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BACKGROUND OF THE INVENTION
The present invention relates to the synthesis and placement of materials
at known locations. In particular, one embodiment of the invention
provides a method and associated apparatus for the selective application
of an array of oligonucleotides on a substrate by way of standard
dimethoxytrityl (DMT) based chemistry. The invention may be applied in the
field of preparation of an oligomer, a peptide, a nucleic acid, an
oligosaccharide, a phospholipid, a polymer, or a drug congener
preparation, especially to create sources of chemical diversity for use in
screening for biological activity.
Industry utilizes or has proposed various techniques to synthesize arrays
of oligonucleotides. One such technique is the use of small rubber tubes
as reaction chambers to make up a single dimensional array by the
sequential addition of reagents. This technique has advantages by the use
of standard DMT based chemistry. However, a limitation with resolution
often exists with such technique. Typically the smallest cell size is
about 1 millimeter in dimension. This method also does not enable the
synthesis of a sufficiently large number of polymer sequences for
effective economical screening. A further limitation is an inability to
form an array of, for example, oligonucleotides at selected regions of a
substrate.
Other representative techniques are described in U.S. Pat. No. 5,143,854
and WO93/09668 which is hereby incorporated by reference for all purposes.
Such techniques are finding wide use and are considered pioneering in the
industry. In some applications, however, it is desirable to have
alternative techniques and chemistries for synthesis of compound
libraries.
It would be desirable to have a method and apparatus for making high
density arrays of oligonucleotides using DMT-based chemistry and other
suitable oligonucleotide synthesis chemistries, as is a method and
apparatus for conventional phosphoramidite-based synthesis of a spatially
defined array of oligomers (e.g., polynucleotides, polypeptides,
oligosaccharides, and the like) each having a substantially predetermined
sequence of residues (i.e., polymerized monomer units).
SUMMARY OF THE INVENTION
According to the present invention, a method and apparatus to form an array
of polymers, such as oligonucleotides and related polymers (e.g., peptide
nucleic acids) at selected regions of a substrate using conventional
linkage chemistries (e.g., standard DMT-based oligonucleotide synthesis
chemistry) is provided. The method and apparatus includes use of selected
printing techniques in distributing materials such as barrier materials,
deprotection agents, base groups, nucleosides, nucleotides, nucleotide
analogs, amino acids, imino acids, carrier materials, and the like to
selected regions of a substrate. Each of the printing techniques may be
used in some embodiments with, for example, standard DMT-based chemistry
for synthesis of oligonucleotides, and in particular selected deprotecting
agents in vapor form.
In a specific embodiment, the present invention provides a method of
forming polymers having diverse monomer sequences on a substrate. In an
embodiment, the method is used to synthesize oligonucleotides having
predetermined polynucleotide sequence(s) on a solid substrate, typically
in the form of a spatially defined array, wherein the sequence(s) of an
oligonucleotide is positionally determined. The present method includes
steps of providing a substrate with a linker molecule layer thereon. The
linker molecule layer has a linker molecule and a protective group. The
present method also includes a step of applying a barrier layer overlying
at least a portion of the linker molecule layer. The barrier layer shields
the underlying portion from contact with a reagent capable of otherwise
reacting with the underlying portion and applied subsequent to application
of the barrier layer, thereby substantially precluding a predetermined
chemical reaction from occurring on areas of the substrate overlaid with
the barrier material. The applying step forms selected exposed regions of
the linker molecule layer. A step of exposing the selected exposed regions
of the linker molecule layer (e.g., regions not overlaid with the barrier
material) to a reagent, typically in vapor phase, and often comprising a
deprotecting agent is also included.
In an alternative specific embodiment, the present method includes a method
of applying a medium in selected regions of a substrate. The present
method includes steps of providing a substrate with a top surface, and
selectively applying a medium having an element selected from a group
consisting of a barrier material, a receptor, a deprotection agent, a
monomer group, a carrier material, and an activator to selected regions of
the substrate top surface.
In an embodiment, the invention provides a method for synthesizing a
spatial array of polymers of diverse monomeric sequence (e.g., such as a
collection of oligonucleotides having unique sequences), wherein the
composition (e.g., nucleotide sequence) of each polymer is positionally
defined by its location in the spatial array. In general, the method
employs a masking step whereby a spatially distributed barrier material is
applied to a substrate to block at least one step of a monomer addition
cycle from occurring on a portion of the substrate overlaid by the barrier
material. The method comprises applying a barrier material to a first
spatially defined portion of a substrate, said substrate optionally also
comprising a layer of linker molecules and/or nascent polymers (e.g.,
nascent oligonucleotides), whereby the barrier material overlaying said
first spatially defined portion of said substrate shields the underlying
portion from contact with a subsequently applied reagent capable of
otherwise reacting with the underlying portion and necessary for a
complete monomer addition cycle whereby a monomer unit is covalently
linked to a nascent polymer or linker, thereby substantially precluding a
chemical reaction from occurring on said first spatially defined portion
which is overlaid with the barrier material and providing a remaining
unshielded portion of said substrate (i.e., portion(s) not overlaid with
the barrier material) available for contacting said subsequently applied
reagent and undergoing said chemical reaction necessary for a complete
monomer addition cycle (i.e., polymer elongation). The subsequently
applied reagent is typically a monomer (e.g., nucleotide, nucleoside,
nucleoside derivative, amino acid, and the like), a deprotecting agent for
removing protecting group(s) which block polymer elongation (e.g., removal
of DMT groups by acid hydrolysis), a coupling agent (e.g.,
phosphoramidites, such as cyanoethyl phosphoramidite nucleosides), a
capping agent (e.g., acetic anhydride and 1-methylimidazole), and/or an
oxidation agent (e.g., iodine; such as in
iodine:water:pyridine:tetrahydrofuran mixture). The method further
provides that, subsequent to the application of the barrier material, the
reagent(s) is/are applied and permitted to chemically react with the
unshielded portion of the substrate for a suitable time period and under
suitable reaction conditions. Following reaction of the unshielded portion
with the reagent(s), monomer addition is completed and the barrier
material is removed (not necessarily in that order), resulting in a
monomer addition to polymer(s) in the unshielded portion of the substrate
and substantial lack of monomer addition to polymer(s) in the shielded
portion of the substrate, during said monomer addition cycle.
In an embodiment, the masking step, wherein a barrier material is applied
to a spatially defined portion of the substrate and used to shield said
spatially defined portion to block a monomer addition cycle on said
spatially defined portion, is employed repetitively. A first barrier mask
is applied to overlay a first spatially defined portion of a substrate
creating: (1) a first shielded portion overlain by said barrier mask, and
(2) a first unshielded portion comprising the portion of the substrate not
overlain by said barrier mask. The application of the first barrier mask
is followed by completion of a first monomer addition cycle, whereby a
monomer unit is covalently added to the first unshielded portion to extend
or initiate a nascent polymer bound To said substrate, typically
covalently, and whereby said first monomer addition cycle substantially
fails to result in addition of a monomer unit to nascent polymers in the
first shielded portion. The first barrier mask is removed, concomitant
with, prior to, or subsequent to the completion of said first monomer
addition cycle, and one or more subsequent cycles of applying a subsequent
barrier mask, which may overlay subsequent shielded portions which is/are
spatially distinct from said first shielded portion, and performing at
least one subsequent monomer addition cycle(s) followed after each cycle
by barrier removal, and optionally, reapplication of a barrier mask and
initiation of a further monomer addition cycle until polymers of a
predetermined length (number of incorporated monomer units) are produced.
In an aspect of the invention, a repetitive masking/synthesis process can
be comprised of the following steps:
(1) application of barrier material to substrate having a reactive surface
capable of covalently bonding to a monomer unit or reacting with a
deprotecting agent or other reagent necessary for completion of a monomer
addition cycle, said reactive surface being derivatived with a linker
and/or a monomer unit or nascent polymer (e.g., a 3'-linked nucleoside or
3'-linked polynucleotide), wherein said barrier material covers a portion
of said reactive surface creating a covered portion, said covered portion
being a shielded portion and being substantially incapable of reacting
with a monomer unit or reagent necessary for completion of a monomer
addition cycle, and the remaining portion of the substrate being an
unshielded portion capable of reacting with a monomer unit or reagent
necessary for completion of a monomer addition cycle;
(2) contacting the substrate with reagents necessary for completion of a
monomer addition cycle, wherein a monomer unit is covalently attached to
the reactive surface of the substrate (e.g., a linker, a 3'-linked
nucleoside, or 3'-linked nascent polynucleotide) in an unshielded portion;
(3) removing the barrier material; and
(4) repeating steps 1, 2, and 3 from 0 to 5000 cycles, preferably from 2 to
250 cycle, more usually from 4 to 100 cycles, and typically from about 7
to 50 cycles, until a predetermined polymer length is produced on a
portion of the substrate. The pattern of barrier material applied in each
cycle may be different that the prior or subsequent cycle(s), if any, or
may be the same. Often, in step (2), at least one reagent necessary for
completion of a monomer addition cycle is applied in vapor phase.
In an embodiment of the invention is provided a substrate having a spatial
array of polymers of predetermined length produced by the method described
supra.
In one aspect of the invention is provided a method for applying a barrier
material or reagent necessary for a monomer addition cycle to a substrate,
said method comprising transferring the barrier material or reagent as a
charged droplet by electrostatic interaction, such as, for example, in an
inkier or bubble jet print head or similar device. In an embodiment, the
barrier material or reagent is suitable for use in polynucleotide
(oligonucleotide) synthesis. In an embodiment, the substrate is a silicon
or glass substrate or a charged membrane (e.g., nylon 66 or
nitrocellulose).
An aspect of the invention provides a method for synthesizing
polynucleotides on a substrate, said method comprising application of at
least one reagent necessary for addition of a nucleotide to a nascent
polynucleotide or linker molecule bound to a substrate, wherein said
application is performed with the reagent present substantially in vapor
phase.
A further understanding of the nature and advantages of the present
invention may be realized by reference to the latter portions of the
specification and attached drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1-3 illustrate simplified cross-sectional views of a substrate being
processed according to the present invention;
FIGS. 4-13 illustrate selected printing techniques according to the present
invention;
FIG. 14 illustrates a simplified cross-sectional view of an apparatus used
to achieve local selectivity;
FIG. 15 illustrates a jig used for contacting a mask to a substrate without
smearing;
FIG. 16 is a photograph of a fluorescent image of a fluoreprimed workpiece
that was selectively shielded from liquid deprotection by a lacquer;
FIG. 17 is a photograph of dots of uncured epoxy and pump oil overlying a
workpiece;
FIG. 18 illustrates a SEM photograph of a liquid uncured epoxy pattern on a
glass workpiece;
FIG. 19 illustrates a photograph of a 100 micron resolution sample with an
epoxy barrier pattern;
FIG. 20 illustrates a photograph of a 75 micron resolution sample with an
epoxy barrier pattern;
FIG. 21 is a photograph of a fluorescent pattern from vapor deprotection
through an uncoated silicon stencil mask;
FIG. 22 is a close-up version of the photograph of FIG. 21;
FIG. 23 is a photograph of an epoxy paint pattern transferred from a nickel
grid;
FIGS. 24 and 25 are photographs of fluorescent images resulting from vapor
phase deprotection through an epoxy pattern;
FIG. 26 illustrates a 2.times.2 array of oligonucleotides formed by masking
out deprotection agents after A (vertical mask) and a first T in the
synthesis of 3'-CGCATTCCG;
FIG. 27 is a scanned output of an array after hybridizing with 10 nM target
oligonucleotide 5'-GCGTAGGC-fluorescein for 15 minutes at 15.degree. C.;
FIGS. 28 and 29 are scanned outputs after hybridizing to a newly-made
sample of the same target sequence of FIGS. 26 and 27;
FIG. 30 is an array of same oligos as in FIGS. 26 and 27 made by displacing
the reaction chamber when added bases A and the first T in the sequence
3'-CGCATTCCG;
FIGS. 31 and 32 illustrate scanned outputs after hybridizing with 10 nM
5'-GCGTAGGC-fluorescein.
DESCRIPTION OF THE SPECIFIC EMBODIMENT
Glossary
The following terms are intended to have the following general meanings as
they are used herein:
1. Ligand: A ligand is a molecule that is recognized by a particular
receptor. Examples of ligand that can be investigated by this invention
include, but are not restricted to, agonists and antagonists for cell
membrane receptors, toxins and venoms, viral epitopes, hormones (e.g.,
opiates, steroids, etc.), hormone receptors, peptides, enzymes, enzyme
substrates, cofactors, drugs, lectins, sugars, oligonucleotides, nucleic
acids, oligosaccharides, proteins, and monoclonal antibodies.
2. Monomer: A member of the set of small molecules which are or can be
joined together to form a polymer. The set of monomers includes but is not
restricted to, for example, the set of common L-amino acids, the set of
D-amino acids, the set of synthetic and/or natural amino acids, the set of
nucleotides and the set of pentoses and hexoses. The particular ordering
of monomers within a polymer is referred to herein as the "sequence" of
the polymer. As used herein, monomers refers to any member of a basis set
for synthesis of a polymer, which include for example and not limitation,
polynucleotides, polypeptides, and small molecules such as
benzodiazepines, .beta.-turn mimetics, and protoprostaglandins, among
others. For example, dimers of the 20 naturally occurring L-amino acids
form a basis set of 400 monomers for synthesis of polypeptides. Different
basis sets of monomers may be used at successive steps in the synthesis of
a polymer. Furthermore, each of the sets may include protected members
which are modified after synthesis. The invention is described herein
primarily with regard to the preparation of molecules containing sequences
of monomers such as amino acids, but could readily be applied in the
preparation of other polymers. Such polymers include, for example, both
linear and cyclic polymers of nucleic acids, polysaccharides,
phospholipids, and peptides having either .alpha.-, .beta.-, OR
.omega.-amino acids, heteropolymers in which a known drug is covalently
bound to any of the above, polynucleotides, polyurethanes, polyesters,
polycarbonates, polyureas, polyamides, polyethyleneimines, polyarylene
sulfides, polysiloxanes, polyimides, polyacetates, or other polymers which
will be apparent upon review of this disclosure. Such polymers are
"diverse" when polymers having different monomer sequences are formed at
different predefined regions of a substrate. Methods of cyclization and
polymer reversal of polymers are disclosed in application Ser. No.
07/796,727 filed Nov. 22, 1991 (now U.S. Pat. No. 5,242,974 issued Sep. 7,
1993, entitled "POLYMER REVERSAL ON SOLID SURFACES," incorporated herein
by reference for all purposes. One set of polymers is polynucleotides and
peptide nucleic acids.
3. Peptide: A polymer in which the monomers are alpha amino acids and which
are joined together through amide bonds, alternatively referred to as a
polypeptide. In the context of this specification it should be appreciated
that the amino acids may be the L-optical isomer or the D-optical isomer.
Peptides are often two or more amino acid monomers long, and often more
than 20 amino acid monomers long. Standard abbreviations for amino acids
are used (e.g., P for proline). These abbreviations are included in
Stryer, Biochemistry, Third Ed., 1988, which is incorporated herein by
reference for all purposes. Peptide analogs are commonly used in the
pharmaceutical industry as non-peptide drugs with properties analogous to
those of the template peptide. These types of non-peptide compound are
termed "peptide mimetics" or "peptidomimetics" (Fauchere, J. (1986) Adv.
Drug Res. 15: 29; Veber and Freidinger (1985) TINS p.392 and Evans et al.
(1987) J. Med. Chem 30: 1229, which are incorporated herein by reference)
and are often developed with the aid of computerized molecular modeling.
Peptide mimetics that are structurally similar to therapeutically useful
peptides may be used to produce an equivalent therapeutic or prophylactic
effect. Generally, peptidomimetics have one or more peptide linkages
optionally replaced by a linkage selected from the group consisting of:
--CH.sub.2 NH--, --CH.sub.2 S--, --CH.sub.2 --CH.sub.2 --,
--CH.dbd.CH--(cis and trans), --COCH.sub.2 --, --CH(OH)CH.sub.2 --, and
--CH.sub.2 SO--, by methods known in the art and further described in the
following references: Spatola, A. F. in "Chemistry and Biochemistry of
Amino Acids, Peptides, and Proteins," B. Weinstein, eds., Marcel Dekker,
New York, p. 267 (1983); Spatola, A. F., Vega Data (March 1983), Vol. 1,
Issue 3, "Peptide Backbone Modificaticns" (general review); Morley, J. S.,
Trends Pharm Sci (1980) pp. 463-468 (general review); Hudson, D. et al.,
Int J Pept Prot Res (1979) 14:177-185 (--CH.sub.2 NH--, CH.sub.2 CH.sub.2
--); Spatola, A. F. et al., Life Sci (1986) 38:1243-1249 (--CH.sub.2 --S);
Hann, M. M., J Chem Soc Perkin Trans I (1982) 307-314 (--CH--CH--, cis and
trans); Almquist, R. G. et al., J Med Chem (1980) 23:1392-1398
(--COCH.sub.2 --); Jennings-White, C. et al., Tetrahedron Lett (1982)
23:2533 (--COCH.sub.2 --); Szelke, M. et al., European Appln. EP 45665
(1982) CA: 97:39405 (1982) (--CH(OH)CH.sub.2 --); Holladay, M. W. et al.,
Tetrahedron Lett (1983) 24:4401-4404 (--C(OH)CH.sub.2 --); and Hruby, V.
J., Life Sci (1982) 31:189-199 (--CH.sub.2 --S--); each of which is
incorporated herein by reference. A particularly preferred non-peptide
linkage is --CH.sub.2 NH--. Such peptide mimetics may have significant
advantages over polypeptide embodiments, including, for example: more
economical production, greater chemical stability, enhanced
pharmacological properties (half-life, absorption, potency, efficacy,
etc.), altered specificity (e.g., a broad-spectrum of biological
activities), reduced antigenicity, and others. Systematic substitution of
one or more amino acids of a consensus sequence with a D-amino acid of the
same type (e.g., D-lysine in place of L-lysine) may be used to generate
more stable peptides. In addition, constrained peptides (including
cyclized peptides) comprising a consensus sequence or a substantially
identical consensus sequence variation may be generated by methods known
in the art (Rizo and Gierasch (1992) Ann. Rev. Biochem. 61: 387,
incorporated herein by reference); for example, by adding internal
cysteine residues capable of forming intramolecular disulfide bridges
which cyclize the peptide.
4. Receptor: A molecule that has an affinity for a given ligand. Receptors
may be naturally-occurring or manmade molecules. Also, they can be
employed in their unaltered state or as aggregates with other species.
Receptors may be attached, covalently or noncovalently, to a binding
member, either directly or via a specific binding substance. Examples of
receptors which can be employed by this invention include, but are not
restricted to, antibodies, cell membrane receptors, monoclonal antibodies
and antisera reactive with specific antigenic determinants (such as on
viruses, cells or other materials), drugs, polynucleotides, nucleic acids,
peptides, cofactors, lectins, sugars, polysaccharides, cells, cellular
membranes, and organelles. Receptors are sometimes referred to in the art
as anti-ligands. As the term receptors is used herein, no difference in
meaning is intended. A "Ligand Receptor Pair" is formed when two
macromolecules have combined through molecular recognition to form a
complex. Specific examples of receptors which can be investigated by this
invention include but are not restricted to:
a) Microorganism receptors: Determination of ligands which bind to
receptors, such as specific transport proteins or enzymes essential to
survival of microorganisms, is useful in a new class of antibiotics. Of
particular value would be antibiotics against opportunistic fungi,
protozoa, and those bacterial resistant to the antibiotics in current use.
b) Enzymes: For instance, the binding site of enzymes such as the enzymes
responsible for cleaving neurotransmitters; determination of ligands which
bind to certain receptors to modulate the action of the enzymes which
cleave the different neurotransmitters is useful in the development of
drugs which can be used in the treatment of disorders of
neurotransmission.
c) Antibodies: For instance, the invention may be useful in investigating
the ligand-binding site on the antibody molecule which combines with the
epitope of an antigen of interest; determining a sequence that mimics an
antigenic epitope may lead to the development of vaccines of which the
immunogen is based on one or more of such sequences or led to the
development of related diagnostic agents or compounds useful in
therapeutic treatments such as for auto-immune diseases (e.g., by blocking
the binding of the "self" antibodies).
d) Nucleic Acids: Sequences of nucleic acids may be synthesized to
establish DNA or RNA binding sequences. Polynucleotides, which include
oligonucleotides, are composed of nucleotides, typically linked 5' to 3'
by a phosphodiester bond or phosphorothiolate bond or the like. The term
"corresponds to" is used herein to mean that a polynucleotide sequence is
homologous (i.e., is identical, not strictly evolutionarily related) to
all or a portion of a reference polynucleotide sequence, or that a
polypeptide sequence is identical to a reference polypeptide sequence. In
contradistinction, the term "complementary to" is used herein to mean that
the complementary sequence is homologous to all or a portion of a
reference polynucleotide sequence. For illustration, the nucleotide
sequence "TATAC" corresponds to a reference sequence "TATAC" and is
complementary to a reference sequence "GTATA". Polynucleotides can include
nucleotides having a variety of bases, including but not limited to:
adenine, thymine, cytosine, guanine, uridine, inosine, deazaguanosine,
N.sup.2 -dimethylguanosine, 7-methylguanosine, N.sup.6 -.DELTA..sup.2
isopentenyl-2-methylthioadenosine, 2'-O-methyladenine, 2'-O-methylthymine,
2'-O-methylcytosine, 2'-O-methylguanine, pseudouridine, dihydrouridine,
4-thiouridine, and the like.
e) Catalytic Polypeptides: Polymers, preferably polypeptides, which are
capable of promoting a chemical reaction involving the conversion of one
or more reactants to one or more products. Such polypeptides generally
include a binding site specific for at least one reactant or reaction
intermediate and an active functionality proximate to the binding site,
which functionality is capable of chemically modifying the bound reactant.
Catalytic polypeptides and others are described in, for example, PCT
Publication No. WO 90/05746, WO 90/05749, and WO 90/05785, which are
incorporated herein by reference for all purposes.
f) Hormone Receptors: For instance, the receptors for insulin and growth
hormone. Determination of the ligands which bind with high affinity to a
receptor is useful in the development of, for example, an oral replacement
of the daily injections which diabetics must take to relieve the symptoms
of diabetes, and in the other case, a replacement for the scarce human
growth hormone which can only be obtained from cadavers or by recombinant
DNA technology. Other examples are the vasoconstrictive hormone receptors;
determination of those ligands which bind to a receptor may lead to the
development of drugs to control blood pressure.
g) Opiate receptors: Determination of ligands which bind to the opiate
receptors in the brain is useful in the development of less-addictive
replacements for morphine and related drugs.
5. Substrate: A material having a rigid or semi-rigid surface. In many
embodiments, at least one surface of the substrate will be substantially
flat, although in some embodiments it may be desirable to physically
separate synthesis regions for different polymers with, for example,
wells, raised regions, etched trenches, or the like. According to other
embodiments, small beads may be provided on the surface which may be
released upon completion of the synthesis. Often, the substrate is a
silicon or glass surface, or a charged membrane, such as nylon 66 or
nitrocellulose.
6. Protective Group: A material which is bound to a monomer unit and which
may be selectively removed therefrom to expose an active site such as, in
the specific example of an amino acid, an amine group. In the specific
example of a polynucleotide synthesized via phosphoramidite chemistry, a
protecting group can be a trityl ether (DMT ether) group linked to a
nucleotide via a 5'-hydroxyl position.
7. Predefined Region: A predefined region is a localized area on a
substrate which is, was, or is intended to be used for formation of a
selected polymer and is otherwise referred to herein in the alternative as
a "selected" region or simply a "region." The predefined region may have
any convenient shape, e.g., circular, rectangular, elliptical,
wedge-shaped, etc. In some embodiments, a predefined region and,
therefore, the area upon which each distinct polymer sequence is
synthesized is smaller than about 1 cm.sup.2, more preferably less than 1
mm.sup.2, still more preferably less than 0.5 mm.sup.2, and in some
embodiments about 0.125 to 0.5 mm.sup.2. In most preferred embodiments the
regions have an area less than about 10,000 .mu.m.sup.2 or, more
preferably, less than 100 .mu.m.sup.2. Within these regions, the polymer
synthesized therein is preferably synthesized in a substantially pure
form. A shielded portion or unshielded portion can be a predefined region.
8. Substantially Pure: A polymer is considered to be "substantially pure"
within a predefined region of a substrate when it exhibits characteristics
that distinguish it from other predefined regions. Typically, purity will
be measured in terms of biological activity or function as a result of
uniform sequence. such characteristics will typically be measured by way
of binding with a selected ligand or receptor. Preferably the region is
sufficiently pure such that the predominant species in the predefined
region is the desired sequence. According to preferred aspects of the
invention, the polymer is 5% pure, more preferably more than 10% pure,
preferably more than 20% pure, and more preferably more than 80% pure,
more preferably more than 90% pure, more preferably more than 95% pure,
where purity for this purpose refers to the ratio of the number of ligand
molecules formed in a predefined region having a desired sequence to the
total number of molecules formed in the predefined region.
9. Monomer Addition Cycle: A monomer addition cycle is a cycle comprising
the chemical reactions necessary to produce covalent attachment of a
monomer to a nascent polymer or linker, such as to elongate the polymer
with the desired chemical bond (e.g., 5'-3' phosphodiester bond, peptide
bond, etc.). For example and not to limit the invention, the following
steps typically comprise a monomer additon cycle in phosphoramidite-based
oligonucleotide synthesis: (1) deprotection, comprising removal of the DMT
group from a 5' -protected nucleoside (which may be part of a nascent
polynucleotide) wherein the 5'-hydroxyl is blocked by covalent attachment
of DMT, such deprotection is usually done with a suitable deprotection
agent (e.g., a protic acid: trichloroacetic acid or dichloroacetic acid),
and may include physical removal (e.g., washing, such as with
acetonitrile) of the removed protecting group (e.g., the cleaved
dimethyltrityl group), (2) coupling, comprising reacting a phosphoramidite
nucleoside(s), often activated with tetrazole, with the deprotected
nucleoside, (3) optionally including capping, to truncate unreacted
nucleosides from further participation in subsequent monomer addition
cycles, such as by reaction with acetic anhydride and N-methylimidazole to
acetylate free 5'-hydroxyl groups, and (4) oxidation, such as by iodine in
tetrahydrofuran/water/pyridine, to convert the trivalent phosphite
triester linkage to a pentavalent phosphite triester, which in turn can be
converted to a phosphodiester via reaction with ammonium hydroxide. Thus,
with respect to phosphoramidite synthesis of polynucleotides, the
following reagents are typically necessary for a complete monomer addition
cycle: trichloroacetic acid or dichloroacetic acid, a phosphoramidite
nucleoside, an oxidizing agent, such as iodine (e.g.,
iodine/water/THF/pyridine), and optionally N-methylimidazole for capping.
10. Specific hybridization is defined herein as the formation of hybrids
between a probe polynucleotide (e.g., a polynucleotide of the invention
which may include substitutions, deletion, and/or additions) and a
specific target polynucleotide (e.g., an analyte polynucleotide) wherein
the probe preferentially hybridizes to the specific target polynucleotide
and substantially does not hybridize to polynucleotides consisting of
sequences which are not substantially identical to the target
polynucleotide. However, it will be recognized by those of skill that the
minimum length of a polynucleotide required for specific hybridization to
a target polynucleotide will depend on several factors: G/C content,
positioning of mismatched bases (if any), degree of uniqueness of the
sequence as compared to the population of target polynucleotides, and
chemical nature of the polynucleotide (e.g., methylphosphonate backbone,
phosphorothiolate, etc.), among others.
General
The present invention provides for the use of a substrate with a surface.
In preferred embodiments, linker molecules are provided on a surface of
the substrate. The purpose of the linker molecules, in certain
embodiments, is to facilitate receptor recognition of the synthesized
polymers. In preferred embodiments, the linker molecules each include a
protecticn group. A layer of barrier material may be applied to the
surface of the substrate, and in particular the linker molecule layer. The
barrier material is selectively applied by way of a variety of printing
techniques to form exposed regions. A step of deprotection by way of
deprotection agents may then be applied to the exposed regions.
Preferably, the deprotection step occurs with use of deprotection agents
in the vapor phase. This sequence of steps may be used for the selected
synthesis of an array of oligonucleotides.
The present invention also provides for use of selected printing techniques
to apply deprotection agents, barrier materials, nucleosides, and the like
for the synthesis of an array of oligonucleotides. Preferably, the type of
printing technique should be able to transfer a sufficient volume of print
material to selected regions of the substrate in an easy, accurate, and
cost effective manner. Examples of various printing techniques for the
synthesis of for example an array of oligonucleotides are described
herein. Further examples of these embodiments of the present invention may
be applied to the synthesis of arrays of DNA as explained by application
Ser. No. 07/796,243 in the name of Winkler et al., and U.S. Pat. No.
5,143,854 in the name of Pirrung et al., which are both hereby
incorporated by reference for all purposes.
Examples of suitable phosphoramidite synthesis methods are described in the
User Manual for Applied Biosystems Model 391, pp. 6-1 to 6-24, available
from Applied Biosystems, 850 Lincoln Center Dr., Foster City, Calif.
94404, and are generally known by those skilled in the art.
Chemical synthesis of polypeptides is known in the art and are described
further in Merrifield, J. (1969) J. Am. Chem. Soc. 91: 501; Chaiken I. M.
(1981) CRC Crit. Rev. Biochem. 11: 255; Kaiser et al.(1989) Science 243:
187; Merrifield, B. (1986) Science 232: 342; Kent, S. B. H. (1988) Ann.
Rev. Biochem. 57: 957; and Offord, R. E. (1980) Semisynthetic Proteins,
Wiley Publishing, which are incorporated herein by reference).
Once synthesized, polynucleotide arrays of the invention have many
art-recognized uses. For example and not limitation, the synthesized
sequences may be used as hybridization probes or PCR amplimers to detect
the presence of a specific DNA or mRNA, for example to diagnose a disease
characterized by the presence of an elevated mRNA level in cells, to
identify a disease allele, or to perform tissue typing (i.e., identify
tissues characterized by the expression of a particular mRNA), and the
like. The sequences may also be used for detecting genomic gene sequences
in a DNA sample, such as for forensic DNA analysis (e.g., by RFLP
analysis, PCR product length(s) distribution, etc.) or for diagnosis of
diseases characterized by amplification and/or rearrangements of a
characteristic gene.
Embodiments of the Present Invention
An embodiment of the present invention may be briefly outlined by way of
the following method.
1. Provide a substrate.
2. Optionally, form a layer of linker molecules on the substrate.
3. Mechanically apply a barrier pattern on the linker molecules with
exposed regions.
4. Deprotect the linker molecules in the exposed regions with standard DMT
chemistry.
5. Strip barrier pattern.
6. Apply remaining synthesis steps.
This sequence of steps provides for an embodiment with use of a barrier
layer with standard DMT chemistry. This provides for a desired
selectivity, easy in synthesis, low costs, high contrast, high resolution,
among other features. Of course, this sequence of steps is shown for
illustrative purposes only, and should not limit the scope of the appended
claims herein.
An alternative embodiment of the present invention may be briefly outlined
by way of the following method.
1. Provide a substrate.
2. Optionally, form a layer of linker molecules on the substrate.
3. Selectively apply a print media by way of a printing technique (not a
photosensitive printing techniques) on the linker molecules.
4. Apply remaining synthesis steps.
This sequence of steps allows for the selective application of a print
medium onto a substrate by way of the various printing techniques
described herein. These printing techniques simply do not use any exotic
photosensitive type materials, although later photosensitive steps can be
combined with the teachings herein. In preferred embodiments, deprotection
agents may be introduced onto the substrate in vapor form. Accordingly,
the present invention provides for the selective application of a variety
of print media onto a substrate without necessitating the use of
conventional photosensitive materials.
FIG. 1 illustrates one embodiment according to the present method. A
substrate 12 is shown in cross-section. The substrate may be 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, and
the like. The substrate may have any convenient shape, such as a disc,
square, sphere, circle, etc. The substrate is preferably flat but may take
on a variety of alternative surface configurations. For example, the
substrate may contain raised or depressed regions on which the synthesis
takes place. The substrate and its surface preferably form a rigid support
on which to carry out the reactions described herein. For instance, the
substrate may be a functionalized glass, Si, Ge, GaAs, GaP, SiO.sub.2,
SiN.sub.4, modified silicon, or any one of a wide variety of gels or
polymers such as (poly)tetrafluoroethylene, polypropylene, polyethylene,
(poly)vinylidenedifluoride, poly-styrene, polycarbonate, or combinations
thereof. Other substrate materials will be readily apparent to those of
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