U.S. patent application number 10/021906 was filed with the patent office on 2002-11-28 for detection of nucleic acid amplification reactions using bead arrays.
This patent application is currently assigned to Illumina, Inc.. Invention is credited to Chee, Mark S., Gunderson, Kevin.
Application Number | 20020177141 10/021906 |
Document ID | / |
Family ID | 27574895 |
Filed Date | 2002-11-28 |
United States Patent
Application |
20020177141 |
Kind Code |
A1 |
Chee, Mark S. ; et
al. |
November 28, 2002 |
Detection of nucleic acid amplification reactions using bead
arrays
Abstract
The invention relates to compositions and methods for detecting
and quantifying a target nucleic acid using a variety of both
signal amplification and target amplification techniques.
Inventors: |
Chee, Mark S.; (Del Mar,
CA) ; Gunderson, Kevin; (Encinitas, CA) |
Correspondence
Address: |
FLEHR HOHBACH TEST ALBRITTON & HERBERT LLP
Suite 3400
Four Embarcadero Center
San Francisco
CA
94111-4187
US
|
Assignee: |
Illumina, Inc.
|
Family ID: |
27574895 |
Appl. No.: |
10/021906 |
Filed: |
December 12, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10021906 |
Dec 12, 2001 |
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09517945 |
Mar 3, 2000 |
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6355431 |
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60161148 |
Oct 22, 1999 |
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60135051 |
May 20, 1999 |
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60160027 |
Oct 18, 1999 |
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60130089 |
Apr 20, 1999 |
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60135053 |
May 20, 1999 |
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60135123 |
May 20, 1999 |
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60160927 |
Oct 22, 1999 |
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60160917 |
Oct 22, 1999 |
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Current U.S.
Class: |
506/5 ; 435/91.2;
506/16; 506/4 |
Current CPC
Class: |
C12Q 1/6813 20130101;
C12Q 1/6874 20130101; C40B 40/06 20130101; B01J 2219/00707
20130101; B01J 2219/00596 20130101; B01J 2219/00659 20130101; Y10S
977/924 20130101; B01J 2219/005 20130101; B01J 2219/00722 20130101;
B01J 2219/00648 20130101 |
Class at
Publication: |
435/6 ;
435/91.2 |
International
Class: |
C12Q 001/68; C12P
019/34 |
Claims
We claim:
1. A method for detecting a first target nucleic acid sequence
comprising: a) hybridizing at least a first primer nucleic acid to
said first target sequence to form a first hybridization complex;
b) contacting said first hybridization complex with a first enzyme
to form a modified first primer nucleic acid; c) disassociating
said first hybridization complex; d) contacting said modified first
primer nucleic acid with an array comprising: i) a substrate with a
surface comprising discrete sites; and ii) a population of
microspheres comprising at least a first subpopulation comprising a
first capture probe; such that said first capture probe and the
modified primer form an assay complex; wherein said microspheres
are distributed on said surface; and e) detecting the presence of
the modified primer nucleic acid.
2. A method according to claim 1 wherein steps a) through c) are
repeated prior to step d).
3. A method according to claim 1 further comprising: a) hybridizing
at least a second primer nucleic acid to a second target sequence
that is substantially complementary to said first target sequence
to form a second hybridization complex; b) contacting said second
hybridization complex with said first enzyme to form a modified
second primer nucleic acid; c) disassociating said second
hybridization complex; and d) forming a second assay complex
comprising said modified second primer nucleic acid and a second
capture probe on a second subpopulation.
4. A method according to claim 3 wherein steps a) through c) are
repeated prior to step d).
5. A method according to claim 2 wherein said first enzyme
comprises a DNA polymerase and said modification is an extension of
said primer such that the polymerase chain reaction (PCR)
occurs.
6. A method according to claim 2 wherein said first enzyme
comprises a ligase and said modification comprises a ligation of
said first primer which hybridizes to a first domain of said first
target sequence to a third primer which hybridizes to a second
adjacent domain of said first target sequence, such that the ligase
chain reaction (LCR) occurs.
7. A method according to claim 3 wherein said first enzyme
comprises a ligase and said modification is a ligation of said
second primer which hybridizes to a first domain of said second
target sequence to a fourth primer which hybridizes to a second
adjacent domain of said second target sequence, such that the
ligase chain reaction (LCR) occurs.
8. A method according to claim 2 wherein said first primer
comprises a first probe sequence, a first scissile linkage and a
second probe sequence, wherein said first enzyme will cleave said
first scissile linkage resulting in the separation of said first
and said second probe sequences and the disassociation of said
first hybridization complex, leaving said first target sequence
intact, such that the cycling probe technology (CPT) reaction
occurs.
9. A method according to claim 4 wherein said second primer
comprises a third probe sequence, a second scissile linkage and a
fourth probe sequence, wherein said first enzyme will cleave said
second scissile linkage resulting in the separation of said third
and said fourth probe sequences and the disassociation of said
second hybridization complex, leaving said second target sequence
intact, such that the cycling probe technology (CPT) reaction
occurs.
10. A method according to claim 2 wherein said first enzyme is a
polymerase that extends said first primer and said modified first
primer comprises a first newly synthesized strand, and said method
further comprises: a) the addition of a second enzyme comprising a
nicking enzyme that nicks said extended first primer leaving said
first target sequence intact; and b) extending from said nick using
said polymerase, thereby displacing said first newly synthesized
strand and generating a second newly synthesized strand; such that
strand displacement amplification (SDA) occurs.
11. A method according to claim 4 wherein said first enzyme is a
polymerase that extends said second primer and said modified first
primer comprises a third newly synthesized strand, and said method
further comprises: a) the addition of a second enzyme comprising a
nicking enzyme that nicks said extended second primer leaving said
second target sequence intact; and b) extending from said nick
using said polymerase, thereby displacing said third newly
synthesized strand and generating a fourth newly synthesized
strand; such that strand displacement amplification (SDA)
occurs.
12. A method according to claim 2 wherein said first target
sequence is a RNA target sequence, said first primer nucleic acid
is a DNA primer comprising an RNA polymerase promoter, said first
enzyme is a reverse-transcriptase that extends said first primer to
form a first newly synthesized DNA strand, and said method further
comprises: a) the addition of a second enzyme comprising an RNA
degrading enzyme that degrades said first target sequence; b) the
addition of a third primer that hybridizes to said first newly
synthesized DNA strand; c) the addition of a third enzyme
comprising a DNA polymerase that extends said third primer to form
a second newly synthesized DNA strand, to form a newly synthesized
DNA hybrid; d) the addition of a fourth enzyme comprising an RNA
polymerase that recognizes said RNA polymerase promoter and
generates at least one newly synthesized RNA strand from said DNA
hybrid; such that nucleic acid sequence-based amplification (NASBA)
occurs.
13. A method according to claim 2 wherein said first primer is an
invader primer, said method further comprises hybridizing a
signalling primer to said target sequence, said enzyme comprises a
structure-specific cleaving enzyme and said modification comprises
a cleavage of said signalling primer, such that the invasive
cleavage reaction occurs.
14. A method for detecting a target nucleic acid sequence
comprising: a) hybridizing a first primer to a first target
sequence to form a first hybridization complex; b) contacting said
first hybridization complex with a first enzyme to extend said
first primer to form a first newly synthesized strand and form a
nucleic acid hybrid that comprises an RNA polymerase promoter; c)
contacting said hybrid with an RNA polymerase that recognizes said
RNA polymerase promoter and generates at least one newly
synthesized RNA strand; d) contacting said newly synthesized RNA
strand with an array comprising: i) a substrate with a surface
comprising discrete sites; and ii) a population of microspheres
comprising at least a first subpopulation comprising a first
capture probe; such that said first capture probe and the modified
primer form an assay complex; wherein said microspheres are
distributed on said surface; and e) detecting the presence of the
newly synthesized RNA strand.
15. A method according to claim 14 wherein steps a) through c) are
repeated prior to step d).
16. A method according to claim 14 wherein said target nucleic acid
sequence is a RNA sequence, and prior to step a), said method
comprises: f) hybridizing a second primer comprising an RNA
polymerase promoter sequence to said RNA sequence to form a second
hybridization complex; g) contacting said second hybridization
complex with a second enzyme to extend said second primer to form a
second newly synthesized strand and form a nucleic acid hybrid; and
h) degrading said RNA sequence to leave said second newly
synthesized strand as said first target sequence.
17. A method according to claim 16 wherein said degrading is done
by the addition of an RNA degrading enzyme.
18. A method according to claim 16 wherein said degrading is done
by RNA degrading activity of said reverse transcriptase.
19. A method according to claim 14 wherein said target nucleic acid
sequence is a DNA sequence, and prior to step a), said method
comprises: f) hybridizing a second primer comprising an RNA
polymerase promoter sequence to said DNA sequence to form a second
hybridization complex; g) contacting said second hybridization
complex with a second enzyme to extend said second primer to form a
second newly synthesized strand and form a nucleic acid hybrid; and
h) denaturing said nucleic acid hybrid such that said second newly
synthesized strand is said first target sequence.
20. A method according to claim 1 wherein said first primer nucleic
acid hybridizes at its 5' end to said target nucleic acid sequence
and at its 3' end to a sequence immediately adjacent to said 5'
end, wherein said first enzyme comprises a ligase and said
modification comprises ligation of said 5' end with said 3' end to
form a circular probe, wherein said second enzyme is a polymerase
and said amplification is an amplification of said circular probe
such that rolling circle amplification occurs.
21. A kit for the detection of a first target nucleic acid sequence
comprising: a) at least a first nucleic acid primer substantially
complementary to at least a first domain of said target sequence;
b) at least a first enzyme that will modify said first nucleic acid
primer; and c) an array comprising: i) a substrate with a surface
comprising discrete sites; and ii) a population of microspheres
comprising at least a first and a second subpopulation, wherein
each subpopulation comprises a bioactive agent; wherein said
microspheres are distributed on said surface.
22. A kit according to claim 21 for the detection of a PCR reaction
wherein said first enzyme is a thermostable DNA polymerase.
23. A kit according to claim 21 for the detection of a LCR reaction
wherein said first enzyme is a ligase and said kit comprises a
first nucleic acid primer substantially complementary to a first
domain of said first target sequence and a third nucleic acid
primer substantially complementary to a second adjacent domain of
said first target sequence.
24. A kit according to claim 21 for the detection of a strand
displacement amplification (SDA) reaction wherein said first enzyme
is a polymerase and said kit further comprises a nicking
enzyme.
25. A kit according to claim 21 for the detection of a NASBA
reaction wherein said first enzyme is a reverse transcriptase, and
said kit comprises a second enzyme comprising an RNA degrading
enzyme, a third primer, a third enzyme comprising a DNA polymerase
and a fourth enzyme comprising an RNA polymerase.
26. A kit according to claim 21 for the detection of an invasive
cleavage reaction wherein said first enzyme is a structure-specific
cleaving enzyme, and said kit comprises a signalling primer.
Description
[0001] The present invention is a continuation-in-part of U.S. Ser.
No. 60/161,148, filed Oct. 22, 1999, which is a
continuation-in-part of U.S. Ser. No. 60/135,051, filed May 20,
1999, and a continuation-in-part of U.S. Ser. No. 60/160,027, filed
Oct. 22, 1999, which is a continuation-in-part of U.S. Ser. No.
60/130,089, filed Apr. 20, 1999, all of which are pending.
FIELD OF THE INVENTION
[0002] The invention relates to compositions and methods useful in
the detection and quantification of a nucleic acid target using a
variety of amplification techniques, including both signal
amplification and target amplification. Detection proceeds through
the use of a label that is associated with the amplified signal or
target, either directly or indirectly, to allow optical detection
of the light absorbing label using a microsphere array sensor.
BACKGROUND OF THE INVENTION
[0003] The detection of specific nucleic acids is an important tool
for diagnostic medicine and molecular biology research. Gene probe
assays currently play roles in identifying infectious organisms
such as bacteria and viruses, in probing the expression of normal
genes and identifying mutant genes such as oncogenes, in typing
tissue for compatibility preceding tissue transplantation, in
matching tissue or blood samples for forensic medicine, and for
exploring homology among genes from different species.
[0004] Ideally, a gene probe assay should be sensitive, specific
and easily automatable (for a review, see Nickerson, Current
Opinion in Biotechnology 4:48-51 (1993)). The requirement for
sensitivity (i.e. low detection limits) has been greatly alleviated
by the development of the polymerase chain reaction (PCR) and other
amplification technologies which allow researchers to amplify
exponentially a specific nucleic acid sequence before analysis as
outlined below (for a review, see Abramson et al., Current Opinion
in Biotechnology, 4:41-47 (1993)).
[0005] Sensitivity, i.e. detection limits, remain a significant
obstacle in nucleic acid detection systems, and a variety of
techniques have been developed to address this issue. Briefly,
these techniques can be classified as either target amplification
or signal amplification. Target amplification involves the
amplification (i.e. replication) of the target sequence to be
detected, resulting in a significant increase in the number of
target molecules. Target amplification strategies include the
polymerase chain reaction (PCR), strand displacement amplification
(SDA), and nucleic acid sequence based amplification (NASBA).
[0006] Alternatively, rather than amplify the target, alternate
techniques use the target as a template to replicate a signalling
probe, allowing a small number of target molecules to result in a
large number of signalling probes, that then can be detected.
Signal amplification strategies include the ligase chain reaction
(LCR), cycling probe technology (CPT), invasive cleavage techniques
such as Invader.TM. technology, Q-Beta replicase (Q.beta.R)
technology, and the use of "amplification probes" such as "branched
DNA" that result in multiple label probes binding to a single
target sequence.
[0007] The polymerase chain reaction (PCR) is widely used and
described, and involves the use of primer extension combined with
thermal cycling to amplify a target sequence; see U.S. Pat. Nos.
4,683,195 and 4,683,202, and PCR Essential Data, J. W. Wiley &
sons, Ed. C. R. Newton, 1995, all of which are incorporated by
reference. In addition, there are a number of variations of PCR
which also find use in the invention, including "quantitative
competitive PCR" or "QC-PCR", "arbitrarily primed PCR" or "AP-PCR",
"immuno-PCR", "Alu-PCR", "PCR single strand conformational
polymorphism" or "PCR-SSCP", allelic PCR (see Newton et al. Nucl.
Acid Res. 17:2503 91989); "reverse transcriptase PCR" or "RT-PCR",
"biotin capture PCR", "vectorette PCR", "panhandle PCR", and "PCR
select cDNA subtraction", among others.
[0008] Strand displacement amplification (SDA) is generally
described in Walker et al., in Molecular Methods for Virus
Detection, Academic Press, Inc., 1995, and U.S. Pat. Nos. 5,455,166
and 5,130,238, all of which are hereby incorporated by
reference.
[0009] Nucleic acid sequence based amplification (NASBA) is
generally described in U.S. Pat. No. 5,409,818 and "Profiting from
Gene-based Diagnostics", CTB International Publishing Inc., N.J.,
1996, both of which are incorporated by reference.
[0010] Cycling probe technology (CPT) is a nucleic acid detection
system based on signal or probe amplification rather than target
amplification, such as is done in polymerase chain reactions (PCR).
Cycling probe technology relies on a molar excess of labeled probe
which contains a scissile linkage of RNA. Upon hybridization of the
probe to the target, the resulting hybrid contains a portion of
RNA:DNA. This area of RNA:DNA duplex is recognized by RNAseH and
the RNA is excised, resulting in cleavage of the probe. The probe
now consists of two smaller sequences which may be released, thus
leaving the target intact for repeated rounds of the reaction. The
unreacted probe is removed and the label is then detected. CPT is
generally described in U.S. Pat. Nos. 5,011,769, 5,403,711,
5,660,988, and 4,876,187, and PCT published applications WO
95/05480, WO 95/1416, and WO 95/00667, all of which are
specifically incorporated herein by reference.
[0011] The oligonucleotide ligation assay (OLA; sometimes referred
to as the ligation chain reaction (LCR)) involve the ligation of at
least two smaller probes into a single long probe, using the target
sequence as the template for the ligase. See generally U.S. Pat.
Nos. 5,185,243, 5,679,524 and 5,573,907; EP 0 320 308 B1; EP 0 336
731 B1; EP 0 439 182 B1; WO 90/01069; WO 89/12696; and WO 89/09835,
all of which are incorporated by reference.
[0012] Invader.TM. technology is based on structure-specific
polymerases that cleave nucleic acids in a site-specific manner.
Two probes are used: an "invader" probe and a "signalling" probe,
that adjacently hybridize to a target sequence with a
non-complementary overlap. The enzyme cleaves at the overlap due to
its recognition of the "tail", and releases the "tail" with a
label. This can then be detected. The Invader.TM. technology is
described in U.S. Pat. Nos. 5,846,717; 5,614,402; 5,719,028;
5,541,311; and 5,843,669, all of which are hereby incorporated by
reference.
[0013] "Rolling circle amplification" is based on extension of a
circular probe that has hybridized to a target sequence. A
polymerase is added that extends the probe sequence. As the
circular probe has no terminus, the polymerase repeatedly extends
the circular probe resulting in concatamers of the circular probe.
As such, the probe is amplified. Rolling-circle amplification is
generally described in Baner et al. (1998) Nuc. Acids Res.
26:5073-5078; Barany, F. (1991) Proc. Natl. Acad. Sci. USA
88:189-193; and Lizardi et al. (1998) Nat Genet. 19:225-232, all of
which are incorporated by reference in their entirety.
[0014] "Branched DNA" signal amplification relies on the synthesis
of branched nucleic acids, containing a multiplicity of nucleic
acid "arms" that function to increase the amount of label that can
be put onto one probe. This technology is generally described in
U.S. Pat. Nos. 5,681,702, 5,597,909, 5,545,730, 5,594,117,
5,591,584, 5,571,670, 5,580,731, 5,571,670, 5,591,584, 5,624,802,
5,635,352, 5,594,118, 5,359,100, 5,124,246 and 5,681,697, all of
which are hereby incorporated by reference.
[0015] Similarily, dendrimers of nucleic acids serve to vastly
increase the amount of label that can be added to a single
molecule, using a similar idea but different compositions. This
technology is as described in U.S. Pat. No. 5,175,270 and Nilsen et
al., J. Theor. Biol. 187:273 (1997), both of which are incorporated
herein by reference.
[0016] In each of these methods, analysis of the amplified target
or amplified signal remains problematic. Accordingly, it is an
object of the invention to provide compositions and methods for the
detection and quantification of the products, either directly or
indirectly, of nucleic acid amplification reactions.
SUMMARY OF THE INVENTION
[0017] In accordance with the objects outlined above, the present
invention provides a method for detecting a first target nucleic
acid sequence. In one aspect the method comprises hybridizing at
least a first primer nucleic acid to the first target sequence to
form a first hybridization complex, contacting the first
hybridization complex with a first enzyme to form a modified first
primer nucleic acid, disassociating the first hybridization
complex, contacting the modified first primer nucleic acid with an
array comprising a substrate with a surface comprising discrete
sites and a population of microspheres comprising at least a first
subpopulation comprising a first capture probe such that the first
capture probe and the modified primer form an assay complex,
wherein the microspheres are distributed on the surface, and
detecting the presence of the modified primer nucleic acid.
[0018] In addition the method further comprises hybridizing at
least a second primer nucleic acid to a second target sequence that
is substantially complementary to the first target sequence to form
a second hybridization complex, contacting the second hybridization
complex with the first enzyme to form modified second primer
nucleic acid, disassociating the second hybridization complex and
forming a second assay complex comprising the modified second
primer nucleic acid and a second capture probe on a second
subpopulation.
[0019] In an additional aspect of the invention the primer forms a
circular probe following hybridization with the target nucleic acid
to form a first hybridization complex and contacting the first
hybridization complex with a first enzyme comprising a ligase such
that the oligonucleotide ligation assay (OLA) occurs. This is
followed by adding the second enzyme, a polymerase, such that the
circular probe is amplified in a rolling circle amplification (RCA)
assay.
[0020] In an additional aspect of the invention, the first enzyme
comprises a DNA polymerase and the modification is an extension of
the primer such that the polymerase chain reaction (PCR) occurs. In
an additional aspect of the invention the first enzyme comprises a
ligase and the modification comprises a ligation of the first
primer which hybridizes to a first domain of the first target
sequence, to a third primer which hybridizes to a second adjacent
domain of the first target sequence such that the ligase chain
reaction (LCR) occurs.
[0021] In an additional aspect of the invention, the first primer
comprises a first probe sequence, a first scissile linkage and a
second probe sequence, wherein the first enzyme will cleave the
scissile linkage resulting in the separation of the first and
second probe sequences and the disassociation of the first
hybridization complex, leaving the first target sequence intact
such that the cycling probe technology (CPT) reaction occurs.
[0022] In addition, wherein the first enzyme is a polymerase that
extends the first primer and the modified first primer comprises a
first newly synthesized strand, the method further comprises the
addition of a second enzyme comprising a nicking enzyme that nicks
the extended first primer leaving the first target sequence intact,
and extending from the nick using the polymerase, and thereby
displacing the first newly synthesized strand and generating a
second newly synthesized strand such that strand displacement
amplification (SBA) occurs.
[0023] In addition, wherein the first target sequence is an RNA
target sequence, the first primer nucleic acid is a DNA primer
comprising an RNA polymerase promoter, the first enzyme is a
reverse-transcriptase that extends the first primer to form a first
newly synthesized DNA strand, the method further comprises the
addition of a second enzyme comprising an RNA degrading enzyme that
degrades the first target sequence, the addition of a third primer
that hybridizes to the first newly synthesized DNA strand, the
addition of a third enzyme comprising a DNA polymerase that extends
the third primer to form a second newly synthesized DNA strand, to
form a newly synthesized DNA hybrid, the addition of a fourth
enzyme comprising an RNA polymerase that recognizes the RNA
polymerase promoter and generates at least one newly synthesized
RNA strand from the DNA hybrid, such that nucleic acid
sequence-based amplification (NASBA) occurs.
[0024] In addition, wherein the first primer is an invader primer,
the method further comprises hybridizing a signalling primer to the
target sequence, the enzyme comprises a structure-specific cleaving
enzyme and the modification comprises a cleavage of said signalling
primer, such that the invasive cleavage reaction occurs.
[0025] An additional aspect of the invention is a method for
detecting a target nucleic acid sequence comprising hybridizing a
first primer to a first target sequence to form a first
hybridization complex, contacting the first hybridization complex
with a first enzyme to extend the first primer to form a first
newly synthesized strand and form a nucleic acid hybrid that
comprises an RNA polymerase promoter, contacting the hybrid with an
RNA polymerase that recognizes the RNA polymerase promoter and
generates at least one newly synthesized RNA strand, contacting the
newly synthesized RNA strand with an array comprising a substrate
with a surface comprising discrete sites and a population of
microspheres comprising at least a first subpopulation comprising a
first capture probe; such that the first capture probe and the
modified primer form an assay complex; wherein the microspheres are
distributed on the surface and detecting the presence of the newly
synthesized RNA strand.
[0026] In addition, when the target nucleic acid sequence is an RNA
sequence, and prior to hybridizing a first primer to a first target
sequence to form a first hybridization complex, method comprises
hybridizing a second primer comprising an RNA polymerase promoter
sequence to the RNA sequence to form a second hybridization
complex, contacting the second hybridization complex with a second
enzyme to extend the second primer to form a second newly
synthesized strand and form a nucleic acid hybrid; and degrading
the RNA sequence to leave the second newly synthesized strand as
the first target sequence. In one aspect of the invention the
degrading is done by the addition of an RNA degrading enzyme. In an
additional aspect of the invention the degrading is done by RNA
degrading activity of reverse transcriptase.
[0027] In addition, when the target nucleic acid sequence is a DNA
sequence, and prior to hybridizing a first primer to a first target
sequence to form a first hybridization complex, the method
comprises hybridizing a second primer comprising an RNA polymerase
promoter sequence to the DNA sequence to form a second
hybridization complex, contacting the second hybridization complex
with a second enzyme to extend the second primer to form a second
newly synthesized strand and form a nucleic acid hybrid, and
denaturing the nucleic acid hybrid such that the second newly
synthesized strand is the first target sequence.
[0028] An additional aspect of the invention is a kit for the
detection of a first target nucleic acid sequence. The kit
comprises at least a first nucleic acid primer substantially
complementary to at least a first domain of the target sequence, at
least a first enzyme that will modify the first nucleic acid
primer, and an array comprising a substrate with a surface
comprising discrete sites, and a population of microspheres
comprising at least a first and a second subpopulation, wherein
each subpopulation comprises a bioactive agent, wherein the
microspheres are distributed on the surface.
[0029] In an additional aspect of the invention, is a kit for the
detection of a PCR reaction wherein the first enzyme is a
thermostable DNA polymerase.
[0030] In an additional aspect of the invention, is a kit for the
detection of a LCR reaction wherein the first enzyme is a ligase
and the kit comprises a first nucleic acid primer substantially
complementary to a first domain of the first target sequence and a
third nucleic acid primer substantially complementary to a second
adjacent domain of the first target sequence.
[0031] In an additional aspect of the invention, is a kit for the
detection of a strand displacement amplification (SDA) reaction
wherein the first enzyme is a polymerase and the kit further
comprises a nicking enzyme.
[0032] In an additional aspect of the invention, is a kit for the
detection of a NASBA reaction wherein the first enzyme is a reverse
transcriptase, and the kit comprises a second enzyme comprising an
RNA degrading enzyme, a third primer, a third enzyme comprising a
DNA polymerase and a fourth enzyme comprising an RNA
polymerase.
[0033] In an additional aspect of the invention, is a kit for the
detection of an invasive cleavage reaction wherein the first enzyme
is a structure-specific cleaving enzyme, and the kit comprises a
signaling primer.
DETAILED DESCRIPTION OF THE FIGURES
[0034] FIGS. 1A, 1B and 1C depict three different embodiments for
attaching a target sequence to an array. The solid support 5 has
microsphere 10 with capture probe 20 linked via a linker 15. FIG.
1A depicts direct attachment; the capture probe 20 hybridizes to a
first portion of the target sequence 25. FIG. 1B depicts the use of
a capture extender probe 30 that has a first portion that
hybridizes to the capture probe 20 and a second portion that
hybridizes to a first domain of the target sequence 25. FIG. 1C
shows the use of an adapter sequence 35, that has been added to the
target sequence, for example during an amplification reaction as
outlined herein.
[0035] FIGS. 2A and 2B depict two preferred embodiments of SBE
amplification. FIG. 2A shows extension primer 40 hybridized to the
target sequence 25. Upon addition of the extension enzyme and
labelled nucleotides, the extension primer is modified to form a
labelled primer 41. The reaction can be repeated and then the
labelled primer is added to the array as above. FIG. 2B depicts the
same reaction but using adapter sequences.
[0036] FIGS. 3A and 3B depict two preferred embodiments of OLA
amplification. FIG. 3A depicts a first ligation probe 45 and a
second ligation probe 50 with a label 55. Upon addition of the
ligase, the probes are ligated. The reaction can be repeated and
then the ligated primer is added to the array as above. FIG. 3B
depicts the same reaction but using adapter sequences.
[0037] FIG. 4 depicts a preferred embodiment of the invasive
cleavage reaction. In this embodiment, the signaling probe 65
comprises two portions, a detection sequence 67 and a signaling
portion 66. The signaling portion can serve as an adapter sequence.
In addition, the signaling portion generally comprises the label
55, although as will be appreciated by those in the art, the label
may be on the detection sequence as well. In addition, for optional
removal of the uncleaved probes, a capture tag 60 may also be used.
Upon addition of the enzyme, the structure is cleaved, releasing
the signaling portion 66. The reaction can be repeated and then the
signaling portion is added to the array as above.
[0038] FIGS. 5A and 5B depict two preferred embodiments of CPT
amplification. A CPT primer 70 comprising a label 55, a first probe
sequence 71 and a second probe sequence 73, separated by a scissile
linkage 72, and optionally comprising a capture tag 60, is
hybridized to the target sequence 25. Upon addition of the enzyme,
the scissile linkage is cleaved. The reaction can be repeated and
then the probe sequence comprising the label is added to the array
as above. FIG. 5B depicts the same reaction but using adapter
sequences.
[0039] FIG. 6 depicts OLA/RCA amplification using a single "padlock
probe" 57. The padlock probe is hybridized with a target sequence
25. When the probe 57 is complementary to the target sequence 26,
ligation of the probe termini occurs forming a circular probe 28.
When the probe 57 is not complementary to the target sequence 27,
ligation does not occur. Addition of polymerase and nucleotides to
the circular probe results amplification of the probe 58. Cleavage
of the amplified probe 58 yields fragments 59 that hybridize with
an identifier probe 21 immobilized on a microsphere 10.
[0040] FIG. 7 depicts an alternative method of OLA/RCA. An
immobilized first OLA primer 45 is hybridized with a target
sequence 25 and a second OLA primer 50. Following the addition of
ligase, the first and second OLA primers are ligated to form a
ligated oligonucleotide 56. Following denaturation to remove the
target nucleic acid, the immobilized ligated oligonucleotide is
distributed on an array. An RCA probe 57 and polymerase are added
to the array resulting in amplification of the circular RCA probe
58.
DETAILED DESCRIPTION OF THE INVENTION
[0041] This invention is directed to the detection (and optionally
quantification) of products of nucleic acid amplification
reactions, using bead arrays for detection of the amplification
products. Suitable amplification methods include both target
amplification and signal amplification and include, but are not
limited to, polymerase chain reaction (PCR), ligation chain
reaction (sometimes referred to as oligonucleotide ligase
amplification OLA), cycling probe technology (CPT), strand
displacement assay (SDA), transcription mediated amplification
(TMA), nucleic acid sequence based amplification (NASBA), rolling
circle amplification (RCA), and invasive cleavage technology. All
of these methods require a primer nucleic acid (including nucleic
acid analogs) that is hybridized to a target sequence to form a
hybridization complex, and an enzyme is added that in some way
modifies the primer to form a modified primer. For example, PCR
generally requires two primers, dNTPs and a DNA polymerase; LCR
requires two primers that adjacently hybridize to the target
sequence and a ligase; CPT requires one cleavable primer and a
cleaving enzyme; invasive cleavage requires two primers and a
cleavage enzyme; etc. Thus, in general, a target nucleic acid is
added to a reaction mixture that comprises the necessary
amplification components, and a modified primer is formed.
[0042] In general, the modified primer comprises a detectable
label, such as a fluorescent label, which is either incorporated by
the enzyme or present on the original primer. As required, the
unreacted primers are removed, in a variety of ways, as will be
appreciated by those in the art and outlined herein. The
hybridization complex is then disassociated, and the modified
primer is detected and optionally quantitated by a microsphere
array. In some cases, the newly modified primer serves as a target
sequence for a secondary reaction, which then produces a number of
amplified strands, which can be detected as outlined herein.
[0043] Accordingly, the present invention provides compositions and
methods for detecting the presence or absence of target nucleic
acid sequences in a sample. As will be appreciated by those in the
art, the sample solution may comprise any number of things,
including, but not limited to, bodily fluids (including, but not
limited to, blood, urine, serum, lymph, saliva, anal and vaginal
secretions, perspiration and semen, of virtually any organism, with
mammalian samples being preferred and human samples being
particularly preferred); environmental samples (including, but not
limited to, air, agricultural, water and soil samples); biological
warfare agent samples; research samples; purified samples, such as
purified genomic DNA, RNA, proteins, etc.; raw samples (bacteria,
virus, genomic DNA, etc.; As will be appreciated by those in the
art, virtually any experimental manipulation may have been done on
the sample.
[0044] The present invention provides compositions and methods for
detecting the presence or absence of target nucleic acid sequences
in a sample. 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
91986)), 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 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., Nucleoside & Nucleotide 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.,
Bioorganic & 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)
pp169-176). 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 labels, or to increase the stability and half-life of
such molecules in physiological environments.
[0045] As will be appreciated by those in the art, all of these
nucleic acid analogs may find use in the present invention. In
addition, mixtures of naturally occurring nucleic acids and analogs
can be made. Alternatively, mixtures of different nucleic acid
analogs, and mixtures of naturally occuring nucleic acids and
analogs may be made.
[0046] Particularly preferred are peptide nucleic acids (PNA) which
includes peptide nucleic acid analogs. These backbones are
substantially non-ionic under neutral conditions, in contrast to
the highly charged phosphodiester backbone of naturally occurring
nucleic acids. This results in two advantages. First, the PNA
backbone exhibits improved hybridization kinetics. PNAs have larger
changes in the melting temperature (Tm) for mismatched versus
perfectly matched basepairs. DNA and RNA typically exhibit a
2-4.degree. C. drop in Tm for an internal mismatch. With the
non-ionic PNA backbone, the drop is closer to 7-9.degree. C. This
allows for better detection of mismatches. Similarly, due to their
non-ionic nature, hybridization of the bases attached to these
backbones is relatively insensitive to salt concentration.
[0047] 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, xathanine hypoxathanine, isocytosine, isoguanine, etc. A
preferred embodiment utilizes isocytosine and isoguanine in nucleic
acids designed to be complementary to other probes, rather than
target sequences, as this reduces non-specific hybridization, as is
generally described in U.S. Pat. No. 5,681,702. As used herein, the
term "nucleoside" includes nucleotides as well as nucleoside and
nucleotide analogs, and modified nucleosides such as amino modified
nucleosides. In addition, "nucleoside" includes non-naturally
occuring analog structures. Thus for example the individual units
of a peptide nucleic acid, each containing a base, are referred to
herein as a nucleoside.
[0048] The compositions and methods of the invention are directed
to the detection of target sequences. The term "target sequence" or
"target nucleic acid" 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. As is
outlined herein, the target sequence may be a target sequence from
a sample, or a secondary target such as a product of a reaction
such as a detection sequence from an invasive cleavage reaction, a
ligated probe from an OLA reaction, an extended probe from a PCR
reaction, etc. Generally, as outlined herein, a target sequence
from a sample is amplified to produce a secondary target that is
detected, as outlined herein. Alternatively, an amplification step
is done using a signal probe that is amplified, again producing a
secondary target that is detected. The target sequence 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. The
target sequence may also be comprised of different target domains;
for example, in "sandwich" type assays as outlined below, a first
target domain of the sample target sequence may hybridize to a
capture probe or a portion of capture extender probe, a second
target domain may hybridize to a portion of an amplifier probe, a
label probe, or a different capture or capture extender probe, etc.
In addition, the target domains may be adjacent (i.e. contiguous)
or separated. For example, when LCR techniques are used, a first
primer may hybridize to a first target domain and a second primer
may hybridize to a second target domain; either the domains are
adjacent, or they may be separated by one or more nucleotides,
coupled with the use of a polymerase and dNTPs, as is more fully
outlined below. The terms "first" and "second" are not meant to
confer an orientation of the sequences with respect to the 5'-3'
orientation of the target sequence. For example, assuming a 5'-3'
orientation of the complementary target sequence, the first target
domain may be located either 5' to the second domain, or 3' to the
second domain.
[0049] If required, the target sequence is prepared using known
techniques. For example, the sample may be treated to lyse the
cells, using known lysis buffers, sonication, electroporation,
etc., with purification occuring as needed, as will be appreciated
by those in the art. In addition, the reactions outlined herein may
be accomplished in a variety of ways, as will be appreciated by
those in the art. Components of the reaction may be added
simultaneously, or sequentially, in any order, with preferred
embodiments outlined below. In addition, the reaction may include a
variety of other reagents which may be included in the assays.
These include reagents like salts, buffers, neutral proteins, e.g.
albumin, detergents, etc., which may be used to facilitate optimal
hybridization and detection, 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, depending on
the sample preparation methods and purity of the target.
[0050] In addition, in most embodiments, double stranded target
nucleic acids are denatured to render them single stranded so as to
permit hybridization of the primers and other probes of the
invention. A preferred embodiment utilizes a thermal step,
generally by raising the temperature of the reaction to about
95.degree. C., although pH changes and other techniques may also be
used.
[0051] A primer nucleic acid is then contacted to the target
sequence to form a hybridization complex. By "primer nucleic acid"
herein is meant a probe nucleic acid that will hybridize to some
portion, i.e. a domain, of the target sequence. Probes of the
present invention are designed to be complementary to a target
sequence (either the target sequence of the sample or to other
probe sequences, as is described below), such that hybridization of
the target sequence and the probes of the present invention occurs.
As outlined below, this complementarity need not be perfect; there
may be any number of base pair mismatches which 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 normal
reaction conditions.
[0052] A variety of hybridization conditions may be used in the
present invention, including high, moderate and low stringency
conditions; see for example Maniatis et al., Molecular Cloning: A
Laboratory Manual, 2d Edition, 1989, and Short Protocols in
Molecular Biology, ed. Ausubel, et al, 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.degree. C. lower than the thermal melting
point (Tm) for the specific sequence at a defined ionic strength
and pH. The Tm 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 Tm,
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.degree. C. for short probes (e.g.
10 to 50 nucleotides) and at least about 60.degree. C. for long
probes (e.g. greater than 50 nucleotides). Stringent conditions may
also be achieved with the addition of helix destabilizing agents
such as formamide. The hybridization conditions may also vary when
a non-ionic backbone, i.e. PNA is used, as is known in the art. In
addition, cross-linking agents may be added after target binding to
cross-link, i.e. covalently attach, the two strands of the
hybridization complex.
[0053] Thus, the assays are generally run under stringency
conditions which allows formation of the hybridization complex only
in the presence of target. Stringency can be controlled by altering
a step parameter that is a thermodynamic variable, including, but
not limited to, temperature, formamide concentration, salt
concentration, chaotropic salt concentration, pH, organic solvent
concentration, etc.
[0054] These parameters may also be used to control non-specific
binding, as is generally outlined in U.S. Pat. No. 5,681,697. Thus
it may be desirable to perform certain steps at higher stringency
conditions to reduce non-specific binding.
[0055] The size of the primer nucleic acid may vary, as will be
appreciated by those in the art, in general varying from 5 to 500
nucleotides in length, with primers of between 10 and 100 being
preferred, between 15 and 50 being particularly preferred, and from
10 to 35 being especially preferred, depending on the use and
amplification technique.
[0056] In addition, the different amplification techniques may have
further requirements of the primers, as is more fully described
below.
[0057] Once the hybridization complex between the primer and the
target sequence has been formed, an enzyme, sometimes termed an
"amplification enzyme", is used to modify the primer. As for all
the methods outlined herein, the enzymes may be added at any point
during the assay, either prior to, during, or after the addition of
the primers. The identity of the enzyme will depend on the
amplification technique used, as is more fully outlined below.
Similarly, the modification will depend on the amplification
technique, as outlined below.
[0058] Once the enzyme has modified the primer to form a modified
primer, the hybridization complex is disassociated. In one aspect,
dissociation is by modification of the assay conditions. In another
aspect, the modified primer no longer hybridizes to the target
nucleic acid and dissociates. Either one or both of these aspects
can be employed in signal and target amplification reactions as
described below. Generally, the amplification steps are repeated
for a period of time to allow a number of cycles, depending on the
number of copies of the original target sequence and the
sensitivity of detection, with cycles ranging from 1 to thousands,
with from 10 to 100 cycles being preferred and from 20 to 50 cycles
being especially preferred.
[0059] After a suitable time of amplification, unreacted primers
are removed, in a variety of ways, as will be appreciated by those
in the art and described below, and the hybridization complex is
disassociated. In general, the modified primer comprises a
detectable label, such as a fluorescent label, which is either
incorporated by the enzyme or present on the original primer, and
the modified primer is added to a microsphere array such is
generally described in U.S. Ser. Nos. 09/189,543; 08/944,850;
09/033,462; 09/287,573; 09/151,877; 09/187,289 and 09/256,943; and
PCT applications US98/09163 and US99/14387; US98/21193; US99/04473
and US98/05025, all of which are hereby incorporated by reference.
The microsphere array comprises subpopulations of microspheres that
comprise capture probes that will hybridize to the modified
primers. Detection proceeds via detection of the label as an
indication of the presence, absence or amount of the target
sequence, as is more fully outlined below.
[0060] Target Amplification
[0061] In a preferred embodiment, the amplification is target
amplification. Target amplification involves the amplification
(replication) of the target sequence to be detected, such that the
number of copies of the target sequence is increased. Suitable
target amplification techniques include, but are not limited to,
the polymerase chain reaction (PCR), strand displacement
amplification (SDA), transcription mediated amplification (TMA) and
nucleic acid sequence based amplification (NASBA).
[0062] Polymerase Chain Reaction Amplification
[0063] In a preferred embodiment, the target amplification
technique is PCR. The polymerase chain reaction (PCR) is widely
used and described, and involves the use of primer extension
combined with thermal cycling to amplify a target sequence; see
U.S. Pat. Nos. 4,683,195 and 4,683,202, and PCR Essential Data, J.
W. Wiley & sons, Ed. C. R. Newton, 1995, all of which are
incorporated by reference. In addition, there are a number of
variations of PCR which also find use in the invention, including
"quantitative competitive PCR" or "QC-PCR", "arbitrarily primed
PCR" or "AP-PCR", "immuno-PCR", "Alu-PCR", "PCR single strand
conformational polymorphism" or "PCR-SSCP", "reverse transcriptase
PCR" or "RT-PCR", "biotin capture PCR", "vectorette PCR",
"panhandle PCR", and "PCR select cDNA subtraction",
"allele-specific PCR", among others.
[0064] In general, PCR may be briefly described as follows. A
double stranded target nucleic acid is denatured, generally by
raising the temperature, and then cooled in the presence of an
excess of a PCR primer, which then hybridizes to the first target
strand. A DNA polymerase then acts to extend the primer with dNTPs,
resulting in the synthesis of a new strand forming a hybridization
complex. The sample is then heated again, to disassociate the
hybridization complex, and the process is repeated. By using a
second PCR primer for the complementary target strand, rapid and
exponential amplification occurs. Thus PCR steps are denaturation,
annealing and extension. The particulars of PCR are well known, and
include the use of a thermostable polymerase such as Taq I
polymerase and thermal cycling.
[0065] Accordingly, the PCR reaction requires at least one PCR
primer, a polymerase, and a set of dNTPs. As outlined herein, the
primers may comprise the label, or one or more of the dNTPs may
comprise a label.
[0066] In general, as is more fully outlined below, the capture
probes on the beads of the array are designed to be substantially
complementary to the extended part of the primer; that is,
unextended primers will not bind to the capture probes.
Alternatively, as further described below, unreacted probes may be
removed prior to addition to the array.
[0067] Strand Displacement Amplification (SDA)
[0068] In a preferred embodiment, the target amplification
technique is SDA. Strand displacement amplification (SDA) is
generally described in Walker et al., in Molecular Methods for
Virus Detection, Academic Press, Inc., 1995, and U.S. Pat. Nos.
5,455,166 and 5,130,238, all of which are hereby expressly
incorporated by reference in their entirety.
[0069] In general, SDA may be described as follows. A single
stranded target nucleic acid, usually a DNA target sequence, is
contacted with an SDA primer. An "SDA primer" generally has a
length of 25-100 nucleotides, with SDA primers of approximately 35
nucleotides being preferred. An SDA primer is substantially
complementary to a region at the 3' end of the target sequence, and
the primer has a sequence at its 5' end (outside of the region that
is complementary to the target) that is a recognition sequence for
a restriction endonuclease, sometimes referred to herein as a
"nicking enzyme" or a "nicking endonuclease", as outlined below.
The SDA primer then hybridizes to the target sequence. The SDA
reaction mixture also contains a polymerase (an "SDA polymerase",
as outlined below) and a mixture of all four
deoxynucleoside-triphosphates (also called deoxynucleotides or
dNTPs, i.e. dATP, dTTP, dCTP and dGTP), at least one species of
which is a substituted or modified dNTP; thus, the SDA primer is
modified, i.e. extended, to form a modified primer, sometimes
referred to herein as a "newly synthesized strand". The substituted
dNTP is modified such that it will inhibit cleavage in the strand
containing the substituted dNTP but will not inhibit cleavage on
the other strand. Examples of suitable substituted dNTPs include,
but are not limited, 2'deoxyadenosine 5'-O-(1-thiotriphosphate),
5-methyideoxycytidine 5'-triphosphate, 2'-deoxyuridine
5'-triphosphate, adn 7-deaza-2'-deoxyguanosine 5'-triphosphate. In
addition, the substitution of the dNTP may occur after
incorporation into a newly synthesized strand; for example, a
methylase may be used to add methyl groups to the synthesized
strand. In addition, if all the nucleotides are substituted, the
polymerase may have 5'.fwdarw.3' exonuclease activity. However, if
less than all the nucleotides are substituted, the polymerase
preferably lacks 5'.fwdarw.3' exonuclease activity.
[0070] As will be appreciated by those in the art, the recognition
site/endonuclease pair can be any of a wide variety of known
combinations. The endonuclease is chosen to cleave a strand either
at the recognition site, or either 3' or 5' to it, without cleaving
the complementary sequence, either because the enzyme only cleaves
one strand or because of the incorporation of the substituted
nucleotides. Suitable recognition site/endonuclease pairs are well
known in the art; suitable endonucleases include, but are not
limited to, HincII, HindII, AvaI, Fnu4HI, TthIIII, NcII, BstXI,
BamHI, etc. A chart depicting suitable enzymes, and their
corresponding recognition sites and the modified dNTP to use is
found in U.S. Pat. No. 5,455,166, hereby expressly incorporated by
reference.
[0071] Once nicked, a polymerase (an "SDA polymerase") is used to
extend the newly nicked strand, 5'.fwdarw.3', thereby creating
another newly synthesized strand. The polymerase chosen should be
able to intiate 5'.fwdarw.3' polymerization at a nick site, should
also displace the polymerized strand downstream from the nick, and
should lack 5'.fwdarw.3' exonuclease activity (this may be
additionally accomplished by the addition of a blocking agent).
Thus, suitable polymerases in SDA include, but are not limited to,
the Klenow fragment of DNA polymerase I, SEQUENASE 1.0 and
SEQUENASE 2.0 (U.S. Biochemical), T5 DNA polymerase and Phi29 DNA
polymerase.
[0072] Accordingly, the SDA reaction requires, in no particular
order, an SDA primer, an SDA polymerase, a nicking endonuclease,
and dNTPs, at least one species of which is modified. Again, as
outlined above for PCR, preferred embodiments utilize capture
probes complementary to the newly synthesized portion of the
primer, rather than the primer region, to allow unextended primers
to be removed.
[0073] In general, SDA does not require thermocycling. The
temperature of the reaction is generally set to be high enough to
prevent non-specific hybridization but low enough to allow specific
hybridization; this is generally from about 37.degree. C. to about
42.degree. C., depending on the enzymes.
[0074] In a preferred embodiment, as for most of the amplification
techniques described herein, a second amplification reaction can be
done using the complementary target sequence, resulting in a
substantial increase in amplification during a set period of time.
That is, a second primer nucleic acid is hybridized to a second
target sequence, that is substantially complementary to the first
target sequence, to form a second hybridization complex. The
addition of the enzyme, followed by disassociation of the second
hybridization complex, results in the generation of a number of
newly synthesized second strands.
[0075] Nucleic Acid Sequence Based Amplification (NASBA) and
Transcription Mediated Amplification (TMA)
[0076] In a preferred embodiment, the target amplification
technique is nucleic acid sequence based amplification (NASBA).
NASBA is generally described in U.S. Pat. No. 5,409,818; Sooknanan
et al., Nucleic Acid Sequence-Based Amplification, Ch. 12 (pp.
261-285) of Molecular Methods for Virus Detection, Academic Press,
1995; and "Profiting from Gene-based Diagnostics", CTB
International Publishing Inc., N.J., 1996, all of which are
incorporated by reference. NASBA is very similar to both TMA and
QBR. Transcription mediated amplification (TMA) is generally
described in U.S. Pat. Nos. 5,399,491, 5,888,779, 5,705,365,
5,710,029, all of which are incorporated by reference. The main
difference between NASBA and TMA is that NASBA utilizes the
addition of RNAse H to effect RNA degradation, and TMA relies on
inherent RNAse H activity of the reverse transcriptase.
[0077] In general, these techniques may be described as follows. A
single stranded target nucleic acid, usually an RNA target sequence
(sometimes referred to herein as "the first target sequence" or
"the first template"), is contacted with a first primer, generally
referred to herein as a "NASBA primer" (although "TMA primer" is
also suitable). Starting with a DNA target sequence is described
below. These primers generally have a length of 25-100 nucleotides,
with NASBA primers of approximately 50-75 nucleotides being
preferred. The first primer is preferably a DNA primer that has at
its 3' end a sequence that is substantially complementary to the 3'
end of the first template. The first primer also has an RNA
polymerase promoter at its 5' end (or its complement (antisense),
depending on the configuration of the system). The first primer is
then hybridized to the first template to form a first hybridization
complex. The reaction mixture also includes a reverse transcriptase
enzyme (an "NASBA reverse transcriptase") and a mixture of the four
dNTPs, such that the first NASBA primer is modified, i.e. extended,
to form a modified first primer, comprising a hybridization complex
of RNA (the first template) and DNA (the newly synthesized
strand).
[0078] By "reverse transcriptase" or "RNA-directed DNA polymerase"
herein is meant an enzyme capable of synthesizing DNA from a DNA
primer and an RNA template. Suitable RNA-directed DNA polymerases
include, but are not limited to, avian myloblastosis virus reverse
transcriptase ("AMV RT") and the Moloney murine leukemia virus RT.
When the amplification reaction is TMA, the reverse transcriptase
enzyme further comprises a RNA degrading activity as outlined
below.
[0079] In addition to the components listed above, the NASBA
reaction also includes an RNA degrading enzyme, also sometimes
referred to herein as a ribonuclease, that will hydrolyze RNA of an
RNA:DNA hybrid without hydrolyzing single- or double-stranded RNA
or DNA. Suitable ribonucleases include, but are not limited to,
RNase H from E. coli and calf thymus.
[0080] The ribonuclease activity degrades the first RNA template in
the hybridization complex, resulting in a disassociation of the
hybridization complex leaving a first single stranded newly
synthesized DNA strand, sometimes referred to herein as "the second
template".
[0081] In addition, the NASBA reaction also includes a second NASBA
primer, generally comprising DNA (although as for all the probes
herein, including primers, nucleic acid analogs may also be used).
This second NASBA primer has a sequence at its 3' end that is
substantially complementary to the 3' end of the second template,
and also contains an antisense sequence for a functional promoter
and the antisense sequence of a transcription initiation site.
Thus, this primer sequence, when used as a template for synthesis
of the third DNA template, contains sufficient information to allow
specific and efficient binding of an RNA polymerase and initiation
of transcription at the desired site. Preferred embodiments
utilizes the antisense promoter and transcription initiation site
are that of the T7 RNA polymerase, although other RNA polymerase
promoters and initiation sites can be used as well, as outlined
below.
[0082] The second primer hybridizes to the second template, and a
DNA polymerase, also termed a "DNA-directed DNA polymerase", also
present in the reaction, synthesizes a third template (a second
newly synthesized DNA strand), resulting in second hybridization
complex comprising two newly synthesized DNA strands.
[0083] Finally, the inclusion of an RNA polymerase and the required
four ribonucleoside triphosphates (ribonucleotides or NTPs) results
in the synthesis of an RNA strand (a third newly synthesized strand
that is essentially the same as the first template). The RNA
polymerase, sometimes referred to herein as a "DNA-directed RNA
polymerase", recognizes the promoter and specifically initiates RNA
synthesis at the initiation site. In addition, the RNA polymerase
preferably synthesizes several copies of RNA per DNA duplex.
Preferred RNA polymerases include, but are not limited to, T7 RNA
polymerase, and other bacteriophage RNA polymerases including those
of phage T3, phage .phi.II, Salmonella phage sp6, or Pseudomonase
phage gh-1.
[0084] In some embodiments, TMA and NASBA are used with starting
DNA target sequences. In this embodiment, it is necessary to
utilize the first primer comprising the RNA polymerase promoter and
a DNA polymerase enzyme to generate a double stranded DNA hybrid
with the newly synthesized strand comprising the promoter sequence.
The hybrid is then denatured and the second primer added.
[0085] Accordingly, the NASBA reaction requires, in no particular
order, a first NASBA primer, a second NASBA primer comprising an
antisense sequence of an RNA polymerase promoter, an RNA polymerase
that recognizes the promoter, a reverse transcriptase, a DNA
polymerase, an RNA degrading enzyme, NTPs and dNTPs, in addition to
the detection components outlined below.
[0086] These components result in a single starting RNA template
generating a single DNA duplex; however, since this DNA duplex
results in the creation of multiple RNA strands, which can then be
used to initiate the reaction again, amplification proceeds
rapidly.
[0087] Accordingly, the TMA reaction requires, in no particular
order, a first TMA primer, a second TMA primer comprising an
antisense sequence of an RNA polymerase promoter, an RNA polymerase
that recognizes the promoter, a reverse transcriptase with RNA
degrading activity, a DNA polymerase, NTPs and dNTPs, in addition
to the detection components outlined below.
[0088] These components result in a single starting RNA template
generating a single DNA duplex; however, since this DNA duplex
results in the creation of multiple RNA strands, which can then be
used to initiate the reaction again, amplification proceeds
rapidly.
[0089] As outlined herein, the detection of the newly synthesized
strands can proceed in several ways. Direct detection can be done
when the newly synthesized strands comprise detectable labels,
either by incorporation into the primers or by incorporation of
modified labelled nucleotides into the growing strand.
Alternatively, as is more fully outlined below, indirect detection
of unlabelled strands (which now serve as "targets" in the
detection mode) can occur using a variety of sandwich assay
configurations. As will be appreciated by those in the art, any of
the newly synthesized strands can serve as the "target" for form an
assay complex on a surface with a capture probe. In NASBA and TMA,
it is preferable to utilize the newly formed RNA strands as the
target, as this is where significant amplification occurs.
[0090] In this way, a number of secondary target molecules are
made. As is more fully outlined below, these reactions (that is,
the products of these reactions) can be detected in a number of
ways.
[0091] Signal Amplification Techniques
[0092] In a preferred embodiment, the amplification technique is
signal amplification. Signal amplification involves the use of
limited number of target molecules as templates to either generate
multiple signalling probes or allow the use of multiple signalling
probes. Signal amplification strategies include LCR, CPT, Q.beta.R,
invasive cleavage technology, and the use of amplification probes
in sandwich assays.
[0093] Single Base Extension (SBE)
[0094] In a preferred embodiment, single base extension (SBE;
sometimes referred to as "minisequencing") is used for
amplification. It should also be noted that SBE finds use in
genotyping, as is described in co-pending application entitled
"SEQUENCE DETERMINATION OF NUCLEIC ACIDS USING ARRAYS WITH
MICROSPHERES" filed on Oct. 22, 1999 as U.S. Ser. No. 09/425,633.
Briefly, SBE is a technique that utilizes an extension primer that
hybridizes to the target nucleic acid. A polymerase (generally a
DNA polymerase) is used to extend the 3' end of the primer with a
nucleotide analog labeled a detection label as described herein.
Based on the fidelity of the enzyme, a nucleotide is only
incorporated into the extension primer if it is complementary to
the adjacent base in the target strand. Generally, the nucleotide
is derivatized such that no further extensions can occur, so only a
single nucleotide is added. However, for amplification reactions,
this may not be necessary. Once the labeled nucleotide is added,
detection of the label proceeds as outlined herein. See generally
Sylvanen et al., Genomics 8:684-692 (1990); U.S. Pat. Nos.
5,846,710 and 5,888,819; Pastinen et al., Genomics Res.
7(6):606-614 (1997); all of which are expressly incorporated herein
by reference.
[0095] The reaction is initiated by introducing the assay complex
comprising the target sequence (i.e. the array) to a solution
comprising a first nucleotide, frequently an nucleotide analog. By
"nucleoide analog" in this context herein is meant a
deoxynucleoside-triphosphate (also called deoxynucleotides or
dNTPs, i.e. dATP, dTTP, dCTP and dGTP), that is further derivatized
to be chain terminating. As will be appreciated by those in the
art, any number of nucleotide analogs may be used, as long as a
polymerase enzyme will still incorporate the nucleotide at the
interrogation position. Preferred embodiments utilize
dideoxy-triphosphate nucleotides (ddNTPs). Generally, a set of
nucleotides comprising ddATP, ddCTP, ddGTP and ddTTP is used, at
least one of which includes a label, and preferably all four. For
amplification rather than genotyping reactions, the labels may all
be the same; alternatively, different labels may be used.
[0096] In a preferred embodiment, the nucleotide analogs comprise a
detectable label, which can be either a primary or secondary
detectable label. Preferred primary labels are those outlined
above. However, the enzymatic incorporation of nucleotides
comprising fluorophores is poor under many conditions; accordingly,
preferred embodiments utilize secondary detectable labels. In
addition, as outlined below, the use of secondary labels may also
facilitate the removal of unextended probes.
[0097] In addition to a first nucleotide, the solution also
comprises an extension enzyme, generally a DNA polymerase. Suitable
DNA polymerases include, but are not limited to, the Klenow
fragment of DNA polymerase I, SEQUENASE 1.0 and SEQUENASE 2.0 (U.S.
Biochemical), T5 DNA polymerase and Phi29 DNA polymerase. If the
NTP is complementary to the base of the detection position of the
target sequence, which is adjacent to the extension primer, the
extension enzyme will add it to the extension primer. Thus, the
extension primer is modified, i.e. extended, to form a modified
primer, sometimes referred to herein as a "newly synthesized
strand".
[0098] A limitation of this method is that unless the target
nucleic acid is in sufficient concentration, the amount of
unextended primer in the reaction greatly exceeds the resultant
extended-labeled primer. The excess of unextended primer competes
with the detection of the labeled primer in the assays described
herein. Accordingly, when SBE is used, preferred embodiments
utilize methods for the removal of unextended primers as outlined
herein.
[0099] One method to overcome this limitation is thermocycling
minisequencing in which repeated cycles of annealing, primer
extension, and heat denaturation using a thermocycler and
thermo-stable polymerase allows the amplification of the extension
probe which results in the accumulation of extended primers. For
example, if the original unextended primer to target nucleic acid
concentration is 100:1 and 100 thermocycles and extensions are
performed, a majority of the primer will be extended.
[0100] As will be appreciated by those in the art, the
configuration of the SBE system can take on several forms. As for
the LCR reaction described below, the reaction may be done in
solution, and then the newly synthesized strands, with the
base-specific detectable labels, can be detected. For example, they
can be directly hybridized to capture probes that are complementary
to the extension primers, and the presence of the label is then
detected.
[0101] Alternatively, the SBE reaction can occur on a surface. For
example, a target nucleic acid may be captured using a first
capture probe that hybridizes to a first target domain of the
target, and the reaction can proceed at a second target domain. The
extended labeled primers are then bound to a second capture probe
and detected.
[0102] Thus, the SBE reaction requires, in no particular order, an
extension primer, a polymerase and dNTPs, at least one of which is
labeled.
[0103] Oligonucleotide Ligation Amplification (OLA)
[0104] In a preferred embodiment, the signal amplification
technique is OLA. OLA, which is referred to as the ligation chain
reaction (LCR) when two-stranded substrates are used, involves the
ligation of two smaller probes into a single long probe, using the
target sequence as the template. In LCR, the ligated probe product
becomes the predominant template as the reaction progresses. The
method can be run in two different ways; in a first embodiment,
only one strand of a target sequence is used as a template for
ligation; alternatively, both strands may be used. See generally
U.S. Pat. Nos. 5,185,243, 5,679,524 and 5,573,907; EP 0 320 308 B1;
EP 0 336 731 B1; EP 0 439 182 B1; WO 90/01069; WO 89/12696; WO
97/31256; and WO 89/09835, and U.S. Ser. Nos. 60/078,102 and
60/073,011, all of which are incorporated by reference.
[0105] In a preferred embodiment, the single-stranded target
sequence comprises a first target domain and a second target
domain, which are adjacent and contiguous. A first OLA primer and a
second OLA primer nucleic acids are added, that are substantially
complementary to their respective target domain and thus will
hybridize to the target domains. These target domains may be
directly adjacent, i.e. contiguous, or separated by a number of
nucleotides. If they are non-contiguous, nucleotides are added
along with means to join nucleotides, such as a polymerase, that
will add the nucleotides to one of the primers. The two OLA primers
are then covalently attached, for example using a ligase enzyme
such as is known in the art, to form a modified primer. This forms
a first hybridization complex comprising the ligated probe and the
target sequence. This hybridization complex is then denatured
(disassociated), and the process is repeated to generate a pool of
ligated probes.
[0106] In a preferred embodiment, OLA is done for two strands of a
double-stranded target sequence. The target sequence is denatured,
and two sets of probes are added: one set as outlined above for one
strand of the target, and a separate set (i.e. third and fourth
primer probe nucleic acids) for the other strand of the target. In
a preferred embodiment, the first and third probes will hybridize,
and the second and fourth probes will hybridize, such that
amplification can occur. That is, when the first and second probes
have been attached, the ligated probe can now be used as a
template, in addition to the second target sequence, for the
attachment of the third and fourth probes. Similarly, the ligated
third and fourth probes will serve as a template for the attachment
of the first and second probes, in addition to the first target
strand. In this way, an exponential, rather than just a linear,
amplification can occur.
[0107] As will be appreciated by those in the art, the ligation
product can be detected in a variety of ways. In a preferred
embodiment, the ligation reaction is run in solution. In this
embodiment, only one of the primers carries a detectable label,
e.g. the first ligation probe, and the capture probe on the bead is
substantially complementary to the other probe, e.g. the second
ligation probe. In this way, unextended labeled ligation primers
will not interfere with the assay. That is, in a preferred
embodiment, the ligation product is detected by solid-phase
oligonucleotide probes. The solid-phase probes are preferably
complementary to at least a portion of the ligation product. In a
preferred embodiment, the solid-phase probe is complementary to the
5' detection oligonucleotide portion of the ligation product. This
substantially reduces or eliminates false signal generated by the
optically-labeled 3' primers. Preferably, detection is accomplished
by removing the unligated 5' detection oligonucleotide from the
reaction before application to a capture probe. In one embodiment,
the unligated 5' detection oligonucleotides are removed by
digesting 3' non-protected oligonucleotides with a 3' exonuclease,
such as, exonuclease I. The ligation products are protected from
exo I digestion by including, for example, 4-phosphorothioate
residues at their 3' terminus, thereby, rendering them resistant to
exonuclease digestion. The unligated detection oligonucleotides are
not protected and are digested.
[0108] Alternatively, the target nucleic acid is immobilized on a
solid-phase surface. The ligation assay is performed and unligated
oligonucleotides are removed by washing under appropriate
stringency to remove unligated oligonucleotides. The ligated
oligonucleotides are eluted from the target nucleic acid using
denaturing conditions, such as, 0.1 N NaOH, and detected as
described herein.
[0109] Again, as outlined above, the detection of the LCR reaction
can also occur directly, in the case where one or both of the
primers comprises at least one detectable label, or indirectly,
using sandwich assays, through the use of additional probes; that
is, the ligated probes can serve as target sequences, and detection
may utilize amplification probes, capture probes, capture extender
probes, label probes, and label extender probes, etc.
[0110] Rolling-Circle Amplification (RCA)
[0111] In a preferred embodiment the signal amplification technique
is RCA. Rolling-circle amplification is generally described in
Baner et al. (1998) Nuc. Acids Res. 26:5073-5078; Barany, F. (1991)
Proc. Natl. Acad. Sci. USA 88:189-193; and Lizardi et al. (1998)
Nat. Genet. 19:225-232, all of which are incorporated by reference
in their entirety.
[0112] In general, RCA may be described in two ways. First, as is
outlined in more detail below, a single probe is hybridized with a
target nucleic acid. Each terminus of the probe hybridizes
adjacently on the target nucleic acid and the OLA assay as
described above occurs. When ligated, the probe is circularized
while hybridized to the target nucleic acid. Addition of a
polymerase results in extension of the circular probe. However,
since the probe has no terminus, the polymerase continues to extend
the probe repeatedly. Thus results in amplification of the circular
probe.
[0113] A second alternative approach involves OLA followed by RCA.
In this embodiment, an immobilized primer is contacted with a
target nucleic acid. Complementary sequences will hybridize with
each other resulting in an immobilized duplex. A second primer is
contacted with the target nucleic acid. The second primer
hybridizes to the target nucleic acid adjacent to the first primer.
An OLA assay is performed as described above. Ligation only occurs
if the primer are complementary to the target nucleic acid. When a
mismatch occurs, particularly at one of the nucleotides to be
ligated, ligation will not occur. Following ligation of the
oligonucleotides, the ligated, immobilized, oligonucleotide is then
hybridized with an RCA probe. This is a circular probe that is
designed to specifically hybridize with the ligated oligonucleotide
and will only hybridize with an oligonucleotide that has undergone
ligation. RCA is then performed as is outlined in more detail
below.
[0114] Accordingly, in an preferred embodiment, a single
oligonucleotide is used both for OLA and as the circular template
for RCA (referred to herein as a "padlock probe" or a "RCA probe").
That is, each terminus of the oligonucleotide contains sequence
complementary to the target nucleic acid and functions as an OLA
primer as described above. That is, the first end of the RCA probe
is substantially complementary to a first target domain, and the
second end of the RCA probe is substantially complementary to a
second target domain, adjacent to the first domain. Hybridization
of the oligonucleotide to the target nucleic acid results in the
formation of a hybridization complex. Ligation of the "primers"
(which are the discrete ends of a single oligonucleotide) results
in the formation of a modified hybridization complex containing a
circular probe i.e. an RCA template complex. That is, the
oligonucleotide is circularized while still hybridized with the
target nucleic acid. This serves as a circular template for RCA.
Addition of a polymerase to the RCA template complex results in the
formation of an amplified product nucleic acid. Following RCA, the
amplified product nucleic acid is detected (FIG. 6). This can be
accomplished in a variety of ways; for example, the polymerase may
incorporate labelled nucleotides, or alternatively, a label probe
is used that is substantially complementary to a portion of the RCA
probe and comprises at least one label is used.
[0115] The polymerase can be any polymerase, but is preferably one
lacking 3' exonuclease activity (3' exo.sup.-). Examples of
suitable polymerase include but are not limited to exonuclease
minus DNA Polymerase I large (Klenow) Fragment, Phi29 DNA
polymerase, Taq DNA Polymerase and the like. In addition, in some
embodiments, a polymerase that will replicate single-stranded DNA
(i.e. without a primer forming a double stranded section) can be
used.
[0116] In a preferred embodiment, the RCA probe contains an adapter
sequence as outlined herein, with adapter capture probes on the
array, for example on a microsphere when microsphere arrays are
being used. Alternatively, unique portions of the RCA probes, for
example all or part of the sequence corresponding to the target
sequence, can be used to bind to a capture probe.
[0117] In a preferred embodiment, the padlock probe contains a
restriction site. The restriction endonuclease site allows for
cleavage of the long concatamers that are typically the result of
RCA into smaller individual units that hybridize either more
efficiently or faster to surface bound capture probes. Thus,
following RCA, the product nucleic acid is contacted with the
appropriate restriction endonuclease. This results in cleavage of
the product nucleic acid into smaller fragments. The fragments are
then hybridized with the capture probe that is immobilized
resulting in a concentration of product fragments onto the
microsphere. Again, as outlined herein, these fragments can be
detected in one of two ways: either labelled nucleotides are
incorporated during the replication step, or an additional label
probe is added.
[0118] Thus, in a preferred embodiment, the padlock probe comprises
a label sequence; i.e. a sequence that can be used to bind label
probes and is substantially complementary to a label probe. In one
embodiment, it is possible to use the same label sequence and label
probe for all padlock probes on an array; alternatively, each
padlock probe can have a different label sequence.
[0119] The padlock probe also contains a priming site for priming
the RCA reaction. That is, each padlock probe comprises a sequence
to which a primer nucleic acid hybridizes forming a template for
the polymerase. The primer can be found in any portion of the
circular probe. In a preferred embodiment, the primer is located at
a discrete site in the probe. In this embodiment, the primer site
in each distinct padlock probe is identical, although this is not
required. Advantages of using primer sites with identical sequences
include the ability to use only a single primer oligonucleotide to
prime the RCA assay with a plurality of different hybridization
complexes. That is, the padlock probe hybridizes uniquely to the
target nucleic acid to which it is designed. A single primer
hybridizes to all of the unique hybridization complexes forming a
priming site for the polymerase. RCA then proceeds from an
identical locus within each unique padlock probe of the
hybridization complexes.
[0120] In an alternative embodiment, the primer site can overlap,
encompass, or reside within any of the above-described elements of
the padlock probe. That is, the primer can be found, for example,
overlapping or within the restriction site or the identifier
sequence. In this embodiment, it is necessary that the primer
nucleic acid is designed to base pair with the chosen primer
site.
[0121] Thus, the padlock probe of the invention contains at each
terminus, sequences corresponding to OLA primers. The intervening
sequence of the padlock probe contain in no particular order, an
adapter sequence and a restriction endonuclease site. In addition,
the padlock probe contains a RCA priming site.
[0122] Thus, in a preferred embodiment the OLA/RCA is performed in
solution followed by restriction endonuclease cleavage of the RCA
product. The cleaved product is then applied to an array comprising
beads, each bead comprising a probe complementary to the adapter
sequence located in the padlock probe. The amplified adapter
sequence correlates with a particular target nucleic acid. Thus the
incorporation of an endonuclease site allows the generation of
short, easily hybridizable sequences. Furthermore, the unique
adapter sequence in each rolling circle padlock probe sequence
allows diverse sets of nucleic acid sequences to be analyzed in
parallel on an array, since each sequence is resolved on the basis
of hybridization specificity.
[0123] In an alternative OLA/RCA method, one of the OLA primers is
immobilized on the microsphere; the second primer is added in
solution. Both primers hybridize with the target nucleic acid
forming a hybridization complex as described above for the OLA
assay.
[0124] As described herein, the microsphere is distributed on an
array. In a preferred embodiment, a plurality of microspheres each
with a unique OLA primer is distributed on the array.
[0125] Following the OLA assay, and either before, after or
concurrently with distribution of the beads on the array, a segment
of circular DNA is hybridized to the bead-based ligated
oligonucleotide forming a modified hybridization complex. Addition
of an appropriate polymerase (3' exo.sup.-), as is known in the
art, and corresponding reaction buffer to the array leads to
amplification of the circular DNA. Since there is no terminus to
the circular DNA, the polymerase continues to travel around the
circular template generating extension product until it detaches
from the template. Thus, a polymerase with high processivity can
create several hundred or thousand copies of the circular template
with all the copies linked in one contiguous strand.
[0126] Again, these copies are subsequently detected by one of two
methods; either hybridizing a labeled oligo complementary to the
circular target or via the incorporation of labeled nucleotides in
the amplification reaction. The label is detected using
conventional label detection methods as described herein.
[0127] In one embodiment, when the circular DNA contains sequences
complementary to the ligated oligonucleotide it is preferable to
remove the target DNA prior to contacting the ligated
oligonucleotide with the circular DNA (See FIG. 7). This is done by
denaturing the double-stranded DNA by methods known in the art. In
an alternative embodiment, the double stranded DNA is not denatured
prior to contacting the circular DNA.
[0128] In an alternative embodiment, when the circular DNA contains
sequences complementary to the target nucleic acid, it is
preferable that the circular DNA is complementary at a site
distinct from the site bound to the ligated oligonucleotide. In
this embodiment it is preferred that the duplex between the ligated
oligonucleotide and target nucleic acid is not denatured or
disrupted prior to the addition of the circular DNA so that the
target DNA remains immobilized to the bead.
[0129] Hybridization and washing conditions are well known in the
art; various degrees of stringency can be used. In some embodiments
it is not necessary to use stringent hybridization or washing
conditions as only microspheres containing the ligated probes will
effectively hybridize with the circular DNA; microspheres bound to
DNA that did not undergo ligation (those without the appropriate
target nucleic acid) will not hybridize as strongly with the
circular DNA as those primers that were ligated. Thus,
hybridization and/or washing conditions are used that discriminate
between binding of the circular DNA to the ligated primer and the
unligated primer.
[0130] Alternatively, when the circular probe is designed to
hybridize to the target nucleic acid at a site distinct from the
site bound to the ligated oligonucleotide, hybridization and
washing conditions are used to remove or dissociate the target
nucleic acid from unligated oligonucleotides while target nucleic
acid hybridizing with the ligated oligonucleotides will remain
bound to the beads. In this embodiment, the circular probe only
hybridizes to the target nucleic acid when the target nucleic acid
is hybridized with a ligated oligonucleotide that is immobilized on
a bead.
[0131] As is well known in the art, an appropriate polymerase (3'
exo.sup.-) is added to the array. The polymerase extends the
sequence of a single-stranded DNA using double-stranded DNA as a
primer site. In one embodiment, the circular DNA that has
hybridized with the appropriate OLA reaction product serves as the
primer for the polymerase. In the presence of an appropriate
reaction buffer as is known in the art, the polymerase will extend
the sequence of the primer using the single-stranded circular DNA
as a template. As there is no terminus of the circular DNA, the
polymerase will continue to extend the sequence of the circular
DNA. In an alternative embodiment, the RCA probe comprises a
discrete primer site located within the circular probe.
Hybridization of primer nucleic acids to this primer site forms the
polymerase template allowing RCA to proceed.
[0132] In a preferred embodiment, the polymerase creates more than
100 copies of the circular DNA. In more preferred embodiments the
polymerase creates more than 1000 copies of the circular DNA; while
in a most preferred embodiment the polymerase creates more than
10,000 copies or more than 50,000 copies of the template.
[0133] The amplified circular DNA sequence is then detected by
methods known in the art and as described herein. Detection is
accomplished by hybridizing with a labeled probe. The probe is
labeled directly or indirectly. Alternatively, labeled nucleotides
are incorporated into the amplified circular DNA product. The
nucleotides can be labeled directly, or indirectly as is further
described herein.
[0134] The RCA as described herein finds use in allowing highly
specific and highly sensitive detection of nucleic acid target
sequences. In particular, the method finds use in improving the
multiplexing ability of DNA arrays and eliminating costly sample or
target preparation. As an example, a substantial savings in cost
can be realized by directly analyzing genomic DNA on an array,
rather than employing an intermediate PCR amplification step. The
method finds use in examining genomic DNA and other samples
including mRNA.
[0135] In addition the RCA finds use in allowing rolling circle
amplification products to be easily detected by hybridization to
probes in a solid-phase format (e.g. an array of beads). An
additional advantage of the RCA is that it provides the capability
of multiplex analysis so that large numbers of sequences can be
analyzed in parallel. By combining the sensitivity of RCA and
parallel detection on arrays, many sequences can be analyzed
directly from genomic DNA.
[0136] Chemical Ligation Techniques
[0137] A variation of LCR utilizes a "chemical ligation" of sorts,
as is generally outlined in U.S. Pat. Nos. 5,616,464 and 5,767,259,
both of which are hereby expressly incorporated by reference in
their entirety. In this embodiment, similar to enzymatic ligation,
a pair of primers are utilized, wherein the first primer is
substantially complementary to a first domain of the target and the
second primer is substantially complementary to an adjacent second
domain of the target (although, as for enzymatic ligation, if a
"gap" exists, a polymerase and dNTPs may be added to "fill in" the
gap). Each primer has a portion that acts as a "side chain" that
does not bind the target sequence and acts as one half of a stem
structure that interacts non-covalently through hydrogen bonding,
salt bridges, van der Waal's forces, etc. Preferred embodiments
utilize substantially complementary nucleic acids as the side
chains. Thus, upon hybridization of the primers to the target
sequence, the side chains of the primers are brought into spatial
proximity, and, if the side chains comprise nucleic acids as well,
can also form side chain hybridization complexes.
[0138] At least one of the side chains of the primers comprises an
activatable cross-linking agent, generally covalently attached to
the side chain, that upon activation, results in a chemical
cross-link or chemical ligation. The activatible group may comprise
any moiety that will allow cross-linking of the side chains, and
include groups activated chemically, photonically and thermally,
with photoactivatable groups being preferred. In some embodiments a
single activatable group on one of the side chains is enough to
result in cross-linking via interaction to a functional group on
the other side chain; in alternate embodiments, activatable groups
are required on each side chain.
[0139] Once the hybridization complex is formed, and the
cross-linking agent has been activated such that the primers have
been covalently attached, the reaction is subjected to conditions
to allow for the disassocation of the hybridization complex, thus
freeing up the target to serve as a template for the next ligation
or cross-linking. In this way, signal amplification occurs, and can
be detected as outlined herein.
[0140] Invasive Cleavage Techniques
[0141] In a preferred embodiment, the signal amplification
technique is invasive cleavage technology, which is described in a
number of patents and patent applications, including U.S. Pat. Nos.
5,846,717; 5,614,402; 5,719,028; 5,541,311; and 5,843,669, all of
which are hereby incorporated by reference in their entirety.
[0142] Generally, invasive cleavage technology may be described as
follows. A target nucleic acid is recognized by two distinct
probes. A first probe, generally referred to herein as an "invader"
probe, is substantially complementary to a first portion of the
target nucleic acid. A second probe, generally referred to herein
as a "signal probe", is partially complementary to the target
nucleic acid; the 3' end of the signal oligonucleotide is
substantially complementary to the target sequence while the 5' end
is non-complementary and preferably forms a single-stranded "tail"
or "arm". The non-complementary end of the second probe preferably
comprises a "generic" or "unique" sequence, frequently referred to
herein as a "detection sequence", that is used to indicate the
presence or absence of the target nucleic acid, as described below.
The detection sequence of the second probe preferably comprises at
least one detectable label, although as outlined herein, since this
detection sequence can function as a target sequence for a capture
probe, sandwich configurations utilizing label probes as described
herein may also be done.
[0143] Hybridization of the first and second oligonucleotides near
or adjacent to one another on the target nucleic acid forms a
number of structures. In a preferred embodiment, a forked cleavage
structure forms and is a substrate of a nuclease which cleaves the
detection sequence from the signal oligonucleotide. The site of
cleavage is controlled by the distance or overlap between the 3'
end of the invader oligonucleotide and the downstream fork of the
signal oligonucleotide. Therefore, neither oligonucleotide is
subject to cleavage when misaligned or when unattached to target
nucleic acid.
[0144] In a preferred embodiment, the nuclease that recognizes the
forked cleavage structure and catalyzes release of the tail is
thermostable, thereby, allowing thermal cycling of the cleavage
reaction, if desired. Preferred nucleases derived from thermostable
DNA polymerases that have been modified to have reduced synthetic
activity which is an undesirable side-reaction during cleavage are
disclosed in U.S. Pat. Nos. 5,719,028 and 5,843,669, hereby
expressly by reference. The synthetic activity of the DNA
polymerase is reduced to a level where it does not interfere with
detection of the cleavage reaction and detection of the freed tail.
Preferably the DNA polymerase has no detectable polymerase
activity. Examples of nucleases are those derived from Thermus
aquaticus, Thermus flavus, or Thermus thermophilus.
[0145] In another embodiment, thermostable structure-specific
nucleases are Flap endonucleases (FENs) selected from FEN-1 or
FEN-2 like (e.g. XPG and RAD2 nucleases) from Archaebacterial
species, for example, FEN-1 from Methanococcus jannaschii,
Pyrococcus furiosis, Pyrococcus woesei, and Archaeoglobus fulgidus.
(U.S. Pat. No. 5,843,669 and Lyamichev et al. 1999. Nature
Biotechnology 17:292-297; both of which are hereby expressly by
reference).
[0146] In a preferred embodiment, the nuclease is AfuFEN1 or
PfuFEN1 nuclease. To cleave a forked structure, these nucleases
require at least one overlapping nucleotide between the signal and
invasive probes to recognize and cleave the 5' end of the signal
probe. To effect cleavage the 3'-terminal nucleotide of the invader
oligonucleotide is not required to be complementary to the target
nucleic acid. In contast, mismatch of the signal probe one base
upstream of the cleavage site prevents creation of the overlap and
cleavage. The specificity of the nuclease reaction allows single
nucleotide polymorphism (SNP) detection from, for example, genomic
DNA, as outlined below (Lyamichev et al.).
[0147] In a preferred embodiment invasive cleavage technology is
used. Invasive cleavage technology is based on structure-specific
nucleases that cleave nucleic acids in a site-specific manner. Two
probes are used: an "invader" probe and a "signalling" probe, that
adjacently hybridize to a target sequence with overlap. For
mismatch discrimination, the invader technology relies on
complementarity at the overlap position where cleavage occurs. The
enzyme cleaves at the overlap, and releases the "tail" which may or
may not be labeled. This can then be detected. The Invader.TM.
technology is described in U.S. Pat. Nos. 5,846,717; 5,614,402;
5,719,028; 5,541,311; and 5,843,669, all of which are hereby
incorporated by reference.
[0148] The invasive cleavage assay is preferably performed on an
array format. In a preferred embodiment, the signal probe has a
detectable label, attached 5' from the site of nuclease cleavage
(e.g. within the detection sequence) and a capture tag, as
described below (e.g. biotin or other hapten) 3' from the site of
nuclease cleavage. After the assay is carried out, the 3' portion
of the cleaved signal probe (e.g. the the detection sequence) are
extracted, for example, by binding to streptavidin beads or by
crosslinking through the capture tag to produce aggregates or by
antibody to an attached hapten. By "capture tag" herein is a meant
one of a pair of binding partners as described above, such as
antigen/antibody pairs, digoxygenenin, dinitrophenol, etc.
[0149] The cleaved 5' region, e.g. the detection sequence, of the
signal probe, comprises a label and is detected and optionally
quantitated. In one embodiment, the cleaved 5' region is hybridized
to a probe on an array (capture probe) and optically detected. As
described below, many signal probes can be analyzed in parallel by
hybridization to their complementary probes in an array.
[0150] In a preferred embodiment, the invasive cleavage reaction is
configured to utilize a fluorophore-quencher reaction. A signalling
probe comprising both a fluorophore and a quencher is used, with
the fluorophore and the quencher on opposite sides of the cleavage
site. As will be appreciated by those in the art, these will be
positioned closely together. Thus, in the absence of cleavage, very
little signal is seen due to the quenching reaction. After
cleavage, however, the distance between the two is large, and thus
fluorescence can be detected. Upon assembly of an assay complex,
comprising the target sequence, an invader probe, and a signalling
probe, and the introduction of the cleavage enzyme, the cleavage of
the complex results in the disassociation of the quencher from the
complex, resulting in an increase in fluorescence.
[0151] In this embodiment, suitable fluorophore-quencher pairs are
as known in the art. For example, suitable quencher molecules
comprise Dabcyl.
[0152] As will be appreciated by those in the art, this system can
be configured in a variety of conformations, as discussed in FIG.
4.
[0153] In a preferred embodiment, to obtain higher specificity and
reduce the detection of contaminating uncleaved signal probe or
incorrectly cleaved product, an additional enzymatic recognition
step is introduced in the array capture procedure. For example, the
cleaved signal probe binds to a capture probe to produce a
double-stranded nucleic acid in the array. In this embodiment, the
3' end of the cleaved signal probe is adjacent to the 5' end of one
strand of the capture probe, thereby, forming a substrate for DNA
ligase (Broude et al. 1991. PNAS 91: 3072-3076). Only correctly
cleaved product is ligated to the capture probe. Other incorrectly
hybridized and non-cleaved signal probes are removed, for example,
by heat denaturation, high stringency washes, and other methods
that disrupt base pairing.
[0154] Cycling Probe Techniques (CPT)
[0155] In a preferred embodiment, the signal amplification
technique is CPT. CPT technology is described in a number of
patents and patent applications, including U.S. Pat. Nos.
5,011,769, 5,403,711, 5,660,988, and 4,876,187, and PCT published
applications WO 95/05480, WO 95/1416, and WO 95/00667, and U.S.
Ser. No. 09/014,304, all of which are expressly incorporated by
reference in their entirety.
[0156] Generally, CPT may be described as follows. A CPT primer
(also sometimes referred to herein as a "scissile primer"),
comprises two probe sequences separated by a scissile linkage. The
CPT primer is substantially complementary to the target sequence
and thus will hybridize to it to form a hybridization complex. The
scissile linkage is cleaved, without cleaving the target sequence,
resulting in the two probe sequences being separated. The two probe
sequences can thus be more easily disassociated from the target,
and the reaction can be repeated any number of times. The cleaved
primer is then detected as outlined herein.
[0157] By "scissile linkage" herein is meant a linkage within the
scissile probe that can be cleaved when the probe is part of a
hybridization complex, that is, when a double-stranded complex is
formed. It is important that the scissile linkage cleave only the
scissile probe and not the sequence to which it is hybridized (i.e.
either the target sequence or a probe sequence), such that the
target sequence may be reused in the reaction for amplification of
the signal. As used herein, the scissile linkage, is any connecting
chemical structure which joins two probe sequences and which is
capable of being selectively cleaved without cleavage of either the
probe sequences or the sequence to which the scissile probe is
hybridized. The scissile linkage may be a single bond, or a
multiple unit sequence. As will be appreciated by those in the art,
a number of possible scissile linkages may be used.
[0158] In a preferred embodiment, the scissile linkage comprises
RNA. This system, previously described in as outlined above, is
based on the fact that certain double-stranded nucleases,
particularly ribonucleases, will nick or excise RNA nucleosides
from a RNA:DNA hybridization complex. Of particular use in this
embodiment is RNAseH, Exo III, and reverse transcriptase.
[0159] In one embodiment, the entire scissile probe is made of RNA,
the nicking is facilitated especially when carried out with a
double-stranded ribonuclease, such as RNAseH or Exo III. RNA probes
made entirely of RNA sequences are particularly useful because
first, they can be more easily produced enzymatically, and second,
they have more cleavage sites which are accessible to nicking or
cleaving by a nicking agent, such as the ribonucleases. Thus,
scissile probes made entirely of RNA do not rely on a scissile
linkage since the scissile linkage is inherent in the probe.
[0160] In a preferred embodiment, when the scissile linkage is a
nucleic acid such as RNA, the methods of the invention may be used
to detect mismatches, as is generally described in U.S. Pat. Nos.
5,660,988, and WO 95/14106, hereby expressly incorporated by
reference. These mismatch detection methods are based on the fact
that RNAseH may not bind to and/or cleave an RNA:DNA duplex if
there are mismatches present in the sequence. Thus, in the
NA.sub.1-R-NA.sub.2 embodiments, NA.sub.1 and NA.sub.2 are non-RNA
nucleic acids, preferably DNA. Preferably, the mismatch is within
the RNA:DNA duplex, but in some embodiments the mismatch is present
in an adjacent sequence very close to the desired sequence, close
enough to affect the RNAseH (generally within one or two bases).
Thus, in this embodiment, the nucleic acid scissile linkage is
designed such that the sequence of the scissile linkage reflects
the particular sequence to be detected, i.e. the area of the
putative mismatch.
[0161] In some embodiments of mismatch detection, the rate of
generation of the released fragments is such that the methods
provide, essentially, a yes/no result, whereby the detection of
virtually any released fragment indicates the presence of the
desired target sequence. Typically, however, when there is only a
minimal mismatch (for example, a 1-, 2- or 3-base mismatch, or a
3-base deletion), there is some generation of cleaved sequences
even though the target sequence is not present. Thus, the rate of
generation of cleaved fragments, and/or the final amount of cleaved
fragments, is quantified to indicate the presence or absence of the
target. In addition, the use of secondary and tertiary scissile
probes may be particularly useful in this embodiment, as this can
amplify the differences between a perfect match and a mismatch.
These methods may be particularly useful in the determination of
homozygotic or heterozygotic states of a patient.
[0162] In this embodiment, it is an important feature of the
scissile linkage that its length is determined by the suspected
difference between the target and the probe. In particular, this
means that the scissile linkage must be of sufficient length to
encompass the suspected difference, yet short enough so that the
scissile linkage cannot inappropriately "specifically hybridize" to
the selected nucleic acid molecule when the suspected difference is
present; such inappropriate hybridization would permit excision and
thus cleavage of scissile linkages even though the selected nucleic
acid molecule was not fully complementary to the nucleic acid
probe. Thus in a preferred embodiment, the scissile linkage is
between 3 to 5 nucleotides in length, such that a suspected
nucleotide difference from 1 nucleotide to 3 nucleotides is
encompassed by the scissile linkage, and 0, 1 or 2 nucleotides are
on either side of the difference.
[0163] Thus, when the scissile linkage is nucleic acid, preferred
embodiments utilize from 1 to about 100 nucleotides, with from
about 2 to about 20 being preferred and from about 5 to about 10
being particularly preferred.
[0164] CPT may be done enzymatically or chemically. That is, in
addition to RNAseH, there are several other cleaving agents which
may be useful in cleaving RNA (or other nucleic acid) scissile
bonds. For example, several chemical nucleases have been reported;
see for example Sigman et al., Annu. Rev. Biochem. 1990, 59,
207-236; Sigman et al., Chem. Rev. 1993, 93, 2295-2316; Bashkin et
al., J. Org. Chem. 1990, 55, 5125-5132; and Sigman et al., Nucleic
Acids and Molecular Biology, vol. 3, F. Eckstein and D. M. J.
Lilley (Eds), Springer-Verlag, Heidelberg 1989, pp. 13-27; all of
which are hereby expressly incorporated by reference.
[0165] Specific RNA hydrolysis is also an active area; see for
example Chin, Acc. Chem. Res. 1991, 24, 145-152; Breslow et al.,
Tetrahedron, 1991, 47, 2365-2376; Anslyn et al., Angew. Chem. Int.
Ed. Engl., 1997, 36, 432-450; and references therein, all of which
are expressly incorporated by reference. Reactive phosphate centers
are also of interest in developing scissile linkages, see Hendry et
al., Prog. Inorg. Chem.: Bioinorganic Chem. 1990, 31, 201-258 also
expressly incorporated by reference.
[0166] Current approaches to site-directed RNA hydrolysis include
the conjugation of a reactive moiety capable of cleaving
phosphodiester bonds to a recognition element capable of
sequence-specifically hybridizing to RNA. In most cases, a metal
complex is covalently attached to a DNA strand which forms a stable
heteroduplex. Upon hybridization, a Lewis acid is placed in close
proximity to the RNA backbone to effect hydrolysis; see Magda et
al., J. Am. Chem. Soc. 1994, 116, 7439; Hall et al., Chem. Biology
1994, 1, 185-190; Bashkin et al., J. Am. Chem. Soc. 1994, 116,
5981-5982; Hall et al., Nucleic Acids Res. 1996, 24, 3522; Magda et
al., J. Am. Chem. Soc. 1997, 119, 2293; and Magda et al., J. Am.
Chem. Soc. 1997, 119, 6947, all of which are expressly incorporated
by reference.
[0167] In a similar fashion, DNA-polyamine conjugates have been
demonstrated to induce site-directed RNA strand scission; see for
example, Yoshinari et al., J. Am. Chem. Soc. 1991, 113, 5899-5901;
Endo et al., J. Org. Chem. 1997, 62, 846; and Barbier et al., J.
Am. Chem. Soc. 1992, 114, 3511-3515, all of which are expressly
incorporated by reference.
[0168] In a preferred embodiment, the scissile linkage is not
necessarily RNA. For example, chemical cleavage moieties may be
used to cleave basic sites in nucleic acids; see Belmont, et
al.,New J. Chem. 1997, 21, 47-54; and references therein, all of
which are expressly incorporated herein by reference. Similarly,
photocleavable moieties, for example, using transition metals, may
be used; see Moucheron, et al., Inorg. Chem. 1997, 36, 584-592,
hereby expressly by reference.
[0169] Other approaches rely on chemical moieties or enzymes; see
for example Keck et al., Biochemistry 1995, 34, 12029-12037; Kirk
et al., Chem. Commun. 1998, in press; cleavage of G-U basepairs by
metal complexes; see Biochemistry, 1992, 31, 5423-5429; diamine
complexes for cleavage of RNA; Komiyama, et al., J. Org. Chem.
1997, 62, 2155-2160; and Chow et al., Chem. Rev. 1997, 97,
1489-1513, and references therein, all of which are expressly
incorporated herein by reference.
[0170] The first step of the CPT method requires hybridizing a
primary scissile primer (also called a primary scissile probe) to
the target. This is preferably done at a temperature that allows
both the binding of the longer primary probe and disassociation of
the shorter cleaved portions of the primary probe, as will be
appreciated by those in the art. As outlined herein, this may be
done in solution, or either the target or one or more of the
scissile probes may be attached to a solid support. For example, it
is possible to utilize "anchor probes" on a solid support which are
substantially complementary to a portion of the target sequence,
preferably a sequence that is not the same sequence to which a
scissile probe will bind.
[0171] Similarly, as outlined herein, a preferred embodiment has
one or more of the scissile probes attached to a solid support such
as a bead. In this embodiment, the soluble target diffuses to allow
the formation of the hybridization complex between the soluble
target sequence and the support-bound scissile probe. In this
embodiment, it may be desirable to include additional scissile
linkages in the scissile probes to allow the release of two or more
probe sequences, such that more than one probe sequence per
scissile probe may be detected, as is outlined below, in the
interests of maximizing the signal.
[0172] In this embodiment (and in other amplification techniques
herein), preferred methods utilize cutting or shearing techniques
to cut the nucleic acid sample containing the target sequence into
a size that will allow sufficient diffusion of the target sequence
to the surface of a bead. This may be accomplished by shearing the
nucleic acid through mechanical forces (e.g. sonication) or by
cleaving the nucleic acid using restriction endonucleases.
Alternatively, a fragment containing the target may be generated
using polymerase, primers and the sample as a template, as in
polymerase chain reaction (PCR). In addition, amplification of the
target using PCR or LCR or related methods may also be done; this
may be particularly useful when the target sequence is present in
the sample at extremely low copy numbers. Similarly, numerous
techniques are known in the art to increase the rate of mixing and
hybridization including agitation, heating, techniques that
increase the overall concentration such as precipitation, drying,
dialysis, centrifugation, electrophoresis, magnetic bead
concentration, etc.
[0173] In general, the scissile probes are introduced in a molar
excess to their targets (including both the target sequence or
other scissile probes, for example when secondary or tertiary
scissile probes are used), with ratios of scissile probe:target of
at least about 100:1 being preferred, at least about 1000:1 being
particularly preferred, and at least about 10,000:1 being
especially preferred. In some embodiments the excess of
probe:target will be much greater. In addition, ratios such as
these may be used for all the amplification techniques outlined
herein.
[0174] Once the hybridization complex between the primary scissile
probe and the target has been formed, the complex is subjected to
cleavage conditions. As will be appreciated, this depends on the
composition of the scissile probe; if it is RNA, RNAseH is
introduced. It should be noted that under certain circumstances,
such as is generally outlined in WO 95/00666 and WO 95/00667,
hereby incorporated by reference, the use of a double-stranded
binding agent such as RNAseH may allow the reaction to proceed even
at temperatures above the Tm of the primary probe:target
hybridization complex. Accordingly, the addition of scissile probe
to the target can be done either first, and then the cleavage agent
or cleavage conditions introduced, or the probes may be added in
the presence of the cleavage agent or conditions.
[0175] The cleavage conditions result in the separation of the two
(or more) probe sequences of the primary scissile probe. As a
result, the shorter probe sequences will no longer remain
hybridized to the target sequence, and thus the hybridization
complex will disassociate, leaving the target sequence intact.
[0176] The optimal temperature for carrying out the CPT reactions
is generally from about 5.degree. C. to about 25.degree. C. below
the melting temperatures of the probe:target hybridization complex.
This provides for a rapid rate of hybridization and high degree of
specificity for the target sequence. The Tm of any particular
hybridization complex depends on salt concentration, G-C content,
and length of the complex, as is known in the art and described
herein.
[0177] During the reaction, as for the other amplification
techniques herein, it may be necessary to suppress cleavage of the
probe, as well as the target sequence, by nonspecific nucleases.
Such nucleases are generally removed from the sample during the
isolation of the DNA by heating or extraction procedures. A number
of inhibitors of single-stranded nucleases such as vanadate,
inhibitors it-ACE and RNAsin, a placental protein, do not affect
the activity of RNAseH. This may not be necessary depending on the
purity of the RNAseH and/or the target sample.
[0178] These steps are repeated by allowing the reaction to proceed
for a period of time. The reaction is usually carried out for about
15 minutes to about 1 hour. Generally, each molecule of the target
sequence will turnover between 100 and 1000 times in this period,
depending on the length and sequence of the probe, the specific
reaction conditions, and the cleavage method. For example, for each
copy of the target sequence present in the test sample 100 to 1000
molecules will be cleaved by RNAseH. Higher levels of amplification
can be obtained by allowing the reaction to proceed longer, or
using secondary, tertiary, or quaternary probes, as is outlined
herein.
[0179] Upon completion of the reaction, generally determined by
time or amount of cleavage, the uncleaved scissile probes must be
removed or neutralized prior to detection, such that the uncleaved
probe does not bind to a detection probe, causing false positive
signals. This may be done in a variety of ways, as is generally
described below.
[0180] In a preferred embodiment, the separation is facilitated by
the use of beads containing the primary probe. Thus, when the
scissile probes are attached to beads, removal of the beads by
filtration, centrifugation, the application of a magnetic field,
electrostatic interactions for charged beads, adhesion, etc.,
results in the removal of the uncleaved probes.
[0181] In a preferred embodiment, the separation is based on strong
acid precipitation. This is useful to separate long (generally
greater than 50 nucleotides) from smaller fragments (generally
about 10 nucleotides). The introduction of a strong acid such as
trichloroacetic acid into the solution causes the longer probe to
precipitate, while the smaller cleaved fragments remain in
solution. The solution can be centrifuged or filtered to remove the
precipitate, and the cleaved probe sequences can be
quantitated.
[0182] In a preferred embodiment, the scissile probe contains both
a detectable label and an affinity binding ligand or moiety, such
that an affinity support is used to carry out the separation. In
this embodiment, it is important that the detectable label used for
detection is not on the same probe sequence that contains the
affinity moiety, such that removal of the uncleaved probe, and the
cleaved probe containing the affinity moiety, does not remove all
the detectable labels. Alternatively, the scissile probe may
contain a capture tag; the binding partner of the capture tag is
attached to a solid support such as glass beads, latex beads,
dextrans, etc. and used to pull out the uncleaved probes, as is
known in the art. The cleaved probe sequences, which do not contain
the capture tag, remain in solution and then can be detected as
outlined below.
[0183] In a preferred embodiment, similar to the above embodiment,
a separation sequence of nucleic acid is included in the scissile
probe, which is not cleaved during the reaction. A nucleic acid
complementary to the separation sequence is attached to a solid
support such as a bead and serves as a catcher sequence.
Preferably, the separation sequence is added to the scissile
probes, and is not recognized by the target sequence, such that a
generalized catcher sequence may be utilized in a variety of
assays.
[0184] After removal of the uncleaved probe, as required, detection
proceeds via the addition of the cleaved probe sequences to the
array compositions, as outlined below. In general, the cleaved
probe is bound to a capture probe, either directly or indirectly,
and the label is detected. In a preferred embodiment, no higher
order probes are used, and detection is based on the probe
sequence(s) of the primary primer. In a preferred embodiment, at
least one, and preferably more, secondary probes (also referred to
herein as secondary primers) are used; the secondary probes
hybridize to the domains of the cleavage probes; etc.
[0185] Thus, CPT requires, again in no particular order, a first
CPT primer comprising a first probe sequence, a scissile linkage
and a second probe sequence; and a cleavage agent.
[0186] In this manner, CPT results in the generation of a large
amount of cleaved primers, which then can be detected as outlined
below.
[0187] Labeling Techniques
[0188] In general, either direct or indirect detection of the
target products can be done. "Direct" detection as used in this
context, as for the other amplification strategies outlined herein,
requires the incorporation of a label, in this case a detectable
label, preferably an optical label such as a fluorophore, into the
target sequence, with detection proceeding as outlined below. In
this embodiment, the label(s) may be incorporated in three ways:
(1) the primers comprise the label(s), for example attached to the
base, a ribose, a phosphate, or to analogous structures in a
nucleic acid analog; (2) modified nucleosides are used that are
modified at either the base or the ribose (or to analogous
structures in a nucleic acid analog) with the label(s); these
label-modified nucleosides are then converted to the triphosphate
form and are incorporated into the newly synthesized strand by a
polymerase; (3) modified nucleotides are used that comprise a
functional group that can be used to add a detectable label; or (4)
modified primers are used that comprise a functional group that can
be used to add a detectable label. Any of these methods result in a
newly synthesized strand that comprises labels, that can be
directly detected as outlined below.
[0189] Thus, the modified strands comprise a detection label. By
"detection label" or "detectable label" herein is meant a moiety
that allows detection. This may be a primary label or a secondary
label.
[0190] In a preferred embodiment, the detection label is a primary
label. A primary label is one that can be directly detected, such
as a fluorophore. In general, labels fall into three classes: a)
isotopic labels, which may be radioactive or heavy isotopes; b)
magnetic, electrical, thermal labels; and c) colored or luminescent
dyes. Labels can also include enzymes (horseradish peroxidase,
etc.) and magnetic particles. Preferred labels include chromophores
or phosphors but are preferably fluorescent dyes. Suitable dyes for
use in the invention 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, alexa dyes,
phycoerythin, bodipy, and others described in the 6th Edition of
the Molecular Probes Handbook by Richard P. Haugland, hereby
expressly incorporated by reference.
[0191] In a preferred embodiment, a secondary detectable label is
used. Accordingly, detection labels may be primary labels (i.e.
directly detectable) or secondary labels (indirectly detectable). A
secondary label is one that is indirectly detected; for example, a
secondary label can bind or react with a primary label for
detection, or may allow the separation of the compound comprising
the secondary label from unlabeled materials, etc. Secondary labels
find particular use in systems requiring separation of labeled and
unlabeled probes, such as SBE reactions. Secondary labels include,
but are not limited to, one of a binding partner pair; chemically
modifiable moieties; nuclease inhibitors, etc.
[0192] In a preferred embodiment, the secondary label is a binding
partner pair. For example, the label may be a hapten or antigen,
which will bind its binding partner. In a preferred embodiment, the
binding partner can be attached to a solid support to allow
separation of extended and non-extended primers. For example,
suitable binding partner pairs include, but are not limited to:
antigens (such as proteins (including peptides)) and antibodies
(including fragments thereof (FAbs, etc.)); proteins and small
molecules, including biotin/streptavidin; enzymes and substrates or
inhibitors; other protein-protein interacting pairs;
receptor-ligands; and carbohydrates and their binding partners.
Nucleic acid-nucleic acid binding proteins pairs are also useful.
In general, the smaller of the pair is attached to the NTP for
incorporation into the extension primer.
[0193] In a preferred embodiment, the binding partner pair
comprises biotin or imino-biotin and streptavidin. Imino-biotin is
particularly preferred as imino-biotin disassociates from
streptavidin in pH 4.0 buffer while biotin requires harsh
denaturants (e.g. 6 M guanidinium HCl, pH 1.5 or 90% formamide at
95.degree. C.).
[0194] In a preferred embodiment, the binding partner pair
comprises a primary detection label (for example, attached to the
NTP and therefore to the extended primer) and an antibody that will
specifically bind to the primary detection label. By "specifically
bind" herein is meant that the partners bind with specificity
sufficient to differentiate between the pair and other components
or contaminants of the system. The binding should be sufficient to
remain bound under the conditions of the assay, including wash
steps to remove non-specific binding. In some embodiments, the
dissociation constants of the pair 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.
[0195] For removal of unextended primers, it is preferred that the
other half of the binding pair is attached to a solid support. In
this embodiment, the solid support may be any as described herein
for substrates and microspheres, and the form is preferably
microspheres as well; for example, a preferred embodiment utilizes
magnetic beads that can be easily introduced to the sample and
easily removed, although any affinity chromatography formats may be
used as well. Standard methods are used to attach the binding
partner to the solid support, and can include direct or indirect
attachment methods. For example, biotin labeled antibodies to
fluorophores can be attached to streptavidin coated magnetic
beads.
[0196] Thus, in this embodiment, the extended primers comprise a
binding partner that is contacted with its binding partner under
conditions wherein the extended primers are separated from the
unextended primers. These extended primers can then be added to the
array comprising capture probes as described herein.
[0197] In a preferred embodiment, the secondary label is a
chemically modifiable moiety. In this embodiment, labels comprising
reactive functional groups are incorporated into the nucleic acid.
The functional group can then be subsequently labeled with a
primary label. Suitable functional groups include, but are not
limited to, amino groups, carboxy groups, maleimide groups, oxo
groups and thiol groups, with amino groups and thiol groups being
particularly preferred.
[0198] Removal of Unextended Primers
[0199] In a preferred embodiment, it is desirable to remove the
unextended or unreacted primers from the assay mixture, and
particularly from the array, as unextended primers will compete
with the extended (labeled) primers in binding to capture probes,
thereby diminishing the signal. The concentration of the unextended
primers relative to the extended primer may be relatively high,
since a large excess of primer is usually required to generate
efficient primer annealing. Accordingly, a number of different
techniques may be used to facilitate the removal of unextended
primers. While the discussion below applies specifically to SBE,
these techniques may be used in any of the methods described
herein.
[0200] In a preferred embodiment, the NTPs (or, in the case of
other methods, one or more of the probes) comprise a secondary
detectable label that can be used to separate extended and
non-extended primers. As outlined above, detection labels may be
primary labels (i.e. directly detectable) or secondary labels
(indirectly detectable). A secondary label is one that is
indirectly detected; for example, a secondary label can bind or
react with a primary label for detection, or may allow the
separation of the compound comprising the secondary label from
unlabeled materials, etc. Secondary labels find particular use in
systems requiring separation of labeled and unlabeled probes, such
as SBE, OLA, invasive cleavage, etc. reactions; in addition, these
techniques may be used with many of the other techniques described
herein. Secondary labels include, but are not limited to, one of a
binding partner pair; chemically modifiable moieties; nuclease
inhibitors, etc.
[0201] In a preferred embodiment, the secondary label is a binding
partner pair. For example, the label may be a hapten or antigen,
Which will bind its binding partner (generally attached to a solid
support) and thus allow separation of extended and non-extended
primers. For example, suitable binding partner pairs include, but
are not limited to: antigens (such as proteins (including
peptides)) and antibodies (including fragments thereof (FAbs,
etc.)); proteins and small molecules, including biotin/streptavidin
and digoxygenin and antibodies; enzymes and substrates or
inhibitors; other protein-protein interacting pairs;
receptor-ligands; and carbohydrates and their binding partners, are
also suitable binding pairs. Nucleic acid-nucleic acid binding
proteins pairs are also useful. In general, the smaller of the pair
is attached to the NTP (or the probe) for incorporation into the
extension primer.
[0202] In a preferred embodiment, the binding partner pair
comprises biotin or imino-biotin and streptavidin. Imino-biotin is
particularly preferred when the methods require the later
separation of the pair, as imino-biotin disassociates from
streptavidin in pH 4.0 buffer while biotin requires harsh
denaturants (e.g. 6 M guanidinium HCl, pH 1.5 or 90% formamide at
95.degree. C.).
[0203] In addition, the use of streptavidin/biotin systems can be
used to separate unreacted and reacted probes (for example in SBE,
invasive cleavage, etc.). For example, the addition of streptavidin
to a nucleic acid greatly increases its size, as well as changes
its physical properties, to allow more efficient separation
techniques. For example, the mixtures can be size fractionated by
exclusion chromatography, affinity chromatography, filtration or
differential precipitation. Alternatively, an 3' exonuclease may be
added to a mixture of 3' labeled biotin/streptavidin; only the
unreacted oligonucleotides will be degraded. Following exonuclease
treatment, the exonuclease and the streptavidin can be degraded
using a protease such as proteinase K. The surviving nucleic acids
(i.e. those that were biotinylated) are then hybridized to the
array.
[0204] In a preferred embodiment, the binding partner pair
comprises a primary detection label (attached to the NTP and
therefore to the extended primer) and an antibody that will
specifically bind to the primary detection label. By "specifically
bind" herein is meant that the partners bind with specificity
sufficient to differentiate between the pair and other components
or contaminants of the system. The binding should be sufficient to
remain bound under the conditions of the assay, including wash
steps to remove non-specific binding. In some embodiments, the
dissociation constants of the pair 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.
[0205] In this embodiment, it is preferred that the other half of
the binding pair is attached to a solid support. In this
embodiment, the solid support may be any as described herein for
substrates and microspheres, and the form is preferably
microspheres as well; for example, a preferred embodiment utilizes
magnetic beads that can be easily introduced to the sample and
easily removed, although any affinity chromatography formats may be
used as well. Standard methods are used to attach the binding
partner to the solid support, and can include direct or indirect
attachment methods. For example, biotin labeled antibodies to
fluorophores can be attached to streptavidin coated magnetic
beads.
[0206] Thus, in this embodiment, the extended primers comprise a
binding member that is contacted with its binding partner under
conditions wherein the extended primers are separated from the
unextended primers. These extended primers can then be added to the
array comprising capture probes as described herein.
[0207] In a preferred embodiment, the secondary label is a
chemically modifiable moiety. In this embodiment, labels comprising
reactive functional groups are incorporated into the nucleic
acid.
[0208] In a preferred embodiment, the secondary label is a nuclease
inhibitor. In this embodiment, the chain-terminating NTPs are
chosen to render extended primers resistant to nucleases, such as
3'-exonucleases. Addition of an exonuclease will digest the
non-extended primers leaving only the extended primers to bind to
the capture probes on the array. This may also be done with OLA,
wherein the ligated probe will be protected but the unprotected
ligation probe will be digested.
[0209] In this embodiment, suitable 3'-exonucleases include, but
are not limited to, exo I, exo III, exo VII, etc.
[0210] Sandwich Assay Techniques
[0211] In a preferred embodiment, the signal amplification
technique is a "sandwich" assay, as is generally described in U.S.
Ser. No. 60/073,011 and in U.S. Pat. Nos. 5,681,702, 5,597,909,
5,545,730, 5,594,117, 5,591,584, 5,571,670, 5,580,731, 5,571,670,
5,591,584, 5,624,802, 5,635,352, 5,594,118, 5,359,100, 5,124,246
and 5,681,697, all of which are hereby incorporated by reference.
Although sandwich assays do not result in the alteration of
primers, sandwich assays can be considered signal amplification
techniques since multiple signals (i.e. label probes) are bound to
a single target, resulting in the amplification of the signal.
Sandwich assays may be used when the target sequence does not
contain a label; or when adapters are used, as outlined below.
[0212] As discussed herein, it should be noted that the sandwich
assays can be used for the detection of primary target sequences
(e.g. from a patient sample), or as a method to detect the product
of an amplification reaction as outlined above; thus for example,
any of the newly synthesized strands outlined above, for example
using PCR, LCR, NASBA, SDA, etc., may be used as the "target
sequence" in a sandwich assay.
[0213] As will be appreciated by those in the art, the systems of
the invention may take on a large number of different
configurations. In general, there are three types of systems that
can be used: (1) "non-sandwich" systems (also referred to herein as
"direct" detection) in which the target sequence itself is labeled
with detectable labels (again, either because the primers comprise
labels or due to the incorporation of labels into the newly
synthesized strand); (2) systems in which label probes directly
bind to the target sequences; and (3) systems in which label probes
are indirectly bound to the target sequences, for example through
the use of amplifier probes.
[0214] The anchoring of the target sequence to the bead is done
through the use of capture probes and optionally either capture
extender probes (sometimes referred to as "adapter sequences"
herein). When only capture probes are utilized, it is necessary to
have unique capture probes for each target sequence; that is, the
surface must be customized to contain unique capture probes; e.g.
each bead comprises a different capture probe. Alternatively,
capture extender probes may be used, that allow a "universal"
surface, i.e. a surface containing a single type of capture probe
that can be used to detect any target sequence. "Capture extender"
probes have a first portion that will hybridize to all or part of
the capture probe, and a second portion that will hybridize to a
first portion of the target sequence. This then allows the
generation of customized soluble probes, which as will be
appreciated by those in the art is generally simpler and less
costly. As shown herein, two capture extender probes may be used.
This has generally been done to stabilize assay complexes for
example when the target sequence is large, or when large amplifier
probes (particularly branched or dendrimer amplifier probes) are
used.
[0215] Detection of the amplification reactions of the invention,
including the direct detection of amplification products and
indirect detection utilizing label probes (i.e. sandwich assays),
is preferably done by detecting assay complexes comprising
detectable labels, which can be attached to the assay complex in a
variety of ways, as is more fully described below.
[0216] Once the target sequence has preferably been anchored to the
array, an amplifier probe is hybridized to the target sequence,
either directly, or through the use of one or more label extender
probes, which serves to allow "generic" amplifier probes to be
made. As for all the steps outlined herein, this may be done
simultaneously with capturing, or sequentially. Preferably, the
amplifier probe contains a multiplicity of amplification sequences,
although in some embodiments, as described below, the amplifier
probe may contain only a single amplification sequence, or at least
two amplification sequences. The amplifier probe may take on a
number of different forms; either a branched conformation, a
dendrimer conformation, or a linear "string" of amplification
sequences. Label probes comprising detectable labels (preferably
but not required to be fluorophores) then hybridize to the
amplification sequences (or in some cases the label probes
hybridize directly to the target sequence), and the labels
detected, as is more fully outlined below.
[0217] Accordingly, the present invention provides compositions
comprising an amplifier probe. By "amplifier probe" or "nucleic
acid multimer" or "amplification multimer" or grammatical
equivalents herein is meant a nucleic acid probe that is used to
facilitate signal amplification. Amplifier probes comprise at least
a first single-stranded nucleic acid probe sequence, as defined
below, and at least one single-stranded nucleic acid amplification
sequence, with a multiplicity of amplification sequences being
preferred.
[0218] Amplifier probes comprise a first probe sequence that is
used, either directly or indirectly, to hybridize to the target
sequence. That is, the amplifier probe itself may have a first
probe sequence that is substantially complementary to the target
sequence, or it has a first probe sequence that is substantially
complementary to a portion of an additional probe, in this case
called a label extender probe, that has a first portion that is
substantially complementary to the target sequence. In a preferred
embodiment, the first probe sequence of the amplifier probe is
substantially complementary to the target sequence.
[0219] In general, as for all the probes herein, the first probe
sequence is of a length sufficient to give specificity and
stability. Thus generally, the probe sequences of the invention
that are designed to hybridize to another nucleic acid (i.e. probe
sequences, amplification sequences, portions or domains of larger
probes) are at least about 5 nucleosides long, with at least about
10 being preferred and at least about 15 being especially
preferred.
[0220] In a preferred embodiment, several different amplifier
probes are used, each with first probe sequences that will
hybridize to a different portion of the target sequence. That is,
there is more than one level of amplification; the amplifier probe
provides an amplification of signal due to a multiplicity of
labelling events, and several different amplifier probes, each with
this multiplicity of labels, for each target sequence is used.
Thus, preferred embodiments utilize at least two different pools of
amplifier probes, each pool having a different probe sequence for
hybridization to different portions of the target sequence; the
only real limitation on the number of different amplifier probes
will be the length of the original target sequence. In addition, it
is also possible that the different amplifier probes contain
different amplification sequences, although this is generally not
preferred.
[0221] In a preferred embodiment, the amplifier probe does not
hybridize to the sample target sequence directly, but instead
hybridizes to a first portion of a label extender probe. This is
particularly useful to allow the use of "generic" amplifier probes,
that is, amplifier probes that can be used with a variety of
different targets. This may be desirable since several of the
amplifier probes require special synthesis techniques. Thus, the
addition of a relatively short probe as a label extender probe is
preferred. Thus, the first probe sequence of the amplifier probe is
substantially complementary to a first portion or domain of a first
label extender single-stranded nucleic acid probe. The label
extender probe also contains a second portion or domain that is
substantially complementary to a portion of the target sequence.
Both of these portions are preferably at least about 10 to about 50
nucleotides in length, with a range of about 15 to about 30 being
preferred. The terms "first" and "second" are not meant to confer
an orientation of the sequences with respect to the 5'-3'
orientation of the target or probe sequences. For example, assuming
a 5'-3' orientation of the complementary target sequence, the first
portion may be located either 5' to the second portion, or 3' to
the second portion. For convenience herein, the order of probe
sequences are generally shown from left to right.
[0222] In a preferred embodiment, more than one label extender
probe-amplifier probe pair may be used, that is, n is more than 1.
That is, a plurality of label extender probes may be used, each
with a portion that is substantially complementary to a different
portion of the target sequence; this can serve as another level of
amplification. Thus, a preferred embodiment utilizes pools of at
least two label extender probes, with the upper limit being set by
the length of the target sequence.
[0223] In a preferred embodiment, more than one label extender
probe is used with a single amplifier probe to reduce non-specific
binding, as is generally outlined in U.S. Pat. No. 5,681,697,
incorporated by reference herein. In this embodiment, a first
portion of the first label extender probe hybridizes to a first
portion of the target sequence, and the second portion of the first
label extender probe hybridizes to a first probe sequence of the
amplifier probe. A first portion of the second label extender probe
hybridizes to a second portion of the target sequence, and the
second portion of the second label extender probe hybridizes to a
second probe sequence of the amplifier probe. These form structures
sometimes referred to as "cruciform" structures or configurations,
and are generally done to confer stability when large branched or
dendrimeric amplifier probes are used.
[0224] In addition, as will be appreciated by those in the art, the
label extender probes may interact with a preamplifier probe,
described below, rather than the amplifier probe directly.
[0225] Similarly, as outlined above, a preferred embodiment
utilizes several different amplifier probes, each with first probe
sequences that will hybridize to a different portion of the label
extender probe. In addition, as outlined above, it is also possible
that the different amplifier probes contain different amplification
sequences, although this is generally not preferred.
[0226] In addition to the first probe sequence, the amplifier probe
also comprises at least one amplification sequence. An
"amplification sequence" or "amplification segment" or grammatical
equivalents herein is meant a sequence that is used, either
directly or indirectly, to bind to a first portion of a label probe
as is more fully described below. Preferably, the amplifier probe
comprises a multiplicity of amplification sequences, with from
about 3 to about 1000 being preferred, from about 10 to about 100
being particularly preferred, and about 50 being especially
preferred. In some cases, for example when linear amplifier probes
are used, from 1 to about 20 is preferred with from about 5 to
about 10 being particularly preferred.
[0227] The amplification sequences may be linked to each other in a
variety of ways, as will be appreciated by those in the art. They
may be covalently linked directly to each other, or to intervening
sequences or chemical moieties, through nucleic acid linkages such
as phosphodiester bonds, PNA bonds, etc., or through interposed
linking agents such amino acid, carbohydrate or polyol bridges, or
through other cross-linking agents or binding partners. The site(s)
of linkage may be at the ends of a segment, and/or at one or more
internal nucleotides in the strand. In a preferred embodiment, the
amplification sequences are attached via nucleic acid linkages.
[0228] In a preferred embodiment, branched amplifier probes are
used, as are generally described in U.S. Pat. No. 5,124,246, hereby
incorporated by reference. Branched amplifier probes may take on
"fork-like" or "comb-like" conformations. "Fork-like" branched
amplifier probes generally have three or more oligonucleotide
segments emanating from a point of origin to form a branched
structure. The point of origin may be another nucleotide segment or
a multifunctional molecule to which at least three segments can be
covalently or tightly bound. "Comb-like" branched amplifier probes
have a linear backbone with a multiplicity of sidechain
oligonucleotides extending from the backbone. In either
conformation, the pendant segments will normally depend from a
modified nucleotide or other organic moiety having the appropriate
functional groups for attachment of oligonucleotides. Furthermore,
in either conformation, a large number of amplification sequences
are available for binding, either directly or indirectly, to
detection probes. In general, these structures are made as is known
in the art, using modified multifunctional nucleotides, as is
described in U.S. Pat. Nos. 5,635,352 and 5,124,246, among
others.
[0229] In a preferred embodiment, dendrimer amplifier probes are
used, as are generally described in U.S. Pat. No. 5,175,270, hereby
expressly incorporated by reference. Dendrimeric amplifier probes
have amplification sequences that are attached via hybridization,
and thus have portions of double-stranded nucleic acid as a
component of their structure. The outer surface of the dendrimer
amplifier probe has a multiplicity of amplification sequences.
[0230] In a preferred embodiment, linear amplifier probes are used,
that have individual amplification sequences linked end-to-end
either directly or with short intervening sequences to form a
polymer. As with the other amplifier configurations, there may be
additional sequences or moieties between the amplification
sequences. In one embodiment, the linear amplifier probe has a
single amplification sequence.
[0231] In addition, the amplifier probe may be totally linear,
totally branched, totally dendrimeric, or any combination
thereof.
[0232] The amplification sequences of the amplifier probe are used,
either directly or indirectly, to bind to a label probe to allow
detection. In a preferred embodiment, the amplification sequences
of the amplifier probe are substantially complementary to a first
portion of a label probe. Alternatively, amplifier extender probes
are used, that have a first portion that binds to the amplification
sequence and a second portion that binds to the first portion of
the label probe.
[0233] In addition, the compositions of the invention may include
"preamplifier" molecules, which serves a bridging moiety between
the label extender molecules and the amplifier probes. In this way,
more amplifier and thus more labels are ultimately bound to the
detection probes. Preamplifier molecules may be either linear or
branched, and typically contain in the range of about 30-3000
nucleotides.
[0234] Thus, label probes are either substantially complementary to
an amplification sequence or to a portion of the target
sequence.
[0235] Detection of the amplification reactions of the invention,
including the direct detection of amplification products and
indirect detection utilizing label probes (i.e. sandwich assays),
is done by detecting assay complexes comprising labels.
[0236] Arrays
[0237] Detection of the amplified products described above
preferably employs arrays, as defined herein. The arrays are
preferably high density arrays that can allow simultaneous
analysis, i.e. parallel rather than serial processing, on a number
of samples. This is preferably done by forming an "array of
arrays", i.e. a composite array comprising a plurality of
individual arrays, that is configured to allow processing of
multiple samples, as is generally outlined in U.S. Ser. No.
09/256,943, hereby expressly incorporated by reference. For
example, each individual array is present within each well of a
microtiter plate. Thus, depending on the size of the microtiter
plate and the size of the individual array, very high numbers of
assays can be run simultaneously; for example, using individual
arrays of 2,000 and a 96 well microtiter plate, 192,000 experiments
can be done at once; the same arrays in a 384 microtiter plate
yields 768,000 simultaneous experiments, and a 1536 microtiter
plate gives 3,072,000 experiments.
[0238] Generally, the array of array compositions of the invention
can be configured in several ways. 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.
[0239] 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.
[0240] 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. The beads are generally put onto the substrate
randomly, and thus several different methodologies can be used to
"decode" the arrays. In one embodiment, unique optical signatures
are incorporated into the beads, generally fluorescent dyes, that
could be used to identify the chemical functionality on any
particular bead. This allows the synthesis of the nucleic acids to
be divorced from their placement on an array, i.e. the capture
probes 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 probe
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. These methods are generally outlined
in PCTs US98/05025 and US99/14387 and U.S. Ser. Nos. 08/818,199 and
09/151,877, all of which are expressly incorporated herein by
reference.
[0241] However, the drawback to these methods is that for a very
high density array, the system requires a large number of different
optical signatures, which may be difficult or time-consuming to
utilize. Accordingly, the present invention also 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 probes is generally random,
and thus a coding/decoding system is required to identify the
probes 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 a decoding binding ligand (DBL), generally directly
labeled, that binds to either the capture probes 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.
[0242] Once the identity (i.e. the actual agent) and location of
each microsphere in the array has been fixed, the array is exposed
to samples containing the target sequences, such as, the products
of amplification reactions described above, although as outlined
below, this can be done prior to or during the analysis as well.
The target sequences will bind to the capture probes as is more
fully outlined below, and results (in the case of optical labels)
in a change in the optical signal of a particular bead.
[0243] In the present invention, "decoding" can use optical
signatures, decoding binding ligands that are added during a
decoding step, or a combination of these methods. The decoding
binding ligands will bind either to a distinct identifier binding
ligand partner that is placed on the beads, or to the capture
probes, with the latter being preferred. 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.
[0244] Accordingly, the present invention provides composite array
compositions comprising at least a first substrate with a surface
comprising a plurality of assay locations. 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 probes (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 per square cm,
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, (with all numbers being per
square centimeter) 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.
[0245] 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 .mu.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 250,000 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.
[0246] 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.
[0247] 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, TeflonJ, 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 fluorescese.
[0248] 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.
[0249] The first substrate comprises a surface comprising a
plurality of assay locations, i.e. the location where the assay for
the detection of a target analyte will occur. The assay locations
are generally physically separated from each other, for example as
assay wells in a microtiter plate, although other configurations
(hydrophobicity/hydrophilicity, etc.) can be used to separate the
assay locations.
[0250] In a preferred embodiment, the second 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.
[0251] In a preferred embodiment, the array comprises a plurality
of discrete sites. Thus, in the former case, the assay location is
the same as the array location, as described herein. In the latter
case, the array location is fitted into the assay location
separately. In these embodiments, 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.
[0252] 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 attachment of beads at any position. That
is, the surface of the substrate is modified to allow attachment 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.
[0253] 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
microetching techniques. As will be appreciated by those in the
art, the technique used will depend on the composition and shape of
the substrate. When the first substrate comprises both the assay
locations and the individual arrays, a preferred method utilizes
molding techniques that form the bead wells in the bottom of the
assay wells in a microtiter plate.
[0254] In a preferred embodiment, physical alterations are made in
a surface of the substrate to produce the sites. In a preferred
embodiment, for example when the second substrate is a fiber optic
bundle, the surface of the substrate is a terminal end of the fiber
bundle, as is generally described in 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.
[0255] 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.
[0256] In a preferred embodiment, the surface of the substrate is
modified to contain chemically modified sites, that can be used to
attach, 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 attachment 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 attachment 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.
[0257] 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.
[0258] 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 graphited, 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 IN
is a helpful guide.
[0259] 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 assay. 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.
[0260] 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.
[0261] Each microsphere comprises a capture probe although as will
be appreciated by those in the art, there may be some microspheres
which do not contain a capture probe, depending the on the
synthetic methods. By "capture probe" or "capture nucleic acid"
herein is meant a probe for the direct or indirect binding of the
target sequence to a bead. By "nucleic acid" or "oligonucleotide"
or grammatical equivalents herein means at least two nucleotides
covalently linked together, as described above.
[0262] In a preferred embodiment, each bead comprises a single type
of capture probes, although a plurality of individual probes are
preferably attached to each bead. Similarly, preferred embodiments
utilize more than one microsphere containing a unique capture
probe; that is, there is redundancy built into the system by the
use of subpopulations of microspheres, each microsphere in the
subpopulation containing the same probe.
[0263] As will be appreciated by those in the art, the probes 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 probes to the beads, to allow both
good attachment, sufficient flexibility to allow good interaction
with the target sequence, and to avoid undesirable binding
reactions.
[0264] In a preferred embodiment, the probes are synthesized
directly on the beads. As is known in the art, many classes of
chemical compounds are currently synthesized on solid supports,
such as peptides, organic moieties, and nucleic acids. It is a
relatively straightforward matter to adjust the current synthetic
techniques to use beads.
[0265] In a preferred embodiment, the probes 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.
[0266] In some embodiments, the beads may additionally comprise an
optical signature, that can be used to identify the bioactive
agent; see for example U.S. Ser. Nos. 08/818,199 and 09/151,877,
and PCT US98/05025, all of which are expressly incorporated herein
by reference.
[0267] 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.
[0268] 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). 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. 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, and carbohydrates
and their binding partners are also suitable binding pairs. Nucleic
acid-nucleic acid binding proteins pairs are also useful.
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
"aptomers" can be developed for binding to virtually any target;
such a aptomer-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.
[0269] In a preferred embodiment, the IBL is a molecule whose color
or luminescence properties change in the presence of a
selectively-binding DBL. For example, the IBL may be a fluorescent
pH indicator whose emission intensity changes with pH. Similarly,
the IBL may be a fluorescent ion indicator, whose emission
properties change with ion concentration.
[0270] Alternatively, the IBL is a molecule whose color or
luminescence properties change in the presence of various solvents.
For example, the IBL may be a fluorescent molecule such as an
ethidium salt whose fluorescence intensity increases in hydrophobic
environments. Similarly, the IBL may be a derivative of fluorescein
whose color changes between aqueous and nonpolar solvents.
[0271] 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.
[0272] 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.
[0273] 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.
[0274] In a preferred embodiment, the microspheres may 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 capture probe 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 probe a unique optical signature such that any
microspheres comprising that probe 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.
[0275] While generally, the present invention does not rely solely
on the use of optical properties to decode the arrays, 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 of optical signatures one some 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.
[0276] In a preferred embodiment, each subpopulation of beads
comprises a plurality of different IBLs. By using a plurality of
different IBLs to encode each probe, the number of possible unique
codes is substantially increased. That is, by using one unique IBL
per probe, 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 probe in the array is assigned a
combination of IBLs, which can be added to the beads prior to the
addition of the probe, after, or during the synthesis of the probe,
i.e. simultaneous addition of IBLs and probes.
[0277] In some embodiment, the combination of different IBLs can be
used to elucidate the sequence of the probe.
[0278] 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.
[0279] 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 probe.
[0280] 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 labelled DBL allows the user to distinguish
between the two beads.
[0281] Once the microspheres comprising the probes and the unique
tags are generated, they are added to the substrate to form an
array. 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 attachment 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.
[0282] 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.
[0283] 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.
[0284] 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.
[0285] 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.
[0286] 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.
[0287] 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.
[0288] 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.
[0289] In some embodiments, for example when chemical attachment is
done, it is possible to attach 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.
[0290] 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.
[0291] Thus, after the array is made, it is "decoded" in order to
identify the location of one or more of the probes, i.e. each
subpopulation of beads, on the substrate surface.
[0292] 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
mass spectral tags.
[0293] 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 the
positives are fluorescent, and sorting can proceed. The
characterization of the attached probe may be done directly, for
example using mass spectroscopy.
[0294] 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 capture probe 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.
[0295] 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 distinquish 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).
[0296] In an alternate preferred embodiment, the sites of
attachment 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
distinquish 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.
[0297] In a preferred embodiment, the location of every capture
probe 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 capture probes
themselves.
[0298] In a preferred embodiment, as outlined above, the DBL binds
to the IBL.
[0299] In a preferred embodiment, the capture probes are
single-stranded nucleic acids and the DBL is a substantially
complementary single-stranded nucleic acid that binds (hybridizes)
to the capture probe, 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.
[0300] 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. 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.
[0301] 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 capture probe (i.e. a hybridization
between the capture probe and the decoder probe). 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 capture probe, 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 to in an assay, during the assay, or after the assay.
[0302] In one embodiment, a single decoding step is done. In this
embodiment, each DBL is labeled with a unique label, such that the
the number of unique tags is equal to or greater than the number of
capture probe (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 capture probe or IBL, a DBL is made that will specifically
bind to it and contains a unique tag, 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 capture probe; the
random array has been decoded. The DBLs can then be removed, if
necessary, and the target sample applied.
[0303] 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 capture probe 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. The
decoder probes are pooled so that each pool 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. 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 tags 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).
[0304] 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. 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.
[0305] In addition, since the size of the array will be set by the
number of unique decoding binding ligands, it is possible to
"reuse" a set of unique DBLs to allow for a greater number of test
sites. This may be done in several ways; for example, by using some
subpopulations that comprise optical signatures. Similarly, the use
of a positional coding scheme within an array; different
sub-bundles may reuse the set of DBLs. Similarly, one embodiment
utilizes bead size as a coding modality, thus allowing the reuse of
the set of unique DBLs for each bead size. Alternatively,
sequential partial loading of arrays with beads can also allow the
reuse of DBLs. Furthermore, "code sharing" can occur as well.
[0306] 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, 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, and others described in the 6th
Edition of the Molecular Probes Handbook by Richard P. Haugland,
hereby expressly incorporated by reference.
[0307] 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.
[0308] 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 capture probe, 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.
[0309] In a preferred embodiment, the dyes are non-covalently
associated with the beads, generally by entrapping the dyes in the
pores of the beads.
[0310] 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.
[0311] 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 tags (i.e. telephone numbers) of other
subarrays, that are separated by virtue of the location of the
subarray. Thus, for example, the same unique tags can be reused
from bundle to bundle. Thus, the use of 50 unique tags 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, i.e. beads containing unique tags for each subarray.
[0312] 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.
[0313] 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 sublibrary 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.
[0314] 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.
[0315] 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 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.
[0316] 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.
[0317] 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. Furthermore, while these techniques are generally
described for nucleic acid systems, these techniques are useful in
the detection of other binding ligand/target analyte systems as
well.
[0318] 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.
[0319] 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.
[0320] 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.
[0321] 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 substracted 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.
[0322] 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.
[0323] 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.
[0324] In a preferred embodiment, cummulative 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.
[0325] In a preferred embodiment, the mean of the subpopulation
(i.e. the plurality of identical beads) is determined, using the
well known Equation 1:
[0326] Equation 1 1 = x i n Equation 1
[0327] In some embodiments, the subpopulation may be redefined to
exclude some beads if necessary (for example for obvious outliers,
as discussed below).
[0328] 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):
[0329] Equation 2 2 = ( x i - ) 2 n Equation 2
[0330] Equation 3 3 s = ( x i - x _ ) 2 n - 1 Equation 3
[0331] As for the mean, the subpopulation may be redefined to
exclude some beads if necessary (for example for obvious outliers,
as discussed below).
[0332] 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:
[0333] Equation 4 4 t = x _ - s / n Equation 4
[0334] 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.
[0335] 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.
[0336] 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:
[0337] Equation 5 5 z = x _ 1 - x _ 2 1 2 n 1 + 2 2 n 2 Equation
5
[0338] Furthermore, analyzing the distribution of individual
members of a subpopulation of sensor elements may be done. For
example, a subpopulation distribution can be evaluated to determine
whether the distribution is binomial, Poisson, hypergeometric,
etc.
[0339] 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.
[0340] 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.
[0341] As above, any number of statistical analyses may be run on
the data from target redundant sensors.
[0342] 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.
[0343] Once made, the compositions of the invention find use in a
number of applications.
[0344] 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.
[0345] 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.
[0346] 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.
[0347] In a further embodiment, the probes are used for forensic
"DNA fingerprinting" to match crime-scene DNA against samples taken
from victims and suspects.
[0348] In an additional embodiment, the probes in an array are used
for sequencing by hybridization.
[0349] The present invention also finds use as a methodology for
the detection of mutations or mismatches in target nucleic acid
sequences. 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),
which 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.
[0350] In a preferred embodiment, a 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
nucleic acid 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, as outlined herein. 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.
[0351] 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.
[0352] 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.
[0353] 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.
[0354] 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. An advantage of this quality control procedure is that it
can be implemented immediated prior to the assay itself, 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.
[0355] 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. The
approach described here can be used to do this by treating the
reagents (formulated as the DBLs) as variable instead of the
arrays.
[0356] 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. 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. Thus, in this
embodiment, the sequential decoding scheme is 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.
[0357] 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.
[0358] 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, this 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 chosen
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.
[0359] All references cited herein are incorporated by reference in
their entirety.
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