Title: Methods and compositions for monitoring polymer array synthesis
Abstract: VLSIPS™ manufacturing processes are of increasing commercial importance. The present invention provides methods and compositions for monitoring the efficiency and quality of polymer synthesis in VLSIPS™ arrays. Methods for monitoring polymer synthesis in an array on a substrate are provided. Monoisomeric labels for the labeling of synthetic polymer arrays are provided. Methods and compositions for post-synthetically labeling polymers in polymer arrays are also provided.
Patent Number: 7,026,114 Issued on 04/11/2006 to Barone, et al.
| Inventors:
|
Barone; Anthony D. (Santa Clara, CA);
Mc Gall; Glenn H. (Mountain View, CA);
Chai; Evelyn (Foster City, CA);
Ngo; Nam Quoc (Campbell, CA)
|
| Assignee:
|
Affymetrix, Inc. (Santa Clara, CA)
|
| Appl. No.:
|
574461 |
| Filed:
|
November 30, 1995 |
| Current U.S. Class: |
435/6; 435/4; 435/287.1; 435/288.7; 435/DIG.1; 435/DIG.34; 435/DIG.49; 436/518; 530/333; 530/334; 530/335; 536/24.3; 536/25.3; 536/25.31; 536/25.32 |
| Current Intern'l Class: |
C12Q 1/68 (20060101); G01N 33/54.3 (20060101); A61K 38/00 (20060101); C07H 21/04 (20060101) |
| Field of Search: |
536/231,253,253.2,243,253.1
435/71,6,DIG.1,DIG.2,DIG.14,DIG.15,DIG.16,DIG.17,DIG.18,DIG.46,DIG.49,4, 287.1,288.7,DIG.34
436/518
530/333,334,335
|
References Cited [Referenced By]
U.S. Patent Documents
Other References
Pease AC, et al, (1994) Light-generated oligonucleotide arrays for rapid DNA
sequence analysis. Proc.Natl.Acad.Sci.U.S.A. 91:5022-5026.
Reynolds MA, et al, (1992) A non-nucleotide-based linking method for the preparation
of psoralen-derivatized methylphosphonate oligonucleotides. Bioconjug.Chem. 3:366-374.
Silverstein and Bassler, Spectrophotmetric Identification of Organic Compounds,
John Wiley and Sons, New York pp. 161-162, 1963.
Fodor et al. Light-Directed, spatially addressable parallel chemical synthesis.
Science 251:767-773, Feb. 15, 1991.
Fodor et al. (1991) Science 251:767-777.
Frank and Doring (1988) Tetrahedron 44:6031-6040.
Advanced Organic Chemistry: Reactions, Mechanisms and Structure, Fourth
Edition (Mar. 1992), Chapter 4, John Wiely and Sons.
Hochuli (1989) Chemische Industrie 12:69-70.
Hochuli (1990) "Purification of Recombinant Proteins with Metal Chelate Absorbent"
In: Genetic Engineering, Principle and Methods, Setlow (ed.) Plenum Press,
N.Y. 12:87-98.
Barone et al. (1984) Nucleic Acids Research 12:4051.
Reynolds et al. (1992) Bioconjugate Chemistry 3:366.
Pease et al. (1994) Proc. Natl. Acad. Sci. USA 91:5022.
Cho et al., "An Unnatural Biopolymer," Science, 261:1303-1305 (1993).
Nestler et al., "A General Method for Molecular Tagging of Encoded Combinatorial
Chemistry Libraries," J. Org. Chem., 59:4723-4724 (1994).
|
Primary Examiner: Ponnaluri; Padmashri
Attorney, Agent or Firm: Banner & Witcoff, Ltd.
Parent Case Text
CROSS REFERENCE TO RELATED APPLICATIONS
This Application claims the benefit of the U.S. Provisional Application by Barone
et al. (U.S. Ser. No. 60/003,726) filed Sep. 13, 1995.
Claims
What is claimed is:
1. A method of monitoring polymer array synthesis on a solid substrate comprising:
(i) synthesizing a preselected array of diverse biological polymers connected
to a solid substrate, whereby the diverse biological polymers occupy different
regions of the substrate and are spatially defined on the solid substrate on which
the preselected array is synthesized, and wherein the diverse biological polymers
comprise nucleotides, nucleosides, phosphoramidites, carbohydrates or natural or
synthetic amino acids;
(ii) cleaving the diverse biological polymers from the solid substrate thereby
creating a mixture of diverse unbound biological polymers; and
(iii) predicting a quantity of diverse biological polymers formed and comparing
a measurement of quantity of diverse cleaved biological polymers from the array
with the predicted quantity of diverse biological polymers formed as an indicator
of the efficiency of the synthesis procedure, thereby determining the efficiency
of the synthesis procedure.
2. The method of claim 1, wherein each of the polymers further comprises a label,
thereby forming labeled polymers.
3. The method of claim 2, wherein each of the labeled polymers comprises a single
isomeric label.
4. The method of claim 2, wherein the labeled unbound polymers are heterogeneous
by number of monomeric units, and wherein the method further comprises separating
the labeled unbound polymers by number of monomeric units.
5. The method of claim 2, wherein the labeled unbound polymers are heterogeneous
by number of monomeric units, and wherein the method further comprises separating
the labeled unbound polymers by charge using ion exchange chromatography.
6. The method of claim 2, wherein each of the labeled unbound polymers are heterogeneous
by number of monomeric units, and wherein the method further comprises separating
the labeled unbound polymers by number of monomeric units using capillary gel electrophoresis.
7. The method of claim 5, wherein the ion exchange chromatography is performed
by HPLC.
8. The method of claim 5, wherein the ion exchange chromatography is performed
by HPLC, and wherein the labeled unbound polymers are detected as they exit an
ion exchange column.
9. The method of claim 1, wherein the polymer is an oligonucleotide.
10. A method for measuring the effect of altering a polymer array synthesis protocol, comprising:
(i) synthesizing a preselected array of diverse biological polymers occupying
different regions on a solid support by a first synthesis protocol, wherein the
diverse biological polymers are spatially defined on the solid support on which
the preselected array is synthesized, thereby creating a reference array of biological
polymers, wherein the diverse biological polymers comprise nucleotides, nucleosides,
phosphoramidites, carbohydrates or natural or synthetic amino acids;
(ii) synthesizing a preselected array of diverse biological polymers occupying
different regions on a solid support synthesized by a second synthesis protocol,
wherein the diverse biological polymers are spatially defined on the solid support
on which the preselected array is synthesized, and wherein the second synthesis
protocol is different than the first synthesis protocol, thereby creating a test
array of biological polymers; wherein biological polymers of the test array are
preselected to be the same as preselected biological polymers of the reference array;
(iii) cleaving separately the reference array of biological polymers and the
test array of biological polymers, thereby creating a mixture of diverse cleaved
biological polymers from the reference array and a mixture of diverse cleaved biological
polymers from the test array;
(iv) comparing a measurement of presence of diverse cleaved biological polymers
from the test array as an indicator of the efficiency of the second synthesis procedure
with a measurement of presence of diverse cleaved biological polymers from the
reference array as an indicator of the efficiency of the first synthesis procedure,
thereby determining whether a difference between the first and second synthesis
procedure affects the efficiency of the second synthesis procedure.
11. The method of claim 10, wherein the test and reference polymers are oligonucleotides.
12. The method of claim 10, wherein the first synthesis protocol differs from
the second synthesis protocol by a single variation.
13. The method of claim 10, wherein the reference polymers and the test polymers
are attached to the solid substrate by a cleavable linker.
14. The method of claim 10, wherein the test and reference polymers comprise
a detectable label.
15. The method of claim 14, wherein the label is a single isomeric label.
16. The method of claim 2, wherein the labeled polymers comprise a label comprising
a fluorescent moiety.
17. The method of claim 14, wherein the detectable label comprises a fluorescent moiety.
18. A method of monitoring polymer array synthesis on a solid substrate comprising:
(i) synthesizing a preselected array of diverse polymers on a solid substrate,
whereby the diverse polymers occupy different regions of the solid substrate and
are spatially defined on the solid substrate on which the preselected array is synthesized;
(ii) cleaving the diverse polymers from the solid substrate thereby creating
a mixture of diverse unbound polymers; and
(iii) predicting a quantity of diverse polymers formed and comparing a measurement
of quantity of diverse cleaved polymers from the array with the predicted quantity
of diverse polymers formed as an indicator of the efficiency of the synthesis procedure,
thereby determining the efficiency of the synthesis procedure.
19. The method of claim 18, wherein each of the polymers further comprises a
label, thereby forming labeled polymers.
20. The method of claim 19, wherein the labeled polymers comprise a label comprising
a fluorescent moiety.
21. The method of claim 19, wherein each of the labeled polymers comprises a
single isomeric label.
22. The method of claim 19, wherein the labeled unbound polymers are heterogeneous
by number of monomeric units, and wherein the method further comprises separating
the labeled unbound polymers by number of monomeric units.
23. The method of claim 19, wherein the labeled unbound polymers are heterogeneous
by number of monomeric units, and wherein the method further comprises separating
the labeled unbound polymers by charge using ion exchange chromatography.
24. The method of claim 19, wherein each of the labeled unbound polymers is heterogeneous
by number of monomeric units, and wherein the method further comprises separating
the labeled unbound polymers by number of monomeric units using capillary gel electrophoresis.
25. The method of claim 23, wherein the ion exchange chromatography is performed
by HPLC.
26. The method of claim 23, wherein the ion exchange chromatography is performed
by HPLC, and wherein the labeled unbound polymers are detected as they exit an
ion exchange column.
27. The method of claim 18, wherein the polymer is an oligonucleotide.
28. A method for measuring the effect of altering a polymer array synthesis protocol, comprising:
(i) synthesizing a preselected array of diverse polymers occupying different
regions on a solid support by a first synthesis protocol, wherein the diverse polymers
are spatially defined on the solid support on which the preselected array is synthesized,
thereby creating a reference array of polymers;
(ii) synthesizing a preselected array of diverse polymers occupying different
regions on a solid support synthesized by a second synthesis protocol, wherein
the diverse polymers are spatially defined on the solid support on which the preselected
array is synthesized, and wherein the second synthesis protocol is different than
the first synthesis protocol, thereby creating a test array of polymers;
(iii) cleaving separately the reference array of polymers and the test array
of polymers, thereby creating a mixture of diverse cleaved polymers from the reference
array and a mixture of diverse cleaved polymers from the test array;
(iv) comparing a measurement of presence of diverse cleaved polymers from the
test array as an indicator of the efficiency of the second synthesis procedure
with a measurement of presence of the mixture of diverse cleaved polymers from
the reference array as an indicator of the efficiency of the first synthesis procedure,
thereby determining whether a difference between the first and second synthesis
procedures affects the efficiency of the second synthesis procedure.
29. The method of claim 28, wherein the test and reference polymers are oligonucleotides.
30. The method of claim 28, wherein the first synthesis protocol differs from
the second synthesis protocol by a single variation.
31. The method of claim 28, wherein the reference polymers and the test polymers
are attached to the solid substrate by a cleavable linker.
32. The method of claim 28, wherein the test and reference polymers comprise
a detectable label.
33. The method of claim 32, wherein the label is a single isomeric label.
34. The method of claim 32, wherein the detectable label comprises a fluorescent moiety.
Description
COPYRIGHT NOTICE
A portion of the disclosure of this patent document contains material which is
subject to copyright protection. The copyright owner has no objection to the facsimile
reproduction by anyone of the patent document or the patent disclosure as it appears
in the Patent and Trademark Office patent file or records, but otherwise reserves
all copyright rights whatsoever.
BACKGROUND OF THE INVENTION
Methods of forming large arrays of oligonucleotides, peptides and other polymers
on a solid substrate are known. Pirrung et al., U.S. Pat. No. 5,143,854 (see also
PCT Application No. WO 90/15070), McGall et al., U.S. Ser. No. 06/440742, Chee
et al., SN PCT/US94/12305, and Fodor et al., PCT Publication No. WO 92/10092 describe
methods of forming vast arrays of peptides, oligonucleotides and other polymers
using, for example, light-directed synthesis techniques.
In the Fodor et al. PCT application, methods are described for using computer-controlled
systems to direct polymer array synthesis. Using the Fodor approach, one heterogeneous
array of polymers is converted, through simultaneous coupling at multiple reaction
sites, into a different heterogeneous array. See also, U.S. Ser. No. 07/796,243
and U.S. Ser. No. 07/980,523 and Fodor et al. (1991)
Science, 251: 767-777.
The arrays are typically placed on a solid surface with an area less than 1 inch
2,
although much larger surfaces are optionally used.
More recently, U.S. applications U.S. Ser. No. 06/440,742, U.S. Ser. No. 08/284,064,
U.S. Ser. No. 08/143,312, U.S. Ser. No. 08/082,937 and PCT application (designating
the United States) SN PCT/U594/12305, describe methods of making arrays of oligonucleotide
and oligonucleotide analogue probes, e.g., to check or determine a partial or complete
sequence of a target nucleic acid, or to detect the presence of a nucleic acid
containing a specific oligonucleotide sequence. U.S. application Ser. No. 08/327,687
and U.S. application Ser. No. 06/440,742 describe methods of creating libraries
of nucleic acid probes for the analysis of nucleic acid hybridization, and for
screening nucleic acid binding molecules, e.g., as potential therapeutic agents.
Additional methods applicable to polymer synthesis on a substrate are
described in co-pending Applications U.S. Ser. No. 07/980,523, filed Nov. 20, 1992,
and U.S. Ser. No. 07/796,243, filed Nov. 22, 1991, incorporated herein by reference
for all purposes. In the methods disclosed in these applications, reagents are
delivered to the substrate by flowing or spotting polymer synthesis reagents on
predefined regions of the solid substrate. In each instance, certain activated
regions of the substrate are physically separated from other regions when the monomer
solutions are delivered to the various reaction sites, e.g., by means of grooves,
wells and the like.
SUMMARY OF THE INVENTION
VLSIPS™ procedures are of increasing commercial importance,
providing powerful compositions and methods, e.g., for detecting genetic disorders,
screening potential therapeutics, facilitating basic research and rapid sequencing
of nucleic acids. Accordingly, the manufacturing processes which produce VLSIPS™
arrays benefit from quality control and synthesis optimization methods for measuring
and improving the efficiency of polymer and polymer array synthesis and coupling
of monomers and polymers to solid substrates.
The present invention provides methods and compositions to monitor the synthesis
and coupling of monomers and polymers to solid substrates, e.g., in VLSIPS™
arrays. The methods typically operate by incorporating a detectable label (typically
an isomeric label, e.g., as provided by the compositions herein) into the polymers
of an array. The polymers are cleaved from the array, and the efficiency of polymer
synthesis assessed by monitoring the detectable label in an appropriate assay.
In one class of embodiments, the present invention provides a method of monitoring
polymer array synthesis on a solid substrate by providing a preselected array of
labeled polymers connected to cleavable linkers on a solid substrate, cleaving
the array of labeled polymers from the solid substrate by cleaving the cleavable
linkers, thereby creating labeled unbound polymers, and detecting the labeled unbound
polymers. In this embodiment, the labeled polymers each typically comprise a single
isomeric label, although any detectable label can also be used. The polymers cleaved
from the array are separated by physical properties such as size and/or charge,
using known analytical techniques such as HPLC, standard column chromatography
(anion, cation, size exclusion, etc.), gel-electrophoresis, centrifugation, capillary
gel electrophoresis and the like.
The methods are generally suitable to any polymer array, regardless of the type
of polymer. Thus, the efficiency of synthesis for biological polymers such as proteins,
nucleic acids, antigens, and venoms are monitored using the above method. Non-biological
polymers such as carbon chains, vinyls, alcohols, and other polymers are similarly
monitored. The polymer array is typically provided by synthesizing the array on
the solid substrate, but the array can also be provided by synthesizing the polymers
to be attached to the array in solution, and subsequently attaching the polymers
to pre-selected sites in the array.
In a second class of embodiments, the invention provides a method of measuring
and improving the synthesis of polymer arrays, by synthesizing an array of polymers
on a solid support by a first synthesis protocol, creating a reference array of
polymers; synthesizing an array of polymers on a solid support by a second synthesis
protocol, wherein the second synthesis protocol is different than the first synthesis
protocol, thereby creating a test array of polymers; cleaving separately the reference
array of polymers and the test array of polymers, thereby creating cleaved reference
polymers and cleaved test polymers; detecting the cleaved test polymers and the
cleaved reference polymers, and comparing the cleaved test polymers to the cleaved
reference polymers. By repeating the process and altering different synthesis parameters
between the test and the reference array of polymers, the optimal method of synthesizing
a particular array is determined.
Typically, the polymers of an array comprise a detectable label to facilitate
analysis of the cleaved polymers, although the polymers themselves are also detectable,
and the method can, therefore, be performed without incorporating a detectable
label. Where the method used for detecting the label discriminates between optical
isomers of a label (e.g., HPLC) the label will most often comprise a single optical
isomer. Although it is most preferred that a single synthetic parameter is altered
for the test polymers relative to the control polymers, multiple parameters can
be altered in each synthetic protocol. Once again, the method is generally applicable
to biological and artificial polymers, each of which are typically connected to
a solid substrate by a cleavable linker.
The methods of the present invention are typically performed using a detectable
monomeric monoisomeric polymer synthesis reagent with the structure A-B, wherein
A comprises a detectable chromogenic moiety and B comprises a polymer integration
element. The polymer integration element typically includes a group which can be
joined to one end of the polymer, or incorporated into the polymer as it is synthesized
(i.e., as a monomeric unit of the polymer). The precise nature of the integration
element depends on the polymer which the detectable moiety is to be integrated
into. For instance, where the polymer is a peptide, the integration element will
typically comprise an amino or a carboxy group, or both, similar to the amino acids
which comprise the peptide. Similarly, where the polymer is an oligonucleotide,
the polymer will typically comprise a phosphate or hydroxyl, or both, similar to
the nucleotides which constitute the oligonucleotide polymer. The chromogenic moiety
is most typically a fluorophore, although other chromogenic agents are also suitable.
In one embodiment, the polymer synthesis reagent is a nucleic acid synthesis
reagent,
and B (the polymer integration element) comprises the structure
##STR1##
wherein
L is an alkyl linking group from 1 to 30 carbons in length, wherein one or more
carbon is optionally replaced or substituted with a heteroatom selected from the
group consisting of N, S, O and P, and is optionally part of a ring system, and
wherein the alkyl linking chain optionally includes one or more site of unsaturation;
R
1 is selected from the group consisting of hydrogen, alkyl,
and aryl;
R
2 is selected from the group consisting of hydrogen, alkyl,
and aryl;
X is a nucleic acid integration element comprising a phosphorous atom;
Y is selected from the group consisting of hydrogen, alkyl, and aryl;
Y
2 is an alkyl chain; and
Z comprises a protecting group.
In one class of preferred embodiments, the polymer synthesis reagent is a nucleic
acid synthesis reagent, and B (the polymer integration element) comprises the structure
##STR2##
wherein
L is an alkyl linking group from 1 to 30 carbons in length, wherein one or more
carbon is optionally replaced or substituted with a heteroatom selected from the
group consisting of N, S, O and P, and is optionally part of a ring system, and
wherein the alkyl linking chain optionally includes one or more site of unsaturation; and
R
1 is selected from the group consisting of hydrogen, alkyl,
and aryl.
This polymer integration element is typically joined to a fluorophore.
In another class of preferred embodiments, the invention provides an array of
polymers, such as an array of oligonucleotides or proteins, or non-biological polymers,
with a monoisomeric detectable label incorporated into each polymer. For instance,
in one embodiment where the array is an oligonucleotide, the invention provides
an array of oligonucleotides attached to a solid substrate, wherein the label is
a monoisomeric label comprising the structure wherein F comprises a fluorescent group;
L is an alkyl linking group from 1 to 30 carbons in length, wherein one or more
carbon is optionally replaced or substituted with a heteroatom selected from the
group consisting of N, S, O and P, and is optionally part of a ring system, and
wherein the alkyl linking chain optionally includes one or more site of unsaturation;
R
1 is selected from the group consisting of hydrogen, alkyl,
and aryl;
R
2 is selected from the group consisting of hydrogen, alkyl,
and aryl;
X is a nucleotide, nucleic acid or a cleavable linker;
Y is selected from the group consisting of hydrogen, alkyl, and aryl;
Y
2 is an alkyl chain; and
Z is a nucleotide or nucleic acid.
In one preferred group of embodiments, the nucleic acid synthesis reagent has
the structure
wherein R
1 is selected from the group consisting of alkyl, aryl,
and hydrogen; R
2 is selected from the group consisting of alkyl, and
aryl; and FL is a fluorescent moiety.
An example compound is fluorescein phosphoramidite 7.
##STR3##
In another preferred group of embodiments, the reagent has the structure
##STR4##
wherein
L is an alkyl linking group from 1 to 30 carbons in length, wherein one or more
carbon is optionally replaced or substituted with a heteroatom selected from the
group consisting of N, S, O and P, and is optionally part of a ring system, and
wherein the alkyl linking chain optionally includes one or more site of unsaturation.
Most typically, the polymer arrays of the invention further comprise cleavable
linkers, often located proximal to the substrate which the array is formed upon,
to facilitate cleavage of the polynucleotide from the array.
In a preferred embodiment of the invention, methods of post-synthetically labeling
an oligonucleotide array are provided. In these methods, a polymer array which
comprises a plurality of polymers is provided, wherein each polymer in the array,
or a plurality of polymers in the array, include a labeling site to which a detectable
label such as a fluorophore is attached.
Most typically in this preferred embodiment, the polymers in the array are synthesized
on labeling linkers, which are most typically attached to cleavable linkers proximal
to the surface upon which the array is synthesized. The labeling linkers include
attachment sites for the detectable label. During polymer synthesis the labeling
linker includes a protected site for the attachment of the detectable label which
is deprotected at a defined point in the synthesis of the array (typically after
the polymers in the array are completely synthesized, and often after the polymers
are cleaved from the array at the cleavable linker) so that the detectable moiety
can be attached. For instance, where the detectable reagent is a fluorescent phosphoramidite,
the protected site on the labeling linker will typically comprise an oxygen with
which the phosphate on the phosphoramidite will react to form a phosphodiester
linkage (i.e., after the oxygen is deprotected). DMT is a preferred protecting
group, although many others are also suitable, depending on the nature of the group
to be protected, the polymer and the detectable moiety.
The post-synthetic labeling linker used in the method typically has a site for
polymer elongation, a site for attaching a polymer to a substrate and an attachment
site for attaching a detectable label. Preferred labeling linkers are described
herein. In general, where the labeling linker is a nucleic acid synthesis reagent,
the labeling linker has the structure
##STR5##
wherein:
R
1 is selected from the group consisting of hydrogen, alkyl
and aryl;
R
2 is selected from the group consisting of hydrogen, alkyl
and aryl;
R
3 is selected from the group consisting of hydrogen, alkyl
and aryl;
L
1 is a linking chain selected from the group of alkyl linking
chains consisting of an alkyl linking chain from 1 to 30 carbons in length, wherein
one or more carbon is optionally substituted with a heteroatom selected from the
group consisting of N, S, O and P, and wherein the alkyl linking group optionally
includes one or more sites of unsaturation, and an alkyl linking chain from 1 to
30 carbons in length, wherein one or more carbon is optionally replaced with a
heteroatom selected from the group consisting of N, S, O and P, and wherein the
alkyl linking group optionally includes one or more sites of unsaturation;
L
2 is a linking chain selected from the group of alkyl linking
chains consisting of an alkyl linking chain from 1 to 30 carbons in length, wherein
one or more carbon is optionally substituted with a heteroatom selected from the
group consisting of N, S, O and P, and wherein the alkyl linking group optionally
includes one or more sites of unsaturation, and an alkyl linking chain from 1 to
30 carbons in length, wherein one or more carbon is optionally replaced with a
heteroatom selected from the group consisting of N, S, O and P, and wherein the
alkyl linking group optionally includes one or more sites of unsaturation;
Y is selected from the group consisting of a dimethoxytrityl protecting group
and
a photoclevable protecting group;
Z is selected from the group consisting of a dimethoxytrityl (DMT) protecting
group
and a photoclevable protecting group; and
X is a nucleic acid integration element comprising a phosphorous atom.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 provides a synthesis scheme for the synthesis of fluorescent amidite (7).
FIG. 2 provides a post-synthetic labeling scheme for oligonucleotide probe arrays.
FIG. 3 provides a chromatogram of a fluorescein-labeled T
16 homopolymer.
DEFINITIONS
Unless defined otherwise, technical and scientific terms used herein have
the same meaning as commonly understood by one of ordinary skill in the art to
which this invention belongs. Singleton et al. (1994)
Dictionary of Microbiology
and Molecular Biology, second edition, John Wiley and Sons (New York) and March
(March,
Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th
ed J. Wiley and Sons (New York, 1992) provides one of skill with a general guide
to many of the terms used in this invention.
Although one of skill will recognize many methods and materials similar
or equivalent to those described herein which can be used in the practice of the
present invention, the preferred methods and materials are described. For purposes
of the present invention, the following terms are defined below.
An "activating agent" refers to those groups which, when attached to a particular
functional group or reactive site, render that site more reactive toward covalent
bond formation with a second functional group or reactive site. For example, the
group of activating groups which are useful for a carboxylic acid include simple
ester groups and anhydrides. The ester groups include alkyl, aryl and alkenyl esters,
and in particular such groups as 4-nitrophenyl, N-succinimidyl and pentafluorophenyl.
Other activating agents are known to those of skill in the art.
An "alkyl" or "lower alkyl" group refers to a saturated hydrocarbon or hydrocarbon
radical which includes a straight or branched chain (for example, methyl, ethyl,
isopropyl, t-amyl, or 2,5-dimethylhexyl). Preferred alkyl groups are those containing
1 to 15 carbon atoms or more preferably contain 1-6 carbon atoms unless otherwise
indicated (e.g., for certain alkyl linking groups described herein, the optimal
length of the alkyl chain is from 1 to 30 carbons in length). When "alkyl" or "alkylene"
is used to refer to a linking group or a spacer moiety, it is taken to be a group
having at least two available valences for covalent attachment, for example, —CH
2CH
2—,
—CH
2CH
2CH
2—, —CH
2CH
2CH(CH
3)CH
2—
and —CH
2(CH
2CH
2)
2CH
2—.
The hydrocarbon chain is optionally substituted with heteroatoms such as N, O,
P, and S, or similarly, one or more carbons can be replaced with a heteroatom.
The alkyl chain optionally includes one or more site of unstauration (e.g., a C═C bond).
An "aryl" group as used herein, refers to an aromatic substituent which is a
single
or multiple ring structure, linked covalently or linked to a common group such
as an ethylene or methylene moiety. The aromatic rings may each contain heteroatoms,
for example, phenyl, naphthyl, biphenyl, diphenylmethyl, 2,2-diphenyl-1-ethyl,
thienyl, pyridyl and quinoxalyl. The aryl moieties may also be optionally substituted
with halogen atoms, or other groups such as nitro, carboxyl, alkoxy, phenoxy and
the like. Additionally, the aryl radicals may be attached to other moieties at
any position on the aryl radical which would otherwise be occupied by a hydrogen
atom (such as, for example, 2-pyridyl, 3-pyridyl and 4-pyridyl). As used herein,
the term "aralkyl" refers to an alkyl group bearing an aryl substituent (for example,
benzyl, phenylethyl, 3-(4-nitrophenyl)propyl, and the like). All numerical ranges
in this specification and claims are intended to be inclusive of their upper and
lower limits.
The term "capping" in the context of synthesizing an array of polymers refers
to a step in which unreacted groups that fail to condense and successfully couple
with the next polymer synthesis reagent (e.g., a monomer such as a phosphoramidite
or amino acid) are blocked. This insures that subsequent reactions proceed only
by propagating chains of desired sequence. For instance, capping typically involves
the acetylation of 5′-hydroxyl functions on oligonucleotides. This is accomplished,
e.g., using acetic anhydride catalyzed by 4-dimethylaminopyridine (DMAP). Other
reagents known to those of skill in the art are also suitable.
The term "label" refers to a composition detectable by spectroscopic, photochemical,
biochemical, immunochemical, or chemical means. For example, useful labels include
32P,
35S, fluorescent dyes, chromophores, electron-dense
reagents, enzymes (e.g., as commonly used in an ELISA), biotin, dioxigenin, or
haptens and proteins for which antisera or monoclonal antibodies are available.
A "labeling linker" is a polymer synthesis reagent which contains a labeling
site
and a polymer attachment site. The chemical nature of the polymer attachment site
is selected to be compatible with the polymer to be synthesized. Thus, where the
polymer is an oligonucleotide, the polymer attachment site comprises an oxygen
or phosphate for attachment to the 3′ OH or 5′ phosphate of the oligonucleotide.
A "polymer labeling site" or "labeling site" in the context of the labeling linker,
or polymers in a polymer array is a chemical site which is reactive with the reactive
groups in a label or synthetic labeling reagent. Thus, the precise chemical composition
of the site depends on the chemical nature of the reactive site on the label. Thus,
where the label comprises a phosphate, the labeling site would optionally include
an oxygen (e.g., a double-bond O or hydroxyl), or other phosphate-reactive moiety.
It is assumed that one of skill is familiar with many chemical sites which react
to form a chemical bond, including those which occur during nucleic acid synthesis
and polynucleotide synthesis. In many embodiments, the labeling site is protected
with a protecting group. It is generally understood that nucleic acid reagents
carry a protected phosphate or hydroxyl group in order to form a phosphodiester linkage.
In preferred embodiments, the labeling linker comprises two polymer attachment
sites, such that the polymer is extended from the labeling linker. In many embodiments,
the labeling linker is attached to a cleavable linker at one end, and to a polymer
at the other end. Thus, in this embodiment, the labeling linker has three attachment
sites: one site for forming a chemical bond to the cleavable linker, one site for
forming a chemical bond to a monomer from which the polymer is elongated, and one
site for forming a chemical bond to the detectable label.
A "nucleic acid reagent" is a molecule which can be used for oligonucleotide
synthesis.
The molecules typically carry protected phosphates and/or protected oxygen moieties.
For instance, nucleic acid reagents include nucleotide reagents, nucleoside reagents,
nucleoside phosphates, nucleoside-3′-phosphates, nucleoside phosphoramidites,
phosphoramidites, nucleoside phosphonates, phosphonates, methyl phosphonates, O-methyl
phosphates etc. It is generally understood that nucleotide reagents carry a protected
phosphate or hydroxyl group in order to form a phosphodiester linkage.
A "nucleoside" is a pentose glycoside in which the aglycone is a heterocyclic
base;
upon the addition of a phosphate group the compound becomes a "nucleotide". The
major biological nucleosides are β-glycoside derivatives of D-ribose or D-2-deoxyribose.
Nucleotides are phosphate esters of nucleosides which are generally acidic in solution
due to hydroxy groups on the phosphate. The nucleosides of the polymeric nucleic
acids DNA and RNA are connected together via phosphate units attached to the 3′
position of one pentose and the 5′ position of the next pentose. Nucleotide
analogues and/or nucleoside analogues are molecules with structural similarities
to the naturally occurring nucleotides or nucleosides. Means of converting a nucleoside
to a phosphoramidite are well known to those of skill in the art. See, for example,
Atkinson et al., chapter 3, in Gait, ed.,
Oligonucleotide Synthesis: A Practical
Approach (IRL Press, Washington, D.C., 1984), which is incorporated herein
by reference, and McBride and Caruthers,
Tetrahedron Lett., 24: 245 (1983).
See, also Blackburn and Gait (eds) (1990)
Nucleic Acids in Chemistry and Biology
IRL press, NY.
A "nucleic acid" is a deoxyribonucleotide or ribonucleotide polymer in either
single-
or double-stranded form, and unless otherwise limited, encompasses known analogs
of natural nucleotides that function in a manner similar to naturally occurring
nucleotides (See, copending application U.S. Ser. No. 06/440742 for a description
of nucleic acid analogues).
An "oligonucleotide" is a nucleic acid polymer composed of two or more nucleotides
or nucleotide analogues. An oligonucleotide can be derived from natural sources
but is often synthesized chemically. It is of any size. Copending application U.S.
Ser. No. 06/440742 describes a variety of oligonucleotide analogues.
A "polymer" refers to a chain of monomers, typically connected through chemical
or electrostatic interactions. Monomers include, but are not limited to, biological
monomers such as L-amino acids, D-amino acids, synthetic amino acids, nucleotides,
nucleosides, phosphoramidites, and carbohydrates, as well as non-biological monomers
which are connected to form polymers. As used herein, monomer refers to any unit
of a polymer. For example, dimers of the 20 naturally occurring L-amino acids form
400 monomeric units for synthesis of polypeptides. Different monomeric units are
optionally used at any site in the polymer. Each monomeric unit optionally includes
protected members which are optionally modified after polymerization. Biological
polymers include, but are not limited to, agonists and antagonists of cell membrane
receptors, toxins and venoms, viral epitopes, hormones (e.g., opiates, steroids,
etc.), hormone receptors, peptides, retro-inverso peptides, polymers of α-,
β-, or ω-amino acids (D- or L-), enzymes, enzyme substrates, cofactors,
drugs, lectins, sugars, nucleic acids (both linear and cyclic polymer configurations),
oligosaccharides, proteins, phospholipids and antibodies. Synthetic polymers such
as heteropolymers in which a known drug is covalently bound to any of the above,
such as polyurethanes, polyesters, polycarbonates, polyureas, polyamides, polyethyleneimines,
polyarylene sulfides, polysiloxanes, polyimides, and polyacetates are also included.
Other polymers will also be apparent to one of skill upon review of this disclosure.
A "peptide" or "polypeptide" or "protein" refers to a polymer of amino acids.
Typically
the monomers are alpha amino acids which are joined together through amide bonds.
In the context of this specification, the L-optical isomer and the D-optical isomer
are both contemplated, unless otherwise indicated. Standard abbreviations for amino
acids are used (e.g., P for proline). These abbreviations are included, e.g., in
Stryer,
Biochemistry, Third Edition, 1988.
A "polymer array" is a spatially defined pattern of polymers on a solid support.
A "preselected array of polymers" is a spatially defined pattern of polymers on
a solid support which is designed before being constructed (i.e., the arrangement
of polymers on solid substrate during synthesis is deliberate, and not random).
A "protecting group" as used herein, refers to any of the groups which are designed
to block one reactive site in a molecule while a chemical reaction is carried out
at another reactive site. More particularly, the protecting groups used herein
can be any of those groups described in Greene, et al.,
Protective Groups In
Organic Chemistry, 2nd Ed., John Wiley & Sons, New York, N.Y., 1991, which
is incorporated herein by reference. The proper selection of protecting groups
for a particular synthesis is governed by the overall methods employed in the synthesis.
For example, in "light-directed" synthesis, discussed herein, the protecting groups
are typically photolabile protecting groups such as NVOC, MeNPoc, and those disclosed
in co-pending Application PCT/US93/10162 (filed Oct. 22, 1993), incorporated herein
by reference. In other methods, protecting groups are removed by chemical methods
and include groups such as FMOC, DMT and others known to those of skill in the art.
The term "protected amino acid" refers to an amino acid, typically an α-amino
acid having either or both the amine functionality and the carboxylic acid functionality
suitably protected by one of the groups described above. Additionally, for those
amino acids having reactive sites or functional groups on a side chain (i.e., serine,
tyrosine, glutamic acid), the term "protected amino acid" is meant to refer to
those compounds which optionally have the side chain functionality protected as well.
A "solid substrate" has a fixed organizational support matrix, such as silica,
polymeric materials, or glass. In some embodiments, at least one surface of the
substrate is partially planar. In other embodiments it is desirable to physically
separate regions of the substrate to delineate synthetic regions, for example with
trenches, grooves, wells or the like. Example of solid substrates include slides,
beads and polymeric chips. A solid support is "functionalized" to permit the coupling
of monomers used in polymer synthesis. For example, a solid support is optionally
coupled to a nucleoside monomer through a covalent linkage to the 3′-carbon
on a furanose. Solid support materials typically are unreactive during polymer
synthesis, providing a substratum to anchor the growing polymer. Solid support
materials include, but are not limited to, glass, polacryloylmorpholide, silica,
controlled pore glass (CPG), polystyrene, polystyrene/latex, and carboxyl modified
teflon. The solid substrates are biological, nonbiological, organic, inorganic,
or a combination of any of these, existing as particles, strands, precipitates,
gels, sheets, tubing, spheres, containers, capillaries, pads, slices, films, plates,
slides, etc. depending upon the particular application. In light-directed synthetic
techniques, the solid substrate is often planar but optionally takes on alternative
surface configurations. For example, the solid substrate optionally contains raised
or depressed regions on which synthesis takes place. In some embodiments, the solid
substrate is chosen to provide appropriate light-absorbing characteristics. For
example, the substrate may be a polymerized Langmuir Blodgett film, functionalized
glass, Si, Ge, GaAs, GaP, SiO
2, SiN
4, modified silicon, or
any one of a variety of gels or polymers such as (poly)tetrafluoroethylene, (poly)vinylidendifluoride,
polystyrene, polycarbonate, or combinations thereof. Other suitable solid substrate
materials will be readily apparent to those of skill in the art. Preferably, the
surface of the solid substrate will contain reactive groups, such as carboxyl,
amino, hydroxyl, thiol, or the like. More preferably, the surface is optically
transparent and has surface Si—OH functionalities, such as are found on silica
surfaces. A substrate is a material having a rigid or semi-rigid surface. In spotting
or flowing VLSIPS™ techniques, at least one surface of the solid substrate
is optionally planar, although in many embodiments it is desirable to physically
separate synthesis regions for different polymers with, for example, wells, raised
regions, etched trenches, or the like. In some embodiments, the substrate itself
contains wells, trenches, flow through regions, etc. which form all or part of
the regions upon which polymer synthesis occurs.
DETAILED DESCRIPTION
The present invention provides methods and compositions for monitoring and optimizing
polymer array synthesis. The methods presented herein typically proceed by incorporating
a detectable label into polymers in arrays on a solid substrate. The labeled polymers
in the array are then cleaved from the solid surface, typically by cleaving a cleavable
linker which attaches the polymers of the array to the solid surface. The polymers
are then analyzed by monitoring the detectable label in an appropriate assay, i.e.,
determined by the label. A variety of analytic assays such as HPLC, gel electrophoresis
or capillary gel electrophoresis (CGE) are contemplated.
One aspect of interest during the manufacture of a VLSIPS™ array is the
efficiency of polymer synthesis in the array, including the length distribution
of synthesized species and the presence, nature and extent of truncated species.
Measuring the size and/or electrostatic charge of polymers cleaved from an array,
and comparing the measurements to the predicted size and/or charge of the polymers
provides a measure of the efficiency of polymer synthesis. Varying synthesis protocols
and comparing the resulting polymer arrays also provides an efficient empirical
strategy for optimizing the procedures for producing polymer arrays.
The effect of environmental, synthetic, or experimental conditions on polymer
arrays is also determined by the methods presented herein. In one class of embodiments,
a reference polymer array and a test polymer array are synthesized using identical
procedures. The reference polymer array is cleaved from the solid support after
synthesis and analyzed as described herein. The test polymer array is subjected
to defined additional environmental conditions such as immersion in an aqueous,
acidic, or basic solution, or exposure to nucleases or peptidases, and then analyzed.
By comparing the polymers of the array (or of identical sets of arrays) before
and after exposure to the defined environmental conditions, the stability and durability
of the array is determined.
Although essentially any detectable label can be used, in preferred embodiments
the label is monoisomeric, i.e., the label has only a single optical isomer. The
use of monoisomeric labels avoids any ambiguity in monitoring the size or charge
of polymers in an array caused by having an enantiomeric or diastereomeric label.
The use of mono-isomeric labels is particularly useful when the detection method
is extremely sensitive. For instance, the use of mono-isomeric labels when the
detection method is HPLC is particularly preferred.
Synthesis of Polymer Arrays
The present invention relates to the creation of labeled polymer arrays. The
synthesis of polymer arrays generally is known. The development of very large scale
immobilized polymer synthesis (VLSIPS™) technology provides methods for
arranging large numbers of polymer probes in very small arrays. Pirrung et al.,
U.S. Pat. No. 5,143,854 (see also PCT Application No. WO 90/15070), McGall et al.,
Ser. No. 06/440,742, Chee et al. SN PCT/US94/12305, and Fodor et al., PCT Publication
No. WO 92/10092 describe methods of forming vast arrays of peptides, oligonucleotides
and other polymers using, for example, light-directed synthesis techniques. See
also, U.S. Ser. No. 07/796,243 and 07/980,523, Fodor et al. (1991)
Science 251:767-777,
and Ser. No. 08/327,687.
As described above, diverse methods of making polymer arrays are known; accordingly
no attempt is made to describe or catalogue all known methods. For exemplary purposes,
light directed VLSIPS™ methods are briefly described below. One of skill
will understand that alternate methods of creating polymer arrays, such as spotting
and/or flowing reagents over defined regions of a solid substrate, bead based methods
and pin-based methods are also known and applicable to the present invention (See,
Holmes et al. (filed Jan. 17, 1995) U.S. Ser. No. 08/374,492).
Light directed VLSIPS™ methods are found, e.g., in U.S. Pat. No. 5,143,854.
The light directed methods discussed in the '854 patent typically proceed by activating
predefined regions of a substrate or solid support and then contacting the substrate
with a preselected monomer solution. The predefined regions are activated with
a light source, typically shown through a photolithographic mask. Other regions
of the substrate remain inactive because they are blocked by the mask from illumination.
Thus, a light pattern defines which regions of the substrate react with a given
monomer. By repeatedly activating different sets of predefined regions and contacting
different monomer solutions with the substrate, a diverse array of polymers is
produced on the substrate. Other steps, such as washing unreacted monomer solution
from the substrate, are used as necessary.
The surface of a solid support is typically modified with linking groups having
photolabile protecting groups (e.g., NVOC or MeNPOC) and illuminated through a
photolithographic mask, yielding reactive groups (e.g., typically hydroxyl groups
when the polymer array is an oligonucleotide array) in the illuminated regions.
For instance, during oligonucleotide synthesis, a 3′-O-phosphoramidite (or
other nucleic acid synthesis reagent) activated deoxynucleoside (protected at the
5′-hydroxyl with a photolabile group) is then presented to the surface and
coupling occurs at sites that were exposed to light in the previous step. Following
capping, and oxidation, the substrate is rinsed and the surface illuminated through
a second mask, to expose additional hydroxyl groups for coupling. A second 5′-protected,
3′-O-phosphoramidite activated deoxynucleoside (or other monomer as appropriate)
is then presented to the resulting array. The selective photodeprotection and coupling
cycles are repeated until the desired set of oligonucleotides (or other polymers)
is produced.
Making Polymers to be Coupled into Arrays
As described above, several methods for the synthesis of polymer arrays are known.
In preferred embodiments, the polymers are synthesized directly on a solid surface
as described above. However, in certain embodiments, it is useful to synthesize
the polymers and then couple the polymers to the solid substrate to form the desired
array. In these embodiments, polymers are synthesized (in vitro or in vivo, in
solution, or using solid phase chemistry) and then attached to a solid substrate
in a desired pattern to form the desired array on the solid substrate.
Molecular cloning and expression techniques for the synthesis of biological
and synthetic polymers in solution are known in the art. A wide variety of cloning
and expression and in vitro methods suitable for the construction of polymers are
well-known to persons of skill. Examples of techniques and instructions sufficient
to direct persons of skill through many cloning exercises for the expression and
purification of biological polymers (DNA, RNA, proteins) are found in Berger and
Kimmel,
Guide to Molecular Cloning Techniques, Methods in Enzymology volume
152 Academic Press, Inc., San Diego, Calif. (Berger); Sambrook et al. (1989)
Molecular
Cloning—A Laboratory Manual (2nd ed.) Vol. 1-3, Cold Spring Harbor Laboratory,
Cold Spring Harbor Press, NY, (Sambrook); and
Current Protocols in Molecular
Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between
Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1994 Supplement) (Ausubel).
Examples of techniques sufficient to direct persons of skill through in
vitro methods of polymer synthesis in solution, including enzymatic methods such
as the polymerase chain reaction (PCR), the ligase chain reaction (LCR), Qβ-replicase
amplification (QBR), nucleic acid sequence based amplification (NASBA), strand
displacement amplification (SDA), the cycling probe amplification reaction (CPR),
branched DNA (bDNA) and other DNA and RNA polymerase mediated techniques are known.
Examples of these and related techniques are found in Berger, Sambrook, and Ausubel,
as well as Mullis et al., (1987) U.S. Pat. No. 4,683,202
; PCR Protocols A Guide
to Methods and Applications (Innis et al. eds) Academic Press Inc. San Diego,
Calif. (1990) (Innis); Arnheim & Levinson (Oct. 1, 1990); WO 94/11383; Vooijs et
al. (1993)
Am J. Hum. Genet. 52: 586-597
; C&EN 36-47
; The Journal
Of NIH Research (1991) 3, 81-94; (Kwoh et al. (1989)
Proc. Natl. Acad. Sci.
USA 86, 1173; Guatelli et al. (1990)
Proc. Natl. Acad. Sci. USA 87,
1874; Lomell et al. (1989)
J. Clin. Chem 35, 1826; Landegren et al., (1988)
Science 241, 1077-1080; Van Brunt (1990)
Biotechnology 8, 291-294;
Wu and Wallace, (1989)
Gene 4, 560; Sooknanan and Malek (1995)
Bio/Technology
13, 563-564; Walker et al.
Proc. Natl. Acad. Sci. USA 89, 392-396),
and Barringer et al. (1990)
Gene 89, 117. Improved methods of cloning in
vitro amplified nucleic acids are described in Wallace et al., U.S. Pat. No. 5,426,039.
Methods of producing polymers in vitro and in vivo, such as polyclonal and
monoclonal antibodies are also known to those of skill in the art. See, e.g., Coligan
(1991)
Current Protocols in Immunology Wiley/Greene, NY; and Harlow and
Lane (1989)
Antibodies: A Laboratory Manual Cold Spring Harbor Press, NY;
Stites et al. (eds.)
Basic and Clinical Immunology (4th ed.) Lange Medical
Publications, Los Altos, Calif., and references cited therein; Goding (1986)
Monoclonal
Antibodies: Principles and Practice (2d ed.) Academic Press, New York, N.Y.;
and Kohler and Milstein (1975)
Nature 256: 495-497. Other suitable techniques
for antibody preparation include selection of libraries of recombinant antibodies
in phage or similar vectors. See, Huse et al. (1989)
Science 246: 1275-1281;
and Ward, et al. (1989)
Nature 341: 544-546.
Solid phase synthesis of polymers, including biological polymers is also known.
See, e.g., Merrifield (1963)
J. Am. Chem. Soc. 85: 2149-2154. Solid-phase
synthesis techniques have also been provided for the synthesis of peptide sequences
on, for example, a number of "pins." See e.g., Geysen et al. (1987)
J. Immun.
Meth. 102: 259-274 and Holmes et al (filed Jan. 17, 1995) Ser. No. 08/374,492.
Other solid-phase techniques involve, for example, synthesis of various peptide
sequences on cellulose disks supported in a column. See Frank and Doring (1988)
Tetrahedron 44: 6031-6040. Still other solid-phase techniques are described
in U.S. Pat. No. 4,728,502 (Hamill) and WO 90/00626 (Beattie).
Oligonucleotide synthesis is optionally performed on commercially
available solid phase oligonucleotide synthesis machines (see, Needham-VanDevanter
et al. (1984)
Nucleic Acids Res. 12:6159-6168) or manually synthesized using
the solid phase phosphoramidite triester method described by Beaucage et al. (Beaucage
et al. (1981)
Tetrahedron Letts. 22 (20): 1859-1862) prior to attachment
on a solid substrate. Bead-based synthetic techniques are described in copending
application U.S. Ser. No. 07/762,522 (filed Sep. 18, 1991); U.S. Ser. No. 07/946,239
(filed Sep. 16, 1992); U.S. Ser. No. 08/146,886 (filed Nov. 2, 1993); U.S. Ser.
No. 07/876,792 (filed Apr. 29, 1992); PCT/US93/04145 (filed Apr. 28, 1993); and
Holmes et al. (filed Jan. 17, 1995) U.S. Ser. No. 08/374,492. Finally, as described
above, polymers are optionally synthesized using VLSIPS™ methods in arrays,
or optionally cleaved from the array and then reattached to a solid substrate to
form a second array.
Cleavable Linkers
Cleavable linking groups used in VLSIPS™ and other solid phase synthetic
techniques are known. Typically, linking groups are used to attach polymers or
labeled polymers during the organic synthesis of polymer arrays. In addition, in
some embodiments, polymer arrays are used prior to cleavage from the arrays, typically
in aqueous hybridization experiments. Thus, preferred linkers operate well under
organic and aqueous conditions, but cleave readily under specific cleavage conditions.
The linker is typically provided with a spacer having active cleavable sites. In
the particular case of oligonucleotides, for example, the spacer is selected from
a variety of molecules which can be used in organic environments associated with
synthesis as well as aqueous environments, e.g., associated with nucleic acid binding
studies. Examples of suitable spacers are polyethyleneglycols, dicarboxylic acids,
polyamines and alkylenes, substituted with, for example, methoxy and ethoxy groups.
Linking groups which facilitate polymer synthesis on solid supports and which provide
other advantageous properties for biological assays are known. In some embodiments,
the linker provides for a cleavable function by way of, for example, exposure to
an acid or base.
Additionally, the linkers have an active site on the distal end, relative
to the attachment of the linker to the solid substrate. The active sites are op
