U.S. patent application number 09/777553 was filed with the patent office on 2001-10-11 for minimization of blooming in high-density arrays by using reactive wash reagents.
Invention is credited to Kanemoto, Roy H., Perbost, Michel G. M..
Application Number | 20010029296 09/777553 |
Document ID | / |
Family ID | 22723155 |
Filed Date | 2001-10-11 |
United States Patent
Application |
20010029296 |
Kind Code |
A1 |
Perbost, Michel G. M. ; et
al. |
October 11, 2001 |
Minimization of blooming in high-density arrays by using reactive
wash reagents
Abstract
A wash reagent employed for the bulk washing of the surface of a
high-density array to remove unreacted reactants from cells of the
array while, at the same time, reacting with the unreacted monomer
in order to prevent reaction of the reacted monomer with functional
groups on the surface of the HDA outside of the region of the
surface to which the reactive monomer is applied. The wash reagent
is chosen for a particular solid-state synthesis so that the
unreacted reactants and catalyzing agents are soluble in the wash
reagent, so that the wash reagent does not react with, or catalyze,
reactions of the substrate or the biopolymers bound to the
substrate, and so that the wash reagent reacts with unreacted
reactive monomer in order to prevent subsequent reactions of the
unreacted reactive monomer.
Inventors: |
Perbost, Michel G. M.;
(Cupertino, CA) ; Kanemoto, Roy H.; (Palo Alto,
CA) |
Correspondence
Address: |
AGILENT TECHNOLOGIES
Legal Department, 51UPD
Intellectual Property Administration
P. O. Box 58043
Santa Clara
CA
95052-8043
US
|
Family ID: |
22723155 |
Appl. No.: |
09/777553 |
Filed: |
February 5, 2001 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
09777553 |
Feb 5, 2001 |
|
|
|
09195869 |
Nov 19, 1998 |
|
|
|
6184347 |
|
|
|
|
Current U.S.
Class: |
536/23.1 |
Current CPC
Class: |
B01J 2219/00608
20130101; B01J 2219/00722 20130101; B01J 2219/00659 20130101; B01J
2219/00605 20130101; C07H 19/20 20130101; B01J 2219/00527 20130101;
B01J 2219/00585 20130101; C40B 40/06 20130101; B01J 2219/00596
20130101; B01J 2219/00626 20130101; C07H 21/00 20130101; B01J
2219/00497 20130101; B01J 2219/00617 20130101; B01J 2219/00536
20130101; C07B 2200/11 20130101 |
Class at
Publication: |
536/23.1 |
International
Class: |
C07H 021/02; C07H
021/04 |
Claims
1. A method used, during the solid-state synthesis of surface-bound
polymers on a solid substrate, for removing a reaction solution
including unreacted reactive reagents from the surface of the solid
substrate and from any nascent polymers bound to the solid
substrate, the method comprising: selecting a reactive wash
solution that is not reactive toward, and does not catalyze
reactions with, the solid substrate or any nascent polymers bound
to the solid substrate but that reacts with, and deactivates, the
reactive reagents and that is miscible with the reaction solution;
and applying the reactive wash solution to the surface of the solid
substrate in order to react with, and deactivate, any unreacted
reactive reagents and to remove the reaction solution from the
surface of the solid substrate and from any nascent polymers bound
to the substrate.
2. The method of claim 1 wherein the reaction solution includes
reactive monomers and a catalyzing reagent that catalyzes the
coupling of reactive monomers to the nascent polymers.
3. The method of claim 2 wherein the reactive monomers are
deoxynucleoside phosphoramidite s and the polymers are
oligonucleotides.
4. The method of claim 3 wherein the solid substrate is a
high-density array comprising cells in which different
oligonucleotides are synthesized, the method further including:
applying the reactive wash solution separately to each cell of the
high-density array in order to react with, and deactivate, any
unreacted deoxynucleoside phosphoramidite s; and rinsing the
surface of the solid substrate with the reactive wash solution to
remove the deactivated unreacted deoxynucleoside phosphoramidite s
and catalyzing reagent from the surface of the solid substrate and
from any nascent polymers bound to the substrate.
5. The method of claim 3 wherein the solid substrate is a
high-density array comprising cells in which different
oligonucleotides are synthesized, the method further including:
applying the reactive wash solution separately to each cell of the
high-density array in order to react with, and deactivate, any
unreacted deoxynucleoside phosphoramidite s; allowing the applied
reactive wash solution and reaction solution to evaporate; and
rinsing the surface of the solid substrate with the reactive wash
solution to remove the deactivated deoxynucleoside phosphoramidite
s and catalyzing agent from the surface of the solid substrate and
from any nascent polymers bound to the substrate.
6. The method of claim 3 wherein the reactive wash solution
includes a chemical compound containing a hydroxyl functional
group.
7. The method of claim 6 wherein the reactive wash solution is
methanol.
8. The method of claim 1 wherein the reaction solution includes a
reactive dye.
9. The method of claim 1 wherein the reaction solution includes a
reactive radio-labeled marker.
10. A method for coupling a reactive monomer molecule to a nascent
polymer bound to surface of a solid substrate, the method
comprising: applying monomer molecules and any reagents required to
catalyze the coupling of the reactive monomer molecule with the
nascent polymer to the surface of the solid substrate; and applying
a reactive wash solution to the surface of the solid substrate to
react with, and deactivate, any remaining reactive monomers on the
surface of the solid substrate and to dissolve and remove the
remaining deactivated monomers and any reagents required to
catalyze the coupling of the reactive monomer molecule to the
polymer from the surface of the solid substrate and from any
synthesized polymers bound to the substrate.
11. The method of claim 10 wherein the reactive monomers are
deoxynucleoside phosphoramidite s and the polymers are
oligonucleotides.
12. The method of claim 11 wherein the solid substrate is a
high-density array comprising cells in which different
oligonucleotides are synthesized, the method further including:
applying the reactive wash solution separately to each cell of the
high-density array in order to react with, and deactivate, any
unreacted deoxynucleoside phosphoramidite s; and rinsing the
surface of the solid substrate with the reactive wash solution to
remove the deactivated unreacted deoxynucleoside phosphoramidite s
and catalyzing reagent from the surface of the solid substrate and
from any nascent polymers bound to the substrate.
13. The method of claim 12 wherein the solid substrate is a
high-density array comprising cells in which different
oligonucleotides are synthesized, the method further including:
applying the reactive wash solution separately to each cell of the
high-density array in order to react with, and deactivate, any
unreacted deoxynucleoside phosphoramidite s; allowing the applied
reactive wash solution and solution to evaporate; and rinsing the
surface of the solid substrate with the reactive wash solution to
remove the deactivated deoxynucleoside phosphoramidite s and
catalyzing agent from the surface of the solid substrate and from
any nascent polymers bound to the substrate.
14. The method of claim 11 wherein the reactive wash solution
includes a chemical compound containing a hydroxyl functional
group.
15. The method of claim 14 wherein the reactive wash solution is
methanol.
16. A high-density array comprising cells containing different
polymer species bound to the surface of the high-density array and
synthesized on the surface of the high-density array by the
step-wise coupling of reactive monomers to nascent polymers bound
to the surface of the high-density array, the step-wise coupling of
reactive monomers to nascent polymers comprising the steps of:
applying reactive monomer molecules and any reagents required to
catalyze the coupling of the reactive monomer molecule to the
nascent polymer to the surface of the solid substrate; and applying
a reactive wash solution to the surface of the solid substrate to
react with, and deactivate, any remaining reactive monomers on the
surface of the solid substrate and to dissolve and remove the
remaining deactivated monomers and any reagents required to
catalyze the coupling of the reactive monomer molecule with the
polymer from the surface of the solid substrate and from any
synthesized polymers bound to the substrate.
17. The high-density array of claim 17 wherein the reactive
monomers are deoxynucleoside phosphoramidite s and the polymers are
oligonucleotides.
18. The method of claim 17 further including: applying the reactive
wash solution separately to each cell of the high-density array in
order to react with, and deactivate, any unreacted deoxynucleoside
phosphoramidite s; and rinsing the surface of the solid substrate
with the reactive wash solution to remove the deactivated unreacted
deoxynucleoside phosphoramidite s and catalyzing reagent from the
surface of the solid substrate and from any nascent polymers bound
to the substrate.
19. The method of claim 17 further including: applying the reactive
wash solution separately to each cell of the high-density array in
order to react with, and deactivate, any unreacted deoxynucleoside
phosphoramidite s; allowing the applied reactive wash solution and
solution to evaporate; and rinsing the surface of the solid
substrate with the reactive wash solution to remove the deactivated
deoxynucleoside phosphoramidite s and catalyzing agent from the
surface of the solid substrate and from any nascent polymers bound
to the substrate.
20. The method of claim 17 wherein the reactive wash reagent is
methanol.
Description
TECHNICAL FIELD
[0001] The present invention relates to the preparation of
high-density arrays of surface-bound oligonucleotides by a
step-wise synthesis of oligonucleotides on the surface of a
substrate and, in particular, to a method and system for precisely
confining the application of reactive deoxynucleoside
phosphoramidites to particular cells within the high-density
array.
BACKGROUND OF THE INVENTION
[0002] A combination of synthetic chemical technologies and certain
computer-related technologies has lead to the development of an
important analytical tool in the field of molecular biology
commonly referred to as the "gene chip." Gene chips are
high-density arrays of oligonucleotides bound to a chemically
prepared substrate such as silicon, glass, or plastic. Each cell,
or element, within the array is prepared to contain a single
oligonucleotide species, and the oligonucleotide species in a given
cell may differ from the oligonucleotide species in the remaining
cells of the high-density array. Gene chips may be used in DNA
hybridization experiments in which radioactively, fluorescently, or
chemiluminescently labeled DNA or RNA molecules are applied to the
surface of the gene chip and are bound, via Watson-Crick base pair
interactions, to specific oligonucleotides bound to the gene chip.
The gene chip can then be analyzed by radiometric or optical
methods to determine to which specific cells of the gene chip the
labeled DNA or RNA molecules are bound. Thus, in a single
experiment, a DNA or RNA molecule can be screened for binding to
tens or hundreds of thousands of different oligonucleotides.
[0003] Hybridization experiments can be used to identify particular
gene transcripts in mRNA preparations, to identify the presence of
genes or regulatory sequences in cDNA preparations, or to sequence
DNA and RNA molecules. Particularly in the latter application, the
effectiveness of employing gene chips depends of the precision with
which specific oligonucleotides can be synthesized within discrete
cells of the gene chip. As with any chemical synthetic process,
various factors may cause the yields of specific steps in the
synthesis of oligonucleotides to be less than 100%, leading to
unintended and unwanted intermediate species. During an
oligonucleotide lengthening step in the synthesis of
oligonucleotides on the surface of a gene chip, reactive
deoxynucleoside phosphoramidites are successively applied, in
concentrations exceeding the concentrations of target hydroxyl
groups of the substrate or growing oligonucleotide polymers, to
specific cells of the high-density array. Then, unreacted
deoxynucleoside phosphoramidites from multiple cells of the
high-density array are washed away in a single wash step to prepare
for a subsequent step of oligonucleotide synthesis. Unfortunately,
during the wash step, unreacted deoxynucleoside phosphoramidites
may migrate to regions outside the specific region of the
high-density array to which they were applied and react with
functional groups of the high-density array substrate or bound
oligonucleotides rather than being cleanly removed from the surface
of the gene chip. These unintended deoxynucleoside phosphoramidite
reactions may result in the spreading, or blooming, of the
deoxynucleoside phosphoramidite reaction to adjoining regions of
the gene chip and may even lead to cross contamination of adjoining
cells. As a consequence, the cells of the high-density array may
end up containing a mixture of different oligonucleotides rather
than a single specific oligonucleotide. A related blooming problem
may occur when various phosphoramidite dyes are applied to the
surface of the gene chip to mark specific cells or features.
Blooming of these phosphoramidite dyes may lead to imprecise and
low-resolution marking of cells and features.
[0004] Although a method for successively removing unreacted
phosphoramidite reactants from individual cells of the high-density
array might be envisioned, such successive treatment of individual
cells would greatly increase the time required for preparation of
gene chips, and would increase the complexity of the mechanical
devices that are used to prepare gene chips. Instead, a need has
been recognized in the area of gene chip manufacture for a method
for bulk removal of unreacted phosphoramidite reactants from the
cells of a high-density array without producing the blooming
phenomenon resulting from reactions of phosphoramidite reactants
outside the specific areas of a gene chip to which they are
originally applied.
SUMMARY OF THE INVENTION
[0005] The present invention provides a method for the bulk removal
of unreacted phosphoramidite reactants from the surface of a
high-density array so that the unreacted phosphoramidite reactants
do not react with reactive functional groups of the substrate of
the high-density array or with chemical species bound to the
substrate of the high-density array in areas outside of the
specific area to which the phosphoramidite reactants are originally
applied. In the method of the present invention, a
phosphoramidite-reactive solution is employed to wash unreacted
phosphoramidites from the surface of the high-density array. The
phosphoramidite-reactive solution reacts with, and deactivates,
unreacted phosphoramidites before they have a chance to react with
reactive functional groups of either the substrate of the
high-density array or of chemical species bound to the substrate of
the high-density array. Use of the present invention thus allows
for the preparation of high-density arrays with densities, or
resolutions, determined by the precision with which the
phosphoramidite reactants can be applied to the surface of the
high-density array.
BRIEF DESCRIPTION OF THE DRAWINGS
[0006] FIGS. 1A-1D illustrate small regions of two different types
of high-density arrays containing substrate-bound
oligonucleotides.
[0007] FIG. 2 illustrates, in cross-section, the surface of a
high-density array prior to the synthesis of surface-bound
biopolymers.
[0008] FIG. 3 illustrates the deoxynucleoside phosphoramidite
5'-Dimethoxytrityl-N-benzoyl-2'-deoxyAdenosine,3'-[(O-cyanoethyl)-(N,N-di-
isopropyl)]-phosphoramidite.
[0009] FIG. 4 illustrates the chemical steps employed to link a
first deoxynucleoside phosphoramidite monomer to a free hydroxyl
group on the surface of a high-density array.
[0010] FIG. 5 illustrates the addition of a deoxynucleoside
phosphoramidite monomer to a growing oligonucleotide polymer bound
to the surface of a high-density array.
[0011] FIG. 6 is a flow diagram that shows the sequence of steps
employed to synthesize substrate-bound oligonucleotides on the
surface of a high-density array.
[0012] FIG. 7 illustrates the application of four different
deoxynucleoside phosphoramidites to five cells of a 25-cell region
on the surface of a high-density array.
[0013] FIG. 8 shows the spreading, or blooming, of deoxynucleoside
phosphoramidites on the surface of a high-density array.
[0014] FIG. 9 shows the reaction of a protonated protected
deoxynucleoside phosphoramidite with methanol to produce an
unreactive protected deoxynucleoside phosphite triester.
[0015] FIGS. 10A-10C illustrate three different modifications of
solid-state oligonucleotide synthesis that prevent spreading, or
blooming, of the reaction of phosphoramidite reactants during wash
steps.
DETAILED DESCRIPTION OF THE INVENTION
[0016] FIGS. 1A-1D illustrate two small regions of two different
types of high-density arrays ("HDAs") containing surface-bound
oligonucleotides. The first type of HDA, shown in FIGS. 1A-1B, has
disk-shaped surface regions, or cells, that each contain a
particular synthesized biopolymer like, for example, cell 102. FIG.
1A shows a number of cells of small disk-shaped region of an HDA
viewed in a direction orthogonal to the surface of the HDA, and
FIG. 1B shows a cross-section of the region of the HDA shown in
FIG. 1A. The cells are laid out on the surface of the HDA along
concentric circles, as shown in FIG. 1A, or in a matrix or
grid-like arrangement (not shown). Each cell has a precisely
defined location on the surface of the HDA that can be located by
analytical devices that analyze labeled molecules bound to the
surface of a cell using radiometric or optical methods. Biopolymers
may be synthesized on the surface of the HDA in a step-wise fashion
so that each cell of the HDA may contain a different biopolymer.
Solutions containing the necessary reactants for the synthetic
steps for creating substrate-bound biopolymers are applied in small
droplets to the cells and spread in a disk-shaped region of
increasing radius, by surface tension and adsorption to the
hydrophilic substrate, to define the cells. The inter-cell regions
of the HDA surface are treated with a reagent that chemically
modifies the surface of the HDA to prevent reactive monomers from
covalently bonding to the inter-cell surface of the HDA during
biopolymer synthesis.
[0017] FIG. 1C shows 25 cells of a square region of a second type
of HDA containing surface-bound oligonucleotides viewed in a
direction orthogonal to the surface of the HDA. FIG. 1D shows a
cross-section of the region of the HDA shown in FIG. 1C. The cells
of this type HDA form a regular grid, or matrix, as shown in FIG.
1C, or are laid out along concentric circles like the HDA shown in
FIG. 1A. The reactants required during the synthetic steps are
added to tiny surface tension wells, for example, surface tension
well 104. These surface tension wells lie above circular regions of
the substrate of the HDA that are chemically prepared to provide
reactive groups to which the first monomer of a growing biopolymer
can be chemically bound. Intervening regions of the HDA surface 106
outside the perimeter of the surface tension wells may be
chemically prepared to present a hydrophobic surface that causes
aqueous solutions added to the surface tension wells to bead up
into semi-spherical droplets and to remain constrained within the
circular area of the HDA cell to which the aqueous solutions are
applied.
[0018] In one method for preparing HDAs, a device similar to the
ink jet printers used for the computer-control printing of text and
diagrams onto paper is used to successively deposit tiny droplets,
each containing one or more specific reactants, to cells of an HDA
during each synthetic step of the synthesis of substrate-bound
biopolymers. Using inkjet technology, an HDA may be prepared to
contain 100,000 disk-shaped cells or semi-spherical surface tension
wells, each having a diameter of approximately 100 microns, on the
surface of a circular region of an HDA having a diameter of 75 mm.
Each cells or surface tension well may have a volume on the order
of 100 pl.
[0019] The present invention will be described in terms of a
preferred embodiment related to HDAs of the type shown in FIGS.
1A-1B containing oligonucleotides prepared by step-wise addition of
reactive deoxynucleoside phosphoramidites to the cells. However,
one skilled in the art of the preparation of HDAs will appreciate
that the present invention may find application in the preparation
of different types of HDAs, like, for example, the type of HDA
shown in FIGS. 1C-1D, and in the preparation of HDAs containing
other types of biopolymers prepared by step-wise polymerization of
different types of reactive monomers.
[0020] FIG. 2 illustrates, in cross-section, the surface of the HDA
within a cell prior to the synthesis of the biopolymers that will
be bound to the surface. The substrate of the HDA 202 is prepared
to present reactive functional groups, in the present case,
hydroxyl groups 204, at the surface that will serve as anchors to
which synthesized biopolymers will be bound.
[0021] Deoxynucleoside phosphoramidites are used as reactive
monomers for the step-wise synthesis of oligonucleotides. FIG. 3
illustrates the deoxynucleoside phosphoramidite
5'-Dimethoxytrityl-N-benzoyl-2'-deoxyAden-
osine,3'-[(O-cyanotheyl)-(N,N-diisopropyl)]-phosphoramidite. This
monomer is composed of four different subcomponent groups 302-305,
enclosed in FIG. 3 within dashed lines. The first subcomponent
group 302 is a deoxynucleoside. In FIG. 3, the deoxynucleoside
illustrated is adenosine. Other deoxynucleoside phosphoramidites
used in the synthesis of oligonucleotides contain guanosine,
cytidine, and thymidine in place of the adenosine 302 shown in FIG.
3. A benzoyl group 304 is linked through an amide bond to N.sup.6
of the adenosine group 302. This benzoyl group protects the primary
amine of the adenosine group from reacting with the phosphoramidite
group of a second deoxynucleoside phosphoramidite. The primary
amines of guanosine and cytidine are similarly protected in the
other deoxynucleoside phosphoramidites. Different types of
protecting groups are available, including, for example, acetyl or
isobutyryl groups. A dimethoxytrityl ("DMTr") group 305 is linked
to the 5' end of the deoxynucleoside group in order to protect the
5' hydroxyl group of the deoxynucleoside from reacting with the
phosphoramidite group of another deoxyphosphoramidite. Finally, a
phosphoramidite group 303 is linked to the 3' end of the adenosine
group 302. A variety of different phosphoramidite groups may be
employed in which different types of alkyl groups may be
substituted for the isopropyl groups 311-312 linked to the amine
nitrogen atom 314 of the phosphoramidite group 303 and the
cyanoethyl group 313 linked via a phosphite ester bond to the
phosphorous atom 315 of the phosphoramidite group 303.
[0022] FIG. 4 illustrates the chemical steps employed to link the
first protected deoxynucleoside phosphoramidite monomer to a free
hydroxyl group on the surface of the HDA. A solution containing a
protected deoxynucleoside phosphoramidite 402 and tetrazole,
S-ethyl tetrazole, or dicyanoimidazole is applied to the surface of
the HDA that has been chemically prepared to present free hydroxyl
groups 406. Tetrazole, S-ethyl tetrazole, and dicyanoimidazole are
acids that protonate the amine nitrogen 404 of the phosphoramidite
group of the deoxynucleoside phosphoramidite 402. A free hydroxyl
group 406 on the surface of the substrate displaces the protonated
secondary amine group of the phosphoramidite group by nucleophilic
substitution and results in the protected deoxynucleoside
covalently bound to the substrate via a phosphite triester group
408. After a wash step, in which unreacted deoxynucleoside
phosphoramidites and tetrazole, S-ethyl tetrazole, or
dicyanoimidazole are removed, free hydroxyl groups of the substrate
of the HAD, particularly free hydroxyl groups of the inter-cell
regions of the substrate of the HAD 410, are acetylated 412 by
application of a solution of CAP A, comprising acetic anhydride,
2,6lutidine (2,6-dimethylpyridine), and terahydrofuran ("THF"), and
CAP B, comprising 1-methylimidazole in THF. After a wash step, in
which the CAP A/CAP B solution is removed, the phosphite triester
group is oxidized by the addition of iodine in THF, 2,6-lutidine,
and water to form a phosphotriester group 414.
[0023] FIG. 5 illustrates the addition of a deoxyphosphoramidite
monomer to a growing oligonucleotide polymer attached to the
surface of the HDA. After any unreacted reagents from previous
synthetic steps are removed by washing, the DMTr protecting group
of the 5' terminal nucleosides of the growing oligonucleotides are
removed by treatment with acid to produce a free 5'-hydroxyl group
502-503. Next, a protected deoxynucleoside phosphoramidite
(DMTr-N-benzoyl-deoxyCytidine phosphoramidite in FIG. 5) in
solution with tetrazole is applied to the substrate-bound
oligonucleotide and reacts with the 5' hydroxyl of the
oligonucleotide to covalently link the protected deoxynucleoside
504 to the 5' end of the oligonucleotide via a phosphite triester
group 506. After excess, unreacted protected deoxynucleoside
phosphoramidites and tetrazole are removed by washing, any
unreacted 5' hydroxyl groups 508 of substrate-bound
oligonucleotides are acetylated 510 by application of a CAP A/CAP B
solution. This step is necessary because the previous
oligonucleotide elongation reaction does not proceed to 100%
completion, and it is desirable to terminate any unreacted
nucleotides by acetylation so that oligonucleotides with incorrect
sequences are not produced in subsequent synthetic steps. After the
CAP A/CAP B solution is removed by washing with acetonitrile, the
phosphite triester group 512 is oxidized to a phosphotriester group
514 by the addition of I.sub.2, THF, 2,6-lutidine, and H.sub.2O.
The steps illustrated in FIG. 5 are repeated to add each additional
deoxynucleoside to the 5' end of the growing oligonucleotide.
[0024] A particular deoxynucleoside phosphoramidite reactant can be
added to each cell of the HDA during each synthetic cycle. Thus,
for example, protected deoxyadenosine phosphoramidite may be added
to one cell and protected deoxyguanosine phosphoramidite may be
added to an adjoining cell during the first synthetic cycle. Thus,
the oligonucleotide species synthesized in the first cell will have
adenylic acid at the 3' terminus and the oligonucleotide species
synthesized in the adjoining cell will have guanylic acid at the 3'
terminus. At the completion of the synthetic cycles, each cell of
the HDA may contain an oligonucleotide species having a nucleotide
sequence different from the nucleotide sequences of all the other
oligonucleotides synthesized in the other cells of the HDA.
[0025] FIG. 6 is a flow diagram that shows the sequence of steps
employed to synthesize substrate-bound oligonucleotides on the
surface of an HDA. These steps are illustrated in FIGS. 4 and 5. In
the first step 602, a protected deoxynucleoside phosphoramidite is
applied, along with tetrazole, S-ethyl tetrazole, or
dicyanoimidazole, to those cells of the HDA in which the synthesis
of oligonucleotides will be initiated. Although represented in FIG.
6 as a single step, this step is actually composed of many
thousands of successive applications of protected deoxynucleoside
phosphoramidites to individual cells of an HDA. In step 604,
unreacted deoxynucleoside phosphoramidites and tetrazole, S-ethyl
tetrazole, or dicyanoimidazole are removed from the surface of the
HDA by washing, in a single step, the surface of the HDA with a
solvent. Currently, the solvent acetonitrile is employed as the
wash reagent. In step 606, unreated substrate hydroxyl groups are
acetylated by application of a CAP A/CAP B solution. In step 608,
the CAP A/CAP B solution is removed by washing with acetonitrile.
In step 610, the phosphite triester group linking the newly added
protected deoxynucleoside is oxidized, by the addition of I.sub.2,
THF, 2,6-lutidine, and H.sub.2O, to form a phosphotriester group.
In step 612, the oxidizing solution is removed by washing the
surface of the HDA with the wash reagent acetonitrile. In step 614,
the protecting dimethoxytrityl group on the 5' end of the growing
oligonucleotide within designated cells of the HDA is removed by
the addition of acid. In step 616, the added acid and freed
dimethoxytrityl groups are removed from the surface of the HDA by
washing the surface of the HDA with the wash reagent acetonitrile.
In step 618, the next protected deoxynucleosides to be added to the
oligonucleotides in the designated cells are applied to the
designated cells along with tetrazole, S-ethyl tetrazole, or
dicyanoimidazole in many thousands of successive applications. The
applied protected deoxynucleoside phosphoramidite reacts with the
5' hydroxyl group at the 5' terminus of the growing oligonucleotide
and is thereby covalently linked to the growing oligonucleotide
through a phosphite triester group. In step 620, unreacted
protected deoxynucleoside phosphoramidites and tetrazole, S-ethyl
tetrazole, or dicyanoimidazole are removed from the surface of the
HDA by washing, in a single step, the surface of the HDA with the
wash reagent acetonitrile. In step 622, unreacted 5'-hydroxyl
groups are acetylated by application of a CAP A/CAP B solution. In
step 624, the CAP A/CAP B solution is removed by washing with
acetonitrile. In step 626, the phosphite triester group is oxidized
by the addition of I.sub.2, THF, 2,6-lutidine, and H.sub.2O to form
a phosphotriester group. In step 628, the oxidizing solution is
removed from the surface of the HDA by washing the surface of the
HDA with the wash reagent acetonitrile. If another nucleoside is to
be added to the growing oligonucleotides in any of the cells of the
HDA, as detected in step 630, steps 614-628 are repeated, starting
at step 614. Otherwise, oligonucleotide synthesis is complete.
[0026] FIG. 7 illustrates the application of four different
deoxynucleoside phosphoramidites to five cells of a 25-cell
grid-like region on the surface of an HDA. The different protected
deoxynucleoside phosphoramidite and tetrazole solutions have been
applied, in successive applications, to the five cells 702-706
along the diagonal of the 25-cell region. In one method of HDA
preparation, a device incorporating high speed inkjet technology
successively locates those cells on an HDA that are designated to
receive a protected deoxynucleoside phosphoramidite during the next
synthetic cycle and applies tiny droplets, on the order of 100 pl,
of the designated one of the four different types of protected
deoxynucleoside phosphoramidites to the designated cells. Such
devices can seek and apply the designated deoxynucleoside
phosphoramidite to several hundred cells per second. Thus, the
appropriate protected deoxynucleoside phosphoramidite may be
applied to each cell of a 100,000-cell HDA in the course of a
single synthetic step within a period of between 5 and 10 minutes.
After the protected deoxynucleoside phosphoramidite solutions have
been applied to each of cells 702-706, the protected
deoxynucleoside phosphoramidites will react with, and become
covalently bound through phosphite triester groups to, any free
reactive functional groups of the substrate or growing bound
oligonucleotides. Because the protected deoxynucleoside
phosphoramidites are applied in quantitative excess, the cells
702-706 will contain unreacted protected deoxynucleoside
phosphoramidites.
[0027] FIG. 8 shows the spreading, or blooming, of the regions
containing appreciable concentrations of the unreacted protected
deoxynucleoside phosphoramidites, applied to cells of an HDA in a
synthetic step, during a subsequent washing step in which the
unreacted deoxynucleoside phosphoramidites are removed from the
surface of the HDA in a single bulk wash. As shown in FIG. 8,
during the washing step, rather large, irregularly shaped regions
802-806 of the surface of the HDA are exposed to appreciable
concentrations of unreacted deoxynucleoside phosphoramidites washed
from the cells. For example, cells 808, 810, and 812 are exposed to
appreciable concentrations of the protected deoxynucleoside
phosphoramidite originally applied to cell 814. On certain
substrates, unprotected functional groups of the substrate, or of
molecules bound to the substrate, may react with the unreacted
deoxynucleoside phosphoramidites and end up covalently bound to
them. Thus, the deoxynucleoside phosphoramidites so precisely
applied to the cells of the HDA in FIG. 7 end up, after the washing
step, to be smeared into significantly lower resolution,
irregularly shaped areas across the surface of the HDA, as shown in
FIG. 8.
[0028] This spreading, or blooming, of applied phosphoramidite
reagents may have a number of deleterious effects. First, because a
given synthetic step may not proceed to 100% completion, a
deoxynucleoside phosphoramidite applied to one cell may end up
reacting with a growing oligonucleotide in a different cell that
was either designated not to receive another nucleotide in the
given synthetic step or was designated to receive a different
nucleotide during the given synthetic step. Thus, the cells of the
HDA will end up containing impure mixtures of a number of different
oligonucleotides having potentially different lengths and
potentially different nucleotide sequences. Also, when radioactive
phosphoramidite markers or chemiluminescent phosphoramidite dyes
are applied to cells of the HDA in order to mark particular cells
or features for subsequent detection by radiometric or optical
methods, the spreading, or blooming, of the radioactive markers or
chemiluminescent dyes across the surface of the HDA may result in
incorrect alignment and incorrect detection by the detection
devices. Finally, unintended oligonucleotide or oligonucleotide
derivatives may be deposited outside of the circular surface areas
of the HDA cells. Impure substrate-bound oligonucleotide samples
within cells or unintended oligonucleotides on regions of the
surface of the HDA outside the cells may result in radio-labeled or
chemiluminescent DNA or RNA molecules hybridizing to unintended and
unexpected regions of the surface of the HDA. This will result in a
general loss of signal to noise ratio during optical or radiometric
analysis of the results of a hybridization experiment, and may even
result in an incorrect analysis.
[0029] FIG. 9 shows the reaction of a protonated protected
deoxynucleoside phosphoramidite with methanol to produce the
corresponding unreactive protected deoxynucleoside phosphite
triester. The hydroxyl group of methanol 102 can displace the
protonated secondary amine 904 of the phosphoramidite group 906 in
the same way that the free hydroxyl groups of the chemically
prepared substrate or 5' terminal end of a growing oligonucleotide
displace the protenated secondary amine, as shown in FIGS. 4 and 5.
Both protected deoxynucleoside phosphoramidites and tetrazole (or
S-ethyl tetrazole or dicyanoimidazole) are soluble in methanol and
methanol does not react with, or catalyze reactions of, either the
growing oligonucleotide polymers or the substrate. Thus, in a
preferred embodiment of the present invention, methanol is employed
as a phosphoramidite-reactive wash reagent in place of the
phosphoramidite-unreactive wash reagent acetonitrile in the wash
steps of solid-state surface-bound oligonucleotide synthesis.
[0030] FIGS. 10A-10C illustrate three different modifications of
solid-state oligonucleotide synthesis that prevent spreading, or
blooming, of the protected deoxynucleoside phosphoramidite addition
reaction during wash steps. Steps 1002-1003 of FIG. 10A, 1004-1006
of FIG. 10B, or 1007-1009 of FIG. 10C are substituted for steps 602
and 604 and for steps 618 and 620 of FIG. 6. Any single one of, or
any combination of, the alternate embodiments represented by the
steps of FIG. 10A-10C can be substituted for steps 602 and 604 and
for steps 618 and 620 of FIG. 6 in order to produce a solid-state
oligonucleotide synthetic procedure that eliminates the spreading,
or blooming, of the reaction of phosphoramidite reagents with
functional groups of, or bound to, the surface of HDA outside the
region of the surface of the HDA to which the phosphoramidite
reagents are applied.
[0031] In the first alternative embodiment shown in FIG. 10A, a
protected deoxynucleoside phosphoramidite is applied, along with
tetrazole, S-ethyl tetrazole, or dicyanoimidazole, in successive
substeps to one or more designated cells of an HDA in step 1002. In
step 1003, remaining unreacted protected deoxynucleoside
phosphoramidites and tetrazole, S-ethyl tetrazole, or
dicyanoimidazole are removed by bulk washing the surface of the HDA
with methanol. In the embodiment represented in FIG. 10B, protected
deoxynucleoside phosphoramidite and tetrazole, S-ethyl tetrazole,
or dicyanoimidazole are applied, in successive substeps, to one or
more designated cells of an HDA in step 1004. Then, in step 1005,
methanol is applied to those designated cells in individual
successive steps. Finally, in step 1006, the entire surface of the
HDA is bulk washed with methanol to remove protected
deoxynucleoside phosphite triesters, protonated secondary amines,
and tetrazole, S-ethyl tetrazole, or dicyanoimidazole. In FIG. 10C,
protected deoxynucleoside phosphoramidite and tetrazole, S-ethyl
tetrazole, or dicyanoimidazole are applied, in successive
sub-steps, to one or more designated cells within an HDA. In step
1008, the cells of the HDA are allowed to evaporate to dryness. In
step 1009, the dry surface of the HDA is flooded and washed with
methanol. By allowing the cells to evaporate, the methanol wash
reagent applied in step 1009 will react with unreacted protected
deoxynucleoside phosphoramidites as they enter into solution, prior
to their displacement from the circular area of the substrate onto
which the contents of the cell are deposited during the evaporation
step. Analogous embodiments, using methanol as a wash reagent, may
be applied to removing unreacted phosphoramidite dyes and markers
that have been applied to the surface of an HDA in order to prevent
spreading, or blooming, of the dyes or markers.
[0032] Although the present invention has been described in terms
of a particular embodiment, it is not intended that the invention
be limited to this embodiment. Modifications within the spirit of
the invention will be apparent to those skilled in the art. For
example, any number of different phosphoramidite-reactive wash
agents may be devised. A large number of different types of
solvents containing free hydroxyl or primary amine groups may be
employed as phosphoramidite-reactive wash reagents. Moreover, a
phosphoramidite-reactive wash agent may be produced by adding one
or more phosphoramidite-reactive substances to an otherwise
phosphoramidite-unreactive solvent. As another example, the present
invention may be applied during the preparation of HDAs that
contain biopolymers other than oligonucleotides. Wash reagents that
react with, and quench, unreacted reactive monomers may be used to
prevent blooming of the reaction of the unreacted monomers to areas
of the HDA outside of the areas to which the reactive monomer is
applied.
[0033] The foregoing description, for purposes of explanation, used
specific nomenclature to provide a thorough understanding of the
invention. However, it will be apparent to one skilled in the art
that the specific details are not required in order to practice the
invention. The foregoing descriptions of specific embodiments of
the present invention are presented for purpose of illustration and
description. They are not intended to be exhaustive or to limit the
invention to the precise forms disclosed. Obviously many
modifications and variations are possible in view of the above
teachings. The embodiments are shown and described in order to best
explain the principles of the invention and its practical
applications, to thereby enable others skilled in the art to best
utilize the invention and various embodiments with various
modifications as are suited to the particular use contemplated. It
is intended that the scope of the invention be defined by the
following claims and their equivalents:
* * * * *