U.S. patent application number 15/188173 was filed with the patent office on 2016-12-15 for multiplex decoding of array sensors with microspheres.
The applicant listed for this patent is ILLUMINA, INC.. Invention is credited to Mark S. Chee, Anthony W. Czarnik, John R. Stuelpnagel.
Application Number | 20160362733 15/188173 |
Document ID | / |
Family ID | 26782312 |
Filed Date | 2016-12-15 |
United States Patent
Application |
20160362733 |
Kind Code |
A1 |
Chee; Mark S. ; et
al. |
December 15, 2016 |
MULTIPLEX DECODING OF ARRAY SENSORS WITH MICROSPHERES
Abstract
The invention relates to compositions and methods for multiplex
decoding of microsphere array sensors.
Inventors: |
Chee; Mark S.; (Del Mar,
CA) ; Stuelpnagel; John R.; (Encinitas, CA) ;
Czarnik; Anthony W.; (San Diego, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ILLUMINA, INC. |
San Diego |
CA |
US |
|
|
Family ID: |
26782312 |
Appl. No.: |
15/188173 |
Filed: |
June 21, 2016 |
Related U.S. Patent Documents
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Application
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Patent Number |
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14322224 |
Jul 2, 2014 |
9399795 |
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15188173 |
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13893273 |
May 13, 2013 |
8795967 |
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14322224 |
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11739668 |
Apr 24, 2007 |
8460865 |
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13893273 |
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10187321 |
Jun 28, 2002 |
7226734 |
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11739668 |
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11285722 |
Nov 22, 2005 |
7455971 |
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11739668 |
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09748706 |
Dec 22, 2000 |
7033754 |
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11285722 |
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09344526 |
Jun 24, 1999 |
7060431 |
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10187321 |
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09189543 |
Nov 10, 1998 |
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09344526 |
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09344526 |
Jun 24, 1999 |
7060431 |
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09748706 |
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60302213 |
Jun 28, 2001 |
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60235531 |
Sep 26, 2000 |
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60172106 |
Dec 23, 1999 |
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60090473 |
Jun 24, 1998 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/6837 20130101;
B01J 2219/00605 20130101; B01J 2219/00637 20130101; B01J 2219/00648
20130101; B01J 2219/00596 20130101; B01J 2219/00659 20130101; C12Q
1/6816 20130101; C40B 40/06 20130101; C40B 70/00 20130101; B01J
2219/00621 20130101; C12Q 1/6837 20130101; B01J 2219/00722
20130101; G01N 33/54313 20130101; G06K 9/6202 20130101; B01J
2219/005 20130101; B01J 19/0046 20130101; B01L 3/5085 20130101;
B01J 2219/00619 20130101; C12Q 1/6816 20130101; Y10S 977/924
20130101; B01J 2219/00585 20130101; B01J 2219/0061 20130101; B01J
2219/00317 20130101; C12Q 1/6837 20130101; B01J 2219/00459
20130101; B01J 2219/00626 20130101; B01J 2219/00612 20130101; B01J
2219/00677 20130101; B01J 2219/0063 20130101; C12Q 1/6837 20130101;
C12Q 1/6825 20130101; C12Q 2563/155 20130101; C12Q 2563/107
20130101; C12Q 2537/143 20130101; C12Q 2563/155 20130101; C12Q
2563/155 20130101; C12Q 2563/179 20130101; C12Q 2563/155 20130101;
C12Q 2521/313 20130101; C12Q 2527/137 20130101; C12Q 2563/179
20130101; C12Q 2563/155 20130101; C12Q 2563/107 20130101; C12Q
2563/107 20130101; C12Q 2563/179 20130101; B01J 2219/00466
20130101; C12Q 2563/107 20130101; B01J 2219/00641 20130101; B01J
2219/00644 20130101; B82Y 30/00 20130101; B01J 2219/00576 20130101;
C12Q 1/6816 20130101; B01J 2219/00702 20130101; C12Q 1/6834
20130101; B01J 2219/00617 20130101; B01J 2219/00572 20130101; C40B
60/14 20130101; C12Q 1/6834 20130101; C12Q 1/6837 20130101 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; G06K 9/62 20060101 G06K009/62 |
Claims
1. A system of decoding an array, said system comprising: an array
comprising a plurality of distinct capture probes, wherein said
capture probes are randomly distributed on said array; a plurality
of first sets of decoder probes hybridized with said capture
probes, wherein each of said first sets of decoder probes comprises
a different label, each different label producing a different
signal; a plurality of second sets of decoder probes hybridized
with said capture probes, wherein each of said second sets of
decoder probes is different from said first sets of decoder probes,
and wherein each of said second sets of decoder probes comprises a
different label, each different label producing a different signal;
and a detector capable of detecting said different signals produced
by the hybridization of said first sets of decoder probes and
detecting said different signals produced by the hybridization of
said second sets of decoder probes so as to generate a code
indicative of the location of a capture probe, thereby decoding
said array.
2. The system of claim 1, wherein said array comprises a flat
planar surface.
3. The system of claim 2, wherein said flat planar surface
comprises a depression comprising one or more particles.
4. The system of claim 3, wherein said capture probes are attached
to said one or more particles.
5. The system of claim 4, wherein said one or more particles
comprise microspheres.
6. The system of claim 2, wherein said flat planar surface
comprises at least one well having a microsphere deposited
therein.
7. The system of claim 1, wherein said capture probes and said
decoder probes comprise nucleic acids.
8. The system of claim 1, wherein said labels comprise fluorescent
labels.
9. (canceled)
10. A method of decoding an array, said method comprising:
contacting an array comprising a plurality of particle
subpopulations, wherein each particle subpopulation comprises a
distinct bioactive agent and wherein capture probes are randomly
distributed on said array, with a first population of decoder
probes, said first population comprising a plurality of different
labels; obtaining a first image of the location of said first
population of decoder probes; removing said first population of
decoder probes; contacting said array with a second population of
decoder probes, said second population comprising a plurality of
different labels; obtaining a second image of the location of said
second population of decoder probes; and comparing said first image
with said second image to determine the identity of the label that
is present at a location in said first image and the identity of
the label that is present at the same location in said second image
so as to generate a code indicative of the location of a capture
probe, thereby decoding said array.
11. The method of claim 10, wherein the particles comprise
microspheres.
12. The method of claim 10, wherein the number of labels in said
first population of decoder probes or said second population of
decoder probes is less than the number of subpopulations of
particles.
13. The method of claim 10, wherein said first population of
decoder probes and said second population of decoder probes
comprise the same population of decoder probes, said first
population of decoder probes comprising different labels than said
second population of decoder probes.
14. The method of claim 10, wherein at least one of said bioactive
agents comprises a nucleic acid.
15. The method of claim 10, wherein said first population of
decoder probes and said second population of decoder probes
comprise fluorescent labels.
16. A method of decoding an array, said method comprising:
providing an array comprising a population of microspheres having
capture probes attached thereto, said population of microspheres
being randomly distributed on said array at discrete sites;
hybridizing a plurality of first sets of decoder probes with said
capture probes, wherein each of said first sets of decoder probes
comprises a different label, each different label producing a
different signal; removing said plurality of first sets of decoder
probes; hybridizing a plurality of second sets of decoder probes
with said capture probes, wherein each of said second sets of
decoder probes comprises a different label, each different label
producing a different signal; and detecting said different signals
produced by the hybridization of said first sets of decoder probes
and detecting said different signals produced by the hybridization
of said second sets of decoder probes so as to generate a code
indicative of the location of a capture probe, thereby decoding
said array.
17. The method of claim 16, wherein said discrete sites are
wells.
18. The method of claim 16, wherein said array comprises a flat
planar surface.
19. The method of claim 16, wherein said capture probes and said
decoder probes comprise nucleic acids.
20. (canceled)
21. (canceled)
22. (canceled)
23. (canceled)
24. (canceled)
25. (canceled)
26. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 14/322,224 filed on Jul. 2, 2014 which is a
continuation of U.S. patent application Ser. No. 13/893,273 filed
on May 13, 2013, now U.S. Pat. No. 8,795,967, issued on Aug. 5,
2014, which is a continuation of and claims priority to U.S. patent
application Ser. No. 11/739,668, entitled MULTIPLEX DECODING OF
ARRAY SENSORS WITH MICROSPHERES, filed on Apr. 24, 2007, now U.S.
Pat. No. 8,460,865, issued Jun. 11, 2013, which is a continuation
of and claims priority to U.S. patent application Ser. No.
10/187,321, entitled MULTIPLEX DECODING OF ARRAY SENSORS WITH
MICROSPHERES, filed Jun. 28, 2002, now U.S. Pat. No. 7,226,734,
issued Jun. 5, 2007, which is a nonprovisional application of and
claims priority to U.S. Provisional Application No. 60/302,213,
filed Jun. 28, 2001; U.S. patent application Ser. No. 11/739,668 is
also a continuation of U.S. patent application Ser. No. 11/285,722,
filed Nov. 22, 2005, entitled MULTIPLEX DECODING OF ARRAY SENSORS
WITH MICROSPHERES, now U.S. Pat. No. 7,455,971, issued Nov. 25,
2008, which is a continuation-in-part of and claims priority to
U.S. patent application Ser. No. 09/748,706, entitled DECODING OF
ARRAY SENSORS WITH MICROSPHERES, filed Dec. 22, 2000, now U.S. Pat.
No. 7,033,754, issued Apr. 25, 2006, which is a nonprovisional
application of and claims priority to U.S. Provisional Application
No. 60/235,531, filed Sep. 26, 2000 and U.S. Provisional
Application No. 60/172,106, filed Dec. 23, 1999; both U.S. patent
application Ser. Nos. 10/187,321 and 09/748,706 are
continuation-in-part applications of and claim priority to U.S.
patent application Ser. No. 09/344,526, entitled DECODING OF ARRAY
SENSORS WITH MICROSPHERES, filed Jun. 24, 1999, now U.S. Pat. No.
7,060,431, issued Jun. 13, 2006, which is a continuation-in-part of
and claims priority to U.S. patent application Ser. No. 09/189,543,
entitled DECODING OF ARRAY SENSORS WITH MICROSPHERES, filed Nov.
10, 1998, now abandoned, which is a nonprovisional application of
and claims priority to U.S. Provisional Application No. 60/090,473,
filed Jun. 24, 1998.
REFERENCE TO SEQUENCE LISTING
[0002] The Sequence Listing provided herewith in electronic format
was created on Jun. 21, 2016 with a file size about 1.7 KB entitled
ILLINC041P3C5SEQLISTINGTXT. This Sequence Listing is incorporated
herein by reference in its entirety.
FIELD OF THE INVENTION
[0003] The invention relates to compositions and methods for
decoding microsphere array sensors.
BACKGROUND OF THE INVENTION
[0004] There are a number of assays and sensors for the detection
of the presence and/or concentration of specific substances in
fluids and gases. Many of these rely on specific ligand/antiligand
reactions as the mechanism of detection. That is, pairs of
substances (i.e. the binding pairs or ligand/antiligands) are known
to bind to each other, while binding little or not at all to other
substances. This has been the focus of a number of techniques that
utilize these binding pairs for the detection of the complexes.
These generally are done by labeling one component of the complex
in some way, so as to make the entire complex detectable, using,
for example, radioisotopes, fluorescent and other optically active
molecules, enzymes, etc.
[0005] Of particular use in these sensors are detection mechanisms
utilizing luminescence. Recently, the use of optical fibers and
optical fiber strands in combination with light absorbing dyes for
chemical analytical determinations has undergone rapid development,
particularly within the last decade. The use of optical fibers for
such purposes and techniques is described by Milanovich et al.,
"Novel Optical Fiber Techniques For Medical Application",
Proceedings of the SPIE 28th Annual International Technical
Symposium On Optics and Electro-Optics, Volume 494, 1980; Seitz, W.
R., "Chemical Sensors Based On Immobilized Indicators and Fiber
Optics" in C.R.C. Critical Reviews In Analytical Chemistry, Vol.
19, 1988, pp. 135-173; Wolfbeis, O. S., "Fiber Optical
Fluorosensors In Analytical Chemistry" in Molecular Luminescence
Spectroscopy, Methods and Applications (S. G. Schulman, editor),
Wiley & Sons, New York (1988); Angel, S. M., Spectroscopy 2
(4):38 (1987); Walt, et al., "Chemical Sensors and
Microinstrumentation", ACS Symposium Series, Vol. 403, 1989, p.
252, and Wolfbeis, O. S., Fiber Optic Chemical Sensors, Ed. CRC
Press, Boca Raton, Fla., 1991, 2nd Volume.
[0006] When using an optical fiber in an in vitro/in vivo sensor,
one or more light absorbing dyes are located near its distal end.
Typically, light from an appropriate source is used to illuminate
the dyes through the fiber's proximal end. The light propagates
along the length of the optical fiber; and a portion of this
propagated light exits the distal end and is absorbed by the dyes.
The light absorbing dye may or may not be immobilized; may or may
not be directly attached to the optical fiber itself; may or may
not be suspended in a fluid sample containing one or more analytes
of interest; and may or may not be retainable for subsequent use in
a second optical determination.
[0007] Once the light has been absorbed by the dye, some light of
varying wavelength and intensity returns, conveyed through either
the same fiber or collection fiber(s) to a detection system where
it is observed and measured. The interactions between the light
conveyed by the optical fiber and the properties of the light
absorbing dye provide an optical basis for both qualitative and
quantitative determinations.
[0008] Of the many different classes of light absorbing dyes which
conventionally are employed with bundles of fiber strands and
optical fibers for different analytical purposes are those more
common compositions that emit light after absorption termed
"fluorophores" and those which absorb light and internally convert
the absorbed light to heat, rather than emit it as light, termed
"chromophores."
[0009] Fluorescence is a physical phenomenon based upon the ability
of some molecules to absorb light (photons) at specified
wavelengths and then emit light of a longer wavelength and at a
lower energy. Substances able to fluoresce share a number of common
characteristics: the ability to absorb light energy at one
wavelength, .sub.ab; reach an excited energy state; and
subsequently emit light at another light wavelength, .sub.em. The
absorption and fluorescence emission spectra are individual for
each fluorophore and are often graphically represented as two
separate curves that are slightly overlapping. The same
fluorescence emission spectrum is generally observed irrespective
of the wavelength of the exciting light and, accordingly, the
wavelength and energy of the exciting light may be varied within
limits; but the light emitted by the fluorophore will always
provide the same emission spectrum. Finally, the strength of the
fluorescence signal may be measured as the quantum yield of light
emitted. The fluorescence quantum yield is the ratio of the number
of photons emitted in comparison to the number of photons initially
absorbed by the fluorophore. For more detailed information
regarding each of these characteristics, the following references
are recommended: Lakowicz, J. R., Principles of Fluorescence
Spectroscopy, Plenum Press, New York, 1983; Freifelder, D.,
Physical Biochemistry, second edition, W. H. Freeman and Company,
New York, 1982; "Molecular Luminescence Spectroscopy Methods and
Applications: Part I" (S. G. Schulman, editor) in Chemical
Analysis, vol. 77, Wiley & Sons, Inc., 1985; The Theory of
Luminescence, Stepanov and Gribkovskii, Iliffe Books, Ltd., London,
1968.
[0010] In comparison, substances which absorb light and do not
fluoresce usually convert the light into heat or kinetic energy.
The ability to internally convert the absorbed light identifies the
dye as a "chromophore." Dyes which absorb light energy as
chromophores do so at individual wavelengths of energy and are
characterized by a distinctive molar absorption coefficient at that
wavelength. Chemical analysis employing fiber optic strands and
absorption spectroscopy using visible and ultraviolet light
wavelengths in combination with the absorption coefficient allow
for the determination of concentration for specific analyses of
interest by spectral measurement. The most common use of absorbance
measurement via optical fibers is to determine concentration which
is calculated in accordance with Beers' law; accordingly, at a
single absorbance wavelength, the greater the quantity of the
composition which absorbs light energy at a given wavelength, the
greater the optical density for the sample. In this way, the total
quantity of light absorbed directly correlates with the quantity of
the composition in the sample.
[0011] Many of the recent improvements employing optical fiber
sensors in both qualitative and quantitative analytical
determinations concern the desirability of depositing and/or
immobilizing various light absorbing dyes at the distal end of the
optical fiber. In this manner, a variety of different optical fiber
chemical sensors and methods have been reported for specific
analytical determinations and applications such as pH measurement,
oxygen detection, and carbon dioxide analyses. These developments
are exemplified by the following publications: Freeman, et al.,
Anal Chem. 53:98 (1983); Lippitsch, et al., Anal. Chem. Acta.
205:1, (1988); Wolfbeis, et al., Anal. Chem. 60:2028 (1988);
Jordan, et al., Anal. Chem. 59:437 (1987); Lubbers et al., Sens.
Actuators 1983; Munkholm, et al., Talanta 35:109 (1988); Munkholm,
et al., Anal. Chem. 58:1427 (1986); Seitz, W. R., Anal. Chem.
56:16A-34A (1984); Peterson, et al., Anal. Chem. 52:864 (1980):
Saari, et al., Anal. Chem. 54:821 (1982); Saari, et al., Anal.
Chem. 55:667 (1983); Zhujun, et al., Anal. Chem. Acta. 160:47
(1984); Schwab, et al., Anal. Chem. 56:2199 (1984); Wolfbeis, O.
S., "Fiber Optic Chemical Sensors", Ed. CRC Press, Boca Raton,
Fla., 1991, 2nd Volume; and Pantano, P., Walt, D. R., Anal. Chem.,
481A-487A, Vol. 67, (1995).
[0012] More recently, fiber optic sensors have been constructed
that permit the use of multiple dyes with a single, discrete fiber
optic bundle. U.S. Pat. Nos. 5,244,636 and 5,250,264 to Walt, et
al. disclose systems for affixing multiple, different dyes on the
distal end of the bundle, the teachings of each of these patents
being incorporated herein by this reference. The disclosed
configurations enable separate optical fibers of the bundle to
optically access individual dyes. This avoids the problem of
deconvolving the separate signals in the returning light from each
dye, which arises when the signals from two or more dyes are
combined, each dye being sensitive to a different analyte, and
there is significant overlap in the dyes' emission spectra.
[0013] U.S. Ser. Nos. 08/818,199 and 09/151,877 describe array
compositions that utilize microspheres or beads on a surface of a
substrate, for example on a terminal end of a fiber optic bundle,
with each individual fiber comprising a bead containing an optical
signature. Since the beads go down randomly, a unique optical
signature is needed to "decode" the array; i.e. after the array is
made, a correlation of the location of an individual site on the
array with the bead or bioactive agent at that particular site can
be made. This means that the beads may be randomly distributed on
the array, a fast and inexpensive process as compared to either the
in situ synthesis or spotting techniques of the prior art. Once the
array is loaded with the beads, the array can be decoded, or can be
used, with full or partial decoding occurring after testing, as is
more fully outlined below.
[0014] One drawback with the previous system is that it requires a
set of unique optical signatures. While large sets of such
signatures are available, for example by using different ratios of
different dyes, it would be preferable to use decoding systems that
do not rely on the use of sets of optical signatures. Accordingly,
it is an object of the invention to provide methods to allow
decoding of bead arrays without relying solely on unique optical
signatures.
SUMMARY OF THE INVENTION
[0015] In accordance with the above objects, the present invention
provides array compositions comprising a substrate with a surface
comprising discrete sites. The composition further comprises a
population of microspheres comprising at least a first and a second
subpopulation; each subpopulation comprises a bioactive agent; and
an identifier binding ligand that will bind a decoder binding
ligand such that the identity of the bioactive agent can be
elucidated. The microspheres are distributed on the surface.
[0016] In a further aspect, the present invention provides array
compositions comprising a substrate with a surface comprising
discrete sites, and a population of microspheres comprising at
least a first and a second subpopulation. Each subpopulation
comprises a bioactive agent and does not comprise an optical
signature.
[0017] In an additional aspect, the present invention provides
methods of making an array composition as outlined above. The
methods comprise forming a surface comprising individual sites on a
substrate and distributing microspheres on said surface such that
said individual sites contain microspheres. The microspheres
comprise at least a first and a second subpopulation each
comprising a bioactive agent and do not comprise an optical
signature.
[0018] In a further aspect, the invention provides methods of
making a composition comprising forming a surface comprising
individual sites on a substrate and distributing microspheres on
the surface such that the individual sites contain microspheres.
The microspheres comprise at least a first and a second
subpopulation each comprising a bioactive agent and an identifier
binding ligand that will bind a decoder binding ligand such that
the identification of the bioactive agent can be elucidated.
[0019] In an additional aspect, the invention provides methods of
decoding an array composition comprising providing an array
composition as outlined above, and adding a plurality of decoding
binding ligands to the array composition to identify the location
of at least a plurality of the bioactive agents.
[0020] In a further aspect, the invention provides methods of
determining the presence of a target analyte in a sample. The
methods comprise contacting the sample with an array composition as
outlined herein, and determining the presence or absence of the
target analyte.
[0021] In a further aspect, the invention provides a method
comprising providing an array composition comprising a population
of microspheres comprising at least a first and a second
subpopulation, wherein each subpopulation comprises a bioactive
agent and at least a first and a second decoding attribute, and
detecting each of said first and second decoding attributes to
identify each of said bioactive agents.
[0022] In a further aspect the invention provides a method of
increasing the information obtained in a decoding step. The method
includes the use of degenerate probes as DBL-IBL combinations. In
addition the invention provides the use of multiple decoding
attributes on a bead.
[0023] In further aspect, the invention provides a method of
increasing the confidence of decoding. The method includes using
the decoding as a quality control measure. In addition, the
invention provides quality control fibers. In addition, the
invention provides for parity analysis of decoding data.
[0024] In a further aspect, the invention provides a method of
decoding an array composition comprising providing an array
composition comprising a population of microspheres comprising at
least 50 subpopulations, wherein each subpopulation comprises a
bioactive agent adding a plurality of decoding binding ligands to
said population of microspheres to identify at least 50 of the
bioactive agents.
[0025] In a further aspect, the invention provides a method of
determining the presence of a target analyte in a sample comprising
contacting said sample with a composition comprising a population
of microspheres comprising at least 50 subpopulations, wherein each
subpopulation comprises a bioactive agent adding a plurality of
decoding binding ligands to said population of microspheres to
identify at least 50 of the bioactive agents and determining the
presence or absence of said target analyte.
FIGURES
[0026] FIG. 1 illustrates a two color decoding process wherein
either FAM-labeled or Cy3-labeled oligo complements are use to
"paint" (label) the different bead types on the array.
[0027] FIG. 2 depicts the decoding 128 different bead types with
four colors and four decode stages. (inset shows a single decode
stage using four different dyes to decode 16 bead types.)
[0028] FIGS. 3A, 3B, 3C, 3D, and 3E depict grey scale decoding of
16 different bead types. (A) FIG. 3A depicts a combinatorial
pooling scheme for complementary decoding oligos. (B) FIG. 3B is a
graph in which two independent normalizing images were acquired,
and the resulting bead intensities compared. (C) FIGS. 3C, 3D, and
3E are graphs in which the alpha values (ratio of bead intensity in
indicated decode stage to intensity in normalization image) are
plotted for three decodes stage described in (A).
[0029] FIG. 4 depicts the use of fluorescence resonance energy
transfer as a code for a probe on a random array. Panel A. Depicts
FRET oligos with linkers of different lengths. Panel B. Depicts a
plot of the ration of emission of the fluors from the oligos in
Panel A.
[0030] FIG. 5 Use of fluorescence resonance energy transfer to
discriminate between linkers of varying length. ET1 (SEQ ID NO: 1),
ET2 (SEQ ID NO: 2), ET3 (SEQ ID NO: 3), ET4 (SEQ ID NO. 4), ET5
(SEQ ID NO: 5), ET7 (SEQ ID NO: 6).
[0031] FIG. 6 depicts clustering in hyperspectral alpha space
(.DELTA..sub.1=I.sub.1/6I.sub.I, .DELTA..sub.2=I.sub.2/6I.sub.i,
.DELTA..sub.3=I.sub.3/6I.sub.i, etc.). A set of 128 different bead
types present on a fiber bundle were decoded with hybridizing set
of complementary oligonucleotides labeled with four dyes,
Bodipy-493, Bodipy-R6G, Bodipy-TXR, and Bod-564 (only one dye per
oligonucleotide). Shown is the second stage of a four stage decode
in which 4013 beads were decoded. Ovals are drawn around zones of
hue clusters.
[0032] FIG. 7 depicts an error checking scheme.
[0033] FIG. 8 depicts an example of primer extension labeling of
decoder oligonucleotides.
[0034] FIG. 9 depicts two color decoding of 16 beads with 16
distinct IBLs.
[0035] FIG. 10 depicts primer extension labeling of degenerate
IBL-DBL probes.
[0036] FIG. 11 depicts a scatter plot of signals obtained from
three distinct labels recorded in two channels.
[0037] FIG. 12 depicts decoding by sequencing. A polymerase (e.g.
Sequenase, Thermosequenase) and dye-labeled chain-terminating
nucleotides are used to sequence the encoding region of a DNA
strand present on the bead. The four nucleotides are each labeled
with a discrete label. Because only 4 colors are detected at once,
the colors are removed after each extension step. In subsequent
extension steps, the primers are indexed or extended by one base so
that the primer can query the next base in the encoding region.
[0038] FIG. 13 depicts construction of probes on bead containing
encoding sequences, zipcodes, and a gene-specific sequence. Two
different encoding cassettes are employed to facilitate the primer
extension reaction using primers with universal or degenerate
bases. Using four color sequencing and a single base code, 6 bases
generates 4 6=4096 codes, likewise four color hybridization using
single hybridization colors also generates 4096 codes. The grand
total number of codes is 4096*4096=>16 million. If only a single
color/two state scheme is employed for hybridization, than 2 6=64
codes are generated. The grand total is 4096*64=262,144 codes. The
zipcode sequences can also be constructed so as to be overlapping
to reduce the length of the overall sequence (i.e. cZip#1 vs.
cZiP#2).
[0039] FIG. 14 depicts incorporation of a cleavable linker between
encoding sequences and gene-specific sequence. A disulfide
cleavable linker is shown for illustration purposes. If the linker
is not present, the terminal portion of the gene-specific sequence
can be included as part of the encoding sequence to reduce overall
length of the probe.
[0040] FIG. 15 depicts decoding with exonuclease.
[0041] FIG. 16 depicts a graph showing decoding of about 1500 bead
subpopulation on each of several different fiber bundles.
DETAILED DESCRIPTION OF THE INVENTION
[0042] The present invention is generally based on previous work
comprising a bead-based analytic chemistry system in which beads,
also termed microspheres, carrying different chemical
functionalities are distributed on a substrate comprising a
patterned surface of discrete sites that can bind the individual
microspheres. Since the beads are generally put onto the substrate
randomly, the previous work relied on the incorporation of unique
optical signatures, generally fluorescent dyes, that could be used
to identify the chemical functionality on any particular bead. This
allows the synthesis of the candidate agents (i.e. compounds such
as nucleic acids and antibodies) to be divorced from their
placement on an array, i.e. the candidate agents may be synthesized
on the beads, and then the beads are randomly distributed on a
patterned surface. Since the beads are first coded with an optical
signature, this means that the array can later be "decoded", i.e.
after the array is made, a correlation of the location of an
individual site on the array with the bead or candidate agent at
that particular site can be made. This means that the beads may be
randomly distributed on the array, a fast and inexpensive process
as compared to either the in situ synthesis or spotting techniques
of the prior art.
[0043] However, the drawback to these methods is that for a large
array, the system requires a large number of different optical
signatures, which may be difficult or time-consuming to utilize.
Accordingly, the present invention provides several improvements
over these methods, generally directed to methods of coding and
decoding the arrays. That is, as will be appreciated by those in
the art, the placement of the bioactive agents is generally random,
and thus a coding/decoding system is required to identify the
bioactive agent at each location in the array. This may be done in
a variety of ways, as is more fully outlined below, and generally
includes: a) the use of decoding binding ligands (DBLs), generally
directly labeled, that binds to either the bioactive agent or to
identifier binding ligands (IBLs) attached to the beads; b)
positional decoding, for example by either targeting the placement
of beads (for example by using photoactivatible or photocleavable
moieties to allow the selective addition of beads to particular
locations), or by using either sub-bundles or selective loading of
the sites, as are more fully outlined below; c) selective decoding,
wherein only those beads that bind to a target are decoded; or d)
combinations of any of these. In some cases, as is more fully
outlined below, this decoding may occur for all the beads, or only
for those that bind a particular target analyte. Similarly, this
may occur either prior to or after addition of a target
analyte.
[0044] Once the identity of the bioactive agent and its location in
the array has been fixed, the array is exposed to samples
containing the target analytes, although as outlined below, this
can be done prior to or during the analysis as well. The target
analytes will bind to the bioactive agents as is more fully
outlined below, and result in a change in the optical signal of a
particular bead.
[0045] In the present invention, "decoding" does not rely on the
use of optical signatures, but rather on the use of decoding
binding ligands that are added during a decoding step. The decoding
binding ligands will bind either to a distinct identifier binding
ligand partner that is placed on the beads, or to the bioactive
agent itself, for example when the beads comprise single-stranded
nucleic acids as the bioactive agents. The decoding binding ligands
are either directly or indirectly labeled, and thus decoding occurs
by detecting the presence of the label. By using pools of decoding
binding ligands in a sequential fashion, it is possible to greatly
minimize the number of required decoding steps.
[0046] In one embodiment, the invention provides methods for
multiplex decoding of bead-arrays. That is, microspheres with
bioactive agents are decoded to elucidate the identity of the
bioactive agent on the bead. In a preferred embodiment at least 50
different bead types or subpopufations are simultaneously decoded,
or decoded in a single decoding experiment. More preferably at
least 100; more preferably at least 500; more preferably at least
1000; or preferably at least 1500 bead types are simultaneously
decoded or decoded in a single decoding experiment by the methods
of the invention. Preferably at least 5000 bead types are
simultaneously decoded or decoded in a single decoding
experiment.
[0047] Accordingly, the present invention provides array
compositions comprising at least a first substrate with a surface
comprising individual sites. By "array" herein is meant a plurality
of candidate agents in an array format; the size of the array will
depend on the composition and end use of the array. Arrays
containing from about 2 different bioactive agents (i.e. different
beads) to many millions can be made, with very large fiber optic
arrays being possible. Generally, the array will comprise from two
to as many as a billion or more, depending on the size of the beads
and the substrate, as well as the end use of the array, thus very
high density, high density, moderate density, low density and very
low density arrays may be made. Preferred ranges for very high
density arrays are from about 10,000,000 to about 2,000,000,000
(all numbers are per square cm), with from about 100,000,000 to
about 1,000,000,000 being preferred. High density arrays range
about 100,000 to about 10,000,000, with from about 1,000,000 to
about 5,000,000 being particularly preferred. Moderate density
arrays range from about 10,000 to about 100,000 being particularly
preferred, and from about 20,000 to about 50,000 being especially
preferred. Low density arrays are generally less than 10,000, with
from about 1,000 to about 5,000 being preferred. Very low density
arrays are less than 1,000, with from about 10 to about 1000 being
preferred, and from about 100 to about 500 being particularly
preferred. In some embodiments, the compositions of the invention
may not be in array format; that is, for some embodiments,
compositions comprising a single bioactive agent may be made as
well. In addition, in some arrays, multiple substrates may be used,
either of different or identical compositions. Thus for example,
large arrays may comprise a plurality of smaller substrates.
[0048] In addition, one advantage of the present compositions is
that particularly through the use of fiber optic technology,
extremely high density arrays can be made. Thus for example,
because beads of 200 m or less (with beads of 200 nm possible) can
be used, and very small fibers are known, it is possible to have as
many as 40,000 or more (in some instances, 1 million) different
fibers and beads in a 1 mm.sup.2 fiber optic bundle, with densities
of greater than 15,000,000 individual beads and fibers (again, in
some instances as many as 25-50 million) per 0.5 cm.sup.2
obtainable.
[0049] By "substrate" or "solid support" or other grammatical
equivalents herein is meant any material that can be modified to
contain discrete individual sites appropriate for the attachment or
association of beads and is amenable to at least one detection
method. As will be appreciated by those in the art, the number of
possible substrates is very large. Possible substrates include, but
are not limited to, glass and modified or functionalized glass,
plastics (including acrylics, polystyrene and copolymers of styrene
and other materials, polypropylene, polyethylene, polybutylene,
polyurethanes, Teflon, etc.), polysaccharides, nylon or
nitrocellulose, resins, silica or silica-based materials including
silicon and modified silicon, carbon, metals, inorganic glasses,
plastics, optical fiber bundles, and a variety of other polymers.
In general, the substrates allow optical detection and do not
themselves appreciably fluoresce.
[0050] Generally the substrate is flat (planar), although as will
be appreciated by those in the art, other configurations of
substrates may be used as well; for example, three dimensional
configurations can be used, for example by embedding the beads in a
porous block of plastic that allows sample access to the beads and
using a confocal microscope for detection. Similarly, the beads may
be placed on the inside surface of a tube, for flow-through sample
analysis to minimize sample volume. Preferred substrates include
optical fiber bundles as discussed below, and flat planar
substrates such as glass, polystyrene and other plastics and
acrylics.
[0051] In a preferred embodiment, the substrate is an optical fiber
bundle or array, as is generally described in U.S. Ser. Nos.
08/944,850 and 08/519,062, PCT US98/05025, and PCT US98/09163, all
of which are expressly incorporated herein by reference. Preferred
embodiments utilize preformed unitary fiber optic arrays. By
"preformed unitary fiber optic array" herein is meant an array of
discrete individual fiber optic strands that are co-axially
disposed and joined along their lengths. The fiber strands are
generally individually clad. However, one thing that distinguished
a preformed unitary array from other fiber optic formats is that
the fibers are not individually physically manipulatable; that is,
one strand generally cannot be physically separated at any point
along its length from another fiber strand.
[0052] Generally, the array of array compositions of the invention
can be configured in several ways; see for example U.S. Ser. No.
09/473,904, and WO 00/39587, both of which are hereby expressly
incorporated by reference. In a preferred embodiment, as is more
fully outlined below, a "one component" system is used. That is, a
first substrate comprising a plurality of assay locations
(sometimes also referred to herein as "assay wells"), such as a
microtiter plate, is configured such that each assay location
contains an individual array. That is, the assay location and the
array location are the same. For example, the plastic material of
the microtiter plate can be formed to contain a plurality of "bead
wells" in the bottom of each of the assay wells. Beads containing
the capture probes of the invention can then be loaded into the
bead wells in each assay location as is more fully described
below.
[0053] Alternatively, a "two component" system can be used. In this
embodiment, the individual arrays are formed on a second substrate,
which then can be fitted or "dipped" into the first microtiter
plate substrate. A preferred embodiment utilizes fiber optic
bundles as the individual arrays, generally with "bead wells"
etched into one surface of each individual fiber, such that the
beads containing the capture probes are loaded onto the end of the
fiber optic bundle. The composite array thus comprises a number of
individual arrays that are configured to fit within the wells of a
microtiter plate.
[0054] By "composite array" or "combination array" or grammatical
equivalents herein is meant a plurality of individual arrays, as
outlined above. Generally the number of individual arrays is set by
the size of the microtiter plate used; thus, 96 well, 384 well and
1536 well microtiter plates utilize composite arrays comprising 96,
384 and 1536 individual arrays, although as will be appreciated by
those in the art, not each microtiter well need contain an
individual array. It should be noted that the composite arrays can
comprise individual arrays that are identical, similar or
different. That is, in some embodiments, it may be desirable to do
the same 2,000 assays on 96 different samples; alternatively, doing
192,000 experiments on the same sample (i.e. the same sample in
each of the 96 wells) may be desirable. Alternatively, each row or
column of the composite array could be the same, for
redundancy/quality control. As will be appreciated by those in the
art, there are a variety of ways to configure the system. In
addition, the random nature of the arrays may mean that the same
population of beads may be added to two different surfaces,
resulting in substantially similar but perhaps not identical
arrays.
[0055] At least one surface of the substrate is modified to contain
discrete, individual sites for later association of microspheres.
These sites may comprise physically altered sites, i.e. physical
configurations such as wells or small depressions in the substrate
that can retain the beads, such that a microsphere can rest in the
well, or the use of other forces (magnetic or compressive), or
chemically altered or active sites, such as chemically
functionalized sites, electrostatically altered sites,
hydrophobically/hydrophilically functionalized sites, spots of
adhesive, etc.
[0056] The sites may be a pattern, i.e. a regular design or
configuration, or randomly distributed. A preferred embodiment
utilizes a regular pattern of sites such that the sites may be
addressed in the X-Y coordinate plane. "Pattern" in this sense
includes a repeating unit cell, preferably one that allows a high
density of beads on the substrate. However, it should be noted that
these sites may not be discrete sites. That is, it is possible to
use a uniform surface of adhesive or chemical functionalities, for
example, that allows the association of beads at any position. That
is, the surface of the substrate is modified to allow association
of the microspheres at individual sites, whether or not those sites
are contiguous or non-contiguous with other sites. Thus, the
surface of the substrate may be modified such that discrete sites
are formed that can only have a single associated bead, or
alternatively, the surface of the substrate is modified and beads
may go down anywhere, but they end up at discrete sites.
[0057] In a preferred embodiment, the surface of the substrate is
modified to contain wells, i.e. depressions in the surface of the
substrate. This may be done as is generally known in the art using
a variety of techniques, including, but not limited to,
photolithography, stamping techniques, molding techniques and
microetchlng techniques. As will be appreciated by those in the
art, the technique used will depend on the composition and shape of
the substrate.
[0058] In a preferred embodiment, physical alterations are made in
a surface of the substrate to produce the sites. In a preferred
embodiment, the substrate is a fiber optic bundle and the surface
of the substrate is a terminal end of the fiber bundle, as is
generally described in Ser. Nos. 08/818,199 and 09/151,877, both of
which are hereby expressly incorporated by reference. In this
embodiment, wells are made in a terminal or distal end of a fiber
optic bundle comprising individual fibers. In this embodiment, the
cores of the individual fibers are etched, with respect to the
cladding, such that small wells or depressions are formed at one
end of the fibers. The required depth of the wells will depend on
the size of the beads to be added to the wells.
[0059] Generally in this embodiment, the microspheres are
non-covalently associated in the wells, although the wells may
additionally be chemically functionalized as is generally described
below, cross-linking agents may be used, or a physical barrier may
be used, i.e. a film or membrane over the beads.
[0060] In a preferred embodiment, the surface of the substrate is
modified to contain chemically modified sites, that can be used to
associate, either covalently or non-covalently, the microspheres of
the invention to the discrete sites or locations on the substrate.
"Chemically modified sites" in this context includes, but is not
limited to, the addition of a pattern of chemical functional groups
including amino groups, carboxy groups, oxo groups and thiol
groups, that can be used to covalently attach microspheres, which
generally also contain corresponding reactive functional groups;
the addition of a pattern of adhesive that can be used to bind the
microspheres (either by prior chemical functionalization for the
addition of the adhesive or direct addition of the adhesive); the
addition of a pattern of charged groups (similar to the chemical
functionalities) for the electrostatic association of the
microspheres, i.e. when the microspheres comprise charged groups
opposite to the sites; the addition of a pattern of chemical
functional groups that renders the sites differentially hydrophobic
or hydrophilic, such that the addition of similarly hydrophobic or
hydrophilic microspheres under suitable experimental conditions
will result in association of the microspheres to the sites on the
basis of hydroaffinity. For example, the use of hydrophobic sites
with hydrophobic beads, in an aqueous system, drives the
association of the beads preferentially onto the sites. As outlined
above, "pattern" in this sense includes the use of a uniform
treatment of the surface to allow association of the beads at
discrete sites, as well as treatment of the surface resulting in
discrete sites. As will be appreciated by those in the art, this
may be accomplished in a variety of ways.
[0061] The compositions of the invention further comprise a
population of microspheres. By "population" herein is meant a
plurality of beads as outlined above for arrays. Within the
population are separate subpopulations, which can be a single
microsphere or multiple identical microspheres. That is, in some
embodiments, as is more fully outlined below, the array may contain
only a single bead for each bioactive agent; preferred embodiments
utilize a plurality of beads of each type.
[0062] By "microspheres" or "beads" or "particles" or grammatical
equivalents herein is meant small discrete particles. The
composition of the beads will vary, depending on the class of
bioactive agent and the method of synthesis. Suitable bead
compositions include those used in peptide, nucleic acid and
organic moiety synthesis, including, but not limited to, plastics,
ceramics, glass, polystyrene, methylstyrene, acrylic polymers,
paramagnetic materials, thoria sol, carbon graphite, titanium
dioxide, latex or cross-linked dextrans such as Sepharose,
cellulose, nylon, cross-linked micelles and Teflon may all be used.
"Microsphere Detection Guide" from Bangs Laboratories, Fishers Ind.
is a helpful guide.
[0063] The beads need not be spherical; irregular particles may be
used. In addition, the beads may be porous, thus increasing the
surface area of the bead available for either bioactive agent
attachment or IBL attachment. The bead sizes range from nanometers,
i.e. 100 nm, to millimeters, i.e. 1 mm, with beads from about 0.2
micron to about 200 microns being preferred, and from about 0.5 to
about 5 micron being particularly preferred, although in some
embodiments smaller beads may be used.
[0064] It should be noted that a key component of the invention is
the use of a substrate/bead pairing that allows the association or
attachment of the beads at discrete sites on the surface of the
substrate, such that the beads do not move during the course of the
assay.
[0065] Each microsphere comprises a bioactive agent, although as
will be appreciated by those in the art, there may be some
microspheres which do not contain a bioactive agent, depending on
the synthetic methods. By "candidate bioactive agent" or "bioactive
agent" or "chemical functionality" or "binding ligand" herein is
meant as used herein describes any molecule, e.g., protein,
oligopeptide, small organic molecule, coordination complex,
polysaccharide, polynucleotide, etc. which can be attached to the
microspheres of the invention. It should be understood that the
compositions of the invention have two primary uses. In a preferred
embodiment, as is more fully outlined below, the compositions are
used to detect the presence of a particular target analyte; for
example, the presence or absence of a particular nucleotide
sequence or a particular protein, such as an enzyme, an antibody or
an antigen. In an alternate preferred embodiment, the compositions
are used to screen bioactive agents, i.e. drug candidates, for
binding to a particular target analyte.
[0066] Bioactive agents encompass numerous chemical classes, though
typically they are organic molecules, preferably small organic
compounds having a molecular weight of more than 100 and less than
about 2,500 Daltons. Bioactive agents comprise functional groups
necessary for structural interaction with proteins, particularly
hydrogen bonding, and typically include at least an amine,
carbonyl, hydroxyl or carboxyl group, preferably at least two of
the functional chemical groups. The bioactive agents often comprise
cyclical carbon or heterocyclic structures and/or aromatic or
polyaromatic structures substituted with one or more of the above
functional groups. Bioactive agents are also found among
biomolecules including peptides, nucleic acids, saccharides, fatty
acids, steroids, purines, pyrimidines, derivatives, structural
analogs or combinations thereof. Particularly preferred are nucleic
acids and proteins.
[0067] Bioactive agents can be obtained from a wide variety of
sources including libraries of synthetic or natural compounds. For
example, numerous means are available for random and directed
synthesis of a wide variety of organic compounds and biomolecules,
including expression of randomized oligonucleotides. Alternatively,
libraries of natural compounds in the form of bacterial, fungal,
plant and animal extracts are available or readily produced.
Additionally, natural or synthetically produced libraries and
compounds are readily modified through conventional chemical,
physical and biochemical means. Known pharmacological agents may be
subjected to directed or random chemical modifications, such as
acylation, alkylation, esterification and/or amidification to
produce structural analogs.
[0068] In a preferred embodiment, the bioactive agents are
proteins. By "protein" herein is meant at least two covalently
attached amino acids, which includes proteins, polypeptides,
oligopeptides and peptides. The protein may be made up of naturally
occurring amino acids and peptide bonds, or synthetic
peptidomimetic structures. Thus "amino acid", or "peptide residue",
as used herein means both naturally occurring and synthetic amino
acids. For example, homo-phenylalanine, citrulline and norleucine
are considered amino acids for the purposes of the invention. The
side chains may be in either the (R) or the (S) configuration. In
the preferred embodiment, the amino acids are in the (S) or
L-configuration. If non-naturally occurring side chains are used,
non-amino acid substituents may be used, for example to prevent or
retard in vivo degradations.
[0069] In one preferred embodiment, the bioactive agents are
naturally occurring proteins or fragments of naturally occurring
proteins. Thus, for example, cellular extracts containing proteins,
or random or directed digests of proteinaceous cellular extracts,
may be used. In this way libraries of procaryotic and eukaryotic
proteins may be made for screening in the systems described herein.
Particularly preferred in this embodiment are libraries of
bacterial, fungal, viral, and mammalian proteins, with the latter
being preferred, and human proteins being especially preferred.
[0070] In a preferred embodiment, the bioactive agents are peptides
of from about 5 to about 30 amino acids, with from about 5 to about
20 amino acids being preferred, and from about 7 to about 15 being
particularly preferred. The peptides may be digests of naturally
occurring proteins as is outlined above, random peptides, or
"biased" random peptides. By "randomized" or grammatical
equivalents herein is meant that each nucleic acid and peptide
consists of essentially random nucleotides and amino acids,
respectively. Since generally these random peptides (or nucleic
acids, discussed below) are chemically synthesized, they may
incorporate any nucleotide or amino acid at any position. The
synthetic process can be designed to generate randomized proteins
or nucleic acids, to allow the formation of all or most of the
possible combinations over the length of the sequence, thus forming
a library of randomized bioactive proteinaceous agents.
[0071] In a preferred embodiment, a library of bioactive agents are
used. The library should provide a sufficiently structurally
diverse population of bioactive agents to effect a
probabilistically sufficient range of binding to target analytes.
Accordingly, an interaction library must be large enough so that at
least one of its members will have a structure that gives it
affinity for the target analyte. Although it is difficult to gauge
the required absolute size of an interaction library, nature
provides a hint with the immune response: a diversity of
10.sup.7-10.sup.8 different antibodies provides at least one
combination with sufficient affinity to interact with most
potential antigens faced by an organism. Published in vitro
selection techniques have also shown that a library size of
10.sup.7 to 10.sup.8 is sufficient to find structures with affinity
for the target. Thus, in a preferred embodiment, at least 10.sup.6,
preferably at least 10.sup.7, more preferably at least 10.sup.8 and
most preferably at least 10.sup.9 different bioactive agents are
simultaneously analyzed in the subject methods. Preferred methods
maximize library size and diversity.
[0072] In a preferred embodiment, the library is fully randomized,
with no sequence preferences or constants at any position. In a
preferred embodiment, the library is biased. That is, some
positions within the sequence are either held constant, or are
selected from a limited number of possibilities. For example, in a
preferred embodiment, the nucleotides or amino acid residues are
randomized within a defined class, for example, of hydrophobic
amino acids, hydrophilic residues, sterically biased (either small
or large) residues, towards the creation of cysteines, for
cross-linking, prolines for SH-3 domains, serines, threonines,
tyrosines or histidines for phosphorylation sites, etc., or to
purines, etc.
[0073] In a preferred embodiment, the bioactive agents are nucleic
acids (generally called "probe nucleic acids" or "candidate probes"
herein). By "nucleic acid" or "oligonucleotide" or grammatical
equivalents herein means at least two nucleotides covalently linked
together. A nucleic acid of the present invention will generally
contain phosphodiester bonds, although in some cases, as outlined
below, nucleic acid analogs are included that may have alternate
backbones, comprising, for example, phosphoramide (Beaucage, et
al., Tetrahedron, 49(10):1925 (1993) and references therein;
Letsinger, J. Org. Chem., 35:3800 (1970); Sprinzl, et al., Eur. J.
Biochem., 81:579 (1977); Letsinger, et al., Nucl. Acids Res.,
14:3487 (1986); Sawai, et al., Chem. Lett., 805 (1984), Letsinger,
et al., J. Am. Chem. Soc., 110:4470 (1988); and Pauwels, et al.,
Chemica Scripta, 26:141 (1986)), phosphorothioate (Mag, et al.,
Nucleic Acids Res., 19:1437 (1991); and U.S. Pat. No. 5,644,048),
phosphorodithioate (Briu, et al., J. Am. Chem. Soc., 111:2321
(1989)), O-methylphophoroamidite linkages (see Eckstein,
Oligonucleotides and Analogues: A Practical Approach, Oxford
University Press), and peptide nucleic acid backbones and linkages
(see Egholm, J. Am. Chem. Soc., 114:1895 (1992); Meier, et al.,
Chem. Int. Ed. Engl., 31:1008 (1992); Nielsen, Nature 365:566
(1993); Carlsson, et al., Nature 380:207 (1996), all of which are
incorporated by reference)). Other analog nucleic acids include
those with positive backbones (Denpcy, et al., Proc. Natl. Acad.
Sci. USA, 92:6097 (1995)); non-ionic backbones (U.S. Pat. Nos.
5,386,023; 5,637,684; 5,602,240; 5,216,141; and U.S. Pat. No.
4,469,863; Kiedrowshi, et al., Angew. Chem. Intl. Ed. English,
30:423 (1991); Letsinger, et al., J. Am. Chem. Soc., 110:4470
(1988); Letsinger, et al., Nucleosides & Nucleotides, 13:1597
(1994); Chapters 2 and 3, ASC Symposium Series 580, "Carbohydrate
Modifications in Antisense Research", Ed. Y. S. Sanghui and P. Dan
Cook; Mesmaeker, et al., Biorganic & Medicinal Chem. Lett.,
4:395 (1994); Jeffs, et al., J. Biomolecular NMR, 34:17 (1994);
Tetrahedron Lett., 37:743 (1996)) and non-ribose backbones,
including those described in U.S. Pat. Nos. 5,235,033 and
5,034,506, and Chapters 6 and 7, ASC Symposium Series 580,
"Carbohydrate Modifications in Antisense Research", Ed. Y. S.
Sanghui and P. Dan Cook. Nucleic acids containing one or more
carbocyclic sugars are also included within the definition of
nucleic acids (see Jenkins, et al., Chem. Soc. Rev., (1995) pp.
169-176). Several nucleic acid analogs are described in Rawls, C
& E News, Jun. 2, 1997, page 35. All of these references are
hereby expressly incorporated by reference. These modifications of
the ribose-phosphate backbone may be done to facilitate the
addition of additional moieties such as labels, or to increase the
stability and half-life of such molecules in physiological
environments; for example, PNA is particularly preferred. In
addition, mixtures of naturally occurring nucleic acids and analogs
can be made. Alternatively, mixtures of different nucleic acid
analogs, and mixtures of naturally occurring nucleic acids and
analogs may be made. The nucleic acids may be single stranded or
double stranded, as specified, or contain portions of both double
stranded or single stranded sequence. The nucleic acid may be DNA,
both genomic and cDNA, RNA or a hybrid, where the nucleic acid
contains any combination of deoxyribo- and ribo-nucleotides, and
any combination of bases, including uracil, adenine, thymine,
cytosine, guanine, inosine, xanthanine, hypoxanthanine,
isocytosine, isoguanine, and base analogs such as nitropyrrole and
nitroindole, etc.
[0074] In a preferred embodiment, the bioactive agents are
libraries of clonal nucleic acids, including DNA and RNA. In this
embodiment, individual nucleic acids are prepared, generally using
conventional methods (including, but not limited to, propagation in
plasmid or phage vectors, amplification techniques including PCR,
etc.). The nucleic acids are preferably arrayed in some format,
such as a microtiter plate format, and beads added for attachment
of the libraries.
[0075] Attachment of the clonal libraries (or any of the nucleic
acids outlined herein) may be done in a variety of ways, as will be
appreciated by those in the art, including, but not limited to,
chemical or affinity capture (for example, including the
incorporation of derivatized nucleotides such as AminoLink or
biotinylated nucleotides that can then be used to attach the
nucleic acid to a surface, as well as affinity capture by
hybridization), cross-linking, and electrostatic attachment,
etc.
[0076] In a preferred embodiment, affinity capture is used to
attach the clonal nucleic acids to the beads. For example, cloned
nucleic acids can be derivatized, for example with one member of a
binding pair, and the beads derivatized with the other member of a
binding pair. Suitable binding pairs are as described herein for
IBL/DBL pairs. For example, the cloned nucleic acids may be
biotinylated (for example using enzymatic incorporate of
biotinylated nucleotides, for by photoactivated cross-linking of
biotin). Biotinylated nucleic acids can then be captured on
streptavidin-coated beads, as is known in the art. Similarly, other
hapten-receptor combinations can be used, such as digoxigenin and
anti-digoxigenin antibodies. Alternatively, chemical groups can be
added in the form of derivatized nucleotides, that can them be used
to add the nucleic acid to the surface.
[0077] Preferred attachments are covalent, although even relatively
weak interactions (i.e. non-covalent) can be sufficient to attach a
nucleic acid to a surface, if there are multiple sites of
attachment per each nucleic acid. Thus, for example, electrostatic
interactions can be used for attachment, for example by having
beads carrying the opposite charge to the bioactive agent.
[0078] Similarly, affinity capture utilizing hybridization can be
used to attach cloned nucleic acids to beads. For example, as is
known in the art, polyA+RNA is routinely captured by hybridization
to oligo-dT beads; this may include oligo-dT capture followed by a
cross-linking step, such as psoralen crosslinking). If the nucleic
acids of interest do not contain a polyA tract, one can be attached
by polymerization with terminal transferase, or via ligation of an
oligoA linker, as is known in the art.
[0079] Alternatively, chemical crosslinking may be done, for
example by photoactivated crosslinking of thymidine to reactive
groups, as is known in the art.
[0080] In general, special methods are required to decode clonal
arrays, as is more fully outlined below.
[0081] As described above generally for proteins, nucleic acid
bioactive agents may be naturally occurring nucleic acids, random
nucleic acids, or "biased" random nucleic acids. For example,
digests of procaryotic or eukaryotic genomes may be used as is
outlined above for proteins.
[0082] In general, probes of the present invention are designed to
be complementary to a target sequence (either the target analyte
sequence of the sample or to other probe sequences, as is described
herein), such that hybridization of the target and the probes of
the present invention occurs. This complementarily need not be
perfect; there may be any number of base pair mismatches that will
interfere with hybridization between the target sequence and the
single stranded nucleic acids of the present invention. However, if
the number of mutations is so great that no hybridization can occur
under even the least stringent of hybridization conditions, the
sequence is not a complementary target sequence. Thus, by
"substantially complementary" herein is meant that the probes are
sufficiently complementary to the target sequences to hybridize
under the selected reaction conditions. High stringency conditions
are known in the art; see for example Maniatis, et al., Molecular
Cloning: A Laboratory Manual, 2d Edition, 1989, and Short Protocols
in Molecular Biology, ed. Ausubel, et al., both of which are hereby
incorporated by reference. Stringent conditions are
sequence-dependent and will be different in different
circumstances. Longer sequences hybridize specifically at higher
temperatures. An extensive guide to the hybridization of nucleic
acids is found in Tijssen, Techniques in Biochemistry and Molecular
Biology--Hybridization with Nucleic Acid Probes, "Overview of
principles of hybridization and the strategy of nucleic acid
assays" (1993). Generally, stringent conditions are selected to be
about 5-10 C lower than the thermal melting point (T.sub.m) for the
specific sequence at a defined ionic strength pH. The T.sub.m is
the temperature (under defined ionic strength, pH and nucleic acid
concentration) at which 50% of the probes complementary to the
target hybridize to the target sequence at equilibrium (as the
target sequences are present in excess, at T.sub.m, 50% of the
probes are occupied at equilibrium). Stringent conditions will be
those in which the salt concentration is less than about 1.0 M
sodium ion, typically about 0.01 to 1.0 M sodium ion concentration
(or other salts) at pH 7.0 to 8.3 and the temperature is at least
about 30 C for short probes (e.g. 10 to 50 nucleotides) and at
least about 60 C for long probes (e.g. greater than 50
nucleotides). Stringent conditions may also be achieved with the
addition of destabilizing agents such as formamide. In another
embodiment, less stringent hybridization conditions are used; for
example, moderate or low stringency conditions may be used, as are
known in the art; see Maniatis and Ausubel, supra, and Tijssen,
supra.
[0083] The term "target sequence" or grammatical equivalents herein
means a nucleic acid sequence on a single strand of nucleic acid.
The target sequence may be a portion of a gene, a regulatory
sequence, genomic DNA, cDNA, RNA including mRNA and rRNA, or
others. It may be any length, with the understanding that longer
sequences are more specific. As will be appreciated by those in the
art, the complementary target sequence may take many forms. For
example, it may be contained within a larger nucleic acid sequence,
i.e. all or part of a gene or mRNA, a restriction fragment of a
plasmid or genomic DNA, among others. As is outlined more fully
below, probes are made to hybridize to target sequences to
determine the presence or absence of the target sequence in a
sample. Generally speaking, this term will be understood by those
skilled in the art.
[0084] In a preferred embodiment, the bioactive agents are organic
chemical moieties, a wide variety of which are available in the
literature.
[0085] In a preferred embodiment, each bead comprises a single type
of bioactive agent, although a plurality of individual bioactive
agents are preferably attached to each bead. Similarly, preferred
embodiments utilize more than one microsphere containing a unique
bioactive agent; that is, there is redundancy built into the system
by the use of subpopulations of microspheres, each microsphere in
the subpopulation containing the same bioactive agent.
[0086] As will be appreciated by those in the art, the bioactive
agents may either be synthesized directly on the beads, or they may
be made and then attached after synthesis. In a preferred
embodiment, linkers are used to attach the bioactive agents to the
beads, to allow both good attachment, sufficient flexibility to
allow good interaction with the target molecule, and to avoid
undesirable binding reactions.
[0087] In a preferred embodiment, the bioactive agents are
synthesized directly on the beads. As is known in the art, many
classes of chemical compounds are currently synthesized on solid
supports, including beads, such as peptides, organic moieties, and
nucleic acids.
[0088] In a preferred embodiment, the bioactive agents are
synthesized first, and then covalently attached to the beads. As
will be appreciated by those in the art, this will be done
depending on the composition of the bioactive agents and the beads.
The functionalization of solid support surfaces such as certain
polymers with chemically reactive groups such as thiols, amines,
carboxyls, etc., is generally known in the art. Accordingly,
"blank" microspheres may be used that have surface chemistries that
facilitate the attachment of the desired functionality by the user.
Some examples of these surface chemistries for blank microspheres
include, but are not limited to, amino groups including aliphatic
and aromatic amines, carboxylic acids, aldehydes, amides,
chloromethyl groups, hydrazide, hydroxyl groups, sulfonates and
sulfates.
[0089] These functional groups can be used to add any number of
different candidate agents to the beads, generally using known
chemistries. For example, candidate agents containing carbohydrates
may be attached to an amino-functionalized support; the aldehyde of
the carbohydrate is made using standard techniques, and then the
aldehyde is reacted with an amino group on the surface. In an
alternative embodiment, a sulfhydryl linker may be used. There are
a number of sulfhydryl reactive linkers known in the art such as
SPDP, maleimides, -haloacetyls, and pyridyl disulfides (see for
example the 1994 Pierce Chemical Company catalog, technical section
on cross-linkers, pages 155-200, incorporated herein by reference)
which can be used to attach cysteine containing proteinaceous
agents to the support. Alternatively, an amino group on the
candidate agent may be used for attachment to an amino group on the
surface. For example, a large number of stable bifunctional groups
are well known in the art, including homobifunctional and
heterobifunctionallinkers (see Pierce Catalog and Handbook, pages
155-200). In an additional embodiment, carboxyl groups (either from
the surface or from the candidate agent) may be derivatized using
well known linkers (see the Pierce catalog). For example,
carbodiimides activate carboxyl groups for attack by good
nucleophiles such as amines (see Torchilin et al., Critical Rev.
Therapeutic Drug Carrier Systems, 7(4):275-308 (1991), expressly
incorporated herein). Proteinaceous candidate agents may also be
attached using other techniques known in the art, for example for
the attachment of antibodies to polymers; see Slinkin et al.,
Bioconj. Chem. 2:342-348 (1991); Torchilin et al., supra;
Trubetskoy et al., Bioconj. Chem. 3:323-327 (1992); King et al.,
Cancer Res. 54:6176-6185 (1994); and Wilbur et al., Bioconjugate
Chem. 5:220-235 (1994), all of which are hereby expressly
incorporated by reference). It should be understood that the
candidate agents may be attached in a variety of ways, including
those listed above. Preferably, the manner of attachment does not
significantly alter the functionality of the candidate agent; that
is, the candidate agent should be attached in such a flexible
manner as to allow its interaction with a target.
[0090] Specific techniques for immobilizing enzymes on microspheres
are known in the prior art. In one case, NH.sub.2 surface chemistry
microspheres are used. Surface activation is achieved with a 2.5%
glutaraldehyde in phosphate buffered saline (10 mM) providing a pH
of 6.9. (138 mM NaCl, 2.7 mM, KCl). This is stirred on a stir bed
for approximately 2 hours at room temperature. The microspheres are
then rinsed with ultrapure water plus 0.01% tween 20
(surfactant)-0.02%, and rinsed again with a pH 7.7 PBS plus 0.01%
tween 20. Finally, the enzyme is added to the solution, preferably
after being prefiltered using a 0.45 m amicon micropure filter.
[0091] In some embodiments, the microspheres may additionally
comprise identifier binding ligands for use in certain decoding
systems. By "identifier binding ligands" or "IBLs" herein is meant
a compound that will specifically bind a corresponding decoder
binding ligand (DBL) to facilitate the elucidation of the identity
of the bioactive agent attached to the bead. That is, the IBL and
the corresponding DBL form a binding partner pair. By "specifically
bind" herein is meant that the IBL binds its DBL with specificity
sufficient to differentiate between the corresponding DBL and other
DBLs (that is, DBLs for other IBLs), or other components or
contaminants of the system. The binding should be sufficient to
remain bound under the conditions of the decoding step, including
wash steps to remove non-specific binding. In some embodiments, for
example when the IBLs and corresponding DBLs are proteins or
nucleic acids, the dissociation constants of the IBL to its DBL
will be less than about 10.sup.-4-10.sup.-6 M.sup.-1, with less
than about 10.sup.-5 to 10.sup.-9 M.sup.-1 being preferred and less
than about 10.sup.-7-10.sup.-9 M.sup.-1 being particularly
preferred.
[0092] IBL-DBL binding pairs are known or can be readily found
using known techniques. For example, when the IBL is a protein, the
DBLs include proteins (particularly including antibodies or
fragments thereof (FAbs, etc.)) or small molecules, or vice versa
(the IBL is an antibody and the DBL is a protein). Metal ion-metal
ion ligands or chelators pairs are also useful. Antigen-antibody
pairs, enzymes and substrates or inhibitors, other protein-protein
interacting pairs, receptor-ligands, complementary nucleic acids
(including nucleic acid molecules that form triple helices), and
carbohydrates and their binding partners are also suitable binding
pairs. Nucleic acid-nucleic acid binding proteins pairs are also
useful, including single-stranded or double-stranded nucleic acid
binding proteins, and small molecule nucleic acid binding agents.
Similarly, as is generally described in U.S. Pat. Nos. 5,270,163,
5,475,096, 5,567,588, 5,595,877, 5,637,459, 5,683,867, 5,705,337,
and related patents, hereby incorporated by reference, nucleic acid
"aptamers" can be developed for binding to virtually any target;
such an aptamer-target pair can be used as the IBL-DBL pair.
Similarly, there is a wide body of literature relating to the
development of binding pairs based on combinatorial chemistry
methods.
[0093] In a preferred embodiment, the IBL is a protein,
particularly an enzyme, that is conjugated to a bead. One advantage
of using proteins/enzymes as IBLs is that many proteins and enzymes
are commercially available in relatively pure preparations.
Proteins contain many suitable functional groups for attachment to
beads. Moreover, the identity of many small molecules including
substrates that bind the proteins/enzymes are known. Such small
molecules serve as the DBL. In addition, the small molecule can
serve as the IBL and the enzyme as the DBL. As will be appreciated
by those in the art, combinations of enzymes and small molecules
can be used as well, or multiple enzymes and small molecules.
Another potential advantage relates to the purification of the
labeled proteins. If one conjugates a fluorophore to a
recrystallizable protein, then the unlabeled protein can be removed
from labeled protein by use of crystallization. By filtering off
the crystalline solid, the labeled protein will be concentrated in
the filtrate.
[0094] In an alternative embodiment, antigen-antibody pairs are
used as DBL-IBL combinations. In one embodiment antibodies are
conjugated to different labels, defined below, that are then used
to decode antigen-labeled beads.
[0095] In a preferred embodiment, the IBL is a molecule whose color
or luminescence properties change in the presence of a
selectively-binding DBL.
[0096] In one embodiment, the DBL may be attached to a bead, i.e. a
"decoder bead", that may carry a label such as a fluorophore.
[0097] In a preferred embodiment, the IBL-DBL pair comprise
substantially complementary single-stranded nucleic acids. In this
embodiment, the binding ligands can be referred to as "identifier
probes" and "decoder probes". Generally, the identifier and decoder
probes range from about 4 basepairs in length to about 1000, with
from about 6 to about 100 being preferred, and from about 8 to
about 40 being particularly preferred. What is important is that
the probes are long enough to be specific, i.e. to distinguish
between different IBL-DBL pairs, yet short enough to allow both a)
dissociation, if necessary, under suitable experimental conditions,
and b) efficient hybridization.
[0098] In a preferred embodiment, as is more fully outlined below,
the IBLs do not bind to DBLs. Rather, the IBLs are used as
identifier moieties ("IMs") that are identified directly, for
example through the use of mass spectroscopy.
[0099] Alternatively, in a preferred embodiment, the IBL and the
bioactive agent are the same moiety; thus, for example, as outlined
herein, particularly when no optical signatures are used, the
bioactive agent can serve as both the identifier and the agent. For
example, in the case of nucleic acids, the bead-bound probe (which
serves as the bioactive agent) can also bind decoder probes, to
identify the sequence of the probe on the bead. Thus, in this
embodiment, the DBLs bind to the bioactive agents. This is
particularly useful as this embodiment can give information about
the array or the assay in addition to decoding. For example, as is
more fully described below, the use of the DBLs allows array
calibration and assay development. This may be done even if the
DBLs are not used as such; for example in non-random arrays, the
use of these probe sets can allow array calibration and assay
development even if decoding is not required.
[0100] In a preferred embodiment, the microspheres do not contain
an optical signature. That is, as outlined in U.S. Ser. Nos.
08/818,199 and 09/151,877, previous work had each subpopulation of
microspheres comprising a unique optical signature or optical tag
that is used to identify the unique bioactive agent of that
subpopulation of microspheres; that is, decoding utilizes optical
properties of the beads such that a bead comprising the unique
optical signature may be distinguished from beads at other
locations with different optical signatures. Thus the previous work
assigned each bioactive agent a unique optical signature such that
any microspheres comprising that bioactive agent are identifiable
on the basis of the signature. These optical signatures comprised
dyes, usually chromophores or fluorophores, that were entrapped or
attached to the beads themselves. Diversity of optical signatures
utilized different fluorochromes, different ratios of mixtures of
fluorochromes, and different concentrations (intensities) of
fluorochromes.
[0101] Thus, the present invention does not rely solely on the use
of optical properties to decode the arrays. However, as will be
appreciated by those in the art, it is possible in some embodiments
to utilize optical signatures as an additional coding method, in
conjunction with the present system. Thus, for example, as is more
fully outlined below, the size of the array may be effectively
increased while using a single set of decoding moieties in several
ways, one of which is the use in combination with optical
signatures one beads. Thus, for example, using one "set" of
decoding molecules, the use of two populations of beads, one with
an optical signature and one without, allows the effective doubling
of the array size. The use of multiple optical signatures similarly
increases the possible size of the array.
[0102] In a preferred embodiment, each subpopulation of beads
comprises a plurality of different IBLs. By using a plurality of
different IBLs to encode each bioactive agent, the number of
possible unique codes is substantially increased. That is, by using
one unique IBL per bioactive agent, the size of the array will be
the number of unique IBLs (assuming no "reuse" occurs, as outlined
below). However, by using a plurality of different IBLs per bead,
n, the size of the array can be increased to 2.sup.n, when the
presence or absence of each IBL is used as the indicator. For
example, the assignment of 10 IBLs per bead generates a 10 bit
binary code, where each bit can be designated as "1" (IBL is
present) or "0" (IBL is absent). A 10 bit binary code has 2.sup.10
possible variants. However, as is more fully discussed below, the
size of the array may be further increased if another parameter is
included such as concentration or intensity; thus for example, if
two different concentrations of the IBL are used, then the array
size increases as 3.sup.n. Thus, in this embodiment, each
individual bioactive agent in the array is assigned a combination
of IBLs, which can be added to the beads prior to the addition of
the bioactive agent, after, or during the synthesis of the
bioactive agent, i.e. simultaneous addition of IBLs and bioactive
agent components.
[0103] Alternatively, when the bioactive agent is a polymer of
different residues, i.e. when the bioactive agent is a protein or
nucleic acid, the combination of different IBLs can be used to
elucidate the sequence of the protein or nucleic acid.
[0104] Thus, for example, using two different IBLs (IBL1 and IBL2),
the first position of a nucleic acid can be elucidated: for
example, adenosine can be represented by the presence of both IBL1
and IBL2; thymidine can be represented by the presence of IBL1 but
not IBL2, cytosine can be represented by the presence of IBL2 but
not IBL1, and guanosine can be represented by the absence of both.
The second position of the nucleic acid can be done in a similar
manner using IBL3 and IBL4; thus, the presence of IBL1, IBL2, IBL3
and IBL4 gives a sequence of AA; IBL1, IBL2, and IBL3 shows the
sequence AT; IBL1, IBL3 and IBL4 gives the sequence TA, etc. The
third position utilizes IBL5 and IBL6, etc. In this way, the use of
20 different identifiers can yield a unique code for every possible
10-mer.
[0105] The system is similar for proteins but requires a larger
number of different IBLs to identify each position, depending on
the allowed diversity at each position. Thus for example, if every
amino acid is allowed at every position, five different IBLs are
required for each position. However, as outlined above, for example
when using random peptides as the bioactive agents, there may be
bias built into the system; not all amino acids may be present at
all positions, and some positions may be preset; accordingly, it
may be possible to utilize four different IBLs for each amino
acid.
[0106] In this way, a sort of "bar code" for each sequence can be
constructed; the presence or absence of each distinct IBL will
allow the identification of each bioactive agent.
[0107] In addition, the use of different concentrations or
densities of IBLs allows a "reuse" of sorts. If, for example, the
bead comprising a first agent has a 1.times. concentration of IBL,
and a second bead comprising a second agent has a 10.times.
concentration of IBL, using saturating concentrations of the
corresponding labeled DBL allows the user to distinguish between
the two beads.
[0108] In addition, the use of different ratios of labeled to
unlabeled IBLs allows for a differentiation of the intensity rather
than the concentration of IBLs. For example, if a bead comprising a
first agent has a ratio of 1:1 (labeled:unlabeled IBL), while a
second bead comprising a second agent has a ration of 1:10
(labeled:unlabeled IBL), the user will distinguish the different
intensities between the two beads. This procedure offers the
advantage that a broad spectrum of intensities of labels can be
created using only a single label.
[0109] Once the microspheres comprising the candidate agents and
the unique IBLs are generated, they are added to the substrate to
form an array. It should be noted that while most of the methods
described herein add the beads to the substrate prior to the assay,
the order of making, using and decoding the array can vary. For
example, the array can be made, decoded, and then the assay done.
Alternatively, the array can be made, used in an assay, and then
decoded; this may find particular use when only a few beads need be
decoded. Alternatively, the beads can be added to the assay
mixture, i.e. the sample containing the target analytes, prior to
the addition of the beads to the substrate; after addition and
assay, the array may be decoded. This is particularly preferred
when the sample comprising the beads is agitated or mixed; this can
increase the amount of target analyte bound to the beads per unit
time, and thus (in the case of nucleic acid assays) increase the
hybridization kinetics. This may find particular use in cases where
the concentration of target analyte in the sample is low;
generally, for low concentrations, long binding times must be
used.
[0110] In addition, adding the beads to the assay mixture can allow
sorting or selection. For example, a large library of beads may be
added to a sample, and only those beads that bind the sample may be
added to the substrate. For example, if the target analyte is
fluorescently labeled (either directly (for example by the
incorporation of labels into nucleic acid amplification reactions)
or indirectly (for example via the use of sandwich assays)), beads
that exhibit fluorescence as a result of target analyte binding can
be sorted via Fluorescence Activated Cell Sorting (FACS) and only
these beads added to an array and subsequently decoded. Similarly,
the sorting may be accomplished through affinity techniques;
affinity columns comprising the target analytes can be made, and
only those beads which bind are used on the array. Similarly, two
bead systems can be used; for example, magnetic beads comprising
the target analytes can be used to "pull out" those beads that will
bind to the targets, followed by subsequent release of the magnetic
beads (for example via temperature elevation) and addition to an
array.
[0111] In general, the methods of making the arrays and of decoding
the arrays is done to maximize the number of different candidate
agents that can be uniquely encoded. The compositions of the
invention may be made in a variety of ways. In general, the arrays
are made by adding a solution or slurry comprising the beads to a
surface containing the sites for association of the beads. This may
be done in a variety of buffers, including aqueous and organic
solvents, and mixtures. The solvent can evaporate, and excess beads
removed.
[0112] In a preferred embodiment, when non-covalent methods are
used to associate the beads to the array, a novel method of loading
the beads onto the array is used. This method comprises exposing
the array to a solution of particles (including microspheres and
cells) and then applying energy, e.g. agitating or vibrating the
mixture. This results in an array comprising more tightly
associated particles, as the agitation is done with sufficient
energy to cause weakly-associated beads to fall off (or out, in the
case of wells). These sites are then available to bind a different
bead. In this way, beads that exhibit a high affinity for the sites
are selected. Arrays made in this way have two main advantages as
compared to a more static loading: first of all, a higher
percentage of the sites can be filled easily, and secondly, the
arrays thus loaded show a substantial decrease in bead loss during
assays. Thus, in a preferred embodiment, these methods are used to
generate arrays that have at least about 50% of the sites filled,
with at least about 75% being preferred, and at least about 90%
being particularly preferred. Similarly, arrays generated in this
manner preferably lose less than about 20% of the beads during an
assay, with less than about 10% being preferred and less than about
5% being particularly preferred.
[0113] In this embodiment, the substrate comprising the surface
with the discrete sites is immersed into a solution comprising the
particles (beads, cells, etc.). The surface may comprise wells, as
is described herein, or other types of sites on a patterned surface
such that there is a differential affinity for the sites. This
differential affinity results in a competitive process, such that
particles that will associate more tightly are selected.
Preferably, the entire surface to be "loaded" with beads is in
fluid contact with the solution. This solution is generally a
slurry ranging from about 10,000:1 beads:solution (vol:vol) to 1:1.
Generally, the solution can comprise any number of reagents,
including aqueous buffers, organic solvents, salts, other reagent
components, etc. In addition, the solution preferably comprises an
excess of beads; that is, there are more beads than sites on the
array. Preferred embodiments utilize two-fold to billion-fold
excess of beads.
[0114] The immersion can mimic the assay conditions; for example,
if the array is to be "dipped" from above into a microtiter plate
comprising samples, this configuration can be repeated for the
loading, thus minimizing the beads that are likely to fall out due
to gravity.
[0115] Once the surface has been immersed, the substrate, the
solution, or both are subjected to a competitive process, whereby
the particles with lower affinity can be disassociated from the
substrate and replaced by particles exhibiting a higher affinity to
the site. This competitive process is done by the introduction of
energy, in the form of heat, sonication, stirring or mixing,
vibrating or agitating the solution or substrate, or both.
[0116] A preferred embodiment utilizes agitation or vibration. In
general, the amount of manipulation of the substrate is minimized
to prevent damage to the array; thus, preferred embodiments utilize
the agitation of the solution rather than the array, although
either will work. As will be appreciated by those in the art, this
agitation can take on any number of forms, with a preferred
embodiment utilizing microtiter plates comprising bead solutions
being agitated using microtiter plate shakers.
[0117] The agitation proceeds for a period of time sufficient to
load the array to a desired fill. Depending on the size and
concentration of the beads and the size of the array, this time may
range from about 1 second to days, with from about 1 minute to
about 24 hours being preferred.
[0118] In a preferred embodiment, the substrate is pressed into a
mixture of dry beads (although slurries may be used as well), and
then tapped to remove excess beads.
[0119] It should be noted that not all sites of an array may
comprise a bead; that is, there may be some sites on the substrate
surface which are empty. In addition, there may be some sites that
contain more than one bead, although this is not preferred.
[0120] In some embodiments, for example when chemical attachment is
done, it is possible to associate the beads in a non-random or
ordered way. For example, using photoactivatible attachment linkers
or photoactivatible adhesives or masks, selected sites on the array
may be sequentially rendered suitable for attachment, such that
defined populations of beads are laid down.
[0121] The arrays of the present invention are constructed such
that information about the identity of the candidate agent is built
into the array, such that the random deposition of the beads in the
fiber wells can be "decoded" to allow identification of the
candidate agent at all positions. This may be done in a variety of
ways, and either before, during or after the use of the array to
detect target molecules.
[0122] Thus, after the array is made, it is "decoded" in order to
identify the location of one or more of the bioactive agents, i.e.
each subpopulation of beads, on the substrate surface.
[0123] In a preferred embodiment, a selective decoding system is
used. In this case, only those microspheres exhibiting a change in
the optical signal as a result of the binding of a target analyte
are decoded. This is commonly done when the number of "hits", i.e.
the number of sites to decode, is generally low. That is, the array
is first scanned under experimental conditions in the absence of
the target analytes. The sample containing the target analytes is
added, and only those locations exhibiting a change in the optical
signal are decoded. For example, the beads at either the positive
or negative signal locations may be either selectively tagged or
released from the array (for example through the use of
photocleavable linkers), and subsequently sorted or enriched in a
fluorescence-activated cell sorter (FACS). That is, either all the
negative beads are released, and then the positive beads are either
released or analyzed in situ, or alternatively all the positives
are released and analyzed. Alternatively, the labels may comprise
halogenated aromatic compounds, and detection of the label is done
using for example gas chromatography, chemical tags, isotopic tags,
or mass spectral tags.
[0124] As will be appreciated by those in the art, this may also be
done in systems where the array is not decoded; i.e. there need not
ever be a correlation of bead composition with location. In this
embodiment, the beads are loaded on the array, and the assay is
run. The "positives", i.e. those beads displaying a change in the
optical signal as is more fully outlined below, are then "marked"
to distinguish or separate them from the "negative" beads. This can
be done in several ways, preferably using fiber optic arrays. In a
preferred embodiment, each bead contains a fluorescent dye. After
the assay and the identification of the "positives" or "active
beads", light is shown down either only the positive fibers or only
the negative fibers, generally in the presence of a light-activated
reagent (typically dissolved oxygen). In the former case, all the
active beads are photobleached. Thus, upon non-selective release of
all the beads with subsequent sorting, for example using a
fluorescence activated cell sorter (FACS) machine, the
non-fluorescent active beads can be sorted from the fluorescent
negative beads. Alternatively, when light is shown down the
negative fibers, all the negatives are non-fluorescent and the
positives are fluorescent, and sorting can proceed. The
characterization of the attached bioactive agent may be done
directly, for example using mass spectroscopy.
[0125] Alternatively, the identification may occur through the use
of identifier moieties ("IMs"), which are similar to IBLs but need
not necessarily bind to DBLs. That is, rather than elucidate the
structure of the bioactive agent directly, the composition of the
IMs may serve as the identifier. Thus, for example, a specific
combination of IMs can serve to code the bead, and be used to
identify the agent on the bead upon release from the bead followed
by subsequent analysis, for example using a gas chromatograph or
mass spectroscope.
[0126] Alternatively, rather than having each bead contain a
fluorescent dye, each bead comprises a non-fluorescent precursor to
a fluorescent dye. For example, using photocleavable protecting
groups, such as certain ortho-nitrobenzyl groups, on a fluorescent
molecule, photoactivation of the fluorochrome can be done. After
the assay, light is shown down again either the "positive" or the
"negative" fibers, to distinguish these populations. The
illuminated precursors are then chemically converted to a
fluorescent dye. All the beads are then released from the array,
with sorting, to form populations of fluorescent and
non-fluorescent beads (either the positives and the negatives or
vice versa).
[0127] In an alternate preferred embodiment, the sites of
association of the beads (for example the wells) include a
photopolymerizable reagent, or the photopolymerizable agent is
added to the assembled array. After the test assay is run, light is
shown down again either the "positive" or the "negative" fibers, to
distinguish these populations. As a result of the irradiation,
either all the positives or all the negatives are polymerized and
trapped or bound to the sites, while the other population of beads
can be released from the array.
[0128] In a preferred embodiment, the location of every bioactive
agent is determined using decoder binding ligands (DBLs). As
outlined above, DBLs are binding ligands that will either bind to
identifier binding ligands, if present, or to the bioactive agents
themselves, preferably when the bioactive agent is a nucleic acid
or protein.
[0129] In a preferred embodiment, as outlined above, the DBL binds
to the IBL.
[0130] In a preferred embodiment, the bioactive agents are
single-stranded nucleic acids and the DBL is a substantially
complementary single-stranded nucleic acid that binds (hybridizes)
to the bioactive agent, termed a decoder probe herein. A decoder
probe that is substantially complementary to each candidate probe
is made and used to decode the array. In this embodiment, the
candidate probes and the decoder probes should be of sufficient
length (and the decoding step run under suitable conditions) to
allow specificity; i.e. each candidate probe binds to its
corresponding decoder probe with sufficient specificity to allow
the distinction of each candidate probe.
[0131] In a preferred embodiment, the DBLs are either directly or
indirectly labeled. By "labeled" herein is meant that a compound
has at least one element, isotope or chemical compound attached to
enable the detection of the compound. In general, labels fall into
three classes: a) isotopic labels, which may be radioactive or
heavy isotopes; b) magnetic, electrical, thermal; and c) colored or
luminescent dyes; although labels include enzymes and particles
such as magnetic particles as well. Examples of such labels
include: peroxidase; alkaline phosphatase; biotin; urease; E
alactosidase; BSA/KLH; gold particles; quantum dots; redox
indicators; pH indicators; E-lactamase; luciferase; TSA; SPA;
chemiluminescence; sonoluminescence; fluorophores; phosphors; and,
fluorescent metal ion sensors. Preferred labels include luminescent
labels. In a preferred embodiment, the DBL is directly labeled,
that is, the DBL comprises a label. In an alternate embodiment, the
DBL is indirectly labeled; that is, a labeling binding ligand (LBL)
that will bind to the DBL is used. In this embodiment, the labeling
binding ligand-DBL pair can be as described above for IBL-DBL
pairs. Suitable labels include, but are not limited to, fluorescent
lanthanide complexes, including those of Europium and Terbium,
fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin,
coumarin, methyl-coumarins, pyrene, Malacite green, stilbene,
Lucifer Yellow, Cascade Blue.TM., Texas Red, FITC, PE, cy3, cy5 and
others described in the 6th Edition of the Molecular Probes
Handbook by Richard P. Haugland, hereby expressly incorporated by
reference. In addition, ion-sensitive dyes such as bis-BITC,
Newport green, fluo 3, fura red find use in the invention.
[0132] In one embodiment, the label is a molecule whose color or
luminescence properties change in the presence of the IBL, due to a
change in the local environment. For example, the label may be: (1)
a fluorescent pH indicator whose emission intensity changes with
pH; (2) a fluorescent ion indicator, whose emission properties
change with ion concentration; or (3) a fluorescent molecule such
as an ethidium salt whose fluorescence intensity increases in
hydrophobic environments.
[0133] In one embodiment ion sensitive dyes are used for decoding.
That is, dyes that fluoresce in the presence of certain ions are
used. In a preferred embodiment the ion sensitive dyes are attached
to the beads. An advantage of attaching dyes directly to
microspheres is that the cost associated with oligonucleotide
synthesis is avoided.
[0134] Accordingly, when such dyes are attached to the beads they
can be used to encode a specific nucleotide sequence, or the
identity of the bioactive agent. Once the beads are distributed on
the array, the array is contacted with buffers that include certain
ions. In the presence of appropriate ion, certain dyes will
fluoresce and fluorescence is detected. In a preferred embodiment,
the array is contacted with different buffers sequentially. Each
buffer contains a different ion. Preferably the array is washed
between buffer solutions so that any signal produced as a result of
the contact with the ion solution is diminished.
[0135] In this embodiment multiple different ion sensitive dyes can
be used. That is, preferably, more than 2 with more than 3 or 4
dyes are attached to the microspheres. In some embodiments, more
than 10 dyes are attached to the microsphere.
[0136] One advantage to using ion sensitive dyes is that
fluorescence bleed-through is diminished. That is, because the
response of the dyes is either positive or negative, i.e. they
interact or they don't, bleed-through is avoided.
[0137] In a preferred embodiment, decoding of self-assembled random
arrays is done on the basis of pH titration. In this embodiment, in
addition to bioactive agents, the beads comprise optical
signatures, wherein the optical signatures are generated by the use
of pH-responsive dyes (sometimes referred to herein as "pH dyes")
such as fluorophores. This embodiment is similar to that outlined
in PCT US98/05025 and U.S. Ser. No. 09/151,877, both of which are
expressly incorporated by reference, except that the dyes used in
the present invention exhibits changes in fluorescence intensity
(or other properties) when the solution pH is adjusted from below
the pKa to above the pKa (or vice versa). In a preferred
embodiment, a set of pH dyes is used, each with a different pKa,
preferably separated by at least 0.5 pH units. Preferred
embodiments utilize a pH dye set of pKa's of 2.0, 2.5, 3.0, 3.5,
4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0,
10.5, 11, and 11.5. Each bead can contain any subset of the pH
dyes, and in this way a unique code for the bioactive agent is
generated. Thus, the decoding of an array is achieved by titrating
the array from pH 1 to pH 13, and measuring the fluorescence signal
from each bead as a function of solution pH.
[0138] In one embodiment, the decoder probes are labeled in situ;
that is, they need not be labeled prior to the decoding reaction.
In this embodiment, the incoming decoder probe is shorter than the
candidate probe, creating a 5' "overhang" on the decoding probe.
The addition of labeled ddNTPs (each labeled with a unique tag) and
a polymerase will allow the addition of the tags in a sequence
specific manner, thus creating a sequence-specific pattern of
signals. Similarly, other modifications can be done, including
ligation, etc.
[0139] In one embodiment, a series of sequential extension and
denaturing stages allows for positional decoding of the beads. That
is, following one round of extension, the DBL is removed or
stripped from the IBL and a second round of extension is performed.
The contents of any given primer extension reaction is shown in
FIG. 8.
[0140] In the primer extension method, up to four
fluorescently-labeled dideoxynucleotide terminators each with a
different colored label (e.g. using ABl's dRhodamine.RTM.
terminators such as dR110, dR6G, dTAMRA, dROX or Big Dye.RTM.
Terminators) could be used for `painting.` The number of unique
beads that can be decoded is given by N=y.sup.x, where n=the number
of unique codes, y=the number of colors and x=the number of stages
or primer extension reactions. This formula yields 4096 unique
codes using 6 primer extension stages and four colors (i.e.
4096=4.sup.6).
[0141] In a preferred embodiment the bioactive agent is synthesized
with encoding sequences that allows decoding to be accomplished
independent of the useable portion of the bioactive agent, i.e.
nucleic acid sequence. By "useable" portion is meant the adapter
sequence or target sequence. In a preferred embodiment the
bioactive agent also is synthesized with a primer region to
facilitate sequencing or primer extension analysis of the encoding
sequence (see FIG. 12). As shown in FIG. 12, a primer complementary
to a portion of the ssDNA on the bead is extended by single base
polymerase extension using dye-labeled chain terminating
nucleotides. After incorporation of a labeled, i.e. fluorescent,
chain terminator and reading of the array, i.e. detecting the
label, the fluorescence is removed. Removal of the fluorescence is
accomplished directly when reversible chain terminators are used
(see Canard and Sarfati, Gene, 148(1): 1-6, 1994, which is
expressly incorporated herein by reference). However, in a
preferred embodiment the primer is dissociated from the bead and a
subsequent primer is annealed. Preferably the subsequent primer is
1 nucleotide longer than the former primer so that an additional
nucleotide in the encoding sequence can be interrogated. In a
preferred embodiment the subsequent primer is lengthened by the
addition of a degenerate base. This can be accomplished either by
incorporating all 4 nucleotides into the additional position of the
primer, or by the incorporation of a universal base into the
primer. The new primer is then subject to extension as described
above. The primer extension can be repeated as many times as are
necessary to decode the microsphere.
[0142] Decoding by sequencing has the advantage that encoding a
vast number of distinct codes by using only a small number of
nucleotides. That is, the information content of the encoding
region depends on the number of combinations of the bases used,
i.e. 4 when A, C, G and T are used, that are present at each
position and the length of the encoding region. When 4 nucleotides
are used, there are up to fifteen possible combinations of
nucleotides at each position (A, C, G, T, A/C, A/G, A/T, C/G, C/T,
G/T, A/C/G, A/C/T, A/G/T, C/G/G and A/C/G/T). As such, the
information content is 15.sup.n, where n is equal to the length of
the encoding sequence. As such, for example, with just 3 base
positions, 3375 different codes can be generated.
[0143] In addition an additional preferred embodiment includes the
combination of decoding by sequencing with decoding by
hybridization. In one application of decoding by sequencing,
universal primers, each one base longer than the previous, are
sequentially hybridized to the array. The base adjacent to the 3'
end of the universal primer is determined by a single base
extension reaction employing labeled-chain terminators, preferably
using four different color labels one for each base. Since a
hybridization event must be performed for each base read, once can
improve the decoding power by using the hybridization event to also
decode. However, rather than using universal primers, zipcode
sequences would be used instead (see FIG. 13).
[0144] The placement of the encoding sequences can be either on the
"bead-side" of the gene-specific sequence or on the terminal side.
Placement of the encoding sequences on the terminal side offers
that advantage of incorporating a cleavable linker between the
encoding sequences and the gene-specific sequence (FIG. 14). After
completion of decoding, the encoding sequences can be removed to
prevent possible interference in the analytical assay.
[0145] In one embodiment following decoding, the label is
extinguished. By "extinguished" is meant that the signal from the
label is eliminated. This is accomplished by a variety of
techniques including cleaving the label or destroying the signal of
the label. That is, in one embodiment, the label is attached to the
DBL via a cleavable linker. Following a decoding step, the linker
is cleaved thereby releasing the label. In one embodiment cleavage
of the label results in deprotection of the bioactive agent, for
example when the bioactive agent contains a protecting group.
Alternatively, cleavage of the label preserves the nature of the
bioactive agent. That is, what is important is that cleavage of the
label does not interfere with the bioactive agent.
[0146] In an alternative embodiment, the label is not cleaved, but
rather is eliminated by destroying the signal of the label. That
is, following decoding, the label is contacted with an agent that
degrades or destroys the signal. For example, organic fluorescent
dyes are subject to chemical degradation under appropriate
conditions. Thus, following a decoding step where the fluorescent
signal of the dyes are detected, the fluorophore is destroyed by an
appropriate chemical solution that degrades the fluorescent
molecule sufficient leaving no trace of fluorescence. In this way,
beads are tagged with fluorescent labels, i.e. barcodes of sorts,
their location or identity deciphered and recorded. The beads are
then exposed to the "fluorescent destroying solution" and the
fluorescence is eliminated, allowing an analytical assay to be
conducted. Suitable fluorescent dyes include but are not limited to
various bodipy, or Alexa dyes from Molecular Probes. The
"fluorescent destroying solution" is a reducing or oxidizing
solution, such as 0.01 SM sodium periodate.
[0147] In a preferred embodiment not only is the label cleaved, but
the encoding nucleic acid is cleaved. This method finds particular
use in removing residual signal following a decoding step.
Accordingly, in a preferred embodiment a cleavable linker can be
used to bridge the adapter and beads. After decoding, the adapter
oligonucleotide(s) can be easily removed through cleavable linkers.
There are. many commercially available cleavable linkers. They are
routinely used in the combinatorial solid-phase synthesis field.
The linkers can be cleaved under acid, base or photo radiation
conditions.
[0148] In addition, restriction enzymes can be used to cleave a
linker. Alternatively, because of the sequence specificity of
restriction enzymes, they also are used to cleave encoding nucleic
acids. A change in signal following incubation with a sequence
specific enzyme decodes the bead. An example is illustrative.
[0149] Consider the decoding of 6 bead types with three zip codes
or adapters. In general, the strategy will have B=(2 n)-2. Where B
is the number of types encoded and n is the number of zip codes.
The bead types are z1, z2, z3, z1z2, z1z3, and z2z3. Here zizj
indicates that both codes are on a bead. Every code is already dye
labeled.
[0150] Generally, each code is designed with a site that can be
specifically cut with some agent. This includes, but is not limited
to, restriction enzymes. For ease of description, the process using
restriction enzymes and zip codes that contain specific restriction
sites is described. When restriction enzymes are used, the zip
codes are double stranded. Each stage would consist of sequentially
adding one restriction enzyme. An image is taken prior to stage 1,
and the intensity of every bead is measured. Restriction enzyme 1
(Re1) is added. New intensity measurements are taken. The
information of which beads decrease in intensity, and which remain
the same. Detecting the pattern of beads for which the signal
"decreased" and "stayed the same" uniquely determines the bead
type. If we denote "decreased" by 1 and "stayed the same" by 0,
then the decoding would give the following codes:
z1: 1,0,0 z2: 0,1,0 z3: 0,0,1 z1z2: 1,1,0 z1z3: 1,0,1 z2z3:
0,1,1.
[0151] The general approach uses n codes, which would give (2 n)-2
types. Subtracting 2 leaves off the all off state, and the all zip
codes used state.
[0152] In another embodiment the array is decoded by using
exonuclease enzyme. In this embodiment the microspheres are encoded
with dye-labeled nucleotides. Preferably each nucleotide is labeled
with a discrete label. In this particular embodiment it is
desirable to protect the bioactive agent from cleavage. This can be
accomplished by a variety of known methods including hybridizing
the bioactive agent to a complementary sequence or blocking the
terminus of the bioactive agent. Alternatively, if the exonuclease
enzyme operates from 5' to 3', one can design zipcodes from 5' to
3' and SNP-gene specific oligos from 3' to 5'. This way zipcode
sequences will be operated on by the exonuclease, but the
SNP/gene-specific sequences will remain intact.
[0153] The microsphere is then contacted with an exonuclease that
successively removes the labeled nucleotides of the encoding
sequence. Monitoring of the signal of the microspheres should begin
upon addition of the exonuclease and monitored in all channels that
correspond to the signal emitted by the labels in use. For example,
assume the microsphere has all the zipcodes available (with the
above specifications), and is in a solution. At time T=0, the
exonuclease enzyme is applied to the solution. At the same time,
the acquisition system (CCD) starts taking images every few seconds
(FIG. 15). The assumption is that the reaction of the exonuclease
is slow enough, so one would have ample time to take several
images, as the reaction is proceeding. If this is not the case, the
reaction can be slowed down by increasing the stringency
conditions.
[0154] The exonuclease removes the nucleotides, one at a time.
Therefore, ideally, one would notice a step reduction in the signal
of the appropriate channel, once the nucleotide is removed (and
moved away from the focal zone). The complete set of waveforms
(i.e., the four color channels, and from T0 to Tend) will provide a
unique signature for every zipcode.
[0155] In practice, the removal of the nucleotides will not be
completely synchronized on different oligos (as shown in FIG. 15).
For instance, for two olligos on the same bead (same zipcode), the
exonuclease may have removed one nucleotide from one, and two
nucleotides from the other. However, since one monitors many
olligos from each bead, the overall trend should be preserved,
i.e., in general the nucleotides closer to the end should be
removed first. This means that in general, although the transitions
on the waveforms may not be sharp, the signatures could still be
unique.
[0156] Accordingly, the identification of the location of the
individual beads (or subpopulations of beads) is done using one or
more decoding steps comprising a binding between the labeled DBL
and either the IBL or the bioactive agent (i.e. a hybridization
between the candidate probe (sometimes referred to herein as a
"decoding sequence") and the decoder probe when the bioactive agent
is a nucleic acid). After decoding, the DBLs can be removed and the
array can be used; however, in some circumstances, for example when
the DBL binds to an IBL and not to the bioactive agent, the removal
of the DBL is not required (although it may be desirable in some
circumstances). In addition, as outlined herein, decoding may be
done either before the array is used in an assay, during the assay,
or after the assay.
[0157] In one embodiment, a single decoding step is done. In this
embodiment, each DBL is labeled with a unique label, such that the
number of unique labels is equal to or greater than the number of
bioactive agents (although in some cases, "reuse" of the unique
labels can be done, as described herein; similarly, minor variants
of candidate probes can share the same decoder, if the variants are
encoded in another dimension, i.e. in the bead size or label). For
each bioactive agent or IBL, a DBL is made that will specifically
bind to it and contains a unique label, for example one or more
fluorochromes. Thus, the identity of each DBL, both its composition
(i.e. its sequence when it is a nucleic acid) and its label, is
known. Then, by adding the DBLs to the array containing the
bioactive agents under conditions which allow the formation of
complexes (termed hybridization complexes when the components are
nucleic acids) between the DBLs and either the bioactive agents or
the IBLs, the location of each DBL can be elucidated. This allows
the identification of the location of each bioactive agent; the
random array has been decoded. The DBLs can then be removed, if
necessary, and the target sample applied.
[0158] In a preferred embodiment, the number of unique labels is
less than the number of unique bioactive agents, and thus a
sequential series of decoding steps are used. To facilitate the
discussion, this embodiment is explained for nucleic acids,
although other types of bioactive agents and DBLs are useful as
well. In this embodiment, decoder probes are divided into n sets
for decoding. The number of sets corresponds to the number of
unique tags. Each decoder probe is labeled in n separate reactions
with n distinct tags. All the decoder probes share the same n tags.
Each pool of decoders contains only one of the n tag versions of
each decoder, and no two decoder probes have the same sequence of
tags across all the pools. The number of pools required for this to
be true is determined by the number of decoder probes and the n.
Hybridization of each pool to the array generates a signal at every
address comprising an IBL. The sequential hybridization of each
pool in turn will generate a unique, sequence-specific code for
each candidate probe. This identifies the candidate probe at each
address in the array. For example, if four tag's are used, then
4.times.n sequential hybridizations can ideally distinguish 4.sup.n
sequences, although in some cases more steps may be required. After
the hybridization of each pool, the hybrids are denatured and the
decoder probes removed, so that the probes are rendered
single-stranded for the next hybridization (although it is also
possible to hybridize limiting amounts of target so that the
available probe is not saturated. Sequential hybridizations can be
carried out and analyzed by subtracting pre-existing signal from
the previous hybridization).
[0159] An example is illustrative. Assuming an array of 16 probe
nucleic acids (numbers 1-16), and four unique tags (four different
fluors, for example; labels A-D). Decoder probes 1-16 are made that
correspond to the probes on the beads. The first step is to label
decoder probes 1-4 with tag A, decoder probes 5-8 with tag B,
decoder probes 9-12 with tag C, and decoder probes 13-16 with tag
D. The probes are mixed and the pool is contacted with the array
containing the beads with the attached candidate probes. The
location of each tag (and thus each decoder and candidate probe
pair) is then determined. The first set of decoder probes are then
removed. A second set is added, but this time, decoder probes 1, 5,
9 and 13 are labeled with tag A, decoder probes 2, 6, 10 and 14 are
labeled with tag B, decoder probes 3, 7, 11 and 15 are labeled with
tag C, and decoder probes 4, 8, 12 and 16 are labeled with tag D.
Thus, those beads that contained tag A in both decoding steps
contain candidate probe 1; tag A in the first decoding step and tag
B in the second decoding step contain candidate probe 2; tag A in
the first decoding step and tag C in the second step contain
candidate probe 3; etc.
[0160] As will be appreciated by those in the art, the decoder
probes can be made in any order and added in any order.
[0161] In addition, since the size of the array will be set by the
number of unique decoding binding ligands, there are a number of
techniques that allow new combinations and the "reuse" of DBLs to
allow for a greater number of test sites. This may be done in a
wide variety of ways, as outlined below.
[0162] In a preferred embodiment, a spatial or positional coding
system is done. In this embodiment, there are sub-bundles or
subarrays (i.e. portions of the total array) that are utilized. By
analogy with the telephone system, each subarray is an "area code",
that can have the same labels (i.e. telephone numbers) of other
subarrays, that are separated by virtue of the location of the
subarray. Thus, for example, the same unique labels can be reused
from bundle to bundle. Thus, the use of 50 unique labels in
combination with 100 different subarrays can form an array of 5000
different bioactive agents. In this embodiment, it becomes
important to be able to identify one bundle from another; in
general, this is done either manually or through the use of marker
beads; these can be beads containing unique tags for each subarray,
or the use of the same marker bead in differing amounts, or the use
of two or more marker beads in different ratios.
[0163] In a preferred embodiment, the coding and decoding is
accomplished by sequential loading of the microspheres into the
array. As outlined above for spatial coding, in this embodiment,
the optical signatures can be "reused". In this embodiment, the
library of microspheres each comprising a different bioactive agent
(or the subpopulations each comprise a different bioactive agent),
is divided into a plurality of sublibraries; for example, depending
on the size of the desired array and the number of unique tags, 10
sublibraries each comprising roughly 10% of the total library may
be made, with each sub library comprising roughly the same unique
tags. Then, the first sublibrary is added to the fiber optic bundle
comprising the wells, and the location of each bioactive agent is
determined, generally through the use of DBLs. The second
sublibrary is then added, and the location of each bioactive agent
is again determined. The signal in this case will comprise the
signal from the "first" DBL and the "second" DBL; by comparing the
two matrices the location of each bead in each sublibrary can be
determined. Similarly, adding the third, fourth, etc., sublibraries
sequentially will allow the array to be filled.
[0164] In a preferred embodiment, codes can be "shared" in several
ways. In a first embodiment, a single code (i.e. IBL/DBL pair) can
be assigned to two or more agents if the target analytes different
sufficiently in their binding strengths. For example, two nucleic
acid probes used in an mRNA quantitation assay can share the same
code if the ranges of their hybridization signal intensities do not
overlap. This can occur, for example, when one of the target
sequences is always present at a much higher concentration than the
other. Alternatively, the two target sequences might always be
present at a similar concentration, but differ in hybridization
efficiency.
[0165] Alternatively, a single code can be assigned to multiple
agents if the agents are functionally equivalent. For example, if a
set of oligonucleotide probes are designed with the common purpose
of detecting the presence of a particular gene, then the probes are
functionally equivalent, even though they may differ in sequence.
Similarly, if classes or "families" of analytes are desired, all
probes for different members of a class such as kinases or
G-protein coupled receptors could share a code. Similarly, an array
of this type could be used to detect homologs of known genes. In
this embodiment, each gene is represented by a heterologous set of
probes, hybridizing to different regions of the gene (and therefore
differing in sequence). The set of probes share a common code. If a
homolog is present, it might hybridize to some but not all of the
probes. The level of homology might be indicated by the fraction of
probes hybridizing, as well as the average hybridization intensity.
Similarly, multiple antibodies to the same protein could all share
the same code.
[0166] In a preferred embodiment, the set of codes can be increased
using a kind of degeneracy on the IBLs on the beads. For most of
the systems described herein, particularly when the IBL is a
nucleic acid (including the case where the nucleic acid IBL is also
the candidate agent), any single bead contains a single IBL
sequence (decoding sequence). However, in this embodiment, it is
possible to have a single bead comprise two or more decoding
sequences, that differ at a particular nucleotide position. For a
degeneracy of two at position X, there are 10 different possible
combinations: the decoding sequence has an A at position X, a T, a
C, a G, or a mixture of decoding sequences with either A/T, A/G,
A/C, C/G, C/T or G/T. By using decoder probes that have different
labels for each different nucleotide at the position, the number of
codes can be increased, since beads containing the two different
sequences will exhibit a mixture of signals. In addition, as will
be appreciated by those in the art, rather than a "degeneracy" of
two, degeneracies of three or four can be done as well. Thus, a
bead containing three different decoding sequences will exhibit a
signal comprising three different colors, etc.
[0167] Decoding the degenerate probes is done by a series of
hybridizations to labeled oligonucleotides. Each hybridization
decodes one position in the probe so the total number of
hybridizations needed to decode a library is equal to the length of
the variable region. Each hybridization is done with four
oligonucleotides, each designed to hybridize specifically to a
sequence containing a specific base at the interrogated position
and each tagged with a distinctly colored fluorescent dye.
[0168] In a preferred embodiment, a degenerate probe sequence is
attached to one or more constant probe regions. By "constant" probe
region is meant a portion of a probe that is invariant among a
population of decoder probes. In a particularly preferred
embodiment, the degenerate or variable region is flanked on either
side by constant regions. As such, each oligonucleotide has the
following structure:
##STR00001##
[0169] Although the C1 and C2 regions may be any length, preferably
each is from 5 to 50 nucleotides in length. More preferably each is
from 7 to 30 nucleotides in length and most preferably, each is
about 10 bp in length. In one embodiment, the constant regions do
not vary in sequence among the probes designed to decode a
particular set of beads. As such, by using constant and variable
regions (i.e. "CV` or "CVC" when two constant regions are used) in
a set of probes, an array specific set or library of probes is
generated.
[0170] The variable region contains a sequence that is unique to
each address. Each "N" or variable position within the variable
region can contain one of ten possibilities as described above. As
such, the number of unique addresses in a library is determined by
the length of the variable region: if L is length of the variable
region, the number of unique addresses, A, is A=10.sup.L.
[0171] Accordingly, decoding the first base of the variable region
of a decoding sequence is illustrated below:
##STR00002##
[0172] Any address with an A at the first position would hybridize
with the fourth oligonucleotide and give the corresponding color.
An address with an A/C at the first position would hybridize with
both the third and fourth decoders and give the corresponding
combination of colors. By serial hybridizations, each using four
decoders and each interrogating a different position of the Y
region, the entire sequence of each address can be determined. The
"N" positions in the decoders are synthesized with degeneracies of
the four standard bases.
[0173] In one embodiment, CVC probes are incorporated into the
probes of an oligonucleotide array during synthesis as is described
for the addition of adapters; see U.S. Ser. No. 09/553,993, filed
Apr. 20, 2000, Ser. No. 09/556,463, filed Apr. 21, 2000,
60/160,917, filed Oct. 22, 1999, and 60/135,123, filed May 20, 1999
and WO 00/63437, all of which are expressly incorporated herein by
reference. In a similar fashion, the CVC probes could be
synthesized in the 5-prime ends of PCR primers used to generate a
PCR-product-based array. By incorporating different probes into the
two primers of each PCR reaction, a combinatorial expansion of
addresses is possible:
##STR00003##
[0174] The constant regions in the C.sub.1VC.sub.2 addresses would
be different in sequence from the C.sub.3VC.sub.4 addresses. Each
would therefore be decoded with its own set of CVC complement
decoders. If C.sub.1VC.sub.2 has a 3-base V region (1000 addresses)
and C.sub.3VC.sub.4 has a 3-base V region (1,000 addresses), then
the combination results in 1,000.times.1,000, or one million
addresses.
[0175] In a preferred embodiment, there are additional ways to
increase the number of unique or distinct tags. That is, the use of
distinct attributes on each bead can be used to increase the number
of codes. In addition, sequential decoding allows a reuse of codes
in new ways. These attributes are independent of each other, thus
allowing the number of codes to grow exponentially as a function of
the number of decoding steps and the number of attributes (e.g.
distinct codes). However, by increasing the amount of decoding
information obtained in a single decoding step, the number of
decoding steps is markedly reduced. By increasing the number of
attributes per decoding step, fewer decoding steps are required for
a given number of codes. Thus, in a preferred embodiment, a variety
of methods are used to generate a number of codes for use in the
process of decoding the arrays, while minimizing the necessary
decoding steps. For example, a variety of different coding
strategies can be combined: thus, different "colors", combinations
of colors ("hues"), different intensities of colors or hues or
both, different colors and different bead sizes, etc. can all be
combined. Alternatively, the number of distinct codes is markedly
increased.
[0176] In a preferred embodiment DBLs rely on attaching or
embedding a quantitative or discrete set of physical attributes to
the bead, i.e. labeling the bead. Preferred physical attributes of
a bead include but are not limited to: surface "smoothness" or
"roughness", color (fluorescent and otherwise), color intensity,
size, detectable chemical moieties, chemical reactivity,
magnetization, pH sensitivity, energy transfer efficiency between
dyes present, hydrophobicity, hydrophilicity, absorptivity, charge,
pH sensitivity, etc. In general, physical attributes that can be
measured optically are preferred, including color, size, surface
smoothness etc.
[0177] In a preferred embodiment, the DBLs may be reused by having
some subpopulations of beads comprise optical signatures. In a
preferred embodiment, the optical signature is generally a mixture
of reporter dyes, preferably fluorescent. By varying both the
composition of the mixture (i.e. the ratio of one dye to another)
and the concentration of the dye (leading to differences in signal
intensity), matrices of unique optical signatures may be generated.
This may be done by covalently attaching the dyes to the surface of
the beads, or alternatively, by entrapping the dye within the bead.
The dyes may be chromophores or phosphors but are preferably
fluorescent dyes, which due to their strong signals provide a good
signal-to-noise ratio for decoding. Suitable dyes for use in the
invention include those listed for labeling DBLs, above.
[0178] In a preferred embodiment, the encoding can be accomplished
in a ratio of at least two dyes, although more encoding dimensions
may be added in the size of the beads, for example. In addition,
the labels are distinguishable from one another; thus two different
labels may comprise different molecules (i.e. two different fluors)
or, alternatively, one label at two different concentrations or
intensity.
[0179] In a preferred embodiment, the dyes are covalently attached
to the surface of the beads. This may be done as is generally
outlined for the attachment of the bioactive agents, using
functional groups on the surface of the beads. As will be
appreciated by those in the art, these attachments are done to
minimize the effect on the dye.
[0180] In a preferred embodiment, the dyes are non-covalently
associated with the beads, generally by entrapping the dyes in the
pores of the beads.
[0181] Additionally, encoding in the ratios of the two or more
dyes, rather than single dye concentrations, is preferred since it
provides insensitivity to the intensity of light used to
interrogate the reporter dye's signature and detector
sensitivity.
[0182] In alternative embodiments, additional encoding parameters
can be added, such as microsphere size. For example, the use of
different size beads may also allow the reuse of sets of DBLs; that
is, it is possible to use microspheres of different sizes to expand
the encoding dimensions of the microspheres. Optical fiber arrays
can be fabricated containing pixels with different fiber diameters
or cross-sections; alternatively, two or more fiber optic bundles,
each with different cross-sections of the individual fibers, can be
added together to form a larger bundle; or, fiber optic bundles
with fiber of the same size cross-sections can be used, but just
with different sized beads. With different diameters, the largest
wells can be filled with the largest microspheres and then moving
onto progressively smaller microspheres In the smaller wells until
all size wells are then filled. In this manner, the same dye ratio
could be used to encode microspheres of different sizes thereby
expanding the number of different oligonucleotide sequences or
chemical functionalities present in the array. Although outlined
for fiber optic substrates, this as well as the other methods
outlined herein can be used with other substrates and with other
attachment modalities as well.
[0183] A bead decoding scheme includes assigning/imbuing a single
quantifiable attribute to each bead type wherein each bead type
differs in the quantifiable value of that attribute. For instance,
one can attach a given number of fluorophores to a bead and
quantitate the number of attached fluorophores in the decoding
process; however, in practice, attaching a "given amount" of an
attribute to a bead and accurately measuring the attribute may be
problematic. Typically, the coefficient of variation (CV) seen in
associating an attribute and the resultant measurement of this
attribute is quite large (>20%). By coefficient of variation is
meant the variability in labeling a bead in successive labelings.
This CV can be determined by labeling beads with a defined given
number of label (fluorophore, for example) in multiple tests and
measuring the resulting signal emitted by the bead. This large CV
limits the number of useable and resolvable "levels" for any given
attribute.
[0184] A more robust decoding scheme employs ratiometric rather
than absolute measurements for segmenting a quantitative attribute
into codes. By ratiometric decoding is meant labeling a bead with a
ratio of labels or attributes (i.e. 1:10, 1:1, and 10:1). In theory
any number of ratios can be used so long as the difference in
signals between the ratios is detectable. This process produced
smaller CVs and allowing more attribute segmentation within a given
dynamic range. Thus, in a preferred embodiment, the use of
ratiometric decoding reduces the coefficient of variability.
[0185] In addition, as will be appreciated by those in the art,
ratiometric decoding can be accomplished in a different way. In
this embodiment, rather than add a given number of beads with a
first dye (or dye combination) intensity in the first decoding
reaction and a second number with a second dye intensity in the
sequential second decoding reaction, this ratiometric analysis may
be done by using a ratio of labeled:unlabeled beads. That is, given
a set saturating concentration of decoding beads, for example
100,000 beads/reaction, the first intensity decoding step may be
done by adding 100,000 labeled beads and the second step can be
done by adding 10,000 labeled beads and 90,000 unlabeled beads.
Equilibrium dictates that the second step will give one tenth the
signal intensity.
[0186] Because of the spread in values of a quantitatively measured
attribute value, the number of distinct codes is practically
limited to less than a dozen or so codes. However, by serially
"painting" (i.e. temporarily attaching an attribute level to a
bead) and "stripping" (removing the attribute level) a bead with
different attribute values, the number of possible codes grows
exponentially with the number of serial stages in the decoding
process.
[0187] An example is illustrative. For instance, 9 different bead
types and three distinguishable attribute distributions (Table 1).
"Painting" (labeling) the beads with different attribute values in
a combinatorially distinct pattern in the two different stages,
generates a unique code for each bead type, i.e. nine distinct
codes are generated. Thus, in a preferred embodiment beads are
labeled with different attributes in a combinatorially distinct
pattern in a plurality of stages. This generates unique codes for
each bead type. Examples of different attributes are described
above. Labeling of beads with different attributes is performed by
methods known in the art.
TABLE-US-00001 TABLE 1 Serial decode generates unique codes using a
small number of attribute levels. stage 1 stage 2 Bead Type
Attribute Value Attribute Value Code 1 L L (L, L) 2 L M (L, M) 3 L
H .sup. (L, H) 4 M L (M, L) 5 M M (M, M) 6 M H (M, H).sup. 7 H L
(H, L).sup. 8 H M .sup. (H, M) 9 H H (H, H) Number of unique codes
= Number of attributes - Number of stages
[0188] As outlined herein, fluorescent colors are a particularly
convenient and preferable attribute to use in a decoding scheme.
Fluorescent colors can be attached to any agent that recognizes an
IBL to form a labeled DBL. A fluorescently labeled oligonucleotide
is a particularly useful DBL since it can specifically and
reversibly "paint" (label) any desired subset of beads with a
particular color simply by the process of hybridization and
dehybridization (i.e. to the DBL with a complementary sequence).
Moreover, fluorescence is easily imaged and quantitated using
standard optical hardware and software. In order to "paint" a given
bead type with a particular color, the bead type must be labeled
with a unique hybridizable DNA sequence (IBL) and the decoding
solution must contain the color-labeled complement of that
sequence.
[0189] One consideration in implementing a decoding scheme is to
minimize the number of images collected. In a color-based scheme,
the number of images collected is the product of the number of
colors and the number of stages. The number of images can be
reduced by "painting" a bead with multiple colors for each given
stage. By assigning multiple colors to a bead, the number of
effective codes is increased. As an example, in a 24 bit three
color scheme (e.g. red, green, blue) coloring process used by
computers, a total of 256*256*256=16.7 million different "hues" can
be generated from just three colors (red, green, blue).
[0190] Thus, in a preferred embodiment DBLs are labeled with a
combination of colored fluorophores. As such, this method finds use
in increasing the number of available codes for labeling DBLs using
only a handful of different dyes (colors). Increasing the number of
codes available at each decoding step will greatly decrease the
number of decoding steps required in a given decoding process.
[0191] In one embodiment a population of oligonucleotides encoding
a single DBL is labeled with a defined ratio of colors such that
each bead to which the DBL binds is identified based on a
characteristic "hue" formulated from the combination of the colored
fluorophores. In a preferred embodiment two distinct colors are
used. In a preferred embodiment, three or more distinct dyes
(colors) are available for use. In this instance the number of
differentiable codes generated by labeling a population of
oligonucleotides encoding a single DBL with any given color is
three. However by allowing combinations of colors and color levels
in the labeling, many more codes are generated.
[0192] For decoding by hybridization, a preferred number of
distinguishable color shades is from 2 to 2000; a more preferred
number of distinguishable color shades is from 2 to 200 and a most
preferred number of distinguishable color shades is from 2 to 20.
Utilizing three different color shades (intensities) and three
colors, the number of different hues will be 3.sup.4=81. Combining
a hue with sequential decoding allows a virtually limitless number
of codes to be generated.
[0193] As previously described, the DBL can be any agent that binds
to the IBL. In a preferred embodiment, a single DBL is labeled with
a pre-determined ratio of colors. This ratio is varied for each DBL
thus allowing for a unique "hue" for each DBL labeled as such.
Following treatment of the beads with the DBL, the bead is analyzed
to determine the "hue" associated with each bead, thereby
identifying the bead with its associated bioactive agent.
[0194] For instance, with four primary colors and two intensity
levels (color is present or absent), fifteen different hues/stage
are possible. If four dyes and three different intensity levels are
used (absent, half-present, fully present), then 73 different
hues/stage are possible. In this case, acquisition of only 4 color
images is sufficient to obtain information on 73 different coding
hues.
[0195] In a preferred embodiment the DBL relies on a Fluorescence
Resonance Energy Transfer (FRET) identifier. Fluorescence resonance
energy transfer (FRET) is well known in the art and has been
employed for various biotechnological applications. FRET requires
that the energy of the donor excited state overlap with the ground
state of the acceptor, implying that the donor emission spectrum
overlaps with the acceptor absorbance spectrum. In a FRET
experiment, the donor is excited with light; some of the energy of
the excited state is released as light emission while some of the
energy is transferred to the acceptor, which becomes excited
emitting light at a characteristic wavelength. The distance over
which energy transfer may occur can be varied by the choice of
energy donor and acceptor, wherein the use of various different
donor and acceptor molecules is contemplated. The formula for the
efficiency of energy transfer is:
T=R.sub.0.sup.6/(R.sub.0.sup.6+R.sup.6)
Where R is the distance between the donor and acceptor, and R.sub.0
is a constant related to the characteristic overlap of the
absorption and emission spectra of the donor-acceptor pair.
[0196] A FRET identifier comprises a linker region that is labeled
at each terminus with a fluorophore. The first fluorophore serves
as a donor fluorophore, whose signal is detectable; the other acts
as an acceptor fluorophore. The emission of the acceptor
fluorophore also is a detectable signal, the intensity of which
varies proportionally with the distance between the two
fluorophores. That is, by varying the distance between the two
fluorophores different codes can be generated.
[0197] Identification of the decoding oligonucleotide, therefore,
is accomplished by identifying the "efficiency" or ratio of
intensities of the acceptor fluorophore and the donor fluorophore.
This ratio should be invariant with regard to the number of
decoding oligonucleotides bound to a particle (bead); rather,
without being bound by any theory, the distance between the
fluorophores determines the ratio. Thus, by varying the distance
between the fluorophores (i.e. increasing or decreasing the length
of the linker), unique "codes" can be assigned to the DBLs. Each
code is characterized by the ratio of emission intensities between
the two fluorophores.
[0198] An even greater advantage is realized when different bead
attributes can be collected in a single image rather than from
multiple images. For instance, if the colors of red, green and blue
are used as attributes, three separate images need to be collected.
However, if different shades or intensities of each color are used
in the coding process, a much larger number of attributes can be
realized in a single image. The difficulty with using mixtures of
colors, is that the color ratios can vary somewhat between bead
types since individual components are mixed together to determine
the color ratio. If on the other hand, the color ratio is built
into the decoding label, as with the above described FRET decoding
oligonucleotides, the decoding process should be more reliable and
more efficient.
[0199] In preferred embodiments of the present invention, the first
component may be a lanthanide such as a chelate of europium(III)
(Eu(III)) or terbium(III) (Tb(III)) which serves as a fluorescent
donor and the second component may be an activated ester of cyanine
5 (Cy5) or tetramethylrhodamine (TMR) which serve as energy
acceptors of Eu(III) and Tb(III). Other first and second component
pairs that will find use for FRET include, for example, fluorescein
and rhodamine; FITC and rhodamine; and fluorescein and
trinitrophenyl, and other components as are well known In the
art.
[0200] The linker is comprised of any spacer element including
alkyl linkers, polymer linkers, etc. For ease of synthesis,
preferably the linker comprises nucleic acids (including nucleic
acid analogs).
[0201] The FRET decoding oligonucleotide comprises a spacer or
linker region that is labeled with fluorophores (dyes). In general,
this may be used in two general ways. In a preferred embodiment, as
generally depicted in FIG. 4, the linker is attached to the
terminus of a decoding nucleotide that hybridizes with (is
complementary to) the IBL. In this embodiment, the linker length
can be varied to adjust signal intensity. Thus, in a preferred
embodiment a FRET oligonucleotide is adjoined to an oligonucleotide
DBL. In one embodiment the FRET oligonucleotide is synthesized
independent of the DBL oligo. Subsequently the FRET oligonucleotide
is ligated to the DBL oligo according to methods well known in the
art. Alternatively, the DBL oligo and FRET oligo are synthesized as
a single oligonucleotide comprising the FRET fluorophores at the
desired positions.
[0202] Alternatively, in a second embodiment the linker is itself
the oligonucleotide that hybridizes with (is complementary to) the
IBL. That is, fluorophores are attached to the decoding
oligonucleotide at different basepair lengths; some may be 10
nucleotides apart, some 20, etc. In a preferred embodiment the
oligonucleotide DBL is itself labeled with fluors; as such the
oligonucleotide serves as both DBL and FRET oligonucleotide.
[0203] The linker region of a FRET oligonucleotide comprises any
set of nucleotides; in a preferred embodiment, for example when the
linker does not hybridize to anything, the linker comprises a
single repeated nucleotide. A single repeated nucleotide is less
likely to hybridize with the IBL or bioactive agent on the bead
because of the reduced likelihood of the presence of a
complementary nucleotide sequence in either the IBL or bioagent on
the bead. In a preferred embodiment, the linker comprises a
polynucleotide linker such as poly T, poly A, poly G or poly C, or
combinations.
[0204] The linker can be of any length so long as the emission of
the first fluorophore is absorbed by the second fluorophore. In a
preferred embodiment the linker is formed of from 1 to at least 100
nucleotides. More preferably the linker is formed from 1 to 70
nucleotides. Most preferably the linker is formed from 1 to 40
nucleotides, with from 5 to 20 being especially preferred.
[0205] In a preferred embodiment, for a given DBL sequence, several
codes can be generated by varying the distance of separation of the
two fluorophores and hence generating several energy transfer
"efficiency" ratios.
[0206] In another preferred embodiment, for a given DBL sequence, a
single code can be generated by varying the distance of separation
of the two flourophores and hence generating a single energy
transfer ratio for a single DBL. However, numerous DBLs can be
labeled with distinct FRET oligos thus allowing for the
simultaneous determination or identification of multiple DBLs in a
single imaging session as a result of the plurality of energy
transfer ratios.
[0207] For example, given a 40-mer decoding oligo, the donor and
acceptor fluorophores can be located as close as one or as far
apart as 40 bases. In principle this should allow 39 codes to be
generated for a single decoding sequence. Empirically, the number
of codes generated depends on dispersion in the distributions for
the ratiometric measurements.
[0208] Also, given a 40-mer decoding oligo, 39 unique codes can be
generated which will identify 39 different oligos.
[0209] The present invention provides array compositions comprising
a substrate with a surface comprising discrete sites. A population
of microspheres is distributed on the sites, and the population
comprises at least a first and a second sub population. Each
subpopulation comprises a bioactive agent, and, in addition, at
least one optical dye with a given pKa. The pKas of the different
optical dyes are different.
[0210] In a preferred embodiment, when for example the array
comprises cloned nucleic acids, there are several methods that can
be used to decode the arrays. In a preferred embodiment, when some
sequence information about the cloned nucleic acids is known,
specific decoding probes can be made as is generally outlined
herein.
[0211] In a preferred embodiment, "random" decoding probes can be
made. By sequential hybridizations or the use of multiple labels,
as is outlined above, a unique hybridization pattern can be
generated for each sensor element. This allows all the beads
representing a given clone to be identified as belonging to the
same group. In general, this is done by using random or partially
degenerate decoding probes, that bind in a sequence-dependent but
not highly sequence-specific manner. The process can be repeated a
number of times, each time using a different labeling entity, to
generate a different pattern of signals based on quasi-specific
interactions. In this way, a unique optical signature is eventually
built up for each sensor element. By applying pattern recognition
or clustering algorithms to the optical signatures, the beads can
be grouped into sets that share the same signature (i.e. carry the
same probes).
[0212] In order to identify the actual sequence of the clone
itself, additional procedures are required; for example, direct
sequencing can be done. By using an ordered array containing the
clones, such as a spotted cDNA array, a "key" can be generated that
links a hybridization pattern to a specific clone whose position in
the set is known. In this way the clone can be recovered and
further characterized.
[0213] Alternatively, clonal arrays can be decoded using binary
decoding with vector tags. For example, partially randomized oligos
are cloned into a nucleic acid vector (e.g. plasmid, phage, etc.).
Each oligonucleotide sequence consists of a subset of a limited set
of sequences. For example, if the limited set comprises 10
sequences, each oligonucleotide may have some subset (or all of the
10) sequences. Thus each of the 10 sequences can be present or
absent in the oligonucleotide. Therefore, there are 2.sup.10 or
1,024 possible combinations. The sequences may overlap, and minor
variants can also be represented (e.g. A, C, T and G substitutions)
to increase the number of possible combinations. A nucleic acid
library is cloned into a vector containing the random code
sequences. Alternatively, other methods such as PCR can be used to
add the tags. In this way it is possible to use a small number of
oligo decoding probes to decode an array of clones.
[0214] There are a variety of methods used to evaluate decoding. In
a preferred embodiment, discriminant analysis and cluster
algorithms and computer apparatus are used to analyze the decoding
data from the arrays of the invention. The potentially large number
of codes utilized in the invention, coupled with the use of
different intensities and "hues" of fluorophores in multi-step
decoding processes, requires good classification of the data. The
data, particularly intensity data, is acquired in a multi-step
process during which beads are "painted" (for example by
hybridizing dye-labeled complementary decoding oligonucleotides to
the IBL probes on the beads, or the formation of binding ligand
pairs for non-nucleic acid IBL-DBL pairs) with different colors or
mixtures of colors ("hues") at each stage. The challenge is to
accurately classify a bead as to which color with which it was
painted at each step. The more closely related the "paints"
(colors) are to one another (as determined by the optical imaging
system), the more difficult the classification.
[0215] The proximity of the dyes as seen by the imaging system is
determined by the spectral properties of the decoding dyes and the
spectral channel separation of the imaging system. Better color
separation is achieved by employing fluorescent dyes with narrow
emission spectra, and by employing an optical system with narrow
band pass excitation and emission filters which are designed to
excite the dye "on peak" and measure its emission "on peak". The
process of optically imaging the dyes on the beads is similar to
the human vision process in which our brain sees color by measuring
the ratio of excitation in the three different cone types within
our eye. However, with an optical imaging system, the number of
practical color channels is much greater than the three present in
the human eye. CCD based imaging systems can "see" color from 350
nm up to 850 nm whereas the cones in the eye are tuned to the
visible spectrum from 500-600 nm.
[0216] The problem of decoding bead arrays is essentially a
discriminant analysis classification problem. Thus, in a preferred
embodiment, an analysis of variance in hyperspectral alpha space is
performed on a known set of bead colors or hues. The center of the
bead clusters in alpha space are termed the centroids of the
clusters, and the scatter of the points within a cluster determines
the spread of the cluster. A robust classification scheme requires
that the distance between the centroids of the different bead
classes (hues) is much greater than the spread of any cluster
class. Moreover, the location of the centroids should remain
invariant from substrate to substrate and from experiment to
experiment.
[0217] Thus, in a preferred embodiment, a hue "zone" is defined as
a region in alpha space surrounding the hue centroid and extending
out to the spread radius of the cluster. Given a reference set of
hue centroids and spread radii, as determined empirically, the
classification of a new set of data can be accomplished by asking
whether a given bead point falls closest to or within the "zone" of
a hue cluster. This is accomplished by calculating the Mahalanobis
distance (in this case, it is simply a Euclidean distance metric)
of the bead point from the centroids of the different hue classes.
For the data shown in FIG. 6, the location of the centroids and
their distances from one another are indicated in Table 2.
TABLE-US-00002 TABLE 2 Distance between centroids dye/ Centroid
position Bod- Bod- Bod- Bod- channel Blue Green Yellow Red 493 R6G
564 TXR Bod-493 0.63 0.22 0.11 0.03 0.00 Bod-R6G 0.03 0.51 0.37
0.09 0.72 0.00 Bod-564 0.06 0.04 0.57 0.32 0.81 0.55 0.00 Bod-TXR
0.09 0.05 0.04 0.82 0.99 0.93 0.73 0.00
[0218] For classifying the different beads into a particular hue
class, a Euclidean distance cutoff of 0.3 was chosen. The closest
two centroids, the Bod-R6G and Bod-564 (dist=0.55), have a slight
overlap in their decoding zones when using a Euclidean or
Mahalanobis distance of 0.3. An improvement in classification can
be achieved by decreasing this distance, and by weighting the
different coordinate axes appropriately.
[0219] Accordingly, the present invention provides computer methods
for analyzing and classifying the color of a bead. The
classification of the color of the bead is done by viewing the bead
in hyperspectral "alpha" space (.DELTA..sub.1=I.sub.1/6I.sub.1,
.DELTA..sub.2=I.sub.2/6I.sub.i, .DELTA..sub.3=I.sub.3/6I.sub.i,
etc.) in which each coordinate axis represents the fraction of the
bead intensity within a given imaging channel. For instance, if
four imaging channels are used to image the beads, the color or hue
of a bead can be represented by a point in 3-D alpha space (the
fourth dimension is not necessary since Sa.sub.1=1). Given a set of
different primary dyes by which to "paint" the beads, the number of
hues that can be generated from these dyes is unlimited since the
dyes can be combined in varying ratios and in varying combinatorial
patterns. The number of practical hues is experimentally determined
by the separation of the different hue clusters in hyperspectral
alpha space.
[0220] FIG. 6 shows a hyperspectral alpha plot of beads "painted"
with four different hues imaged in four separate imaging channels.
Note that the beads form four distinct clusters. The fact that
these four clusters are well separated allows a robust decode
classification scheme to be implemented.
[0221] In a preferred embodiment, a quality control analysis of the
decoding process is done. This is achieved by performing a cluster
analysis of alpha space for each decoding stage. The number of
clusters determined will be fixed by the expected number of hues.
The positions of the cluster centroids will be monitored and any
deviations from the expected position will be noted.
[0222] Thus the invention provides an apparatus for decoding the
arrays of the invention. In addition to the compositions outlined
herein, the apparatus includes a central processing unit which
communicates with a memory and a set of input/output devices (e.g.,
keyboard, mouse, monitor, printer, etc.) through a bus. The general
interaction between a central processing unit, a memory,
input/output devices, and a bus is known in the art. One aspect of
the present invention is directed toward the hyperspectral "alpha"
space classification system stored in the memory.
[0223] The classification system program includes a data
acquisition module that receives data from the optical reader or
confocal microscope (or other imaging system). In general, the
classification program also includes an analysis module, that can
analyze the variance in hyperspectral alpha space, calculate the
centroids of the clusters, calculate the scatter of the cluster
(the spread) and define the hue zone and distance cutoff. In
general, the analysis module will further determine whether a data
point falls within the hue zone by calculating the Mahalanobis
distance.
[0224] Finally, the analysis module will analyze the different
sequential decoding information to finally assign a bioactive agent
to a bead location.
[0225] In this way, sequential decoding steps are run, with each
step utilizing the discriminant analysis calculations to assign
each bead in the array to a hue cluster at each step. The buildup
of the sequential decoding information allows the correlation of
the location of a bead and the chemistry contained on it.
[0226] In one embodiment, the invention includes modifying the
number of detection channels available for analysis relative to the
number of labels to be detected. That is, in one embodiment, the
number of detection channels is decreased relative to the number of
labels used. In an alternative embodiment the number of detection
channels is increased relative to the number of labels used.
[0227] When fewer detection channels are used, fewer images are
required during decoding. As such the invention provides a method
of reducing the number of images obtained during decoding.
Fluorescent dyes have broad band absorption and emission spectra.
Accordingly, the emission of certain dyes is detected in multiple
channels, generally by choosing channels to maximize the signal.
However by choosing the dyes and the channels carefully, fewer
channels that dyes may be used. As such, decoding with fewer
channels than dyes, allows for detection of signals that are
maximally detected in a particular channel, and overlapping in
several channels. Thus, for example, beads with dye-1 are read in
channel-1, beads with dye-2 are read in channel-2 and beads with
dye-3 show signals in both channel-1 and channel-2. Example 7 is
illustrative and the results of the experiment are found in FIG.
11. It is noted, however, that three dyes are clearly detected and
distinguished following detection in only two channels.
Accordingly, using fewer detection channels than the number of dyes
to be analyzed, allows for a reduction in the number of images and
steps required for decoding.
[0228] In an alternative embodiment, the invention provides for
using additional channels to increase the number of detectable
codes. That is, additional readout channels are used to increase
the coding/decoding capacity of arrays by analyzing attributes of
the bead in addition to an optical signal such as fluorescence. For
example, bead size, shape, density and surface characteristics can
alter the light-scattering properties of the beads and can be
detected optically. Additional channels that provide information
independent of fluorescence are forward-scatter and side-scatter.
Low-angle forward light scatter provides some information on the
relative size of beads, whereas side light scatter provides some
information on the relative granularity or texture of individual
beads. By analogy these channels are often used in combination to
distinguish the different major categories of white cells in
unseparated mammalian blood, but are useful in a wide variety of
other assays as well.
[0229] With respect to particle size, particles may simply be
varied in diameter. Alternatively, particles could be assembled as
doublets, triplets, or other higher-order multimers. Doublets and
triplets often occur spontaneously in a population, and can be
enriched for by fractionation.
[0230] With respect to surface properties, in one embodiment, the
invention provides the use of metallization. This can dramatically
alter the scattering properties of a surface. One way to accomplish
this is to attach gold particles (e.g. colloidal gold) to an
affinity molecule, such as streptavidin or DNA, and then to bind
the molecule at defined loadings to specific sites on the bead. For
example, ten different loadings could give ten new codes. Together
with 100 fluorescence codes, this would allow the recognition of
1000 bead types in an array. Alternatively, metallization could be
used for the assay readout, and fluorescence for the encoding.
[0231] With respect to magnetism, superparamagnetic beads could be
deflected according to the amount of magnetism, and directed to
different readout channels, i.e. this mechanism could be used as a
"pre-sorter" prior to fluorescence readout. In one version, bead
subpopulations could be localized to different regions of a
reaction vessel (e.g. microtiter well) by the use of small magnets.
Each sub-population could be released sequentially for
analysis.
[0232] Although the previously described method involving
additional detection channels finds use in planar bead arrays, it
also finds particular use in liquid arrays. By "liquid arrays" is
meant a collection of beads that are not spatially organized, but
are typically encoded by fluorescence labeling and "decoded" by
readout on a flow-cytometer or equivalent instrument. Assays are
carried out in bulk in solution. Generally, liquid arrays are
designed such that the assay result and decoding result are
obtained essentially simultaneously. However, because only optical
images are detected simultaneously, the number of codes that can be
analyzed is limited. That is, there is a limit to the number of
codes that can be used since the number of assay signals and
encoding signals must be determined at different wavelengths at
essentially the same time. However, by using additional detection
channels as described above, the number of codes that can be
analyzed is markedly increased.
[0233] This method finds particular use in analyzing the results of
genomics based assays such as gene expression profiling, genotyping
and SNP analysis. In such genomics based assays, it is frequently
desirable to make hundreds to millions of measurements
simultaneously. Thus, by increasing the number of codes that can be
simultaneously detected, analysis of such assays is enhanced.
[0234] This invention provides methods of maximizing the
information that is obtained from a single bead. That is, by
providing ways of multiplexing assays on a single bead, multiple
parameters can be simultaneously detected. That is, after a cycle
of performing a particular assay, decoding and reading the assay
results, the bead population is recovered. The detection molecules
are removed from the bead and the cycle is repeated.
[0235] In a preferred embodiment, parity analysis is used during
decoding to increase the robustness and accuracy of the system. By
"parity analysis" is meant a decoding step wherein the signal of a
particular element is analyzed across a plurality of decoding
stages. That is, following at least one decoding step, but most
preferably following multiple decoding steps, the signal of an
array element across the decoding stages is analyzed. That is, the
signal from a particular bead is evaluated across multiple stages.
Although the analysis includes any parameter that can be obtained
from the signals such as evaluating the total signal obtained
across the stages, in a preferred embodiment, the parity of the
signals across the stages is analyzed.
[0236] By "parity" is meant the digital or modular readout of
signals, i.e. odd or even, when binary signals are used. As
depicted in FIG. 7, the digit sum of the signals across a plurality
of stages is translated into a parity determination. The parity
determination is then useful in evaluating the decoding process. An
example is illustrative. Codes are designed to have an odd number
of a particular signal, for example a red signal, when viewed
across all stages or decoding steps, or a pre-determined plurality
of stages or decoding steps. The detection of an even number of red
stages, provides an indication that an error has occurred at some
point in decoding. When this result is obtained, the faulty code
can either be discarded, or the analysis repeated.
[0237] In a preferred embodiment the invention includes introducing
a "redundant stage" into the decoding system. By "redundant stage"
is meant a stage that serves as a parity check. That is, following
the decoding stages, an additional stage is included to analyze the
parity. This analysis provides an indication of the competence or
validity of the decoding. That is, when codes are designed with a
pre-determined parity, the redundant stage is used to detect the
parity of the signals obtained from the decoding step. That is, the
redundant stage detects errors in parity because if there has been
an error in decoding, the parity detected following the redundant
stage will be different from the parity designed into the
codes.
[0238] An example is illustrative. In the following illustration,
array elements are decoded in two stages with two colors. This
results in the detection of 4 (2.sup.2) codes. The codes are
designed to result in odd parity following the analysis with the
redundant stage. That is, the code sequences can be made such that
each code will contain an odd number of "red" colors (when viewed
across all stages, or when viewed across the pre-determined number
of stages). In the illustration below, only the stage where the
"red" signal is detected is indicated.
TABLE-US-00003 Parity (number of Code Stage 1 Stage 2 Redundant
Stage "red" stages) 1 0 0 red 1 (odd) 2 0 red 0 1 (odd) 3 red 0 0 1
(odd) 4 red red red 3 (odd)
[0239] However, if there is an error during decoding, this will be
reflected as detecting a parity that is different from that
designed into the codes. In the following illustration the same
codes as above are used however, an error is detected. In this case
if one detects an even number of "red" stages it can be inferred
that there has been a decoding error.
TABLE-US-00004 Parity (number of Code Stage 1 Stage 2 Redundant
Stage "red" stages) 1 0 0 red 1 (odd) 2 0 red 0 1 (odd) 3 red 0 0 1
(odd) 4 red .smallcircle. (error) red .sup. 2 (even)
[0240] Although in some embodiments, the parity is binary, i.e. odd
or even, in other embodiments the parity is modular or based on
numbers other than 2. That is, the parity can be based on detection
of 3 or more signals. In a preferred embodiment, the signals are
multiple colors. In a particularly preferred embodiment the colors
are red, green and blue.
[0241] An example is illustrative. In the following illustration,
codes are decoded in two steps with three colors. This results in
the detection of 3.sup.2 (9) signals. In the illustration, red is
indicated by 1, green is indicated by 2 and blue is indicated by 3.
The parity is based on a modulus 3. By including a third stage as a
redundant stage the parity can be checked as described above.
TABLE-US-00005 Code Stage 1 Stage 2 Redundant stage Parity 1 1 1 1
3 2 1 2 3 6 3 1 3 2 6 4 2 1 3 6 5 2 2 2 6 6 2 3 1 6 7 3 1 2 6 8 3 2
1 6 9 3 3 3 9
[0242] However, when there is a decoding error, the parity will be
incorrect; in the illustration the parity readout is not divisible
by 3.
TABLE-US-00006 Code Stage 1 Stage 2 Redundant stage Parity 1 1 1 1
3 2 1 2 3 6 3 1 3 2 6 4 2 1 3 6 5 2 2 2 6 6 2 3 1 6 7 3 1 2 6 8 3 2
1 6 9 3 2 (error) 3 8
[0243] Thus, a redundant stage allows for the detection of errors
on the array or in the signal obtained from an array.
[0244] Although described above for detecting errors on arrays, the
method of using redundant stages is not limited to arrays. That is,
the method finds use in detecting errors In a variety of systems
that send, receive and/or interpret or analyze data such as
communications systems including modems etc. In particular, the
method of using the modular redundant stages allows for processing
of signals of vastly increased complexity as compared to sending
bits of data. That is, when using modular redundant stages,
characters as opposed to bits, can be sent across modem lines in
the communications field. The modular redundant stage is used to
detect errors in the communication or processing of the
signals.
[0245] In an additional embodiment a redundant channel is used
during the decoding. By "redundant channel" is meant either the use
of multiple filters to analyze a particular signal or repeated use
of the same filter to analyze the signal, i.e., multiple signals
from the same channel. Redundant channels find use as a source of a
"back-up" signal of sorts for a particular array element. That is,
when multiple images are obtained as a result of redundant
channels, the likelihood of obtaining the correct signal increases.
That is, when it is realized that one of the results for an element
is compromised for some reason, the image obtained from the
redundant channel is used instead. When the initial image is not
compromised, the result from the redundant channel need not be
used, although it may be used, for example as a way to confirm the
initial signal.
[0246] An example is illustrative. During decoding, when it is
apparent that one stage fails, for example as a result of
mis-registration of a channel, the use of a redundant channel
serves to rescue the decoding at that particular stage.
Mis-registration can also be addressed as outlined in U.S. Ser. No.
09/636,387, filed Aug. 9, 2000, which is expressly incorporated
herein by reference. As noted above, the redundant channel can
either be an image separate filter to analyze the signal, or
alternatively, it can be multiple images taken from the same
filter.
[0247] In one embodiment, the number of redundant channels
increases as the number of decoding stages increases. Because the
investment in decoding increases as each decoding stage or step
proceeds, it is important in this embodiment to ensure the
competence of decoding at later decoding stages.
[0248] In a preferred embodiment, redundant systems are used to
avoid the loss of information. It is appreciated that when "S"
decoding steps (sometimes referred to herein as "stages") and "C"
colors are used, the maximum number of distinct signals is C.sup.S
(C raised to the "S"th power). In some instances, the corruption of
a stage results in the loss of C.sup.S-(C-1).times.C.sup.(S-1)
codes. Depending on the values of C and S, the loss can be
substantial. Accordingly, the present invention provides for
methods of improving the robustness of the decoding system. That is
the invention provides for a method of confirming the results of a
decoding analysis or detecting errors in a decoding analysis during
or after the decoding procedure. The method also decreases the
number of false positive or false negative signals; similarly, the
number of true positives and/or true negatives increases.
[0249] In a preferred embodiment, several levels of redundancy are
built into the arrays of the invention. Building redundancy into an
array gives several significant advantages, including the ability
to make quantitative estimates of confidence about the data and
significant increases in sensitivity. Thus, preferred embodiments
utilize array redundancy. As will be appreciated by those in the
art, there are at least two types of redundancy that can be built
into an array: the use of multiple identical sensor elements
(termed herein "sensor redundancy"), and the use of multiple sensor
elements directed to the same target analyte, but comprising
different chemical functionalities (termed herein "target
redundancy"). For example, for the detection of nucleic acids,
sensor redundancy utilizes of a plurality of sensor elements such
as beads comprising identical binding ligands such as probes.
Target redundancy utilizes sensor elements with different probes to
the same target: one probe may span the first 25 bases of the
target, a second probe may span the second 25 bases of the target,
etc. By building in either or both of these types of redundancy
into an array, significant benefits are obtained. For example, a
variety of statistical mathematical analyses may be done.
[0250] In addition, while this is generally described herein for
bead arrays, as will be appreciated by those in the art, this
techniques can be used for any type of arrays designed to detect
target analytes.
[0251] In a preferred embodiment, sensor redundancy is used. In
this embodiment, a plurality of sensor elements. e.g. beads,
comprising identical bioactive agents are used. That is, each
subpopulation comprises a plurality of beads comprising identical
bioactive agents (e.g. binding ligands). By using a number of
identical sensor elements for a given array, the optical signal
from each sensor element can be combined and any number of
statistical analyses run, as outlined below. This can be done for a
variety of reasons. For example, in time varying measurements,
redundancy can significantly reduce the noise in the system. For
non-time based measurements, redundancy can significantly increase
the confidence of the data.
[0252] In a preferred embodiment, a plurality of identical sensor
elements are used. As will be appreciated by those in the art, the
number of identical sensor elements will vary with the application
and use of the sensor array. In general, anywhere from 2 to
thousands may be used, with from 2 to 100 being preferred, 2 to 50
being particularly preferred and from 5 to 20 being especially
preferred. In general, preliminary results indicate that roughly 10
beads gives a sufficient advantage, although for some applications,
more identical sensor elements can be used.
[0253] Once obtained, the optical response signals from a plurality
of sensor beads within each bead subpopulation can be manipulated
and analyzed in a wide variety of ways, including baseline
adjustment, averaging, standard deviation analysis, distribution
and cluster analysis, confidence interval analysis, mean testing,
etc.
[0254] In a preferred embodiment, the first manipulation of the
optical response signals is an optional baseline adjustment In a
typical procedure, the standardized optical responses are adjusted
to start at a value of 0.0 by subtracting the integer 1.0 from all
data points. Doing this allows the baseline-loop data to remain at
zero even when summed together and the random response signal noise
is canceled out When the sample is a fluid, the fluid pulse-loop
temporal region, however, frequently exhibits a characteristic
change in response, either positive, negative or neutral, prior to
the sample pulse and often requires a baseline adjustment to
overcome noise associated with drift in the first few data points
due to charge buildup in the CCD camera. If no drift is present,
typically the baseline from the first data point for each bead
sensor is subtracted from all the response data for the same bead.
If drift is observed, the average baseline from the first ten data
points for each bead sensor is subtracted from the all the response
data for the same bead. By applying this baseline adjustment, when
multiple bead responses are added together they can be amplified
while the baseline remains at zero. Since all beads respond at the
same time to the sample (e.g. the sample pulse), they all see the
pulse at the exact same time and there is no registering or
adjusting needed for overlaying their responses. In addition, other
types of baseline adjustment may be done, depending on the
requirements and output of the system used.
[0255] Once the baseline has been adjusted, a number of possible
statistical analyses may be run to generate known statistical
parameters. Analyses based on redundancy are known and generally
described in texts such as Freund and Walpole, Mathematical
Statistics, Prentice Hall, Inc. New Jersey, 1980, hereby
incorporated by reference in its entirety.
[0256] In a preferred embodiment, signal summing is done by simply
adding the intensity values of all responses at each time point,
generating a new temporal response comprised of the sum of all bead
responses. These values can be baseline-adjusted or raw. As for all
the analyses described herein, signal summing can be performed in
real time or during post-data acquisition data reduction and
analysis. In one embodiment, signal summing is performed with a
commercial spreadsheet program (Excel, Microsoft, Redmond, Wash.)
after optical response data is collected.
[0257] In a preferred embodiment, cumulative response data is
generated by simply adding all data points in successive time
intervals. This final column, comprised of the sum of all data
points at a particular time interval, may then be compared or
plotted with the individual bead responses to determine the extent
of signal enhancement or improved signal-to-noise ratios.
[0258] In a preferred embodiment, the mean of the subpopulation
(i.e. the plurality of identical beads) is determined, using the
well known Equation 1:
.mu. = .SIGMA. x i n Equation 1 ##EQU00001##
[0259] In some embodiments, the subpopulation may be redefined to
exclude some beads if necessary (for example for obvious outliers,
as discussed below).
[0260] In a preferred embodiment, the standard deviation of the
subpopulation can be determined, generally using Equation 2 (for
the entire subpopulation) and Equation 3 (for less than the entire
subpopulation):
.sigma. = .SIGMA. ( x i - .mu. ) 2 n Equation 2 s = .SIGMA. ( x i -
x _ ) 2 n - 1 Equation 3 ##EQU00002##
[0261] As for the mean, the subpopulation may be redefined to
exclude some beads if necessary (for example for obvious outliers,
as discussed below).
[0262] In a preferred embodiment, statistical analyses are done to
evaluate whether a particular data point has statistical validity
within a subpopulation by using techniques including, but not
limited to, t distribution and cluster analysis. This may be done
to statistically discard outliers that may otherwise skew the
result and increase the signal-to-noise ratio of any particular
experiment. This may be done using Equation 4:
t = x _ - .mu. s / n Equation 4 ##EQU00003##
[0263] In a preferred embodiment, the quality of the data is
evaluated using confidence intervals, as is known in the art.
Confidence intervals can be used to facilitate more comprehensive
data processing to measure the statistical validity of a
result.
[0264] In a preferred embodiment, statistical parameters of a
subpopulation of beads are used to do hypothesis testing. One
application is tests concerning means, also called mean testing. In
this application, statistical evaluation is done to determine
whether two subpopulations are different. For example, one sample
could be compared with another sample for each subpopulation within
an array to determine if the variation is statistically
significant.
[0265] In addition, mean testing can also be used to differentiate
two different assays that share the same code. If the two assays
give results that are statistically distinct from each other, then
the subpopulations that share a common code can be distinguished
from each other on the basis of the assay and the mean test, shown
below in Equation 5:
z = x 1 _ - x 2 _ .sigma. 1 2 n 1 + .sigma. 2 2 n 2 Equation 5
##EQU00004##
[0266] Furthermore, analyzing the distribution of individual
members of a subpopulation of sensor elements may be done. For
example, a sub population distribution can be evaluated to
determine whether the distribution is binomial, Poisson,
hypergeometric, etc.
[0267] In addition to the sensor redundancy, a preferred embodiment
utilizes a plurality of sensor elements that are directed to a
single target analyte but yet are not identical. For example, a
single target nucleic acid analyte may have two or more sensor
elements each comprising a different probe. This adds a level of
confidence as non-specific binding interactions can be
statistically minimized. When nucleic acid target analytes are to
be evaluated, the redundant nucleic acid probes may be overlapping,
adjacent, or spatially separated. However, it is preferred that two
probes do not compete for a single binding site, so adjacent or
separated probes are preferred. Similarly, when proteinaceous
target analytes are to be evaluated, preferred embodiments utilize
bioactive agent binding agents that bind to different parts of the
target. For example, when antibodies (or antibody fragments) are
used as bioactive agents for the binding of target proteins,
preferred embodiments utilize antibodies to different epitopes.
[0268] In this embodiment, a plurality of different sensor elements
may be used, with from about 2 to about 20 being preferred, and
from about 2 to about 10 being especially preferred, and from 2 to
about 5 being particularly preferred, including 2, 3, 4 or 5.
However, as above, more may also be used, depending on the
application.
[0269] As above, any number of statistical analyses may be run on
the data from target redundant sensors.
[0270] One benefit of the sensor element summing (referred to
herein as "bead summing" when beads are used), is the increase in
sensitivity that can occur.
[0271] Once made, the compositions of the invention find use in a
number of applications. In a preferred embodiment, the compositions
are used to probe a sample solution for the presence or absence of
a target analyte, including the quantification of the amount of
target analyte present. By "target analyte" or "analyte" or
grammatical equivalents herein is meant any atom, molecule, ion,
molecular ion, compound or particle to be either detected or
evaluated for binding partners. As will be appreciated by those in
the art, a large number of analytes may be used in the present
invention; basically, any target analyte can be used which binds a
bioactive agent or for which a binding partner (i.e. drug
candidate) is sought.
[0272] Suitable analytes include organic and inorganic molecules,
including biomolecules. When detection of a target analyte is done,
suitable target analytes include, but are not limited to, an
environmental pollutant (including pesticides, insecticides,
toxins, etc.); a chemical (including solvents, polymers, organic
materials, etc.); therapeutic molecules (including therapeutic and
abused drugs, antibiotics, etc.); biomolecules (including hormones,
cytokines, proteins, nucleic acids, lipids, carbohydrates, cellular
membrane antigens and receptors (neural, hormonal, nutrient, and
cell surface receptors) or their ligands, etc); whole cells
(including procaryotic (such as pathogenic bacteria) and eukaryotic
cells, including mammalian tumor cells); viruses (including
retroviruses, herpesviruses, adenoviruses, lentiviruses, etc.); and
spores; etc. Particularly preferred analytes are nucleic acids and
proteins.
[0273] In a preferred embodiment, the target analyte is a protein.
As will be appreciated by those in the art, there are a large
number of possible proteinaceous target analytes that may be
detected or evaluated for binding partners using the present
invention. Suitable protein target analytes include, but are not
limited to, (1) immunoglobulins; (2) enzymes (and other proteins);
(3) hormones and cytokines (many of which serve as ligands for
cellular receptors); and (4) other proteins.
[0274] In a preferred embodiment, the target analyte is a nucleic
acid. These assays find use in a wide variety of applications. Such
applications are described in more detail in U.S. Ser. No.
60/130,089, filed Apr. 20, 1999; 60/160,927, filed Oct. 22, 1999;
Ser. No. 09/513,362, filed Feb. 25, 2000; 60/135,051, filed May 20,
1999; 60/161,148, filed Oct. 22, 1999; Ser. No. 09/517,945, filed
Mar. 3, 2000; Ser. No. 09/425,633, filed Oct. 22, 1999; Ser. No.
09/535,854, filed Mar. 27, 2000; Ser. No. 09/535,993, filed Apr.
20, 2000; Ser. No. 09/556,463, filed Apr. 21, 2000; 60/224,119,
filed Oct. 26, 2000, all of which are expressly incorporated herein
by reference.
[0275] In a preferred embodiment, the probes are used in genetic
diagnosis. For example, probes can be made using the techniques
disclosed herein to detect target sequences such as the gene for
nonpolyposis colon cancer, the BRCA1 breast cancer gene, P53, which
is a gene associated with a variety of cancers, the Apo E4 gene
that indicates a greater risk of Alzheimer's disease, allowing for
easy presymptomatic screening of patients, mutations in the cystic
fibrosis gene, cytochrome p450s or any of the others well known in
the art.
[0276] In an additional embodiment, viral and bacterial detection
is done using the complexes of the invention. In this embodiment,
probes are designed to detect target sequences from a variety of
bacteria and viruses. For example, current blood-screening
techniques rely on the detection of anti-HIV antibodies. The
methods disclosed herein allow for direct screening of clinical
samples to detect HIV nucleic acid sequences, particularly highly
conserved HIV sequences. In addition, this allows direct monitoring
of circulating virus within a patient as an improved method of
assessing the efficacy of anti-viral therapies. Similarly, viruses
associated with leukemia, HTLV-I and HTLV-II, may be detected in
this way. Bacterial infections such as tuberculosis, chlamydia and
other sexually transmitted diseases, may also be detected.
[0277] In a preferred embodiment, the nucleic acids of the
invention find use as probes for toxic bacteria in the screening of
water and food samples. For example, samples may be treated to lyse
the bacteria to release its nucleic acid, and then probes designed
to recognize bacterial strains, including, but not limited to, such
pathogenic strains as, Salmonella, Campylobacter, Vibrio cholerae,
Leishmania, enterotoxic strains of E. coli, and Legionnaire's
disease bacteria. Similarly, bioremediation strategies may be
evaluated using the compositions of the invention.
[0278] In a further embodiment, the probes are used for forensic
"DNA fingerprinting" to match crime-scene DNA against samples taken
from victims and suspects.
[0279] In an additional embodiment, the probes in an array are used
for sequencing by hybridization.
[0280] The present invention also finds use as a methodology for
the detection of mutations or mismatches in target nucleic acid
sequences, as is described in more detail in U.S. Ser. No.
09/425,633, filed Oct. 22, 1999, Ser. No. 09/535,854, filed Mar.
27, 2000, both of which are expressly incorporated herein by
reference. For example, recent focus has been on the analysis of
the relationship between genetic variation and phenotype by making
use of polymorphic DNA markers. Previous work utilized short tandem
repeats (STRs) as polymorphic positional markers; however, recent
focus is on the use of single nucleotide polymorphisms (SNPs).
Common SNPs occur at an average frequency of more than 1 per
kilobase in human genomic DNA. Some SNPs, particularly those in and
around coding sequences, are likely to be the direct cause of
therapeutically relevant phenotypic variants. There are a number of
well known polymorphisms that cause clinically important
phenotypes; for example, the apoE2/3/4 variants are associated with
different relative risk of Alzheimer's and other diseases (see
Cordor, et al., Science 261(1993). Multiplex PCR amplification of
SNP loci with subsequent hybridization to oligonucleotide arrays
has been shown to be an accurate and reliable method of
simultaneously genotyping at least hundreds of SNPs; see Wang, et
al., Science, 280:1077 (1998); see also Schafer, et al., Nature
Biotechnology 16:33-39 (1998). The compositions of the present
invention may easily be substituted for the arrays of the prior
art.
[0281] In a preferred embodiment, the compositions of the invention
are used to screen bioactive agents to find an agent that will
bind, and preferably modify the function of, a target molecule. As
above, a wide variety of different assay formats may be run, as
will be appreciated by those in the art. Generally, the target
analyte for which a binding partner is desired is labeled; binding
of the target analyte by the bioactive agent results in the
recruitment of the label to the bead, with subsequent
detection.
[0282] In a preferred embodiment, the binding of the bioactive
agent and the target analyte is specific; that is, the bioactive
agent specifically binds to the target analyte. By "specifically
bind" herein is meant that the agent binds the analyte, with
specificity sufficient to differentiate between the analyte and
other components or contaminants of the test sample. However, as
will be appreciated by those in the art, it will be possible to
detect analytes using binding which is not highly specific; for
example, the systems may use different binding ligands, for example
an array of different ligands, and detection of any particular
analyte is via its "signature" of binding to a panel of binding
ligands, similar to the manner in which "electronic noses" work.
This finds particular utility in the detection of chemical
analytes. The binding should be sufficient to remain bound under
the conditions of the assay, including wash steps to remove
non-specific binding, although in some embodiments, wash steps are
not desired; i.e. for detecting low affinity binding partners. In
some embodiments, for example in the detection of certain
biomolecules, the dissociation constants of the analyte to the
binding ligand will be less than about 10.sup.-4-10.sup.-6
M.sup.-1, with less than about 10.sup.-5 to 10.sup.-9 M.sup.-1
being preferred and less than about 10.sup.-7-10.sup.-9 M.sup.-1
being particularly preferred.
[0283] Generally, a sample containing a target analyte (whether for
detection of the target analyte or screening for binding partners
of the target analyte) is added to the array, under conditions
suitable for binding of the target analyte to at least one of the
bioactive agents, i.e. generally physiological conditions. The
presence or absence of the target analyte is then detected. As will
be appreciated by those in the art, this may be done in a variety
of ways, generally through the use of a change in an optical
signal. This change can occur via many different mechanisms. A few
examples include the binding of a dye-tagged analyte to the bead,
the production of a dye species on or near the beads, the
destruction of an existing dye species, a change in the optical
signature upon analyte interaction with dye on bead, or any other
optically interrogatable event.
[0284] In a preferred embodiment, the change in optical signal
occurs as a result of the binding of a target analyte that is
labeled, either directly or indirectly, with a detectable label,
preferably an optical label such as a fluorochrome. Thus, for
example, when a proteinaceous target analyte is used, it may be
either directly labeled with a fluor, or indirectly, for example
through the use of a labeled antibody. Similarly, nucleic acids are
easily labeled with fluorochromes, for example during PCR
amplification as is known in the art. Alternatively, upon binding
of the target sequences, a hybridization indicator may be used as
the label. Hybridization indicators preferentially associate with
double stranded nucleic acid, usually reversibly. Hybridization
indicators include intercalators and minor and/or major groove
binding moieties. In a preferred embodiment, intercalators may be
used; since intercalation generally only occurs in the presence of
double stranded nucleic acid, only in the presence of target
hybridization will the label light up. Thus, upon binding of the
target analyte to a bioactive agent, there is a new optical signal
generated at that site, which then may be detected.
[0285] Alternatively, in some cases, as discussed above, the target
analyte such as an enzyme generates a species that is either
directly or indirectly optically detectable.
[0286] Furthermore, in some embodiments, a change in the optical
signature may be the basis of the optical signal. For example, the
interaction of some chemical target analytes with some fluorescent
dyes on the beads may alter the optical signature, thus generating
a different optical signal.
[0287] As will be appreciated by those in the art, in some
embodiments, the presence or absence of the target analyte may be
done using changes in other optical or non-optical signals,
including, but not limited to, surface enhanced Raman spectroscopy,
surface plasmon resonance, radioactivity, etc.
[0288] The assays may be run under a variety of experimental
conditions, as will be appreciated by those in the art. A variety
of other reagents may be included in the screening assays. These
include reagents like salts, neutral proteins, e.g. albumin,
detergents, etc. which may be used to facilitate optimal
protein-protein binding and/or reduce non-specific or background
interactions. Also reagents that otherwise improve the efficiency
of the assay, such as protease inhibitors, nuclease inhibitors,
anti-microbial agents, etc., may be used. The mixture of components
may be added in any order that provides for the requisite binding.
Various blocking and washing steps may be utilized as is known in
the art.
[0289] In a preferred embodiment, two-color competitive
hybridization assays are run. These assays can be based on
traditional sandwich assays. The beads contain a capture sequence
located on one side (upstream or downstream) of the SNP, to capture
the target sequence. Two SNP allele-specific probes, each labeled
with a different fluorophor, are hybridized to the target sequence.
The genotype can be obtained from a ratio of the two signals, with
the correct sequence generally exhibiting better binding. This has
an advantage in that the target sequence itself need not be
labeled. In addition, since the probes are competing, this means
that the conditions for binding need not be optimized. Under
conditions where a mismatched probe would be stably bound, a
matched probe can still displace it. Therefore the competitive
assay can provide better discrimination under those conditions.
Because many assays are carried out in parallel, conditions cannot
be optimized for every probe simultaneously. Therefore, a
competitive assay system can be used to help compensate for
non-optimal conditions for mismatch discrimination.
[0290] In a preferred embodiment, dideoxynucleotide
chain-termination sequencing is done using the compositions of the
invention. In this embodiment, a DNA polymerase is used to extend a
primer using fluorescently labeled ddNTPs. The 3' end of the primer
is located adjacent to the SNP site. In this way, the single base
extension is complementary to the sequence at the SNP site. By
using four different fluorophors, one for each base, the sequence
of the SNP can be deduced by comparing the four base-specific
signals. This may be done in several ways. In a first embodiment,
the capture probe can be extended; in this approach, the probe must
either be synthesized 5'-3' on the bead, or attached at the 5' end,
to provide a free 3' end for polymerase extension. Alternatively, a
sandwich type assay can be used; in this embodiment, the target is
captured on the bead by a probe, then a primer is annealed and
extended. Again, in the latter case, the target sequence need not
be labeled. In addition, since sandwich assays require two specific
interactions, this provides increased specificity which is
particularly helpful for the analysis of complex samples.
[0291] In addition, when the target analyte and the DBL both bind
to the agent, it is also possible to do detection of non-labeled
target analytes via competition of decoding.
[0292] In a preferred embodiment, the methods of the invention are
useful in array quality control. Prior to this invention, no
methods have been described that provide a positive test of the
performance of every probe on every array. Decoding of the array
not only provides this test, it also does so by making use of the
data generated during the decoding process itself. Therefore, no
additional experimental work is required. The invention requires
only a set of data analysis algorithms that can be encoded in
software.
[0293] The quality control procedure can identify a wide variety of
systematic and random problems in an array. For example, random
specks of dust or other contaminants might cause some sensors to
give an incorrect signal--this can be detected during decoding. The
omission of one or more agents from multiple arrays can also be
detected; that is, since the arrays formulated herein are randomly
self-assembled, arrays comprising different statistical
distributions of each sensor elements may be made; the invention
allows actual QC on the arrays, for "sorting" arrays with good
distributions from those with less than ideal distributions. These
procedures also allow the determination of sites that do not
comprise beads. This quality control procedure can also be used in
non-random arrays.
[0294] An advantage of this quality control procedure is that it
can be implemented immediately prior to the assay itself (or after,
in some instances), and is a true functional test of each
individual sensor. Therefore any problems that might occur between
array assembly and actual use can be detected. In applications
where a very high level of confidence is required, and/or there is
a significant chance of sensor failure during the experimental
procedure, decoding and quality control can be conducted both
before and after the actual sample analysis.
[0295] In an alternative method of quality control, a single fiber
bundle of an array is dedicated to quality control. That is a
dedicated bundle or subset of fibers within an array is
independently analyzed for quality control. In one embodiment, the
control fiber(s) are targeted or labeled with a unique code or
label. At each state of hybridization, in addition to the
experimental probes, the quality control probes are hybridized with
the array. Upon taking the image from the first hybridization of
the first stage, the intensity of the control fiber(s) is analyzed.
An aberrant reading or signal from the control fiber indicates a
failed or at least non-optimal hybridization step.
[0296] In a preferred embodiment, the arrays can be used to do
reagent quality control. In many instances, biological
macromolecules are used as reagents and must be quality controlled.
For example, large sets of oligonucleotide probes may be provided
as reagents. It is typically difficult to perform quality control
on large numbers of different biological macromolecules. For
example, when large populations of different random
oligonucleotides are synthesized, it may be desirable to check that
each population is represented. The approach described here can be
used to do this by treating the reagents (formulated as the DBLs)
as variable Instead of the arrays.
[0297] In a preferred embodiment, the methods outlined herein are
used in array calibration. For many applications, such as mRNA
quantitation, it is desirable to have a signal that is a linear
response to the concentration of the target analyte, or,
alternatively, if non-linear, to determine a relationship between
concentration and signal, so that the concentration of the target
analyte can be estimated. For example, different probes can show
differences in hybridization efficiencies, etc., and it may be
desirable to choose appropriate probes under experimental
conditions. Thus, for example, when expression profiling is done,
and quantitation is desirable, calibration curves can be done to
see how individual probes react, and then the probes that give the
best response (i.e. linearity at the concentrations and conditions
of interest) can be chosen for further assays. Accordingly, the
present invention provides methods of creating calibration curves
in parallel for multiple beads in an array. The calibration curves
can be created under conditions that simulate the complexity of the
sample to be analyzed. Each curve can be constructed independently
of the others (e.g. for a different range of concentrations), but
at the same time as all the other curves for the array.
[0298] Thus, in these embodiments, different types of experiments
can be done. For example, the sequential decoding scheme can be
implemented with different concentrations being used as the code
"labels", rather than different fluorophores. In this way, signal
as a response to concentration can be measured for each bead. This
calibration can be carried out just prior to array use, so that
every probe on every array is individually calibrated as needed.
Alternatively, different concentrations of the decoding probe can
have different labels.
[0299] It should be noted that the assay calibration methods find
use in non-random arrays, as well; that is, other types of
support-bound nucleic acid arrays can be calibrated using these
methods as well. Thus, for example, the sequential addition of
different pools of probes, wherein the concentration of the probes
is varied, to biochips can allow calibration of any assay system.
This type of analysis can also be done on non-random arrays for
quality control, to verify the integrity and sequence of the
support bound probes, and in assay development to identify good
probes.
[0300] In a preferred embodiment, the methods of the invention can
be used in assay development as well. Thus, for example, the
methods allow the identification of good and bad probes; as is
understood by those in the art, some probes do not function well
because they do not hybridize well, or because they cross-hybridize
with more than one sequence. These problems are easily detected
during decoding. The ability to rapidly assess probe performance
has the potential to greatly reduce the time and expense of assay
development. Thus, probes that respond linearly with concentration,
show low nonspecific binding, or give signals in a particular
range, can be chosen for addition to a new array for assays.
[0301] Similarly, in a preferred embodiment, the methods of the
invention are useful in quantitation in assay development. Major
challenges of many assays is the ability to detect differences in
analyte concentrations between samples, the ability to quantitate
these differences, and to measure absolute concentrations of
analytes, all in the presence of a complex mixture of related
analytes. An example of this problem is the quantitation of a
specific mRNA in the presence of total cellular mRNA. One approach
that has been developed as a basis of mRNA quantitation makes use
of a multiple match and mismatch probe pairs (Lockhart et al.,
1996), hereby incorporated by reference in its entirety. While this
approach is simple, it requires relatively large numbers of probes.
In this approach, a quantitative response to concentration is
obtained by averaging the signals from a set of different probes to
the gene or sequence of interest. This is necessary because only
some probes respond quantitatively, and it is not possible to
predict these probes with certainty. In the absence of prior
knowledge, only the average response of an appropriately chosen
collection of probes is quantitative. However, in the present
invention, that can be applied generally to nucleic acid based
assays as well as other assays. In essence, the approach is to
identify the probes that respond quantitatively in a particular
assay, rather than average them with other probes. This is done
using the array calibration scheme outlined above, in which
concentration-based codes are used. Advantages of this approach
include: fewer probes are needed; the accuracy of the measurement
is less dependent on the number of probes used; and that the
response of the sensors is known with a high level of certainty,
since each and every sequence can be tested in an efficient manner.
It is important to note that probes that perform well are selected
empirically, which avoids the difficulties and uncertainties of
predicting probe performance, particularly in complex sequence
mixtures. In contrast, in experiments described to date with
ordered arrays, relatively small numbers of sequences are checked
by performing quantitative spiking experiments, in which a known
mRNA is added to a mixture.
[0302] In a preferred embodiment, cDNA arrays are made for RNA
expression profiling. In this embodiment, individual cDNA clones
are amplified (for example, using PCR) from cDNA libraries
propagated in a host-vector system. Each amplified DNA is attached
to a population of beads. Different populations are mixed together,
to create a collection of beads representing the cDNA library. The
beads are arrayed, decoded as outlined above, and used in an assay
(although as outlined herein, decoding may occur after assay as
well). The assay is done using RNA samples (whole cell or mRNA)
that are extracted, labeled if necessary, and hybridized to the
array. Comparative analysis allows the detection of differences in
the expression levels of individual RNAs. Comparison to an
appropriate set of calibration standards allows quantification of
absolute amounts of RNA.
[0303] The cDNA array can also be used for mapping, e.g. to map
deletions/insertions or copy number changes in the genome, for
example from tumors or other tissue samples. This can be done by
hybridizing genomic DNA. Instead of cDNAs (or ESTs, etc.), other
STS (sequence tagged sites), including random genomic fragments,
can also be arrayed for this purpose.
[0304] In one embodiment, the invention provides a method of single
nucleotide polymorphism (SNP) genotyping. As described herein and
in U.S. Ser. No. 09/553,993 filed Apr. 20, 2000, Ser. No.
09/556,463 filed Apr. 21, 2000 and 60/244,119, filed Oct. 26, 2000,
all of which are expressly incorporated herein by reference, a
target nucleic acid is contacted with a probe that is specific for
a particular allele of a single nucleotide polymorphism. The target
nucleic acid can be immobilized prior to contacting with the probe,
or alternatively, the probe:target hybridization can occur in
solution following by immobilization of the probe:target nucleic
acid complex. Alternatively, a capture prove on a bead may be
specific for a particular SNP. What is important in this invention
is the nature of the probe for detecting a SNP.
[0305] There are four possibilities for the bases at a polymorphic
site: A, T, G, or C. However, it is known that most of the human
SNP's are dimorphic, i.e., they only include the combinations of
two of the four bases. As such, the prior art generally relied on
distinguishing between two alleles that are present within a SNP.
and characterized them as either "AA", "AB", or "BB".
[0306] However, according to the present invention, the probes are
designed such that each of the four possible nucleotides that could
be present at a particular position are included in the SNP probes.
In addition, the probes are labeled with a specific color that
corresponds with the nucleotide that hybridizes to the nucleotide
to be interrogated. For instance, all the alleles that correspond
to an A-polymorphic site are labeled with red. Similarly, all the
alleles that correspond to a G-polymorphic site are labeled green,
etc. As such the true identity of the polymorphism is determined.
That is, if the polymorphism is of the type GT, one would not
classify is as "AB" genotype. Instead, the GT code will come up
directly from the genotyping routine.
[0307] An advantage of this method over the prior art is the
capability of independent error checking. That is, knowing that
polymorphisms are typically dimorphic, by using probes that
correspond to all possibilities of polymorphisms at a sites, one
obtains a check against false results, for example of a signal is
detected that does not correspond with the known polymorphisms.
That is, in the prior art, the resultant genotyping would be one of
the following three possibilities: AA, AB, or BB. However, by using
this invention, the number of possible results is increased from 3
to 10: AA, AG, AC, AT, GG, GC, GT, CC, CT, TT. Of these ten
possibilities, only 3 are the possible combinations for each SNP,
e.g., a T/G polymorphism can only render one of the following three
possibilities: TT, TG, or GG. Therefore, in this case, if the
computed genotype falls into any of the remaining 10-3=7
combinations (e.g., either of AA, AG, AC, AT, GC, CC, CT), it would
be a clear indication that an error has occurred.
[0308] All references cited herein are incorporated by reference in
their entirety.
[0309] The following examples serve to more fully describe the
manner of using the above-described invention, as well as to set
forth the best modes contemplated for carrying out various aspects
of the invention. It is understood that these examples in no way
serve to limit the true scope of this invention, but rather are
presented for illustrative purposes. All references cited herein
are incorporated by reference in their entirety.
EXAMPLES
Example 1
[0310] Sixteen microspheres (beads) were labeled combinatorially
with two different fluorophores (FAM and Cy3). In a first round of
labeling, either FAM or Cy3-labeled oligonucleotides that were
complementary to the oligonucleotide (IBL) on the microsphere, were
hybridized with the microsphere. Labeling of oligonucleotides was
performed as is well known in the art. Hybridization conditions are
known in the art.
[0311] Following a first round of hybridization, the two pools of
beads were divided into two pools each and each labeled either with
the FAM or Cy3-labeled oligonucleotide. This process was repeated
two additional times. Thus, following four successive rounds of
labeling, each microsphere was labeled with a unique code (see FIG.
1). The identity of each microsphere was elucidated by determining
the identity of each fluorophore in succession; the terminal
fluorophere was determined and then removed to allow for the
identification of the next fluorophore. In this fashion, with as
few as 4 decoding steps, the identity of 16 microspheres is
determined.
Example 2
[0312] A decoding scheme similar to that described in Example 1 was
implemented for four color decoding. In this example, beads were
labeled as described in Example 1 with the exception that 4 labels
were used at each stage. 4013 beads were labeled using Bod493,
BodR6G, Bod564 and BodTXR labeled oligonucleotides. 128 different
bead types were identified based on the successive decoding of the
four colors.
Example 3
[0313] An alternative method to using multiple colors is to use
ratiometric intensities as a coding scheme. A normalizing image is
acquired in which every bead exhibits its "full" intensity.
Subsequent decode stages generate intensity codes by hybridizing
mixtures of "labeled":"unlabeled" complementary oligonucleotides.
For instance, FIG. 1 depicts three different intensity shades (low,
medium, and high) which can be ratioed to a stage with all
complements present at a "high" shading value. An experiment using
grey scale decoding on 16 different bead types is shown in FIGS.
3A, 3B, 3C, 3D, and 3E.
[0314] FIG. 3A depicts the combinatorial pooling scheme for
labeling beads with different ratios of labeled oligonucleotides. A
particular oligo is present at either 100% Cy3-labeled, 40%
Cy3-labeled (60% unlabeled), or 10% Cy3-labeled (90% unlabeled)
fraction. Decode oligos were hybridized to the array for 2 min. at
a 50 nM concentration. Subsequently, two independent normalizing
images (all oligo complements are present as 100% Cy3-labeled
species) were acquired, and the resulting bead intensities
compared. This is depicted in FIG. 3B as the normalized values are
plotted against each other. Finally, to identify or decode the
beads, the alpha values (ratio of bead intensity in indicated
decode stage to intensity in normalization image) are plotted for
three decode stages described in (A). In stage 1, only two peaks
are observed in the alpha value histogram since only 16 bead types
are present on the array. Three distinguishable peaks are observed
in the second and third decode stages indicating the feasibility of
grey scale decoding.
[0315] Physical attributes and different "levels" of the attributes
can be used as codes by which to distinguish bead types from
another. Thus, for an attribute to act as a robust code, it should
be possible to imbue a bead with different "levels" of a particular
attribute. Each "level" of an attribute should be quantitatively
well separated from other "levels". The important point is to
maximize the dynamic range of the attribute measurement, and
minimize the spread of the measurement.
Example 4
[0316] For a given oligo sequence, several codes can be generated
by varying the distance of separation of the two fluorophores. As
shown in FIG. 4, a complementary sequence containing FRET dyes
separated by a linker of varying lengths allows multiple codes to
be generated (FIG. 4, Panel A). As an example, Probe A and B are
hybridized to a complementary sequence immobilized on a solid
support. Probe A contains dyes separated by an 8 poly T linker,
whereas probe B is hybridized to a linker with a 4 poly T linker.
The efficiency of energy transfer (ratio of intensities of
dye2:dye1) is greater for probe B than for probe A (FIG. 4, Panel
B).
Example 5
[0317] The following FRET oligonucleotides (probes ET1, ET2, ET5
and ET7) were synthesized as is known in the art and labeled with
Cy3 and fluorscein separated by linkers of varying length:
TABLE-US-00007 ET1 (SEQ ID NO: 1) T**G*CACGAGAATGGAGGTATCT ET2 (SEQ
ID NO: 2) C**TGTCGC*ACGAGAATGGAGGTATCT ET5 (SEQ ID NO: 5)
C**TGTCGGGGCACTCATTTGTGC*ACGAGAATGGAGGTATCT ET7 (SEQ ID NO: 6)
C**TGTCGGGGCACTCATTTGTCTGTCGGGGCGC*ACGAGAATGGAGG TATCT. **is Cy3
*is fluorescein
[0318] An aliquot of each FRET oligonucleotide was assayed as is
known in the art. Emission spectra were normalized relative to
emission at 530 nm. As can be seen in FIG. 5 emission of
fluorescein increases as the linker is shortened. The ratio of
intensities of the two fluorophores, therefore, is characteristic
of the distance between the fluorophores.
Example 6
[0319] To illustrate the primer extension decoding, a two-color
model is illustrated in FIG. 9. To decode 16 different bead
addresses using only two colors. 16 different beads are labeled by
attaching 16 different unique oligos (IBLs) to each of 16 batches
of beads and pooling the bead batches together. Along with the
beads 16 oligos (DBLs) that are complementary to these IBLs are
synthesized. Each unique IBL used in this method contains two
domains. The domains are composed of a binding domain and an
extension domain. The pooled bead populations are then
self-assembled onto a fiber optic tip end as described herein.
After array self-assembly a combinatorial color decoding process is
initiated as follows: A primer extension reaction is initiated at
the end of the fiber bundle tip containing the arrayed beads by
simultaneously hybridizing 16 complementary oligos (DBLs) to their
cognate IBL. In the first extension stage, the DBLs would have
exactly the same length (n) as the binding domain of the IBL. The
first stage primer extension reaction would contain 16 primer
oligos, a DNA polymerase, appropriate buffer, and two
fluorescently-labeled dideoxynucleotide terminators. During the
extension reaction, hybridized primers are extended by a single
nucleotide complementary to the first nucleotide in the extension
domain of the immobilized IBL. The described extension reaction
"paints" or labels any given bead with one of two possible
fluorescent terminator colors. After the reaction is over the array
tip is imaged to capture the color of the beads at this stage.
Beads are then immediately stripped of color or label by denaturing
and washing off the first extended primer at each zip code loci.
This process can be repeated through multiple stages. In subsequent
stages, the primer extension reactions contain the same reagents as
the previous extension reaction except that the binding
complementary oligos are one base longer. These (n+1) primers
hybridize with their cognate binding domain and one base into the
"extension domain". FIG. 9 illustrates a four-stage extension
process (2-color) whereby each stage allows for the assignment of a
unique combinatorial color-coded `address` to each bead type.
[0320] In addition, if this primer-based extension method were to
be combined with degenerate DBL-BL combinations for decoding large
combinatorial libraries, for example using the CVC
(constant-variable-constant domain idea), as described herein, the
extension reaction would extend into one of 10 different possible
nucleotide combinations in a "variable extension region". This
would require only three extension reactions to decodify 1000
unique addresses (i.e. A=lot where L is the length of the variable
region). The combination of the degenerate DBL-IBL combinations
such as CVC with the primer extension method is illustrated in FIG.
10.
Example 7
[0321] This method relates to fluorescent based encoding and
decoding process based on the fact that fluorescent dyes have broad
band absorption and emission spectra. Fewer detection channels are
used than the number of encoding dyes used in the process. That is,
for n detection channels, a particular dye, x, can be represented
by a vector I.sub.x in the n-dimensional space.
I x = { l .intg. I i n I i .perp. I 1 I 2 I 3 I n -
##EQU00005##
where I.sub.i is the intensity detected at channel i.
Theoretically, an infinite number of vectors are possible in the
n-dimensional space when n is larger or equal to 2. With an
appropriate dye selection and optical filter design, for any given
number of detection channels, a larger number of dyes can be used
for decoding. For example, to distinguish three species, it is
possible to tag the species with three different dyes, x, y, z and
use only two detection channels, such that [0322] I.sub.x={1 0}
[0323] I.sub.y={a 1-a} 0<a<1 [0324] I.sub.z={0 1}
[0325] FIG. 11 illustrates the experimental results for this
example scheme used in decoding. Three classes of beads were tagged
with Bodipy-493, Bodipy-R6G and Bodipy564. Detection channel 1
consisted of a 410 nm/20 excitation filter and a 540 nm/20 emission
filter. Detection channel 2 consisted of a 535 nm/20 excitation
filter and a 585 nm/nm emission filter. Excited with a xenon arc
lamp, the fluorescence emission of Bodipy-493 can only be detected
in channel 1. Bodipy-564 emission can be detected in channel 2.
Bodipy-R6G can be detected in both channels. As expected, the
scatter plot (FIG. 11) of the intensity in the two channels of the
beads clearly indicates three classes. Within each class, the beads
have the same relative intensity ratio (corresponding to the same
unit vector) with a certain uncertainties.
[0326] Similar design and data analysis procedure can be used for
other dyes and detection channels.
Sequence CWU 1
1
6121DNAArtificial SequenceSynthetic 1tgcacgagaa tggaggtatc t
21225DNAArtificial SequenceSynthetic 2ctgtcgcacg agaatggagg tatct
25330DNAArtificial SequenceSynthetic 3ctgtcggggc gcacgagaat
ggaggtatct 30434DNAArtificial SequenceSynthetic 4ctgtcggggc
actcacacga gaatggaggt atct 34540DNAArtificial SequenceSynthetic
5ctgtcggggc actcatttgt gcacgagaat ggaggtatct 40650DNAArtificial
SequenceSynthetic 6ctgtcggggc actcatttgt ctgtcggggc gcacgagaat
ggaggtatct 50
* * * * *