detection Systems
[0094] In one illustrative embodiment of the invention, a system
and method are adapted to analyze a plurality of labels disposed on
a polymer in a manner that prevents an emission signal of one label
from acting as a disturbance to another emission signal. In this
manner, the labels are less likely to be confused with one another
or other components of the detection signal and are thus more
likely to be detected. As is to be appreciated, it is generally
desirable to distinguish different types of labels of a single
polymer so that greater amounts of information may be extracted
from the polymer than might otherwise be possible.
[0095] In illustrative embodiments of the invention it is desirable
to choose labels having distinct emission signals, as is described
in U.S. patent application Ser. No. 10/246,779, filed on Sep. 18,
2002, which is hereby incorporated by reference in its entirety.
For instance, the labels may be fluorophores having emission
signals that lie substantially within a portion of the
electromagnetic spectrum associated with a particular color. FIG. 2
shows a plot of two such distinct emission signals included within
one common detection signal.
[0096] Detectors used in illustrative embodiments of detection
systems may be adapted to count photons emitted by labels without
regard to the wavelength at which the photons are emitted. In such
embodiments, the detectors themselves may be incapable of
distinguishing between emission signals from different labels
without the assistance of other processing instrumentation.
Examples of detectors that may not be adapted to recognize the
particular wavelength at which photons are emitted include, but are
not limited to, some forms of avalanche photon detectors and Charge
Coupled Devices (CCD's).
[0097] In some embodiments, filtering devices such as dichroic
mirrors may be used to extract and direct emission signals to
detectors that might not otherwise be able to distinguish them. In
this manner, embodiments of the detection system may be able to
detect a plurality of labels that each emit a signal associated
with a distinct portion of the electromagnetic spectrum. Such
detection systems may be said to be capable of detecting multiple
colors, as the emission signals may lie in portions of the visible
spectrum that are associated with a particular color. Such
multi-color systems may also be referred to as multi-channel
systems, as each portion of the wavelength range may be converted
into its own electrical signal and analyzed in its own channel of
the data processor.
[0098] In one embodiment of the invention, dichroic mirrors are
used to separate distinct emission signals from a detection signal
and direct each of the emission signals to a corresponding
detector. Dichroic mirrors operate by allowing certain wavelengths
of light to pass through the mirror, while reflecting other
wavelengths of light away from the mirror. Most of such dichroic
mirrors are characterized by a response curve that defines which
wavelengths of light are transmitted and which are reflected, and
the gain values associated with the transmission or reflection of
each wavelength. An example of a such response curve for a dichroic
mirror is shown in FIG. 3.
[0099] FIG. 4 depicts a pair of emission signals overlaid with a
response curve like that shown in FIG. 3. FIG. 4 also shows the
resulting reflected and transmitted signal 76. As used herein, the
transmitted signal is the portion of the signal that passes through
the mirror while the reflected signal 78 is the portion of the
signal that is reflected by the mirror. Here, the transmitted
signal comprises a signal having a distribution of intensities
substantially equal to the original overlapped signal multiplied by
the gain of the response curve at each wavelength. The reflected
signal comprises a signal having intensities substantially equal to
the original, overlapped signal multiplied by one minus the gain
value of the response curve at each wavelength. The transmitted and
reflected signals are typically directed in two different
directions, such as toward different detectors or different mirrors
in the detection system.
[0100] In illustrative embodiments of the invention, dichroic
mirrors, or other processing instrumentation, may be used to divide
signals from one another such that less expensive detectors can be
used to detect emission signals in a detection system. However,
emission signals 62, although distinct, may still overlap at some
wavelengths like the first and second emission signals shown in
FIG. 2. As is to be appreciated, dichroic mirrors only split the
signal according to a response curve, and are not necessarily
capable of separating overlapped portions 66 of the emissions
signals strictly according to which label they came from. In this
regard, the transmitted signal and/or the reflected signal from a
dichroic mirror may contain photons from both of the distinct
emission signals at wavelengths where the signals are
overlapped.
[0101] Practical limitations in many signal processing instruments
such as dichroic mirrors, may also limit the ability of a system to
separate distinct emission signals. Although ideally the response
of a dichroic mirror transitions abruptly between the transmission
band 68 and the reflection band 70, most mirrors typically have a
transition band that spans anywhere between 30 and 100 nanometers,
as shown in FIG. 3. Portions of a signal that reside within the
transition band will be both transmitted to a degree and reflected
to a degree. This will be defined by the response curve 74, and
will depend on the corresponding gain value of the response curve.
In this regard, the transition band 72 of a dichroic mirror
presents another factor to consider when labels, emitters, and
signal processing instruments, such as dichroic mirrors, are chosen
for a detection system.
[0102] Detection systems may be designed to accommodate overlapped
emission signals, transition bands in signal processing
instrumentation, and other factors that may prevent an emission
signal from reaching a detector in whole form. As represented by
the signal intensity versus wavelength graph of FIG. 2, a greater
number of photons per wavelength (i.e., intensity) are emitted at
and about the emission maximum 64 of a signal. As is to be
appreciated, some embodiments of detectors will "detect" a label
after a predetermined number of photons have been counted. As is to
be further appreciated, the predetermined number may be less than
the total number of photons that comprise an emission signal. In
fact, this is preferred in many embodiments to compensate for
portions of a signal that may be lost during the signal processing
steps or due to other imperfection within the system. The portions
of a signal at wavelengths further from the emission maximum are
less important to signal detection in some embodiments, as they are
of a lower intensity and represent a smaller portion of the photons
that comprise a given emission signal.
[0103] Other factors within the system may also make portions of
the signal that are further from the emission maximum less
important to detection of an emission signal. For instance,
detectors are often limited by their quantum efficiency, which is
defined as the ratio of photons that are actually detected to those
that are incident upon the detector. The quantum efficiency of some
detectors is proportional to the intensity of the incident signal.
Those portions of a signal at wavelengths further from the emission
maximum, which are of a lower intensity, are less likely to be
recognized by a detector if they are incident upon it. In this
regard, quantum efficiency is another factor suggesting that
portions of an emission signal further from the emission maximum
are less critical to detection of an emission signal and
consequently, the associated label.
[0104] As is to be appreciated, it may be desirable to use many
distinct probes and labels on a given polymer to allow more
information to be extracted from a polymer when as it passes
through a detection zone. However, at least for the aforementioned
reasons, it is also to be appreciated that increasing the number of
distinct labels and thus the number of distinct emission signals
that a detection system is required to distinguish may also cause
complications in a system. For example, it may also increase the
likelihood that distinct emission signals will overlap at some
wavelengths and thus might be confused with one another. To
optimize among these factors, embodiments of the invention include
methods for choosing labels to reduce the possibility that emission
signals will be confused with one another during detection while
also providing for more labels in the detection system.
[0105] In one illustrative embodiment, the analysis techniques
provide for distinguishing multiple emission signals based on the
fact that they do not overlap above particular intensities. As
represented in FIG. 2, most emission signals may be characterized
as having an emission maximum at a particular wavelength and
intensities that decrease at wavelengths further from the emission
maximum along either direction of the electromagnetic spectrum. As
discussed above, some detectors rely on photon counts to detect the
presence of an emission signal, and are incapable of determining
where photons emanate from within the electromagnetic spectrum.
Thus, in some embodiments, detection signals are filtered to remove
portions of the signal at wavelengths other than those within a
band associated with a particular emission signal. The signals are
then directed to a detector such that photon counts may be made.
However, where emission signals from different labels overlap over
ranges of wavelengths, it may become difficult to extract an
emission signal from the detection signal without also affecting
the overlapped emission signal.
[0106] As previously discussed, a majority of photons for a given
emission signal emanate at or near the wavelengths of the emission
maximum. Thus, if emission signals overlap at lower intensities, it
is less problematic for a detection system. In this regard,
embodiments of the present invention may be adapted to have labels
with emission signals that do not overlap above intensities of a
particular value. This may assist embodiments of the invention in
distinguishing emission signals from one another.
[0107] As described above, it may be desirable in some embodiments
to use labels characterized by emission signals that do not overlap
at points above a certain intensity. In some embodiments using a
plurality of labels with emission signals that do not overlap above
70% of the maximum, normalized emission signal intensity, as shown
in FIG. 5. Here, intensity is measured in terms of total photon
count per unit wavelength and the emission signals are normalized
to one another by dividing each emission signal intensity curve by
its maximum intensity value, as is reflected by the emission signal
curves shown in FIG. 5, and provides emission signals that can be
separated such that they can be detected by the system with a high
degree of confidence. In other embodiments, labels may be chosen
such that there is no overlap above 50% of the maximum, normalized
intensity to accomplish this effect. Still, in other embodiments
labels may be chosen such that there is no emission signal overlap
at points above 30% of the maximum, normalized emission signal
intensity to allow distinct emission signals to be detected with
confidence. FIG. 6 shows a graph of four emission signals that do
not overlap above 30% of the maximum, normalized signal intensity.
In other embodiments, the emission maximums of the signals, the
labels they represent (fluorophores in this embodiment), their
associated excitation frequencies, and lasers that may be used to
excite the labels may be those listed in Table 1 below.
TABLE-US-00001 TABLE 1 Wave- length Excitation Emission Channel
Laser (nm) Label Max. Max. Blue Sapphire 488 BODIPY FL 504 512
Green YAG 532 Tamra 553 575 2.sup.nd harmonic Red HE-Ne 633 Alexa
647 650 665 IR Alexandrite 750 Alexa 750 749 775 IR 38 778 806
[0108] In one illustrative embodiment of the invention, labels are
chosen such that their emission maximum are separated from one
another by no less than a predetermined number of wavelengths to
prevent portions of any emission signal, such as overlapping
portions, from interfering with another emission signal. For
example, in one embodiment, the labels are chosen such that their
emission maximums are separated by more than substantially 60
nanometers. The labels reflected in Table 1 above also meet this
criteria.
[0109] In some illustrative embodiments, the excitation signal used
to excite the label, typically the coherent light of a laser, may
also be reflected toward the detectors of the system as part of the
detection signal 80. The excitation signal 82, if not removed from
the detection signal, may be incorrectly considered part of an
emission signal. As is to be appreciated, it is desirable to
extract any portions of the excitation signal from the detection
signal and thus any emission signals therein, without impacting the
emission signals so much that the emission signal might be
misinterpreted by, or not detected by the detection system.
[0110] As described above, it is desirable to remove as much of the
excitation signal from the detection signal without impacting any
emission signals too much. Having portions of an excitation signal
present in an emission signal may cause incorrect photon counts as
excitation signals typically include high intensity light may
provide a high number of photons to a detector. The high number of
photons associated with the excitation signal may have a greater
impact on the photon count of a detector than other noise within a
system. To help reduce any impact on the emission signals, lasers
or other excitation signals may be chosen such that they are
located at wavelengths that are substantially separated from the
emission maximums of any labels used within the detection system.
In this manner, removing the excitation signals from the emission
signals may be accomplished without adversely affecting the
emission signal at points near an emission maximum.
[0111] In one illustrative embodiment, combinations of lasers and
fluorophores are chosen such that each excitation signal is
separated by about 20 nanometers or more from the emission maximum
of all fluorophores used in a detection system. For example, FIG. 6
shows excitation signals overlaid on a graph of the emission
signals for four distinct labels. The excitation signals and the
emission signals represented in FIG. 6 are those of Table 1. It is
to be noted that Table 1 includes both an Alexa 750 and an IR 38
label that can be used interchangeably to accomplish similar
effects.
[0112] Choosing excitation signals as described above may require
the excitation signal to deviate from the optimal excitation
wavelength for a particular label. For example, this may mean
having an excitation signal wavelength set at 488 nm to excite a
fluorophore with an optimal excitation wavelength of 504 nm.
However, in many embodiments, the tradeoff is worthwhile, as
otherwise the excitation signal might not be removed from a
detection signal in the system without also removing portions of
the emission signal that are closer to the emission maximum. Table
1 also reflects the excitation maximums of various labels
represented in FIG. 6.
[0113] In one illustrative embodiment of the invention, the
detection system includes an optical instrument for extracting each
of the excitation signals from the detection signal. A polychroic
mirror 84 is an example of an instrument that may be used to
accomplish this. The polychroic mirror, like a dichroic mirror 86,
is adapted to transmit particular wavelengths of light and reflect
other wavelengths of light. However, the polychroic mirror
comprises multiple transmission bands and multiple reflection
bands, whereas a dichroic mirror typically has a single
transmission band and a single reflection band. In this regard, a
single polychroic mirror may be used to remove, or reflect in a
different direction, several distinct bands of a detection signal,
such as the excitation signals.
[0114] As with the dichroic mirrors discussed above, the polychroic
mirror is characterized by a transition band disposed between each
of the transmission and reflection bands. Preferably, the
transition band spans a narrow range of wavelengths such that only
narrow bands of a detection signal associated with the excitation
signals are removed when passing through the polychroic mirror.
Having a shorter transition band in the polychroic mirror is
generally more important when the signal to be separated, in this
case the excitation signal, is closer to the emission maximum of
the emission signal. In this regard, techniques for improving the
cutoff rate between the reflection and transmission bands of the
mirror are typically used. One such technique includes using rugate
technology to manufacture the mirror.
[0115] Polychroic mirrors employing rugate technology, and sold as
discrete rugate match filters, can be purchased from Barr
Associates of Westford, Mass. Such polychroic mirrors may be made
having transition bands sharp enough to remove excitation signals
associated with the lasers of Table 1, or other excitation signals,
without affecting the emission signals also shown in Table 1 to a
such a degree that they are incorrectly detected by the system.
FIG. 7 shows a representative response curve for such a polychroic
mirror. Also, FIG. 8 shows the emission and excitation signals of
FIG. 6 after being passed through a polychroic mirror having a
response curve like that shown in FIG. 7. As can be appreciated,
the polychroic mirror not only reflects the excitation signals, but
also any portion of the detections signal within the reflection
bands of the polychroic mirror, such as portions of an emission
signal.
[0116] In one illustrative embodiment of the invention, once the
excitation signals are removed from the detection signal, the
various wavelength bands associated with the emission signals in
the system are separated for independent detection by detectors of
the system. A detection signal containing emission signals of
multiple labels may be passed through one or more dichroic mirrors
to separate the various emission signals out of the detection
signal and direct them to an appropriate detector. Some
representative schemes for separating the emission signals from a
detection signal are discussed below.
[0117] An example of a system used to separate the emission signals
from a detection signal is shown in FIG. 9. Here, a detection
signal is passed through a series of dichroic mirrors that each
reflect a portion of an emission signal from the detection signal
and direct it toward a detector. The first dichroic mirror receives
the entire detection signal and reflects portions below the cutoff
wavelength of the first dichroic. The cutoff of the first dichroic
is set above the emission maximum wavelength of the emission signal
having the shortest wavelength, yet below the emission maximum of
the emission signal having the next largest set of wavelengths in
the system. In this manner, the first dichroic reflects any
emission signal associated with the shortest wavelength (e.g.,
blue) of the detection signal and toward a detector for the
corresponding (e.g., blue) channel of the system. The remaining
portions of the detection signal are transmitted to the next
dichroic mirror, which then reflects portions of the detection
signal below its cutoff wavelength, which is above the next channel
in the system characterized by the next longest wavelength (e.g.,
the green channel). The green emission signal is, in turn, directed
toward a detector that may be optimized to detect signals within
the green portion of the electromagnetic spectrum. This process
continues with each subsequent mirror having a cutoff wavelength
set to remove the emission signal characterized by the next longest
wavelength. In this manner, each of emission signals may be removed
from the detection signal and provided to detectors that might not
otherwise be able to distinguish between emission signals, and thus
between labels. However, it is to be appreciated that other methods
of separating the detection signal into separate emission signals,
or extracting various emission signals from the detection signal
may also be used, as the present invention is not limited to the
above described method.
[0118] In another illustrative embodiment, as represented in FIG.
10, after having removed excitation signals with a polychroic
mirror or equivalent approach, the detection signal is directed to
a first dichroic mirror having a response curve adapted to reflect
the two emission signals having the two shortest wavelengths. In
the illustrated embodiment, this includes the blue and green
emission signals. The remaining red and infrared signals are
transmitted through the first dichroic mirror. Thereafter, the
portion of the detection signal comprising the blue and green
emission signals is passed through an additional dichroic mirror,
which transmits the blue signal and reflects the green signal.
After passing through this second dichroic mirror, each of the blue
and green signals are directed to a detector adapted to detect each
of the blue and green signals, respectively. Like the portion of
the detection signal comprising the blue and green signals, a
portion containing the red and infrared signals is directed through
an additional dichroic mirror that transmits the red emission
signal while reflecting the infrared signal. Each of the red and
infrared signals are then directed to detectors adapted to detect
the red and infrared emission signals, respectively. Although the
above embodiment is described as having four different emission
signals, each corresponding to a portion of the electromagnetic
spectrum, other numbers of distinct labels and thus emission
signals may be used by the detection system, as the present
invention is not limited to four distinct labels.
[0119] In one illustrative embodiment, the detection signal or
separated emission signals may be passed through an additional
bandpass filter, other than the polychroic mirror designed
specifically to remove excitation signals. In this manner, the
bandpass filter may serve to further reduce noise from the
detection or emission signal, thereby improving the quality of the
analysis performed by the detection system. Such a multiple
bandpass filter may be placed anywhere within the flow stream of
the detection signal. For instance, it may be placed immediately
downstream or upstream from one or more of the polychroic mirrors,
or it may be placed downstream from any one of the dichroic
mirrors. It is to be understood that it may even be omitted from
the detection system altogether as the invention is not limited to
including such a multiple bandpass filter. In one particular
embodiment, a bandpass filter with a high degree of rejection
(10,000 to 100,000 fold) is placed upstream of every detector such
that unwanted scattered light is suppressed or removed from the
optical signals before they are passed to the detectors.
[0120] Various types of detectors known to those of skill may be
used in detection systems of the present invention. In one
illustrative embodiment, each of the emission signals, after being
extracted from the detection signal, is directed to avalanche
photon detectors where photons are counted. The photon counts, or
lack thereof, may be used to determine whether a particular label
is present on a polymer at a given time. These counts may be
collected and measured relative to the time at which they are
collected. Alternatively, they may be collected for a given still
image within the detection zone at a known time. In other
embodiments, emission signals separated from a detection signal may
be directed to a CCD detector, where their intensity may be
detected about an array, either a linear array or a two dimensional
array, of the CCD device. The present invention is not limited to
any specific type of detector.
[0121] Having described several components that may be used in
embodiments of the detection system, such as dichroic mirrors,
polychroic mirrors, bandpass filters, detectors, emitters, and the
like, it is to be appreciated that they may be incorporated into a
detection system in various different schemes. However, aspects of
the present invention are not limited to the embodiments disclosed
and discussed above, as those of skill will recognize that
instrumentation may be substituted and signal processing schemes
may be altered without departing from various aspects of the
invention. For example, although many of the illustrative
embodiments have described a system having four channels, (i.e.,
four distinct labels, each associated with particular wavelengths
in the electromagnetic spectrum and its own channel in the
detection system), other systems having any number of channels are
also contemplated by the invention. For instance, a system having
two channels, three channels, or five or more channels are also
embraced by the present invention.
[0122] Overlapping Detection Zones
[0123] As described generally above, embodiments of the detection
system may be used to identify when a label bound to a polymer
passes through a detection zone and/or the nature of the label. The
time when the label passes through the detection zone, measured
with respect to other features of the polymer, may be used to
discern if, and where, a particular sequence is located on the
polymer. To this end, analysis of a polymer by the detection system
may involve passing polymers, with labels thereon, through the
detection zone within the sample area such that label(s) may be
detected.
[0124] In many embodiments of detection systems, multiple polymers
may be passed through a sample area simultaneously. This may be
done for a variety of reasons. For example, in some systems,
multiple polymers may be directed through the sample area and the
detection zone so that multiple assays may be performed in a single
test set up. In other embodiments, multiple polymers may be
directed through the sample area and towards a detection zone to
increase the probability of a particular polymer passing through
the zone. Still, multiple polymers may be directed toward the
detection zone for other reasons, as the invention is not limited
as to why multiple polymers may exist within the detection zone at
a given time.
[0125] As can be appreciated for at least the above described
reasons, it is advantageous in various embodiments of the invention
to direct multiple polymers to a detection zone. However, as can
also be appreciated, situations may arise where having multiple
polymers within the detection zone at the same time may complicate
the analysis of one or all of the polymers. For example, a
detection signal may end up containing emission signals from labels
of different polymers. Aspects of the present invention may be used
to identify which label and polymer the emission signals are from,
and in some cases, to eliminate unwanted emission signals.
[0126] Other aspects of the invention are useful for distinguishing
between emission signals that emanate from different spatial
regions of a detection zone. For example, multiple labels of the
same or different polymers may reside within different portions of
a detection zone at a common time. It may be desirable to isolate
each component of the emission signal corresponding to each of the
labels to discern their location within a detection zone.
Alternately, it may be desirable to eliminate one of the emission
signals from a detection signal (e.g., if the emission signal is
making it more difficult to discern the presence or type of another
label).
[0127] In one illustrative embodiment, multiple detection zones may
be used to distinguish the spatial origin of a portion of a
detection signal. For example, detection zones may be overlapped
where a first detection zone is located partially or entirely
within a second detection zone. Each detection zone is associated
with its own detection signal for downstream data processing. The
detection signals from each of the detection zones may be compared
with one another to discern which portions of the detection signals
are common to both detection zones and which are unique. Those
portions unique to either detection signals may derive from
non-overlapping portions of the corresponding detection zone.
Similarly, those portions common to both detection signals may
derive from common portions of the detection zones. In this manner,
such an approach may identify the spatial origin of portions of a
detection signal.
[0128] As previously defined, an excitation zone includes the light
emitted by an emitter, such as a laser, or combination of lasers.
In many embodiments, the excitation zone may be circular, as is
typical of laser beams. In other embodiments, the excitation zone
may comprise a substantially rectangular line disposed across the
sample area. It may also comprise multiple lasers beams each within
the excitation wavelengths of a label that is used within the
detection system. By way of example, the lasers may be those listed
in Table 1 above. However, it is to be appreciated that the
excitation zone is not limited to these lasers, or even to lasers,
as it may include any signals adapted to excite a label passing
within the excitation zone.
[0129] As previously discussed, labels passing through the
excitation zone may emit an emission signal that contributes to a
detection signal received by the system, particularly when the
excited label is within the detection zone. An objective lens 88
may be positioned to receive the detection signal emanating from
the detection zone and to magnify the detection signal for
subsequent processing. The objective lens may be part of the
microscope of the system. Additionally, other contents of the
excitation zone may emit signals and some portions of the
excitation signals may be reflected into the detection zone, toward
the objective lens as a part of the detection signal. In one
illustrative embodiment, the objective lens receives the detection
signal, including reflected excitation signals and other noise. The
objective lens then magnifies the signal between 60.times. and
100.times. for processing through a polychroic mirror and multiple
dichroic mirrors of the microscope to extract any emission signals
within the detection signal, as previously discussed. After
extracting emission signals from the detection signal, or
performing other optical signal processing steps, the emission or
detection signals are then passed through a confocal lens of the
microscope that may reduce the optical signal back towards its
original magnification, although other reductions are possible, or
no reductions may be made, as the present invention is not limited
in this regard.
[0130] In many embodiments, a confocal aperture 90 is used to
remove out of focus portions of the detection signal before the
signal is transmitted to a detector. The confocal aperture may be
placed at the focal point of the confocal lens and is often sized
to block any light not within the focal point. In some embodiments,
the confocal aperture may be sized smaller than the entire focal
point, such that it not only prevents out of focus light from
passing, but also prevents some in focus light from being
transmitted. In this regard, portions of the detection signal
associated with peripheral portions of the excitation zone, or
other portions of the excitation zone, may be prevented from
passing there through. In this manner, the confocal aperture may
also be used to define the size of detection zone within the sample
area. As is to be appreciated, the detection zone need not be the
same size or shape as the excitation zone, as the confocal aperture
may be sized and shaped to allow only select portions of signals to
be transmitted beyond the aperture of the microscope.
[0131] After emission signals have passed through the confocal
aperture, they are directed to an optical detector for conversion
into an electrical signal for downstream data processing. Although
many types of detectors may be used, some illustrative embodiments
include point detectors, such as avalanche photodiodes or
photomultiplier tubes, which convert the photons of the optical
signal into an electrical signal for data processing and analysis.
The invention is not limited to these point detectors, as other
point detectors, or even wide field image detectors may be used as
well.
[0132] In many embodiments, after passing though the confocal
aperture, the emission signals are directed through a fiber optic
cable to the detector for conversion into electric signals. For
example, after passing through the confocal aperture, the optical
signal may be directed onto a receiving end of a fiber optic cable
that carries the signal to the detector. The receiving end of the
fiber optic cable is typically sized and shaped consistent with the
confocal aperture and in some embodiments is fixed within the
confocal aperture, although it is not required to be in all
embodiments of the invention. Alternatively, in some other
embodiments, the receiving end of the fiber optic cable may
comprise the confocal aperture, as it may only transmit light that
is incident upon its receiving end, which may be a defined cross
section sized and positioned to reside within the focal point of
the confocal lens. In one illustrative embodiment, the confocal
aperture and the fiber optic cable have a circular cross section
with a 100 micron diameter that is associated with a 1 micron
detection zone (due to 100.times. magnification). In another
embodiment, the confocal aperture and fiber optic cable have a
circular cross section with a 50 micron diameter that is associated
with a 0.5 micron detection zone. However, the confocal aperture
may have any shape or size of cross section, as the present
invention is not limited in this respect.
[0133] In one illustrative embodiment, the detection system also
focuses the detection signal onto the receiving end of an
additional, smaller, confocal aperture and associated fiber optic
cable. In this regard, the smaller aperture focuses the same
detection signal onto a confocal aperture of a smaller size, and
thus defines a smaller detection zone that is completely overlapped
with a detection zone defined by a larger, confocal aperture that
receives the same detection signal. As with the detection signal of
the larger detection zone, the smaller detection signal is
transmitted to a detector through an optical fiber for subsequent
analysis and comparison with the signals transmitted by other
detectors within the system, as is discussed in greater detail
below. As is to be appreciated, although the above is described
with respect to a detection signal, the same may be accomplished
with an emission signal after it has been extracted from the
detection signal, or any other signals within a detection system,
as the invention is not limited in this respect.
[0134] In one illustrative embodiment, as described above, one
detection signal is separated into bands of the electromagnetic
spectrum that are each associated with the emission signals of
labels used in the system. Each of the bands may be passed through
a confocal aperture, such as a 100 micron confocal aperture, and
may then be transmitted to single point detector for converting the
optical signal into an electrical signal. Additionally, the
detection signal may also be delivered to a smaller circular,
confocal aperture having, for example, a 50 micron aperture
centered within the focal point of the focused detection signal. In
this manner, the 50 micron confocal aperture defines a 0.50 micron
detection zone centered within a 1.00 micron detection zone used to
provide emission signals to each channel of the detection system.
However, it is to be appreciated that the detection zone, and
sub-portions of the detection zone may be of any size and/or shape
as the invention is not limited to circular detection zones or
detection portions. Similarly, the detection zone need not overlap
at a central portion of either detection zone, as the present
invention is not limited to circular, concentric detection
zones.
[0135] In one illustrative embodiment, each channel of the
detection system may receive an emission signal corresponding to a
1.00 micron detection zone. Also, one separate detector may receive
a detection signal corresponding to a 0.50 micron detection zone
centered within the larger 1.00 micron detection zone. In this
manner, the data processor will receive a signal associated with
the smaller detection zone that can be compared with any of the
emission signals coming from the larger detection zone. If any of
the emission signals are suspected of containing unwanted
components that emanate from portions of the larger detection zone
not also common with the smaller detection zone, they can be
compared with the detection signal associated only with the smaller
zone. If the suspect portions of the emission signals are common to
both of the detection zones, it may be inferred that they emanate
from within the smaller detection zone. However, if the suspect
portions of an emission signal are not also present in the
detection signal coming from the smaller detection zone, then it
may be inferred that the suspect portions emanate from points of
the larger detection zone that are not common with the smaller
detection zone. The data processor may then decide whether to
reject these suspect portions of the emission signal.
[0136] As discussed briefly above, labels may emit an emission
signal having characteristics that are dependent on the location of
the label within the detection zone. For example, some detection
zones may be illuminated by a Gaussian laser beam having
intensities that vary spatially across a detection zone. A label
located in an area of the detection zone having a higher intensity
may emit a more intense emission signal. In other embodiments, such
as in detection systems where labels are moving relative to the
detection zone during analysis, the emission signal emitted by a
label may be dependent upon the path the label travels through the
detection zone. Such characteristics of an emission signal, taken
with respect to the detection zone, may be used by the present
invention to improve analysis of the label and thus any polymer it
is bound to.
[0137] FIG. 11 shows a three dimensional view of laser intensity as
it varies with position across a laser beam in one illustrative
embodiment. Here, the intensity of the laser follows a
substantially Gaussian, two dimensional distribution about its
circular cross section. It is to be appreciated that other
intensity distributions may also exist, as the Guassian
distribution shown in FIG. 11 is merely exemplary of how
intensities may vary with position in the cross section of a laser
beam, or other excitation signals.
[0138] The intensity of an emission signal emitted by a label may
vary with the intensity of the excitation signal that it is exposed
to. In this regard, a label that is disposed within the low
intensity, peripheral portion of an excitation signal, like that
represented by FIG. 11 may emit an emission signal of a lower
intensity. This is represented in FIG. 12, which shows separate
emission signals 86, 88, superimposed on the same graph, of labels
located in both a high intensity region and a low intensity region
of an excitation signal.
[0139] Illustrative embodiments of the detection system are often
characterized by a system noise level. Many factors may contribute
to the noise level of the system, such as imperfections in the
optical instrumentation, impurities in the polymers and/or labels,
imperfections in the electronics, disturbances from the ambient
atmosphere, and the like. Most systems will be less capable of
distinguishing signals, such as emission signals, that are closer
to the system noise level, and thus have a lower signal to noise
ratio. The emission signal of a label located in the low intensity
region of the laser 86, as shown in FIG. 12, is much closer to the
noise level, and thus more difficult for the system to recognize.
Additionally, the emission signal, if not properly recognized, may
actually be contributing to the noise level of the system in some
embodiments, thus making it more difficult to detect other labels
residing in the detection zone.
[0140] In one illustrative embodiment, the emission signals
detected by the system include information that is dependent on the
path traveled by the label. In many detection systems, the polymers
are moved relative to the detection zone while being detected. Some
systems may not be able to control the precise placement of the
polymer within a detection zone. As such, polymers may pass through
a central portion of the detection zone along a path that spans a
distance substantially equal to the diameter of the detection zone
(for a circular detection zone) and thus pass through a significant
portion of the high intensity region. However, some of the polymers
may only pass through the detection zone along a chordal line,
entering the high intensity region only briefly, if at all. Such
path dependence may add to the variability within some systems,
thereby adding to the relevance of aspects of the invention used to
distinguish between labels passing along only a peripheral edge of
a detection zone.
[0141] The emission signal emitted by a label may also be dependent
on the amount of time the label is resident within the detection
zone. As such, the length of the path of travel that a label
follows through a detection zone affects the emission signal in
addition to the intensity of the detection zone that it passes
through. For example, a label following a diametral path line
through a circular detection zone will reside in the detection zone
longer than a label following a chordal path through the detection
zone, all else being constant. The label residing within the
detection zone longer will generally emit more photons than a label
residing in the detection zone for a shorter period of time. This
presents yet another factor that can cause variability, and thus
uncertainty, in analyzing which emission signals are within a
detection signal.
[0142] In one illustrative embodiment, emission signals are
analyzed within the detection signal at the data processing stage
by evaluating the number of peaks within the detection signal at
each intensity level. FIG. 13 shows a graph representative of such
an analysis. Here, the peaks are binned according to their
intensity level and are then plotted as a histogram. Fitting data
from a detection signal in this manner may help distinguish the
emission signals from noise within the system. However, as can also
be appreciated, labels residing within the low intensity regions of
the excitation signal may increase the number of low intensity
peaks and confuse the analysis. For example, the low intensity
peaks may distort the expected distribution of an emission signal
from the expected form shown on FIG. 13 to one like that
represented in FIG. 14, which includes additional low intensity
peaks. This can cause, in some instances, a misidentification of an
emission signal. For example, contributions from a label within the
low intensity portion of a detection zone may appear to be part of
an emission signal that resides on a polymer within the high
intensity region of the detection zone. As is to be appreciated,
this may cause the structure of the polymer within the high
intensity region to be interpreted incorrectly.
[0143] As previously discussed, illustrative embodiments of the
invention are adapted to provide a first detection signal from a
first detection zone 90 and a second detection signal from a
second, smaller detection zone 92 that is overlapped with the first
detection zone as shown by FIG. 15. The signals are passed to
detectors, typically a point detector like an avalanche photodiode,
which convert them into electrical signals of signal intensity
versus time as represented by FIG. 16. As can be seen in FIG. 16,
the peaks represent periods of high intensity, likely associated
with a label being present within the detection zone.
[0144] The signal represented in FIG. 16 also displays a level of
noise 98, varying in a stochastic manner with time. As previously
discussed, the noise in the system may mask, or make the detection
of emission signals more difficult. In some cases it may also cause
an emission signal to be incorrectly identified. To address this,
much of the noise is filtered out of the signals through filtering
techniques previously discussed and as otherwise known to those of
skill in the art. Additionally, in some embodiments, a threshold
detection level is set such that any peaks below a particular
intensity are ignored by the system.
[0145] As can be appreciated, a label passing through a circular
detection zone along a diametral line, at a known speed, will
produce an emission signal having a shape that may be anticipated
in some illustrative embodiments. Additionally, in some
embodiments, portions of the time varying signal may be analyzed to
better understand whether a label is present and what type of label
it might be. One analysis technique involves making a histogram of
the peaks of a channel or channels over a given period of time,
binned with respect to peak intensity, much like the histograms
represented by FIGS. 13 and 14. As is to be appreciated, the
distribution of peaks for a label passing along a diametral line of
the detection zone should show a distribution somewhat like that of
FIG. 13. Here, the highest intensity peaks associated with the
label being in the most intense portion of the detection zone,
typically the center, are few in number. The mid-level intensity
points are the greatest in number and are generally associated with
the label being disposed in central portions of the detection zone,
but not the center most portion of the highest intensity. The
lowest intensity portions of the histogram are associated with the
label residing in the peripheral portions of the detection zone,
which have the lowest intensity.
[0146] As is to be appreciated, the presence of labels 94 on a
second polymer 96 or of an unbound label within the detection zone
may complicate the analysis of a first label 94 on a first polymer
96 within the zone. This may be the case particularly where the
second label is in a peripheral portion of the detection zone. FIG.
14 shows a histogram of peaks sorted by intensity where the
detection zone has a label passing along a substantially diametral
line and a second label passing along a chord across a peripheral
portion of the detection zone. FIG. 14 deviates from the expected
histogram shown in FIG. 13 due to the additional, low intensity
peaks that are contributed by the label passing through the
peripheral portion of the detection zone.
[0147] In one illustrative embodiment of the invention, the
contributions of the second label passing through the detection
zone along a chord near the periphery are captured in a detection
signal associated with a larger detection zone, but not a detection
zone associated only with a smaller, central portion of the larger
detection zone. FIG. 17 illustrates an overlay of histograms for
peaks witnessed in the larger detection zone, and in the smaller
detection zone. Here, coincident peaks are associated with labels
passing through the common portions of both detection zones. In
this regard, the portions of the detection signal associated with
the second label passing along a peripheral portion of the larger
detection zone may be identified and either separated or ignored,
as desired.
[0148] The above described analysis may be used by embodiments of
the detection system selectively at times when a second polymer is
suspected of residing in a portion of the detection zone. In this
regard, the data points that may be unclear to the system, or
identifications of labels that are made with a lower degree of
confidence, may be checked by the above described methods to verify
the results and/or increase the level of confidence. In other
illustrative embodiments, the detection signal from a larger
detection zone may be continually used as a strategy to reduce the
impact of noise within the detection system. In this manner, only
peaks that are coincident between both detection zones may be
reviewed for the presence or absence of labels. Thus, this scheme
can be used to reduce the effects of systematic noise within the
system, instead of or in addition to determining whether a portion
of a signal is associated with a disturbance, such as a label of a
second polymer lying within a peripheral portion of the detection
zone.
[0149] Systems Using Wide Field Imaging Detectors
[0150] As illustrated above, many embodiments of the present
invention accomplish sensitivity to individual labels within the
detection zone with the assistance of confocal optics. In such
systems, the entire contents of a detection zone may be excited
using an excitation signal, such as a laser beam. A detection
signal that emanates from the detection zone is processed by
various optical signal processing instruments in a confocal
microscope and is then transmitted to a detector where it is
translated into an electrical signal for analysis by a data
processor. Before the optical signal is passed to the detector, it
is passed through a confocal aperture, which may remove out of
focus light that might otherwise be introduced to the detector as
noise. However, the confocal aperture also limits the view of the
system to the focal point, or a subsection of the focal point.
[0151] It is also to be appreciated that some embodiments of
detection systems illuminate an excitation zone that includes
elements, other than labels, that react to the excitation signal.
Some of these elements, such as carrier fluid that may surround a
polymer in the sample area, may also alter any emission signal
produced by contributing noise or other disturbance. For example,
in some detection systems there are elements that lie within the
excitation zone that may fluoresce, not unlike the labels that are
bound to the polymer. Such fluorescence, although not typically as
strong as that of the label, may act as noise in the detection
signal. Also, the photons of the excitation signal, or even of the
emission signals emanating from the labels, may cause Raman
scattering throughout the carrier fluid that the polymer resides
in. Raman scattering involves photons contacting molecules and
bouncing off the molecules with energy at the same wavelength as
the incident photon (elastic scattering) or with slightly less
energy (inelastic scattering) than the incident photon and a
corresponding different wavelength.
[0152] In an illustrative embodiment, polymers are provided to the
detection zone where they are processed in a linear fashion. Only
the polymers located within a relatively small detection zone,
e.g., 1.00 micron in some embodiments, are detected and analyzed.
This allows polymers to be passed through the detection zone in a
relatively rapid manner and may result in relatively fast data
processing. The processing speed may also be relatively fast due to
the use of point detectors, which often process signals quicker
than wide area detectors. Microfluidic devices may be used in
detection systems to provide polymers to a detection zone in such a
manner. Examples of such microfluidic devices are disclosed in U.S.
patent application Ser. No. 10/821,664, filed on Apr. 19, 2004,
which is hereby incorporated by reference in its entirety.
[0153] In other illustrative embodiments, a detection zone may be
moved over, or scanned about a sample area. In this manner, the
detection system can gather a larger image of the sample area by
piecing together the image from all that was detected over the
entire area. However, this may require the polymer to be stationary
during the scanning process, which, in turn may increase the time
it takes to perform an analysis.
[0154] Other approaches are available for imaging an entire sample
area, or substantial portions thereof. In one illustrative
embodiment, wide field imaging detectors may be used to image the
entire sample area instead of scanning a single, confocal detection
point about the sample area. CCD detectors or Complimentary Metal
Oxide Semiconductor detectors (CMOS) are examples of detectors that
may be used to accomplish this in some embodiments of the
invention. Use of such wide field imaging devices allows a much
larger portion of the sample area to be imaged at the same time.
However, since such wide field imaging devices are typically taking
in a greater amount of information representing a larger detection
zone, they typically take longer to process an imaged signal.
Nonetheless, they are still capable of detecting dynamic processes.
In this regard, they can be used for dynamic detection of polymers
passing through an entire sample area, or portions thereof.
[0155] CCDs and CMOS detectors are examples of wide-field imaging
devices that may be used as detectors within embodiments of the
present invention. A CCD is an array of photosensitive elements,
where each element is capable of generating an electrical response
to photons that are incident upon it. Each element may be referred
to as a pixel and is typically a square having side dimensions
between 20 and 30 microns. The pixels of the CCD collect photons
that are incident upon them and convert them to electrical charges
representative of the number of photons counted. The charges are
then passed along a first direction of the two-dimensional array of
pixels until all of the charges are represented in a single linear
array of the CCD. After all of the counts are collected in this
single array, they are passed into a corner of the two-dimensional
array (i.e., an end of the linear array) where they may be passed,
in turn, to the data processor. The data processor interprets the
signal provided by the CCD and may reconstruct it as an array
representing photon counts at each of the pixels over the entire
area of the CCD. As may be appreciated, the processing time for a
detection system that uses a CCD, or other type of wide field
imaging device, may be substantially greater than a system that
uses a point detector due to the additional, above-described
processing steps. It is to be appreciated that although a CCD has
been discussed as an exemplary wide field imaging device, other
devices known to those in the art, such as CMOS detectors and
others may also be used.
[0156] Using wide field imaging devices may require larger
excitation zones 106 to be excited by an emitter. In many
embodiments, this can be accomplished by passing an excitation
beam, such as a laser beam or beams, through spherical lenses that
spread the beam over a greater cross sectional area. However, other
methods and devices can be used to illuminate larger portions of
the sample area as will be appreciated by those of skill, as the
present invention is not limited in this regard. In many
embodiments, illuminating a larger portion of the sample area may
cause an increase in noise contributions to detection signals. For
example, it may introduce additional noise from Raman scattering,
among other sources of noise. Although the confocal systems may
also be subject to such noise, such systems may use a confocal
aperture to reduce its effects. Most wide field detectors are not
compatible with such confocal operations. As such, detection
systems of the present invention may employ other methods to reduce
contributions to the detection signal from areas outside of the
focal plane.
[0157] A schematic representation of both a point detection system
and wide field imaging system are shown in FIG. 18. As can be seen,
the detection zone of a wide field imaging system may be much
larger than that of a point detection system. In the wide field
imaging systems, a confocal aperture would need to be much larger
to allow in focus light of the detection zone to pass to a
detector. However, in many embodiments, using such a confocal
aperture would only reduce a portion of the out of focus
contribution. Such an aperture might not be able to reduce out of
focus portions of the detection signal that are associated with
portions of the sample area are lying in different vertical planes,
both above and below the focal plane, which may be significantly
greater than in systems using a point detector. As such, out of
focus noise may be a problem to some embodiments of such detection
systems.
[0158] To help reduce out of focus noise and prevent it from
adversely affecting the ability of a detection system to detect
emission signals, only portions of the sample area that lie within
the in-focus portions of a sample area may be illuminated by the
excitation signal. In this manner, very little, if any, out of
focus elements are illuminated and thus these are prevented from
contributing noise to the detection signal.
[0159] Various approaches can be used to limit illumination to the
focal plane of the detection zone. In one illustrative embodiment,
the sample area comprises a thin sheet of buffer solution 100
containing polymers and is surrounded by essentially noise free
medium that will not react to an excitation signal. For example,
the sample area may comprise a thin passage of a microfluidic
channel, as represented by the channel shown in FIG. 19. The
microfluidic channel may be defined by walls of glass formed of
fused silica, which is a medium that produces little, if any, noise
or emission signals when illuminated by an excitation signal. In
one embodiment, the channel has a depth of 0.50 microns, a width of
50 microns, and a length of 50 microns. In another embodiment, the
channel has a depth of 0.10 microns. Still, in another embodiment,
the channel may have a depth of 0.080 microns or smaller. These
channels may be manufactured through etching techniques or other
processes known to those of skill, such as soft lithography and the
like. In these embodiments, a carrier solution containing polymers
flows through a microchannel and ultimately through the sample
area. The entire microchannel is not required to have a thin cross
sectional area like the sample area, but may transition to such a
cross section, as shown in FIG. 19. Various other aspects of
microfluidic systems, like those described in U.S. patent
application Ser. No. 10/821,664, filed Apr. 19, 2004, which is
hereby incorporated by reference in its entirety, may be employed
by detection systems of the present invention.
[0160] In another illustrative embodiment, the sample area is only
illuminated in the focal plane as the excitation signal is directed
only to points within the focal plane. In one illustrative
embodiment a sheet of light 102 may be projected into the sample
area such that it lies substantially orthogonally to the detector
entirely within the focal plane. Such a thin sheet of illumination
may be formed through various methods. One such method includes
directing an excitation signal, such as a coherent laser beam or
collection of laser beams, through a cylindrical optical lens 104
that spreads the beam substantially in a single dimension. In such
a system, the cylindrical lens may be positioned to direct the
excitation signal, in the form of a thin sheet, into the sample
area, such as a microfluidic channel, while the detector is
positioned above the sample area. One such embodiment is shown in
FIG. 20.
[0161] As can be appreciated, in embodiments where illumination by
the excitation signal is limited to the focal plane, physical
constraints about the polymer or the carrier fluid containing the
polymer, like a thin microfluidic channel, are not required.
However, many embodiments may couple some physical constraints with
illumination of the sample area in this manner to help direct
polymers into the illumination plane. Otherwise, in some
embodiments, polymers pass by the detection zone without being
illuminated.
[0162] As is to be appreciated, the above described embodiments of
excitation signals may employ any of the previously described
aspects of the detection system. For example, the excitation signal
may comprise multiple lasers each adapted to excite a particular
label used within the detection system. In one such embodiment, the
lasers and labels described in Table 1 may be used, although others
may be used in addition to, or in place of these as well. Also, the
optical signal processing techniques for separating various
emission signals of the detection signal may be combined with
detection systems using wide field detectors, as the present
invention is not limited in this regard. For instance, the system
of polychroic and dichroic mirrors may be used to divide the
detection signal into different channels, each associated with a
particular portion of the electromagnetic spectrum.
[0163] As is to be appreciated, single point detectors, such as
avalanche photodiodes or photomultipliers, may not be best suited
to capture all of the information in a wide field image. As such,
wide field imaging detectors 108, such as CCD's or CMOS detectors,
may replace the single point detectors when such an approach is
used. In one illustrative embodiment, a plurality of detectors used
for each channel of a multi-channel detection system is replaced
with a single, wide field imaging detector. As is to be
appreciated, wide field imaging detectors are often more expensive
than single point detectors. In this regard, it may be desirable to
reduce the number of wide field detectors used in a system to also
reduce the overall system cost. However, the wide field imaging
detector, in many embodiments, should be capable of discerning
different wavelengths of a detection signal in order to distinguish
various emission signals contained within the detection system.
[0164] In one illustrative embodiment, different portions of the
detection signal associated with different portions of the
electromagnetic spectrum are projected onto different spatial
portions of the wide field imaging detector to allow them to be
distinguished from one another. A system like that shown in FIG. 21
may accomplish this through the use of dichroic mirrors. FIG. 21
shows a wide field image being transmitted through a series of
dichroic mirrors that divide the image based on the response curve
of the given mirror, as previously discussed. In this manner, a
first mirror may be used to extract an emission signal from the
detection signal. In one embodiment, the detection signal is passed
through a first dichroic mirror which removes a spectral band
associated with an emission signal having the shortest wavelength
(e.g., the blue label). The remaining portions of the detection
signal are then transmitted to a second dichroic mirror which
reflects the next larger wavelength band, which may be associated
with another label (e.g., the green label). The process continues
until all wavelength bands have been separated from the detection
signal. As is to be appreciated, the manner in which the emission
signals are separated from the detection signal may be performed
according to other schemes, and other signal processing steps may
be performed in between, before, or after separation steps, as the
aforementioned process is merely exemplary for how emission signals
may be separated from the detection signal.
[0165] As previously mentioned, after each of the separated
emission signals, or similarly, the bands of wavelengths in which
emission signals may be expected to reside, have been extracted
from the detection signal, they are each directed onto a portion of
a wide field imaging device as depicted in FIG. 21. Each band
associated with a potential emission signal is projected onto the
wide field imaging detector, such as a CCD, in a designated portion
of the detector. The whole image detected by the CCD is converted
to an electrical signal for subsequent data processing to
understand whether a label is present and, if so, what type of
label is present in the sample area at a given time. Although the
signal transmitted by the CCD will suggest that various different
polymers were present at different positions, corrections can be
made during data processing to correct for the offset. In most
embodiments, the offset is defined by the detection system setup;
therefore correcting for the positions is made simpler as the valve
remains constant for a system.
[0166] Linear Imaging Detector
[0167] As previously discussed, some detection systems use point
detectors and confocal optics to detect emission signals, while
others utilize a wide field imaging approach. There are benefits to
each type of system. For example, point detection systems may
collect detection and/or emission signals very rapidly, allowing
for reduced assay time. Additionally, they can focus in on a small
detection zone while filtering out noise from out of focus areas by
passing the detection signal through a confocal aperture, or by
using other noise reduction approaches. Although the point
detection systems may be used, in some embodiments to collect data
from a wide area, this may require scanning of the sample area,
which may be difficult if polymers are passing through the sample
area in a dynamic manner.
[0168] Wide field image detectors may capture a view of a much
larger area in a single step. However, the rate at which images are
collected and processed is typically slower than that of a single
point detection system. The wide field imaging systems provide a
larger target area to capture polymers that may be passing through
the sample area. In this manner, it is easier to extend a detection
zone across a greater portion of a sample area, if not all of the
sample area. Here, the possibility of a polymer passing through a
sample area undetected is reduced, if not eliminated, because the
detection zone may be able to cover all potential locations where a
polymer may reside. Additionally, although approaches may be taken
to remove out of focus noise from the detection signal, these
systems do not generally allow the use of a confocal aperture to
remove out of focus noise from the detection signal as previously
discussed.
[0169] Some embodiments of detection systems optimize between
benefits of both point detection systems and wide field imaging
systems by utilizing a detection zone that is extending in only one
dimension. For example, in one illustrative embodiment, the
detection zone extends in a linear array from one end of the sample
area to the opposite end of the sample area. A linear CCD array 110
is one type of detector that may be used in this manner. A linear
CCD array is constructed much like a two-dimensional CCD array and
shares many of its characteristics; however, the linear CCD array
has only a single row of photo receptors instead of a
two-dimensional array of photo receptors. As such, the processing
time associated with a linear CCD array is typically much faster
than that of a two-dimensional CCD array, although it might not be
as fast as that of a single point detector. As an example, some
linear CCD arrays can read out data at the rate of 10,000 lines per
second.
[0170] In one illustrative embodiment of the invention, a linear
CCD array is extended across an entire path where polymers may be
expected to pass. For instance, this arrangement may be used in
embodiments of microfluidic detection systems. Here, the linear
array may be used with an optical microscope to provide a detection
zone that extends across a microfluidic channel in a direction
substantially orthogonal to the flow of the carrier fluid in the
system. In this manner, polymers residing within the carrier fluid
will likely be detected as they pass through the detection zone.
This may not be the case in embodiments using confocal microscopy
where the detection zone is likely to be substantially smaller
(e.g., it may be smaller than the distance across the sample area
of the microfluidic channel).
[0171] Although systems using the linear CCD array may not lend
themselves to use with a confocal aperture to reduce out of focus
noise, such noise is less likely to be a problem in such systems.
Since the linear CCD array may not require an excitation zone that
is as large as a two-dimensional CCD array, not as much surrounding
media will be excited. In this regard, there will be less material
excited to contribute noise to the detection signal, thus reducing
the need for a confocal aperture.
[0172] As described above, the excitation zone may be smaller than
those of detection systems using a two-dimensional CCD array for
other wide area imaging devices. For this reason, the excitation
zone may be provided by a thin sheet of illumination, like that
described above, which can be created using cylindrical lenses.
This sheet of light may contain multiple, different lasers or a
single laser, as the invention is not limited in this regard. In
embodiments where multiple excitation signals are included within a
single sheet of illumination, the above-described signal processing
schemes may be used to separate emission signals from a detection
signal that is received by the detection zone of the linear CCD
array. Similarly, other schemes may be used as the invention is not
limited in this respect.
[0173] Although embodiments using linear array detectors may not be
amenable to the use of a confocal aperture to remove out of focus
noise from the system, the same approaches discussed above with
respect to removing noise from wide field imaging detection systems
may be employed. For example, only the focal plane may be
illuminated by the excitation signal(s) or the sample area may be
contained to a very thin region with the surrounding features
comprising essentially noise-free media. In this regard, noise may
be dealt with adequately by embodiments using linear array
detectors.
[0174] The methods of the invention can be used to generate unit
specific information about a polymer by capturing polymer dependent
impulses from the polymer using the devices described herein and
elsewhere to manipulate the polymer. As used herein the term "unit
specific information" refers to any structural information about
one, some, or all of the units of the polymer. The structural
information obtained by analyzing a polymer may include the
identification of characteristic properties of the polymer which
(in turn) allows, for example, for the identification of the
presence of a polymer in a sample or a determination of the
relatedness of polymers, identification of the size of the polymer,
identification of the proximity or distance between two or more
individual units of a polymer, identification of the order of two
or more individual units within a polymer, and/or identification of
the general composition of the units of the polymer. Since the
structure and function of biological molecules are interdependent,
the structural information can reveal important information about
the function of the polymer. As used herein "analyzing a polymer"
means obtaining information about the structure of the polymer,
including but not limited to the information recited above.
[0175] A "polymer dependent impulse" as used herein is a detectable
physical quantity which transmits or conveys information about the
structural characteristics of a unit of a polymer. The physical
quantity may be in any form which is capable of being detected. For
instance the physical quantity may be electromagnetic radiation,
chemical conductance, electrical conductance, etc. The polymer
dependent impulse may arise from energy transfer, quenching,
changes in conductance, radioactivity, mechanical changes,
resistance changes, or any other physical changes.
[0176] The method used for detecting the polymer dependent impulse
depends on the type of physical quantity generated. For instance if
the physical quantity is electromagnetic radiation, then the
polymer dependent impulse is optically detected. An "optically
detectable" polymer dependent impulse as used herein is a light
based signal in the form of electromagnetic radiation which can be
detected by light detecting imaging systems. In some embodiments
the intensity of this signal is measured. When the physical
quantity is chemical conductance, then the polymer dependent
impulse is chemically detected. A "chemically detected" polymer
dependent impulse is a signal in the form of a change in chemical
concentration or charge such as ion conductance which can be
detected by standard means for measuring chemical conductance. If
the physical quantity is an electrical signal, then the polymer
dependent impulse is in the form of a change in resistance or
capacitance. These types of signals and detection mechanisms are
described in U.S. Pat. No. 6,355,420 B1.
[0177] The polymer dependent impulses may provide any type of
structural information about the polymer. For instance these
signals may provide the entire or portions of the entire sequence
of the polymer (e.g., by the order of polymer dependent
impulses).
[0178] A "polymer" as used herein is a compound having a linear
backbone of individual units which are linked together by linkages.
In some cases, the backbone of the polymer may be branched.
Preferably the backbone is unbranched. The term "backbone" is given
its usual meaning in the field of polymer chemistry. The polymers
may be heterogeneous in unit and backbone composition. In one
embodiment the polymers are, for example, nucleic acids,
polypeptides, polysaccharides, or carbohydrates. In the most
preferred embodiments, the polymer is a nucleic acid or a
polypeptide. A polypeptide as used herein is a biopolymer comprised
of linked amino acids.
[0179] The invention can be applied to various forms of nucleic
acids including DNAs and RNAs. Examples of DNAs include genomic
DNA, mitochondrial DNA or cDNA. In some important embodiments, the
nucleic acid is not amplified in vitro prior to analysis. It may
however be a nucleic acid that has been amplified in vivo (e.g., in
a subject). Examples of RNAs include but are not limited to
messenger RNA (mRNA), ribosomal RNA (rRNA), microRNA (miRNA), small
interfering RNA (siRNA), and the like. MicroRNA is a class of
noncoding RNAs generally about 22 nucleotides in size that are
believed involved in the regulation of gene expression. siRNA is a
double stranded RNA involved in RNA interference. It reportedly
induces the formation of a ribonucleoprotein complex, which in turn
mediates sequence-specific cleavage of a transcript target. It is
to be understood that miRNA and siRNA can be used as either targets
or as probes in the invention.
[0180] The term "nucleic acid" is used herein to mean multiple
nucleotides (i.e., molecules comprising a sugar (e.g., ribose or
deoxyribose) linked to an exchangeable organic base, which is
either a substituted pyrimidine (e.g., cytosine (C), thymidine (T)
or uracil (U)) or a substituted purine (e.g., adenine (A) or
guanine (G)). "Nucleic acid" and "nucleic acid molecule" are used
interchangeably, and used to refer to oligoribonucleotides as well
as oligodeoxyribonucleotides. The terms shall also include
polynucleosides (i.e., a polynucleotide minus a phosphate) and any
other organic base containing polymer. Nucleic acids can be
obtained from existing nucleic acid sources (e.g., genomic or
cDNA), or by synthetic means (e.g., produced by nucleic acid
synthesis). The nucleic acids can include substituted purines and
pyrimidines such as C-5 propyne modified bases (Wagner et al.,
Nature Biotechnology 14:840-844, 1996). Purines and pyrimidines
include but are not limited to adenine, cytosine, guanine,
thymidine, 5-methylcytosine, 2-aminopurine, 2-amino-6-chloropurine,
2,6-diaminopurine, hypoxanthine, 2-thiouracil, pseudoisocytosine,
and other naturally and non-naturally occurring nucleobases,
substituted and unsubstituted aromatic moieties. Other such
modifications are well known to those of skill in the art.
[0181] The nucleic acids may also encompass substitutions or
modifications, such as in the bases and/or sugars. For example,
they include nucleic acids having backbone sugars which are
covalently attached to low molecular weight organic groups other
than a hydroxyl group at the 3' position and other than a phosphate
group at the 5' position. Thus, modified nucleic acids may include
a 2'-O-alkylated ribose group. In addition, modified nucleic acids
may include sugars such as arabinose instead of ribose. Thus the
nucleic acids may be heterogeneous in backbone composition. In some
embodiments, the nucleic acids are homogeneous in backbone
composition.
[0182] As used herein with respect to linked units of a polymer,
"linked" or "linkage" means two entities are bound to one another
by any physicochemical means. Any linkage known to those of
ordinary skill in the art, covalent or non-covalent, is embraced.
Natural linkages, which are those ordinarily found in nature
connecting the individual units of a particular polymer, are most
common. Natural linkages include, for instance, amide, ester and
thioester linkages. The individual units of a polymer analyzed by
the methods of the invention may be linked, however, by synthetic
or modified linkages. Polymers in which the units are linked by
covalent bonds will be most common but may also include hydrogen
bonded units are also embraced by the invention.
[0183] The polymer is made up of a plurality of individual units.
An "individual unit" as used herein is a building block (e.g., a
monomer) which can be linked directly or indirectly to other
building blocks or monomers to form a polymer. The polymer
preferably is a polymer of at least two different linked units. The
at least two different linked units may produce or be labeled to
produce different signals.
[0184] The "label" or "detectable moiety" may be, for example,
light emitting, energy accepting, fluorescent, radioactive, or
quenching as the invention is not limited in this respect. Many
naturally occurring units of a polymer are light emitting compounds
or quenchers, and thus are intrinsically labeled. Guidelines for
selecting the appropriate labels, and methods for adding extrinsic
labels to polymers are provided in more detail in U.S. Pat. No.
6,355,420 B1.
[0185] It is to be understood that a polymer that is said to "have"
a label is a polymer that may have a label intrinsically as a part
of the polymer. It is also to be understood that a polymer that is
said to "have" a label may be a polymer that is bound to an
extrinsic element that comprises the label, such as a fluorophor, a
radio opaque marker, and the like.
[0186] Some detectable moieties can be detected directly by their
ability to emit and/or absorb light of a particular wavelength.
Other detectable moieties can be detected indirectly by their
ability to bind, recruit and, in some cases, cleave another moiety
which itself may emit or absorb light of a particular wavelength.
An example of indirect detection is the use of a first enzyme label
which cleaves a substrate into visible products. The label may be
chemical, peptide or nucleic acid nature, although it is not so
limited. Detectable moieties may be conjugated to a polymer using
thiol, amino or carboxylic groups. Because it may be desirable to
attach as many detectable labels to the polymer or to a polymer
binding molecule (e.g., a unit specific marker or probe), such
labels may be attached to amino or carboxylic groups which are
common on proteins.
[0187] The detectable moieties described herein are referred to
according to the systems by which they are detected. As an example,
a fluorophore molecule is a molecule that can be detected using a
system of detection that relies on fluorescence.
[0188] Generally, the detectable moiety can be selected from the
group consisting of an electron spin resonance molecule (such as
for example nitroxyl radicals), a fluorescent molecule, a
chemiluminescent molecule, a radioisotope, an enzyme substrate, a
biotin molecule, a streptavidin molecule, a peptide, an electrical
charge transferring molecule, a semiconductor nanocrystal, a
semiconductor nanoparticle, a colloid gold nanocrystal, a ligand, a
microbead, a magnetic bead, a paramagnetic particle, a quantum dot,
a chromogenic substrate, an affinity molecule, a protein, a
peptide, nucleic acid, a carbohydrate, an antigen, a hapten, an
antibody, an antibody fragment, and a lipid.
[0189] As used herein, the terms "charge transducing" and "charge
transferring" are used interchangeably.
[0190] Examples of detectable labels include radioactive isotopes
such as P.sup.32 or H.sup.3, epitope tags such as the FLAG or HA
epitope, and enzyme tags such as alkaline phosphatase, horseradish
peroxidase, .beta.-galactosidase, etc. Other labels include
chemiluminescent substrates, chromogenic substrates, fluorophores
such as fluorescein (e.g., fluorescein succinimidyl ester), TRITC,
rhodamine, tetramethylrhodamine, R-phycoerythrin, Cy-3, Cy-5, Cy-7,
Texas Red, Phar-Red, allophycocyanin (APC), etc. Also envisioned by
the invention is the use of semiconductor nanocrystals such as
quantum dots, described in U.S. Pat. No. 6,207,392 as labels.
Quantum dots are commercially available from Quantum Dot
Corporation.
[0191] In some embodiments, the polymers are labeled with
detectable moieties that emit distinguishable signals that can all
be detected by one type of detection system. For example, the
detectable moieties can all be fluorescent labels or radioactive
labels. In other embodiments, the polymers are labeled with
moieties that are detected using different detection systems. For
example, one unit of a polymer may be labeled with a fluorophore
while another may be labeled with radioactivity.
[0192] In one embodiment, analysis of the polymer involves
detecting signals from the labels (potentially through the use of a
secondary label, as the case may be), and determining the relative
position of those labels relative to one another. In some
instances, it may be desirable to further label the polymer with a
standard marker that facilitates comparing the information so
obtained with that from other polymers analyzed. For example, the
standard marker may be a backbone label, or a label that binds to a
particular sequence of nucleotides (be it a unique sequence or
not), or a label that binds to a particular location in the nucleic
acid molecule (e.g., an origin of replication, a transcriptional
promoter, a centromere, etc.).
[0193] One subset of backbone labels for nucleic acids are nucleic
acid stains that bind nucleic acids in a sequence independent
manner. Examples include intercalating dyes such as phenanthridines
and acridines (e.g., ethidium bromide, propidium iodide, hexidium
iodide, dihydroethidium, ethidium homodimer-1 and -2, ethidium
monoazide, and ACMA); minor grove binders such as indoles and
imidazoles (e.g., Hoechst 33258, Hoechst 33342, Hoechst 34580 and
DAPI); and miscellaneous nucleic acid stains such as acridine
orange (also capable of intercalating), 7-AAD, actinomycin D,
LDS751, and hydroxystilbamidine. All of the aforementioned nucleic
acid stains are commercially available from suppliers such as
Molecular Probes, Inc. Still other examples of nucleic acid stains
include the following dyes from Molecular Probes: cyanine dyes such
as SYTOX Blue, SYTOX Green, SYTOX Orange, POPO-1, POPO-3, YOYO-1,
YOYO-3, TOTO-1, TOTO-3, JOJO-1, LOLO-1, BOBO-1, BOBO-3, PO-PRO-1,
PO-PRO-3, BO-PRO-1, BO-PRO-3, TO-PRO-1, TO-PRO-3, TO-PRO-5,
JO-PRO-1, LO-PRO-1, YO-PRO-1, YO-PRO-3, PicoGreen, OliGreen,
RiboGreen, SYBR Gold, SYBR Green I, SYBR Green II, SYBR DX,
SYTO-40, -41, -42, -43, -44, -45 (blue), SYTO-13, -16, -24, -21,
-23, -12, -11, -20, -22, -15, -14, -25 (green), SYTO-81, -80, -82,
-83, -84, -85 (orange), SYTO-64, -17, -59, -61, -62, -60, -63
(red).
[0194] Polymers can be labeled using antibodies or antibody
fragments and their corresponding antigen or hapten binding
partners. Detection of such bound antibodies and proteins or
peptides is accomplished by techniques well known to those
ordinarily skilled in the art. Antibody/antigen complexes are
easily detected by linking a label to the antibodies which
recognize the polymer and then observing the site of the label.
Alternatively, the antibodies can be visualized using secondary
antibodies or fragments thereof that are specific for the primary
antibody used. Polyclonal and monoclonal antibodies may be used.
Antibody fragments include Fab, F(ab).sub.2, Fd and antibody
fragments which include a CDR3 region.
[0195] The polymer may also be labeled with a unit specific marker
such as a sequence specific probe or tag. "Sequence specific" when
used in the context of a nucleic acid molecule means that the tag
molecule recognizes a particular linear arrangement of nucleotides
or derivatives thereof. An analogous definition applies to
non-nucleic acid polymers. In preferred embodiments, the linear
arrangement includes contiguous nucleotides or derivatives thereof
that each bind to a corresponding complementary nucleotide on the
target nucleic acid. In some embodiments, however, the sequence may
not be contiguous as there may be one, two, or more nucleotides
that do not have corresponding complementary residues on the
target.
[0196] It is to be understood that any nucleic acid analog that is
capable of recognizing a nucleic acid molecule with structural or
sequence specificity can be used as a nucleic acid probe. In most
instances, the nucleic acid probes will form at least a
Watson-Crick bond with the nucleic acid molecule being analyzed
(i.e., the target). In other instances, the nucleic acid probe can
form a Hoogsteen bond with the target nucleic acid, thereby forming
a triplex with the target nucleic acid. A nucleic acid sequence
that binds by Hoogsteen binding enters the major groove of a target
nucleic acid and hybridizes with the bases located there. Examples
of these latter probes include molecules that recognize and bind to
the minor and major grooves of nucleic acids (e.g., some forms of
antibiotics). In preferred embodiments, the nucleic acid probes can
form both Watson-Crick and Hoogsteen bonds with the target nucleic
acid. Bis PNA probes, for instance, are capable of both
Watson-Crick and Hoogsteen binding to a target nucleic acid
molecule. In some embodiments, probes with strong sequence
specificity are preferred.
[0197] In some embodiments, the nucleic acid probe is comprised of
a peptide nucleic acid (PNA), a bis PNA clamp, a
pseudocomplementary PNA, a locked nucleic acid (LNA), DNA, RNA, or
co-polymers of the above such as DNA-LNA co-polymers.
[0198] PNAs are DNA analogs having their phosphate backbone
replaced with 2-aminoethyl glycine residues linked to nucleotide
bases through glycine amino nitrogen and methylenecarbonyl linkers.
PNAs can bind to both DNA and RNA targets by Watson-Crick base
pairing, and in so doing form stronger hybrids than would be
possible with DNA or RNA based tag probes.
[0199] Peptide nucleic acid is synthesized from monomers connected
by a peptide bond (Nielsen, P. E. et al. Peptide Nucleic Acids,
Protocols and Applications, Norfolk: Horizon Scientific Press, p.
1-19 (1999)). It can be built with standard solid phase peptide
synthesis technology.
[0200] PNA chemistry and synthesis allows for inclusion of amino
acids and polypeptide sequences in the PNA design. For example,
lysine residues can be used to introduce positive charges in the
PNA backbone. All chemical approaches available for the
modifications of amino acid side chains are directly applicable to
PNAs.
[0201] Several types of PNA designs exist, and these include single
strand PNA (ssPNA), bis PNA, pseudocomplementary PNA (pcPNA).
[0202] The structure of PNA/DNA complex depends on the particular
PNA and its sequence. Single stranded PNA (ssPNA) binds to ssDNA
preferably in antiparallel orientation (i.e., with the N-terminus
of the ssPNA aligned with the 3' terminus of the ssDNA) and with a
Watson-Crick pairing. PNA also can bind to DNA with a Hoogsteen
base pairing, and thereby forms triplexes with dsDNA (Wittung, P.
et al., Biochemistry 36:7973 (1997)).
[0203] Bis PNAs have multiple modes of binding to nucleic acids
(Hansen, G. I. et al., J. Mol. Biol. 307(1):67-74 (2001)). One
isomer includes two bis PNA molecules instead of one. It is formed
at higher bis PNA concentration and has tendency to rearrange into
the complex with a single bis PNA molecule. Other isomers differ in
positioning of the linker around the target DNA strands. All the
identified isomers still bind to the same binding site/target.
[0204] Pseudocomplementary PNA (pcPNA) (Izvolsky, K. I. et al.,
Biochemistry 10908-10913 (2000)) also delivers two base pairs per
every nucleotide of the target sequence. Hence, it can bind to
short sequences similar to those that are bis PNA targets. The
pcPNA strands are not connected by a hinge, and they have different
sequences.
[0205] Hybridization of pcPNA can be less efficient than that of
bis PNA because it needs three molecules to form the complex.
However, the pseudocomplementary stands can be connected by a
sufficiently long and flexible hinge.
[0206] Another bis PNA-based approach involves use of the displaced
DNA strand (Demidov, V. V. et al., Methods: A Companion to Methods
in Enzymology 23(2):123-131 (2001)). If the second bis PNA is
hybridized close enough to the first one, then a run of DNA (up to
25 bp) is displaced, forming an extended P-loop. This run is long
enough to be tagged. This combination is referred to as a PD-loop
(Demidov, V. V. et al., Methods: A Companion to Methods in
Enzymology 23(2):123-131 (2001)). Other applications for the
opening are also designed including topological labels or
"earrings". Tagging based on PD-loop has important advantages,
including increased specificity.
[0207] Locked nucleic acid (LNA) molecules form hybrids with DNA,
which are at least as stable as PNA/DNA hybrids (Braasch, D. A. et
al., Chem & Biol. 8(1):1-7 (2001)). Therefore, LNA can be used
just as PNA molecules would be. LNA binding efficiency can be
increased in some embodiments by adding positive charges to it.
LNAs have been reported to have increased binding affinity
inherently.
[0208] The probes can also be stabilized in part by the use of
other backbone modifications. The invention intends to embrace the
other backbone modifications such as but not limited to
phosphorothioate linkages, phosphodiester modified nucleic acids,
combinations of phosphodiester and phosphorothioate nucleic acid,
methylphosphonate, alkylphosphonates, phosphate esters,
alkylphosphonothioates, phosphoramidates, carbamates, carbonates,
phosphate triesters, acetamidates, carboxymethyl esters,
methylphosphorothioate, phosphorodithioate, p-ethoxy, and
combinations thereof.
[0209] One limitation of the stability of nucleic acid hybrids is
the length of the probe, with longer probes leading to greater
stability than shorter probes. Notwithstanding this proviso, the
probes of the invention can be any length ranging from at least 4
nucleotides long to in excess of 1000 nucleotides long. In
preferred embodiments, the probes are 5-100 nucleotides in length,
more preferably between 5-25 nucleotides in length, and even more
preferably 5-12 nucleotides in length. The length of the probe can
be any length of nucleotides between and including the ranges
listed herein, as if each and every length was explicitly recited
herein. It should be understood that not all residues of the probe
need hybridize to complementary residues in the target nucleic
acid. For example, the probe may be 50 residues in length, yet only
25 of those residues hybridize to the target nucleic acid.
Preferably, the residues that hybridize are contiguous with each
other.
[0210] The probes are preferably single stranded, but they are not
so limited. For example, when the probe is a bis PNA it can adopt a
secondary structure with the target nucleic acid resulting in a
triple helix conformation, with one region of the bis PNA clamp
forming Hoogsteen bonds with the backbone of the target molecule
and another region of the bis PNA clamp forming Watson-Crick bonds
with the nucleotide bases of the target molecule.
[0211] The specificity of nucleic acid probe binding to a target
nucleic acid can be manipulated based on the hybridization
conditions. For example, salt concentration and temperature can be
modulated in order to vary the range of sequences recognized by the
nucleic acid probes.
[0212] The polymers are analyzed using linear polymer analysis
systems. A linear polymer analysis system is a system that analyzes
polymers in a linear manner (i.e., starting at one location on the
polymer and then proceeding linearly in either direction
therefrom). As a polymer is analyzed, the detectable labels
attached to it are detected in either a sequential or simultaneous
manner. When detected simultaneously, the signals usually form an
image of the polymer, from which distances between labels can be
determined. When detected sequentially, the signals are viewed in
histogram (signal intensity vs. time), that can then be translated
into a map, with knowledge of the velocity of the polymer. It is to
be understood that in some embodiments, the polymer is attached to
a solid support, while in others it is free flowing. In either
case, the velocity of the polymer as it moves past, for example, an
interaction station or a detector, will aid in determining the
position of the labels, relative to each other and relative to
other detectable markers that may be present on the polymer.
[0213] Accordingly, the linear polymer analysis systems are able to
deduce not only the total amount of label on a polymer, but perhaps
more importantly in some embodiments, the location of such labels.
The ability to locate and position the labels allows these patterns
to be superimposed on other genetic maps, in order to orient and/or
identify the regions of the genome being analyzed. In preferred
embodiments, the linear polymer analysis systems are capable of
analyzing nucleic acids individually (i.e., they are single
molecule detection systems).
[0214] An example of such a system is the Gene Engine.TM. system
described in PCT patent applications WO98/35012 and WO00/09757,
published on Aug. 13, 1998, and Feb. 24, 2000, respectively, and in
issued U.S. Pat. No. 6,355,420 B1, issued Mar. 12, 2002. The
contents of these applications and patent, as well as those of
other applications and patents, and references cited herein are
incorporated by reference in their entirety. This system allows
single nucleic acids to be passed through an interaction station in
a linear manner, whereby nucleotides are interrogated individually
in order to determine whether there is a detectable label
conjugated to the nucleic acid molecule. Interrogation involves
exposing the nucleic acid molecule to an energy source such as
optical radiation of a set wavelength. In response to the energy
source exposure, the detectable label on the nucleotide (if one is
present) emits a detectable signal. The mechanism for signal
emission and detection will depend on the type of label sought to
be detected.
[0215] Other single molecule nucleic acid analytical methods which
involve elongation of DNA molecule can also be used in the methods
of the invention. These include optical mapping (Schwartz, D. C. et
al., Science 262(5130):110-114 (1993); Meng, X. et al., Nature
Genet. 9(4):432-438 (1995); Jing, J. et al., Proc. Natl. Acad. Sci.
USA 95(14):8046-8051 (1998); and Aston, C. et al., Trends
Biotechnol. 17(7):297-302 (1999)) and fiber-fluorescence in situ
hybridization (fiber-FISH) (Bensimon, A. et al., Science
265(5181):2096-2098 (1997)). In optical mapping, nucleic acids are
elongated in a fluid sample and fixed in the elongated conformation
in a gel or on a surface. Restriction digestions are then performed
on the elongated and fixed nucleic acids. Ordered restriction maps
are then generated by determining the size of the restriction
fragments. In fiber-FISH, nucleic acids are elongated and fixed on
a surface by molecular combing. Hybridization with fluorescently
labeled probes allows determination of sequence landmarks on the
nucleic acids. Both methods require fixation of elongated molecules
so that molecular lengths and/or distances between probes can be
measured. Pulse field gel electrophoresis can also be used to
analyze the labeled nucleic acids. Pulse field gel electrophoresis
is described by Schwartz, D. C. et al., Cell 37(1):67-75 (1984).
Other nucleic acid analysis systems are described by Otobe, K. et
al., Nucleic Acids Res. 29(22):E109 (2001), Bensimon, A. et al. in
U.S. Pat. No. 6,248,537, issued Jun. 19, 2001, Herrick, J. et al.,
Chromosome Res. 7(6):409:423 (1999), Schwartz in U.S. Pat. No.
6,150,089 issued Nov. 21, 2000 and U.S. Pat. No. 6,294,136, issued
Sep. 25, 2001. Other linear polymer analysis systems can also be
used, and the invention is not intended to be limited to solely
those listed herein.
[0216] The nature of such detection systems will depend upon the
nature of the detectable moiety used to label the polymer. The
detection system can be selected from any number of detection
systems known in the art. These include an electron spin resonance
(ESR) detection system, a charge coupled device (CCD) detection
system, a fluorescent detection system, an electrical detection
system, a photographic film detection system, a chemiluminescent
detection system, an enzyme detection system, an atomic force
microscopy (AFM) detection system, a scanning tunneling microscopy
(STM) detection system, an optical detection system, a nuclear
magnetic resonance (NMR) detection system, a near field detection
system, and a total internal reflection (TIR) detection system,
many of which are electromagnetic detection systems.
[0217] Optical detectable signals are generated, detected and
stored in a database. The signals can be analyzed to determine
structural information about the polymer. The signals can be
analyzed by assessing the intensity of the signal to determine
structural information about the polymer. The computer may be the
same computer used to collect data about the polymers, or may be a
separate computer dedicated to data analysis. A suitable computer
system to implement embodiments of the present invention typically
includes an output device which displays information to a user, a
main unit connected to the output device and an input device which
receives input from a user. The main unit generally includes a
processor connected to a memory system via an interconnection
mechanism. The input device and output device also are connected to
the processor and memory system via the interconnection mechanism.
Computer programs for data analysis of the detected signals are
readily available from CCD (Charge Coupled Device)
manufacturers.
[0218] Other interactions involved in methods of the invention will
produce a nuclear radiation signal. As a radiolabel on a polymer
passes through the defined region of detection, nuclear radiation
is emitted, some of which will pass through the defined region of
radiation detection. A detector of nuclear radiation is placed in
proximity of the defined region of radiation detection to capture
emitted radiation signals. Many methods of measuring nuclear
radiation are known in the art including cloud and bubble chamber
devices, constant current ion chambers, pulse counters, gas
counters (i.e., Geiger-Muller counters), solid state detectors
(surface barrier detectors, lithium-drifted detectors, intrinsic
germanium detectors), scintillation counters, Cerenkov detectors,
to name a few.
[0219] Other types of signals generated are well known in the art
and have many detections means which are known to those of skill in
the art. Some of these include opposing electrodes, magnetic
resonance, and piezoelectric scanning tips. Opposing nanoelectrodes
can function by measurement of capacitance changes. Two opposing
electrodes create an area of energy storage, located effectively
between the two electrodes. It is known that the capacitance of
such a device changes when different materials are placed between
the electrodes. This dielectric constant is a value associated with
the amount of energy a particular material can store (i.e., its
capacitance). Changes in the dielectric constant can be measured as
a change in the voltage across the two electrodes. In the present
example, different nucleotide bases or unit specific markers of a
polymer may give rise to different dielectric constants. The
capacitance changes as the dielectric constant of the unit specific
marker of the polymer per the equation: C=KC.sub.o, where K is the
dielectric constant and C.sub.o is the capacitance in the absence
of any bases. The voltage deflection of the nanoelectrodes is then
outputted to a measuring device, recording changes in the signal
with time.
[0220] The present invention is further illustrated by the
following Examples, which in no way should be construed as further
limiting. The entire contents of all of the references (including
literature references, issued patents, published patent
applications, and co-pending patent applications) cited throughout
this application are hereby expressly incorporated by
reference.
[0221] The foregoing written specification is considered to be
sufficient to enable one skilled in the art to practice the
invention. The present invention is not to be limited in scope by
examples provided, since the examples are intended as a single
illustration of one aspect of the invention and other functionally
equivalent embodiments are within the scope of the invention.
Various modifications of the invention in addition to those shown
and described herein will become apparent to those skilled in the
art from the foregoing description and fall within the scope of the
appended claims. The advantages and objects of the invention are
not necessarily encompassed by each embodiment of the
invention.
* * * * *