U.S. patent application number 15/661456 was filed with the patent office on 2018-01-11 for compositions and methods for preparing oligonucleotide solutions.
The applicant listed for this patent is Illumina, Inc.. Invention is credited to Mark S. Chee, John R. Stuelpnagel.
Application Number | 20180010123 15/661456 |
Document ID | / |
Family ID | 22529862 |
Filed Date | 2018-01-11 |
United States Patent
Application |
20180010123 |
Kind Code |
A1 |
Stuelpnagel; John R. ; et
al. |
January 11, 2018 |
COMPOSITIONS AND METHODS FOR PREPARING OLIGONUCLEOTIDE
SOLUTIONS
Abstract
The present invention is directed to methods and compositions
for generating a pool of oligonucleotides. The invention finds use
in preparing a population or subpopulations of oligonucleotides in
solution. The pool of oligonucleotides finds use in a variety of
nucleic acid detection and/or amplification assays.
Inventors: |
Stuelpnagel; John R.; (San
Jose, CA) ; Chee; Mark S.; (Encinitas, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Illumina, Inc. |
San Diego |
CA |
US |
|
|
Family ID: |
22529862 |
Appl. No.: |
15/661456 |
Filed: |
July 27, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15237490 |
Aug 15, 2016 |
9745573 |
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15661456 |
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14157323 |
Jan 16, 2014 |
9416411 |
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15237490 |
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12558440 |
Sep 11, 2009 |
8669053 |
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14157323 |
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09642068 |
Aug 18, 2000 |
7604996 |
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12558440 |
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60149344 |
Aug 18, 1999 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/6837 20130101;
G11C 7/1048 20130101; C12Q 1/6834 20130101; C12Q 1/6876 20130101;
H03K 19/0966 20130101; C12Q 1/686 20130101; C12N 15/1093 20130101;
C12Q 1/6837 20130101; C12Q 1/6806 20130101; C12Q 1/6834 20130101;
Y10S 977/792 20130101; C12Q 2525/197 20130101; C12Q 2523/107
20130101; C12Q 2523/107 20130101; C12Q 2525/197 20130101 |
International
Class: |
C12N 15/10 20060101
C12N015/10; H03K 19/096 20060101 H03K019/096; G11C 7/10 20060101
G11C007/10; C12Q 1/68 20060101 C12Q001/68 |
Claims
1. (canceled)
2. A system for detecting a plurality of target nucleic acids
comprising: (a) a solution comprising at least a first
oligonucleotide capable of hybridizing to a target nucleic acid;
(b) at least a second oligonucleotide linked to the first substrate
via a second cleavable linker, wherein the second oligonucleotide
is different from the first oligonucleotide and capable of
hybridizing to a target nucleic acid; (c) a plurality of probes
attached to a second substrate and adapted to capture target
nucleic acids hybridized to the first or second oligonucleotide;
and (d) a detector adapted to detect captured target nucleic
acids.
3. The system of claim 2, wherein the first oligonucleotide is
obtained by: (i) providing a first substrate and the first
oligonucleotide linked to the first substrate via a first cleavable
linker; and (ii) cleaving the first cleavable linker.
4. The system of claim 2 comprising a cleaving agent capable of
cleaving the first or second cleavable linker.
5. The system of claim 4, wherein the cleaving agent is selected
from the group consisting of an enzyme, a chemical agent, and a
light source.
6. The system of claim 2, wherein the at least a first
oligonucleotide comprises at least 50 different
oligonucleotides.
7. The system of claim 2, wherein the first and second
oligonucleotides together comprise at least 50 different
oligonucleotides.
8. The system of claim 2, wherein (i) comprises providing the first
and second oligonucleotides linked to the first substrate.
9. The system of claim 8, wherein the first and second
oligonucleotides are linked to the first substrate at discrete
sites.
10. The system of claim 2, wherein the solution comprises the first
oligonucleotide hybridized to the target nucleic acid.
11. The system of claim 2, wherein the solution comprises reagents
for amplifying the first oligonucleotide.
12. The system of claim 11, wherein the amplifying comprises a
polymerase chain reaction.
13. The system of claim 11, wherein the amplifying comprises
rolling circle amplification.
14. A system for detecting a plurality of target nucleic acids
comprising: (a) a solution comprising a pool of oligonucleotides
capable of hybridizing to target nucleic acids; (b) a plurality of
probes attached to a second substrate and adapted to capture target
nucleic acids hybridized to the pool of oligonucleotides; and (c) a
detector adapted to detect captured hybridized target nucleic
acids.
15. The system of claim 14, wherein the pool of oligonucleotides is
obtained by: (i) providing a first substrate comprising the
oligonucleotides linked to the first substrate via cleavable
linkers, wherein the oligonucleotides are different from one
another; and (ii) cleaving the cleavable linkers.
16. The system of claim 14, wherein the pool of oligonucleotides
comprises at least 50 different oligonucleotides.
17. The system of claim 14, wherein the pool of oligonucleotides
are linked to the first substrate at discrete sites.
18. The system of claim 14, wherein the solution comprises the pool
of oligonucleotide hybridized to target nucleic acids.
19. The system of claim 14, wherein the solution comprises reagents
for amplifying the pool of oligonucleotides.
20. The system of claim 14 comprising a cleaving agent capable of
cleaving the cleavable linkers.
21. The system of claim 20, wherein the cleaving agent is selected
from the group consisting of an enzyme, a chemical agent, and a
light source.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 15/237,490 filed Aug. 15, 2016 which is a continuation of U.S.
application Ser. No. 14/157,323 filed Jan. 16, 2014 now U.S. Pat.
No. 9,416,411 issued Aug. 16, 2016 which is a continuation of U.S.
application Ser. No. 12/558,440 filed Sep. 11, 2009 now U.S. Pat.
No. 8,669,053 issued Mar. 11, 2014 which is a continuation of U.S.
application Ser. No. 09/642,068 filed Aug. 18, 2000 now U.S. Pat.
No. 7,604,996 issued Oct. 20, 2009 which claims the benefit of U.S.
Provisional App. No. 60/149,344 filed Aug. 18, 1999 the contents of
which are incorporated herein by reference in their entireties.
FIELD OF THE INVENTION
[0002] The present invention is directed to methods and
compositions for generating a pool of oligonucleotides. The
invention finds use in preparing a pool of oligonucleotides in
solution. The pool of oligonucleotides finds use in a variety of
nucleic acid detection and/or amplification assays.
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
and mutant 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] A variety of techniques for the detection of nucleic acids
have been developed and include techniques that can be classified
as either target amplification or signal amplification. Target
amplification strategies include the polymerase chain reaction
(PCR), strand displacement amplification (SDA), and nucleic acid
sequence based amplification (NASBA).
[0005] Alternatively, rather than amplify the target, alternate
techniques use the target as a template to replicate a signaling
probe, allowing a small number of target molecules to result in a
large number of signaling 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.
[0006] 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.
[0007] 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.
[0008] 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.
[0009] 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.
[0010] 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.
[0011] 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 "signaling" 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.
[0012] "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.
[0013] "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.
[0014] Similarly, 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.
[0015] 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 and/or disease
predisposition. 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 facilitate
multiplex assays.
[0016] There are a variety of particular techniques that are used
to detect sequence, including mutations and SNPs. These include,
but are not limited to, ligation based assays, cleavage based
assays (mismatch and invasive cleavage such as Invader.TM.), single
base extension methods (see WO 92/15712, EP 0 371 437 B1, EP 0317
074 B1; Pastinen et al., Genome Res. 7:606-614 (1997); Syvanen,
Clinica Chimica Acta 226:225-236 (1994); and WO 91/13075), and
competitive probe analysis (e.g. competitive sequencing by
hybridization; see below).
[0017] In addition, DNA sequencing is a crucial technology in
biology today, as the rapid sequencing of genomes, including the
human genome, is both a significant goal and a significant hurdle.
Thus there is a significant need for robust, high-throughput
methods. Traditionally, the most common method of DNA sequencing
has been based on polyacrylamide gel fractionation to resolve a
population of chain-terminated fragments (Sanger et al., Proc.
Natl. Acad. Sci. USA 74:5463 (1977); Maxam & Gilbert). The
population of fragments, terminated at each position in the DNA
sequence, can be generated in a number of ways. Typically, DNA
polymerase is used to incorporate dideoxynucleotides that serve as
chain terminators.
[0018] Several alternative methods have been developed to increase
the speed and ease of DNA sequencing. For example, sequencing by
hybridization has been described (Drmanac et al., Genomics 4:114
(1989); Koster et al., Nature Biotechnology 14:1123 (1996); U.S.
Pat. Nos. 5,525,464; 5,202,231 and 5,695,940, among others).
Similarly, sequencing by synthesis is an alternative to gel-based
sequencing. These methods add and read only one base (or at most a
few bases, typically of the same type) prior to polymerization of
the next base. This can be referred to as "time resolved"
sequencing, to contrast from "gel-resolved" sequencing. Sequencing
by synthesis has been described in U.S. Pat. No. 4,971,903 and
Hyman, Anal. Biochem. 174:423 (1988); Rosenthal, International
Patent Application Publication 761107 (1989); Metzker et al., Nucl.
Acids Res. 22:4259 (1994); Jones, Biotechniques 22:938 (1997);
Ronaghi et al., Anal. Biochem. 242:84 (1996), Nyren et al., Anal.
Biochem. 151:504 (1985). Detection of ATP sulfurylase activity is
described in Karamohamed and Nyren, Anal. Biochem. 271:81
(1999).
[0019] Sequencing using reversible chain terminating nucleotides is
described in U.S. Pat. Nos. 5,902,723 and 5,547,839, and Canard and
Arzumanov, Gene 11:1 (1994), and Dyatkina and Arzumanov, Nucleic
Acids Symp Ser 18; 117 (1987). Reversible chain termination with
DNA ligase is described in U.S. Pat. No. 5,403,708. Time resolved
sequencing is described in Johnson et al., Anal. Biochem. 136:192
(1984). Single molecule analysis is described in U.S. Pat. No.
5,795,782 and Elgen and Rigler, Proc. Natl Acad Sci USA 91(13):5740
(1994), all of which are hereby expressly incorporated by reference
in their entirety.
[0020] One promising sequencing by synthesis method is based on the
detection of the pyrophosphate (PPi) released during the DNA
polymerase reaction. As nucleotriphosphates are added to a growing
nucleic acid chain, they release PPi. This release can be
quantitatively measured by the conversion of PPi to ATP by the
enzyme sulfurylase, and the subsequent production of visible light
by firefly luciferase.
[0021] Several assay systems have been described that capitalize on
this mechanism. See for example WO93/23564, WO 98/28440 and
WO98/13523, all of which are expressly incorporated by reference. A
preferred method is described in Ronaghi et al., Science 281:363
(1998). In this method, the four deoxynucleotides (dATP, dGTP, dCTP
and dTTP; collectively dNTPs) are added stepwise to a partial
duplex comprising a sequencing primer hybridized to a single
stranded DNA template and incubated with DNA polymerase, ATP
sulfurylase, luciferase, and optionally a nucleotide-degrading
enzyme such as apyrase. A dNTP is only incorporated into the
growing DNA strand if it is complementary to the base in the
template strand. The synthesis of DNA is accompanied by the release
of PPi equal in molarity to the incorporated dNTP. The PPi is
converted to ATP and the light generated by the luciferase is
directly proportional to the amount of ATP. In some cases the
unincorporated dNTPs and the produced ATP are degraded between each
cycle by the nucleotide degrading enzyme.
[0022] In some cases the DNA template is associated with a solid
support. To this end, there are a wide variety of known methods of
attaching DNAs to solid supports. Recent work has focused on the
attachment of binding ligands, including nucleic acid probes, to
microspheres that are randomly distributed on a surface, including
a fiber optic bundle, to form high density arrays. See for example
PCTs US98/21193, PCT US99/14387 and PCT US98/05025; WO98150782; and
U.S. Ser. Nos. 09/287,573, 09/151,877, 09/256,943, 09/316,154,
60/119,323, 09/315,584; all of which are expressly incorporated by
reference.
[0023] An additional technique utilizes sequencing by
hybridization. For example, sequencing by hybridization has been
described (Drmanac et al., Genomics 4:114 (1989); U.S. Pat. Nos.
5,525,464; 5,202,231 and 5,695,940, among others, all of which are
hereby expressly incorporated by reference in their entirety). In
addition, sequencing using mass spectrometry techniques has been
described; see Koster et al., Nature Biotechnology 14:1123
(1996).
[0024] Finally, the use of adapter-type sequences that allow the
use of universal arrays has been described in limited contexts; see
for example Chee et al., Nucl. Acid Res. 19:3301 (1991); Shoemaker
et al., Nature Genetics 14:450 (1998); Barany, F. (1991) Proc.
Natl. Acad. Sci. USA 88:189-193, EP 0 799 897 A1 WO 97/31256, all
of which are expressly incorporated by reference.
[0025] PCTs US98/21193, PCT US99/14387 and PCT US98/05025;
WO98/50782; and U.S. Ser. Nos. 09/287,573, 09/151,877, 09/256,943,
09/316,154, 60/119,323, 09/315,584; all of which are expressly
incorporated by reference, describe novel compositions utilizing
substrates with microsphere arrays, which allow for novel detection
methods of nucleic acid hybridization.
[0026] A common feature of all of these assays and techniques is
the requirement for a large number of oligonucleotides. In
addition, as multiplex experiments are performed, solutions
containing multiple types of oligonucleotides must be prepared.
[0027] The prior art describes methods of synthesizing
oligonucleotides. Generally, synthesis methods can be divided into
directed and non-directed methods. For non-directed, combinatorial
methods, bead-based or tea bag synthesis methods have been
described using split and mix procedures. Split and mix synthesis
is described in Peptide and Peptidomimetic Libraries, Molecular
Biotechnology, Vol. 9, 1998, which ex expressly incorporated herein
by reference. A limitation of this method is that all combinations
of polymers are synthesized.
[0028] Alternatively, the prior art describes directed synthesis
methods in which a particular polymer is separated from other
polymers during the synthesis process. A limitation to this
approach is the necessity for separate reactions and the
requirement to mix the polymers together to form pools of
oligonucleotides.
[0029] Accordingly, it is an object of the present invention to
provide compositions and methods for generating a pool of
oligonucleotides.
SUMMARY OF THE INVENTION
[0030] In accordance with the objects outlined above, the present
invention provides methods of generating pools of oligonucleotides.
The methods include providing a substrate and at least first and
second different oligonucleotides linked to said substrate through
first and second cleavable linkers, respectively. In addition, the
method includes cleaving the first and second linkers, thereby
releasing the first and second oligonucleotides from the substrate
thereby generating a pool of oligonucleotides comprising the first
and second oligonucleotides.
[0031] In an additional aspect the invention includes a method for
generating a pool of oligonucleotides comprising providing an array
comprising a substrate and a population of oligonucleotides. The
population comprises at least first and second subpopulations. The
subpopulations comprise at least first and second different
oligonucleotides of known sequence. The first and second
oligonucleotides are immobilized to first and second beads,
respectively, through first and second cleavable linkers,
respectively. The first and second beads are distributed on the
substrate. Subsequently, the first and second linkers are cleaved
thereby releasing the first and second subpopulations from the
first and second beads, thereby generating a pool of
oligonucleotides comprising the first and second
oligonucleotides.
[0032] In an additional aspect the invention includes a method for
generating a pool of oligonucleotides comprising providing an array
comprising a substrate and a population of oligonucleotides. The
population comprises at least first and second subpopulations. The
subpopulations comprise at least first and second different
oligonucleotides of known sequence. The first and second
oligonucleotides are immobilized to a chip through first and second
cleavable linkers, respectively. The first and second linkers are
cleaved thereby releasing the first and second subpopulations from
the chip, thereby generating a pool of oligonucleotides comprising
the first and second oligonucleotides.
[0033] In addition the invention includes a composition comprising
a substrate and at least first and second different
oligonucleotides of known sequence linked to the substrate through
first and second cleavable linkers, respectively. The composition
also includes at least one linker cleaving agent.
[0034] In addition the invention includes a kit comprising a
substrate and at least first and second different oligonucleotides
of known sequence linked to the substrate through first and second
cleavable linkers, respectively. The kit also includes at least one
linker cleaving agent.
BRIEF DESCRIPTION OF THE DRAWINGS
[0035] FIG. 1 depicts an embodiment of a method of generating a
pool of oligonucleotides. Different subpopulations of
oligonucleotides 10, 11 and 12 are immobilized on a substrate 20 by
a cleavable linker 5. Following the addition of a cleavage agent,
the oligonucleotides 10, 11 and 12 are released into the solution
phase.
[0036] FIG. 2 depicts an embodiment of a method of generating a
pool of oligonucleotides. Different subpopulations of
oligonucleotides 10, 11 and 12 are immobilized on a substrate 20 by
different cleavable linkers 5, 6 and 7. Following the addition of
multiple site-specific cleavage agents, the oligonucleotides
immobilized by the respective linkers are released into the
solution phase.
[0037] FIG. 3 depicts an embodiment of a method of generating a
pool of oligonucleotides. Different subpopulations of
oligonucleotides 10, 12 and 13 are immobilized to an association
moiety 30 via a linker 5. The association moiety 30 is distributed
in wells 21 in the substrate 20. Following the addition of a
cleavage agent, the oligonucleotides 10, 11, 12 and 13 are released
into the solution phase.
DETAILED DESCRIPTION OF THE INVENTION
[0038] The present invention is directed to compositions and
methods for preparing oligonucleotide solutions. In particular the
invention includes preparing an array of oligonucleotides. The
oligonucleotides are attached either directly or indirectly to a
substrate through a cleavable linker. Upon cleavage of the linker,
a pool of oligonucleotides is formed. Pools of oligonucleotides
find use in a number of solution-phase nucleic acid detection
and/or amplification reactions.
[0039] Accordingly the present invention provides compositions and
methods for generating pools of oligonucleotides. The method
includes providing a substrate and a plurality of oligonucleotides
attached to the substrate by a cleavable linker and then cleaving
the linker to release the oligonucleotides from the substrate
thereby generating a pool of oligonucleotides.
[0040] In one embodiment the oligonucleotide is directly attached
to the substrate via a cleavable linker. In an alternative
embodiment, the oligonucleotide is indirectly attached to the
substrate. In this embodiment, the oligonucleotide is attached to
an association moiety via a linker. The association moiety is then
distributed on the substrate.
[0041] By "pool" is meant a plurality or more than one
solution-phase oligonucleotide. Preferably, a pool includes two or
more different oligonucleotides. More preferably a pool includes 20
or more different oligonucleotides. Most preferably a pool includes
greater than 50 different oligonucleotides.
[0042] By "population" herein is meant a plurality of
oligonucleotides. In one embodiment, within the population are
separate subpopulations, which can be a single oligonucleotide or
multiple identical oligonucleotides. That is, the oligonucleotides
within a subpopulation are the same. Alternatively, a subpopulation
may be defined by the linker. That is, in this embodiment, each
subpopulation can be defined by the linker used to immobilize the
oligonucleotide to the substrate and/or association moiety. That
is, in this embodiment, the linkers within a subpopulation are the
same. In one embodiment when the linkers within a subpopulation are
the same, the oligonucleotides within the subpopulation are the
same: in an alternative embodiment the oligonucleotides within the
subpopulation need not be the same.
[0043] 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 at, Chem. Soc. Rev. (1995) pp
169-176). Several nucleic acid analogs are described in Rawls, C
& E News Jun. 2, 1997 page 35. All of these references are
hereby expressly incorporated by reference. These modifications of
the ribose-phosphate backbone may be done to facilitate the
addition of labels, or to increase the stability and half-life of
such molecules in physiological environments.
[0044] 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 occurring nucleic acids and
analogs may be made.
[0045] 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-tonic nature, hybridization of the bases attached to these
backbones is relatively insensitive to salt concentration.
[0046] 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
occurring analog structures. Thus for example the individual units
of a peptide nucleic acid, each containing a base, are referred to
herein as a nucleoside.
[0047] The oligonucleotides can be of any length although in a
preferred embodiment they are from 2 to 200 nucleotides in length,
in a preferred embodiment they are from 5 to 100 nucleotides in
length and in a particularly preferred embodiment they are from 7
to 50 nucleotides in length.
[0048] In a preferred embodiment the oligonucleotide is attached to
the substrate via linker. That is, when attached to a substrate or
association moiety, the oligonucleotide is bound or conjugated to a
cleavable linker. By "cleavable linker" is meant a linker that is
susceptible to cleavage with a specific agent and mediates binding
of the substrate and/or the association moiety to the
oligonucleotide. In one embodiment the linker is part of the
nucleic acid. Alternatively, the linker can be a modification of
the nucleic acid. Alternatively, the linker is an additional
moiety.
[0049] Generally, the linker is separable or distinct from the
region of the molecule comprising the desired oligonucleotide. That
is, upon cleavage of the linker, the nature i.e. structure or
sequence of the desired oligonucleotide is not altered. However, in
some embodiments the structure or sequence of the oligonucleotide
is altered.
[0050] In one embodiment the oligonucleotide is linked directly to
a substrate through the linker. In an alternative embodiment the
oligonucleotide is indirectly linked to the substrate, for example
by attachment of the linker to a bead.
[0051] A cleavable linker is susceptible to cleavage with agents
such as but not limited to light, base, acid and enzymes such as
sequence specific restriction enzymes or proteases. In a preferred
embodiment the linker is a nucleotide linker and comprises a site
for cleavage by a sequence specific restriction endonuclease. In an
additionally preferred embodiment the restriction site is a
substrate for a "rare-cutting" enzyme. Rare-cutting restriction
endonucleases are known in the art and include, for example, those
enzymes that recognize 6 or more nucleotides. In some instances it
is preferable to use more frequent restriction sites such as those
that contain a 2, 3, 4 or 5 nucleotide recognition sequence.
[0052] In a preferred embodiment when the linker is an
oligonucleotide, the linker sequences do not have significant
homology to the oligonucleotide to which they are attached. That
is, the linker sequences are substantially unique relative to the
oligonucleotides. Thus, in this embodiment, the linker sequences
can be specifically cleaved relative to the oligonucleotides.
Cleavage of the linker results in release of the oligonucleotides
into the solution-phase to form a pool of oligonucleotides.
[0053] Accordingly, preferred embodiments utilize some method to
select useful linker sequences. Such methods include the use of
computer searching or comparison programs to find unique cleavage
sequences relative to the oligonucleotide sequence. Sequence
comparisons are known in the art and include, but are not limited
to, the local homology algorithm of Smith & Waterman, Adv.
Appl. Math. 2:482 (1981), by the homology alignment algorith of
Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search
for similarity method of Pearson & Lipman, PNAS USA 85:2444
(1988), by computerized implementations of these algorithms (GAP,
BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software
Package, Genetics Computer Group, 575 Science Drive, Madison,
Wis.), the Best Fit sequence program described by Devereux et al.,
Nucl. Acid Res. 12:387-395 (1984), preferably using the default
settings, or by inspection.
[0054] The linker sequences are added to the oligonucleotides in a
variety of ways, as will be appreciated by those in the art. In one
embodiment, the linker sequence and oligonucleotide are synthesized
contiguously. That is, using standard oligonucleotide synthesis
methods, the oligonucleotide and linker are synthesized as one
continuous oligonucleotide.
[0055] In an alternative embodiment, nucleic acid amplification
reactions are done, as is generally outlined in "Detection of
Nucleic Acid Amplification Reactions Using Bead Arrays" and
"Sequence Determination of Nucleic Acids using Arrays with
Microspheres" both of which were filed on Oct. 22, 1999, (U.S. Ser.
Nos. 60/161,148 and 09/425,633, respectively), and "Detection of
Nucleic Acid Reactions on Bead Arrays" filed on Apr. 20, 2000, and
Apr. 21, 2000 (U.S. Ser. Nos. 09/553,993 and 09/556,463,
respectively), all of which are hereby incorporated by reference in
their entirety. In general, the techniques can be described as
follows. Most amplification techniques require one or more primers
hybridizing to the target sequence. The linker sequences can be
added to one or more primers that are complementary to the
oligonucleotide to which the linker is to be added (depending on
the configuration/orientation of the system and need) and the
amplification reactions are run. Thus, for example, PCR primers
comprising at least one linker sequence may be used.
[0056] In an alternative embodiment, non-nucleic acid reactions are
used to add linker sequences to the oligonucleotides. In this
embodiment, binding partner pairs or chemical methods may be used.
For example, one member of a binding partner pair may be attached
to the linker sequence and the other member attached to the
oligonucleotide. For example, the binding partner can be a hapten
or antigen, which will bind its binding partner. 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. Preferred binding partner pairs include, but are not
limited to, biotin (or imino-biotin) and streptavidin, digeoxinin
and Abs, and Prolinx.TM. reagents.
[0057] In a preferred embodiment, chemical attachment methods are
used. In this embodiment, chemical functional groups on each of the
oligonucleotides and linker sequences are used. As is known in the
art, this may be accomplished in a variety of ways. Preferred
functional groups for attachment are amino groups, carboxy groups,
oxo groups and thiol groups, with amino groups being particularly
preferred. Using these functional groups, the two sequences are
joined together; for example, amino groups on each nucleic acid may
be attached, for example using linkers as are known in the art; for
example, homo- or hetero-bifunctional linkers as are well known
(see 1994 Pierce Chemical Company catalog, technical section on
cross-linkers, pages 155-200, incorporated herein by
reference).
[0058] In a preferred embodiment, aptamers are used in the system.
Aptamers are nucleic acids that can be made to bind to virtually
any target; see Bock et al., Nature 355:564 (1992); Femulok et al.,
Current Op. Chem. Biol. 2:230 (1998); and 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, all of which are expressly incorporated herein
by reference.
[0059] In one embodiment linkers are added prior to immobilization
to the substrate and/or bead. That is, a linker-conjugated or
linker-bound oligonucleotide is attached to the substrate or
association moiety. In an alternative embodiment, the
oligonucleotide is attached to the linker while the linker is
immobilized to the substrate or association moiety. Accordingly,
when describing attachment of nucleic acids to a substrate or
association moiety and attachment of linker-bound or
linker-conjugated oligonucleotides to a substrate or association
moiety it is understood that linkers mediate the attachment.
[0060] In addition, the present invention is directed to the use of
linker sequences to assemble arrays comprising other molecules.
That is, cleavable linkers can be used to assemble arrays of
molecules other than oligonucleotides. Other molecules include but
are not limited to other polymers. Thus, upon cleavage of the
linker, pools of solution-phase polymers are generated. Such
polymers include but are not limited to peptides, polysaccharides,
polymers of small molecules and the like.
[0061] In an alternative embodiment the linker comprises amino
acids and thus forms a peptide linker. Peptide linkers are cleaved
by agents that include but are not limited to proteases or
chemicals including bases, acids or CNBr.
[0062] In one embodiment, the oligonucleotides comprise labels. By
"label" or "detectable label" herein is meant a moiety that allows
detection. This may be a primary label or a secondary label.
Accordingly, detection labels may be primary labels (i.e. directly
detectable) or secondary labels (indirectly detectable).
[0063] 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, quantum dots (also referred to as
"nanocrystals": see U.S. Ser. No. 09/315,584, hereby incorporated
by reference), pyrene, Malacite green, stilbene, LUCIFER
YELLOW.TM., CASCADE BLUE.TM., TEXAS RED.TM., Cy dyes (CY3.TM.
CYS.TM., etc.), 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.
[0064] In a preferred embodiment, a secondary detectable label is
used. A secondary label is one that is indirectly detected; for
example, a secondary label can bind or react with a primary label
for detection, can act on an additional product to generate a
primary label (e.g. enzymes), 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 reactions, etc; 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,
enzymes such as horseradish peroxidase, alkaline phosphatases,
lucifierases, etc.
[0065] 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. 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. Preferred binding partner
pairs include, but are not limited to, biotin (or imino-biotin) and
streptavidin, digeoxinin and Abs, and PROLINX.TM. reagents (see
www.prolinxinc.com/ie4/home.hmtl).
[0066] 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.).
[0067] In a preferred embodiment, the binding partner pair
comprises a primary detection label 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.
[0068] 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. For example, primary labels containing
amino groups can be attached to secondary labels comprising amino
groups, for example using linkers as are known in the art; for
example, home- or hetero-bifunctional linkers as are well known
(see 1994 Pierce Chemical Company catalog, technical section on
cross-linkers, pages 155-200, incorporated herein by
reference).
[0069] Thus, when labeled oligonucleotides are synthesized on an
array or synthesized and associated with a substrate, labeled
arrays are formed. In a preferred embodiment, each member of a
population of oligonucleotides is labeled with the same label. In
an alternative embodiment each member of a subpopulation of
oligonucleotides is labeled with the same label. That is, in making
the labeled array, the label serves to identify the oligonucleotide
to which it is attached. In a sense, the label serves as a code for
the sequence of the oligonucleotide.
[0070] In a preferred embodiment, the oligonucleotide is attached
directly to the substrate as is described in more detail herein.
Alternatively, the oligonucleotide is indirectly associated with
the substrate. That is, the oligonucleotide associates with the
substrate via an association moiety as described herein.
[0071] By "substrate" or "solid support" or other grammatical
equivalents herein is meant any material that can be modified for
the attachment or association of nucleic acids. As will be
appreciated by those in the art, the number of possible substrates
is very large. Possible substrates include, but are not limited to,
glass and modified or functionalized glass, plastics (including
acrylics, polystyrene and copolymers of styrene and other
materials, polypropylene, polyethylene, polybutylene,
polyurethanes, Teflon, etc.), polysaccharides, nylon or
nitrocellulose, resins, silica or silica-based materials including
silicon and modified silicon, carbon, metals, inorganic glasses,
plastics, optical fiber bundles, and a variety of other
polymers.
[0072] By "association moiety" (AM) is meant any material to which
an oligonucleotide can be attached that serves as an intermediate
for association of an oligonucleotide to a substrate. As will be
appreciated by those in the art, the number of possible AMs is
large. Possible AMs include any number of solid supports such as
beads or microspheres.
[0073] 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, when oligonucleotides
are associated with the substrate via a bead as described below,
three dimensional configurations can be used, for example by
embedding the beads in a porous block of plastic that allows sample
or reagent access to the beads. Similarly, the beads may be placed
on the inside surface of a tube, for flow-through sample analysis
to minimize sample or reagent volume. Preferred substrates include
optical fiber bundles as discussed below, and flat planar
substrates such as glass, polystyrene and other plastics and
acrylics.
[0074] In a preferred embodiment the substrate is a chip or
biochip. By "chip" or "biochip" herein is meant a planar substrate
to which nucleic acids are directly or indirectly attached. In a
preferred embodiment, the surface of the biochip and the nucleic
acid may be derivatized with chemical functional groups for
subsequent attachment of the two. Thus, for example, the biochip is
derivatized with a chemical functional group including, but not
limited to, amino groups, carboxy groups, oxo groups and thiol
groups, with amino groups being particularly preferred. Using these
functional groups, the oligonucleotides can be attached using
functional groups on the oligonucleotides. For example, nucleic
acids containing amino groups can be attached to surfaces
comprising amino groups, for example using linkers as are known in
the art; for example, homo- or hetero-bifunctional linkers as are
well known (see 1994 Pierce Chemical Company catalog, technical
section on cross-linkers, pages 155-200, incorporated herein by
reference). in addition, in some cases, additional linkers, such as
alkyl groups (including substituted and heteroalkyl groups) may be
used.
[0075] In one embodiment, the substrate is an optical fiber bundle
or array, as is generally described in U.S. Ser. Nos. 08/944,850
and 08/519,062, PCT US98/05025, and PCT US98/09163, all of which
are expressly incorporated herein by reference. Preferred
embodiments utilize preformed unitary fiber optic arrays. By
"preformed unitary fiber optic array" herein is meant an array of
discrete individual fiber optic strands that are co-axially
disposed and joined along their lengths. The fiber strands are
generally individually clad. However, one thing that distinguished
a preformed unitary array from other fiber optic formats is that
the fibers are not individually physically manipulatable; that is,
one strand generally cannot be physically separated at any point
along its length from another fiber strand.
[0076] In one embodiment at least one surface of the substrate is
modified to contain discrete, individual sites for later
association of nucleic acids or oligonucleotides. These sites may
comprise physically altered sites, i.e. physical configurations
such as wells or small depressions in the substrate that can retain
AMs such as 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.
[0077] The sites may be arranged in 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 nucleic acids 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 nucleic
acids at any position. That is, the surface of the substrate is
modified to allow attachment of the nucleic acids 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 retain a
single nucleic acid, or alternatively, the surface of the substrate
is modified and nucleic acids, for example, when attached to beads
may be placed anywhere, but eventually end up at discrete
sites.
[0078] 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 and the nature of any association moieties to be
used, if any.
[0079] In a preferred embodiment, physical alterations are made in
a surface of the substrate to produce the sites. In a preferred
embodiment, the substrate is a fiber optic bundle and the surface
of the substrate is a terminal end of the fiber bundle, as is
generally described in U.S. Pat. No. 6,023,540 and U.S. Ser. No.
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 moiety i.e. beads, to be added
to the wells.
[0080] Generally in this embodiment, the microspheres or beads 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.
[0081] 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
oligonucleotide 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 sal, carbon graphite, titanium
dioxide, latex or cross-linked dextrans such as Sepharose,
cellulose, nylon, cross-linked micelles and Teflon may all be used.
"Microsphere Defection Guide" from Bangs Laboratories, Fishers Ind.
is a helpful guide.
[0082] The beads need not be spherical; irregular particles may be
used. In addition, the beads may be porous, thus increasing the
surface area of the bead available for either capture probe
attachment or tag attachment. The bead sizes range from nanometers,
i.e. 100 nm, to millimeters, i.e. 1 mm, with beads from about 0.2
micron to about 200 microns being preferred, and from about 0.5 to
about 5 micron being particularly preferred, although in some
embodiments smaller beads may be used.
[0083] It should be noted that when beads are used, 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 or
dislodge during the course of the assembly or cleavage.
[0084] Attachment of the nucleic acids to the substrate may be done
in a variety of ways, as will be appreciated by those in the art,
including, but not limited to, chemical or affinity capture (for
example, including the incorporation of derivatized nucleotides
such as AminoLink or biotinylated nucleotides that can then be used
to attach the nucleic acid to a surface, as well as affinity
capture by hybridization), cross-linking, and electrostatic
attachment, etc. In a preferred embodiment, affinity capture is
used to attach the nucleic acids to the substrate. For example,
nucleic acids can be derivatized, for example with one member of a
binding pair, and the substrate or association moiety derivatized
with the other member of a binding pair. Suitable binding pairs
include complementary nucleic acids. In addition, the nucleic acids
may be biotinylated (for example using enzymatic incorporate of
biotinylated nucleotides, for by photoactivated cross-linking of
biotin). Biotinylated nucleic acids can then be captured on
streptavidin-coated substrate or beads, as is known in the art.
Similarly, other hapten-receptor combinations can be used, such as
digoxigenin and antiwdigoxigenin antibodies. Alternatively,
chemical groups can be added in the form of derivatized
nucleotides, that can them be used to add the nucleic acid to the
surface.
[0085] In this embodiment, the oligonucleotides are previously
synthesized as is known in the art, and then attached to the
surface of the solid support. As will be appreciated by those
skilled in the art, either the 5' or 3' terminus may be attached to
the solid support, or attachment may be via an internal
nucleoside.
[0086] Preferred attachments are covalent, although even relatively
weak interactions (i.e. non-covalent) can be sufficient to attach a
nucleic acid to a surface. Thus, for example, electrostatic
interactions can be used for attachment, for example by having
substrates carrying the opposite charge to the oligonucleotide.
[0087] Similarly, affinity capture utilizing hybridization can be
used to attach nucleic acids to substrates or association moieties.
For example, as is known in the art, polyA+RNA is routinely
captured by hybridization to oligo-dT beads; this may include
oligo-dT capture followed by a cross-linking step, such as psoralen
crosslinking). If the nucleic acids of interest do not contain a
polyA tract, one can be attached by polymerization with terminal
transferase, or via ligation of an oligoA linker, as is known in
the art.
[0088] Alternatively, chemical crosslinking may be used to attach
nucleic acids to the substrate, for example by photoactivated
crosslinking of thymidine to reactive groups, as is known in the
art.
[0089] 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 nucleic acids 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 attach nucleic acids, which generally
also contain corresponding reactive functional groups; the addition
of a pattern of charged groups (similar to the chemical
functionalities) for the electrostatic attachment of the nucleic
acids, i.e. when the nucleic acids comprise charged groups opposite
to the sites. As outlined above, "pattern" in this sense includes
the use of a uniform treatment of the surface to allow attachment
of the nucleic acids 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.
[0090] Alternatively, the oligonucleotides may be synthesized in
situ on the substrate, as is known in the art. For example,
photoactivation techniques utilizing photopolymerization compounds
and techniques are used. In a preferred embodiment, the nucleic
acids can be synthesized in situ using well known photolithographic
techniques, such as those described in WO 95/25116; WO 95/35505;
U.S. Pat. Nos. 5,700,637 and 5,445,934; and references cited
within, all of which are expressly incorporated by reference; these
methods of attachment form the basis of the Affymetrix GENECHIP.TM.
technology.
[0091] Alternatively, the oligonucleotides may be synthesized on
the substrate using printing technology as described in U.S. Pat.
No. 5,831,070, which is expressly incorporated herein by reference.
Alternatively, the oligonucleotides may be synthesized by spotting
as described in U.S. Pat. No. 5,807,522 which is expressly
incorporated herein by reference.
[0092] In an alternative embodiment the oligonucleotides are
synthesized on association moieties or solid support such as
microspheres that are then distributed on a substrate. 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.
[0093] In one embodiment the oligonucleotides are synthesized
randomly i.e. with no bias or restriction at any of the positions
in the oligonucleotide. That is, synthesis is non-directed. As
such, pools comprising random oligonucleotides are generated by the
method. Methods of randomly synthesizing oligonucleotides are known
in the art and as described in U.S. Pat. No. 5,504,190, which is
expressly incorporated herein by reference. Other combinatorial
techniques are summarized in Peptide and Peptidomimetic Libraries,
Molecular Biotechnology, Vol. 9, 1998, which is expressly
incorporated herein by reference.
[0094] In an alternative embodiment, the oligonucleotides are not
randomly produced, but rather are synthesized with an eye to
targeting a particular molecule. That is, synthesis of the
oligonucleotides is directed. As is known in the art,
oligonucleotides hybridize with a complementary strand; thus, the
oligonucleotides are designed to target a particular complementary
molecule. This complementarity need not be perfect; there may be
any number of base pair mismatches that will interfere with
hybridization between the target sequence and the single stranded
nucleic acids of the present invention. However, if the number of
mutations is so great that no hybridization can occur under even
the least stringent of hybridization conditions, the sequence is
not a complementary target sequence. Thus, by "substantially
complementary" herein is meant that the probes are sufficiently
complementary to the target sequences to hybridize under the
selected reaction conditions.
[0095] In one embodiment oligonucleotides are designed to hybridize
with DNA, for example, for genotyping, single nucleotide
polymorphism (SNP) detection or for use as primers in
amplification, in particular multiplex amplification,
reactions.
[0096] Alternatively, oligonucleotides are synthesized with only
certain degenerate positions. That is, some of the positions are
fixed or biased for a particular nucleotide while other positions
are degenerate or synthesized with random nucleotides.
[0097] Accordingly the present invention provides array
compositions comprising a substrate comprising oligonucleotides and
a linker. By "array" herein is meant a plurality of nucleic acids
in an array format; the size of the array will depend on the
composition and end use of the array. Nucleic acids arrays are
known in the art, and can be classified in a number of ways; both
ordered arrays (e.g, the ability to resolve chemistries at discrete
sites), and random arrays are included. Ordered arrays include, but
are not limited to, those made using photolithography techniques
(Affymetrix GENECHIP.TM.), spotting techniques (Synteni and
others), printing techniques (Hewlett Packard and Rosetta), three
dimensional "gel pad" arrays, etc.
[0098] In a preferred embodiment the array compositions further
comprise a linker cleaving agent. As described herein, linker
cleaving agents include but are not limited to light, chemicals
including base and acid, enzymes such as proteases and nucleases.
In a particularly preferred embodiment the nucleases include
sequence specific restriction endonucleases as are known in the art
and described herein. Additional cleavage agents are described in
Promega Catalog, 1997, pp. 293-297 and 34-74, and Pierce Catalog
and Handbook, 1994, pp. 0-209 to 0-221, both of which are expressly
incorporated herein by reference.
[0099] In an additional embodiment, the compositions further
comprise solution-phase oligonucleotides. That is, once cleavage of
the linker has begun and the oligonucleotides are cleaved from the
substrate, the oligonucleotides are released into the
solution-phase. Accordingly, a pool of oligonucleotides in solution
is formed.
[0100] Once formed, the array of oligonucleotides finds use in a
number of aspects. In a particularly preferred embodiment the
arrays are contacted with a cleaving agent that cleaves the linker.
That is, the substrate to which the population oligonucleotides is
attached is contacted with a cleaving agent thereby releasing the
oligonucleotides into the solution phase (FIG. 1). As one of
ordinary skill in the art appreciates cleavage conditions will vary
with the nature of the cleavage agent. Generally, when cleavage
agents are enzymes, conditions will vary with respect to metal,
temperature, pH and salt concentration. The duration or time of
cleavage reactions also will vary depending on the cleavage agent
selected.
[0101] In an alternative embodiment, the cleaving agent recognizes
only a subset of linkers. That is, as described above, each
subpopulation of oligonucleotides contains a different linker.
Accordingly, incubation of the array with a particular
site-specific cleaving agent results in release of only the
oligonucleotide immobilized with the respective linker (FIG. 2).
Moreover, incubation with multiple site-specific cleaving agents
results in the release of multiple subpoputations of
oligonucleotides.
[0102] In an alternative embodiment, the oligonucleotides are
indirectly attached to the substrate. That is, linkers can
immobilize the oligonucleotides either directly to the substrate or
indirectly. When indirectly attached to the substrate,
oligonucleotides are attached to AMs via linkers as outlined
herein. The AMs are distributed on the substrate forming an array.
Subsequently, the array is contacted with a cleaving agent as
described herein resulting in the release of the oligonucleotides
into the solution phase (FIG. 3).
[0103] In an additional embodiment, the array of oligonucleotides
finds use in kits. That is, kits can be formulated to include an
array of oligonucleotides. As described herein, the
oligonucleotides may comprise random oligonucleotides;
alternatively, the oligonucleotides may comprise known sequences.
In addition, the oligonucleotides may comprise a label. In this
embodiment, the kit comprises a labeled array.
[0104] The kit also includes a linker cleaving agent. That is, to
facilitate the formation of a pool of oligonucleotides, the kit
includes at least one but may also include as many cleaving agents
as necessary to release the desired oligonucleotides from the
substrate.
[0105] In addition, the kit may also include at least one control
oligonucleotide. The control oligonucleotide is designed to be
complementary to a subpopulation of immobilized oligonucleotides or
a population of control immobilized oligonucleotides. In a
preferred embodiment the control oligonucleotide comprises a label
as described herein.
[0106] In one embodiment the control oligonucleotide finds use in
determining the quality of the array of oligonucleotides, That is,
in one embodiment, the control oligonucleotide is contacted with
the array of oligonucleotides prior to cleavage of the linkers. The
labeled control oligonucleotide is then detected, for example by
viewing the array under a microscope. Other detection methods are
described in more detail in U.S. Ser. No. 09/556,463, filed Apr.
21, 2000, which is expressly incorporated herein by reference. The
presence of the label provides an indication of the quality or
identity of the array. As such, the array of oligonucleotides also
facilitates sample handling, tracking and storage.
[0107] Once formed, the pool of oligonucleotides finds use in a
number of assays. In addition, as nucleic acid experiments are
performed in multiplex, a solution that contains many types of
oligonucleotides must be prepared. Examples of experiments that may
require pools of oligonucleotides when performed in solution
include assays for genotyping, such as OIA, Single Base Extension,
Invader and the like, assays for the detection of single nucleotide
polymorphisms, sequencing, multiplex amplification including
polymerase chain reactions, and the like.
[0108] Preferably, the assays are conducted in solution. Once the
solution phase is performed, the experiments may include an array
detection step. Arrays for detecting nucleic acids and nucleic acid
reactions are more fully described in U.S. Ser. No. 09/556,463,
filed Apr. 21, 2000, which is expressly incorporated herein by
reference.
[0109] Pools of oligonucleotides find use in decoding arrays as
described in more detail in U.S. Ser. No. 09/344,526, and U.S. Ser.
No. 09/574,117, both of which are expressly incorporated herein by
reference. In addition, pools of oligonucleotides find use in
microfluidic systems as described in U.S. Ser. No. 09/306,369 which
is expressly incorporated herein by reference. In addition, pools
of oligonucleotides find use in composite array systems as
described in U.S. Ser. No. 09/606,369, which is expressly
incorporated herein by reference.
[0110] All references cited herein are incorporated by reference in
their entirety.
* * * * *
References