U.S. patent application number 10/674766 was filed with the patent office on 2004-05-27 for microarrays having multiple oligonucleotides in single array features.
Invention is credited to Albert, Thomas, Green, Roland, Norton, Jason.
Application Number | 20040101894 10/674766 |
Document ID | / |
Family ID | 32069803 |
Filed Date | 2004-05-27 |
United States Patent
Application |
20040101894 |
Kind Code |
A1 |
Albert, Thomas ; et
al. |
May 27, 2004 |
Microarrays having multiple oligonucleotides in single array
features
Abstract
The present invention is a method for synthesizing microarrays
having different oligonucleotides present within one feature area
of the array. The method utilizes the techniques common to
microarray synthesis, but limits the duration in which selected
feature areas on the array are initially dosed with light so as to
only deprotect a calculated ratio of the compounds forming the
array's binding layer. The compounds initially deprotected are
capped with a non-photosensitive protecting group, such as
di-methoxy-trityl, to inhibit their involvement in the synthesis of
a first group of DNA strands built onto the array. Once the first
group of DNA strands have been synthesized, the original
deprotected group may then be further processed to build one or
more groups of DNA strands in the same feature area as the first
group of DNA strands. The present invention also includes
microarrays manufactured using the method.
Inventors: |
Albert, Thomas; (Madison,
WI) ; Norton, Jason; (Madison, WI) ; Green,
Roland; (Madison, WI) |
Correspondence
Address: |
Nicholas J. Seay
Quarles & Brady LLP
P O Box 2113
Madison
WI
53701-2113
US
|
Family ID: |
32069803 |
Appl. No.: |
10/674766 |
Filed: |
September 30, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60415046 |
Oct 1, 2002 |
|
|
|
Current U.S.
Class: |
435/6.11 ;
435/287.2 |
Current CPC
Class: |
B01J 2219/00722
20130101; C07H 21/00 20130101; C12Q 1/6837 20130101; B01J
2219/00711 20130101; C40B 40/06 20130101; B01J 2219/00626 20130101;
Y02P 20/55 20151101; B01J 2219/00675 20130101; B01J 2219/00612
20130101; B01J 2219/00608 20130101; B82Y 30/00 20130101; C12Q
1/6837 20130101; C12Q 2565/513 20130101; C12Q 2565/543 20130101;
C12Q 2537/143 20130101 |
Class at
Publication: |
435/006 ;
435/287.2 |
International
Class: |
C12Q 001/68; C12M
001/34 |
Claims
We claim:
1. A microarray comprising a plurality of features, each of the
features formed of single stranded oligonucleotides, at least some
of the features including in the same feature oligonucleotides of
more than one sequence.
2. A microarray as claimed in claim 1 wherein each feature includes
oligonucleotides of two different sequences.
3. A microarray as claimed in claim 1 wherein at least some of the
oligonucleotides in the microarray are oriented both 3' to 5' and
at least some other oligonucleotides are oriented 5' to 3'.
4. A microarray as claimed in claim 1 wherein the two
oligonucleotides in a single feature are each designed to hybridize
to different exons in the same eukaryotic gene.
5. A microarray as claimed in claim 1 wherein the two probes each
make up about 50% of the probes in the feature.
6. A method for synthesizing different oligonucleotides in the same
feature area, the method comprising the steps of: providing a
substrate for manufacturing a microarray, the substrate having
photo-labile protecting groups formed on its surface, the
microarray having at least one feature area; exposing the feature
area to a light source for a period of time sufficient to cleave
the photo-labile protecting group from only a portion of feature
area; coupling a second protecting group to the unprotected area of
the feature, the second protective group not being photo-labile;
exposing the feature area to a light source for a period of time to
cleave the remaining photo-labile protecting groups from the
feature area to leave an unprotected area of the feature; building
a first group of oligonucleotides in the unprotected area of the
feature; capping the first group of oligonucleotides with a capping
compound that is not photo-labile; removing the second protecting
group from the feature area to leave an unprotected area of the
feature; building a second group of oligonucleotides in the
unprotected area of the feature.
7. The method of claim 6 wherein the portion of the feature area in
which the first light exposing step is conducted is about 50% of
the feature area, so that each of the oligonucleotides is about 50%
of the oligonucleotides in the feature.
8. The method of claim 6 wherein the portion of the feature area in
which the first light exposing step is conducted is about 33% of
the feature area, so that one of the oligonucleotides is about 33%
of the oligonucleotides in the feature.
9. The method of claim 6 wherein the second protecting group is
acid labile.
10. The method of claim 9 wherein the second protective group is
di-methoxy-trityl
11. The method of claim 9 wherein the capping compound is acetic
anhydride and tetrahydrofuran.
12. A method of using a microarray to analyze the splicing of an
mRNA transcript from a gene having more than one exon, the method
incorporating the steps of providing a microarray with at least one
feature two oligonucleotides in the feature, a first
oligonucleotide being complementary to the mRNA in a portion of the
mRNA corresponding to one exon and a second oligonucleotide
corresponding in sequence to the mRNA in a portion corresponding to
another exon; hybridizing the microarray to the mRNA so that mRNA
if present will bind to the nucleotide complementary to the mRNA;
extending the first oligonucleotide using the bound mRNA as a
template; removing the bound mRNA; hybridizing the extended first
oligonucleotide to the second oligonucleotide; and extending the
second oligonucleotide against first oligonucleotide using labeled
nucleotides so that the feature can be detected if the
hybridizations occurred.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority from U.S. provisional
patent application Ser. No. 60/415,046 filed Oct. 1, 2002.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] Not applicable.
BACKGROUND OF THE INVENTION
[0003] The advent of DNA microarray technology makes it possible to
build an array of hundreds of thousands of DNA sequences in a very
small area, such as the size of a microscopic slide. See, e.g.,
U.S. Pat. No. 6,375,903 and U.S. Pat. No. 5,143,854, each of which
is hereby incorporated by reference in its entirety. The disclosure
of U.S. Pat. No. 6,375,903 enables the construction of so-called
maskless array synthesizer (MAS) instruments in which light is used
to direct synthesis of the DNA sequences, the light direction being
performed using a digital micromirror device (DMD). Using an MAS
instrument, the selection of DNA sequences to be constructed in the
microarray is under software control so that individually
customized arrays can be built to order. In general, MAS based DNA
microarray synthesis technology allows for the parallel synthesis
of over 800,000 unique oligonucleotides in a very small area of a
standard microscope slide. The microarrays are generally
synthesized by using light to direct the addition of single
nucleotides to the oligonucleotides under construction at specific
locations on an array, these locations being called features.
Typically, the objective is to synthesize many identical
oligonucleotides, each having the same nucleotide sequence, in each
feature of the array, i.e. there are multiple probes in each
feature, but all those probes have the same nucleotide sequence.
For certain applications it would be advantageous to have
oligonucleotides of different sequences present within one feature
of the array, and be able to control the ratio and direction
(5'-3', or 3'-5') of these oligonucleotides.
BRIEF SUMMARY OF THE INVENTION
[0004] The present invention is summarized as a method for
synthesizing a microarray having oligonucleotides of different
sequences present within one feature of the array. The present
invention also includes a method for synthesizing a microarray to
control the ratio and direction (5'-3', or 3'-5') of the
oligonucleotides. The present invention also includes microarrays
manufactured using the disclosed methods.
[0005] The method utilizes the techniques common to microarray
synthesis, but limits the duration in which selected feature areas
on the array are initially dosed with light so as to only deprotect
a calculated ratio of the compounds forming the array's binding
layer. The compounds initially deprotected are capped with a
non-photosensitive protecting group, such as di-methoxy-trityl, to
inhibit their involvement in the synthesis of the first group of
DNA strands built onto the array. Once the first group of DNA
strands have been synthesized, the original deprotected group may
then be further processed to build a second group of DNA strands in
the same feature area as the first group of DNA strands. The same
concept may also be employed to build additional groups of DNA
strands in order to provide feature areas containing more than two
different groups of DNA strands. The DNA strands can be constructed
5' to 3' or 3' to 5', depending solely on the orientation of the
photo-labile conjugated nucleosides used in the process.
[0006] Other objects, advantages and features of the present
invention will become apparent from the following specification and
drawings.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0007] FIGS. 1 to 4 are an schematic illustration process of
microarray synthesis including two oligonucleotides synthesized in
the same feature area.
[0008] FIG. 5 is an illustration of a type of assay that is made
possible, for the first time, from the fact that the microarray has
two oligonucleotides in each feature.
DETAILED DESCRIPTION OF THE INVENTION
[0009] The present invention provides a method for synthesizing a
microarray having a number of features and in which one or more
features can have multiple probes of different sequences present
within them. The method contemplates that two or more probes or
oligonucleotides can be constructed in single features of the
microarray, and further provides a method for controlling the ratio
of the relative numbers of the two probes in the feature. The
method also permits the direction (5'-3', or 3'-5') of these
oligonucleotides to be controlled so that each of the two
oligonucleotides in a single feature can be in the same direction
or they can be in opposite directions.
[0010] It is an advantage of the methods described here that they
use the techniques common to microarray synthesis, but uses
modifications of those method to achieve new products. For example,
the method makes use of the now well-understood process of using
light directed de-protection to select areas on the microarray for
a de-protection step, but limits the duration in which selected
feature areas on the array are initially dosed with light so as to
only de-protect not entire features, but only a calculated portion
of the area of each feature. The area of the array thus
de-protected is then capped with a non-photosensitive protecting
group to inhibit the participation of those areas in the synthesis
of the first group of oligonucleotides built onto the feature of
the array. Once the first group of oligonucleotides have been
synthesized, the original de-protected and capped area may then be
de-capped. This can be done by making the protecting cap be acid or
base labile, and introducing an acid or base into the microarray to
de-cap the capped areas. Then, after restoring appropriate pH, the
nucleotide addition process can be re-started to synthesize a
second group of oligonucleotides in the same feature area as the
first group of oligonucleotides. The same concept may also be
employed to build additional groups of DNA strands in order to
provide feature areas containing more than two different groups of
DNA strands.
[0011] For the purpose of this invention, the term feature is used
for an area on the array that has been intended in prior art
microarrays to have the same nucleotide probe or probes throughout
its area. In the past, microarrays have had features in which the
feature contains only probes or oligonucleotides of the same
nucleotide sequence. This is the first known instance in which it
has been proposed or enabled to put probes of two or more different
sequences in the same feature of a microarray. So for the purposes
of this invention, a feature means a portion of a microarray in
which two or more probes are synthesized in the same general
physical region of the microarray, a region that is preferable
distinct from the region in which other probes are constructed.
[0012] The terms probe and oligonucleotides are used
interchangeably here to refer to the molecules of single stranded
DNA (or RNA) which are synthesized on the microarray.
[0013] Again, the method by which the construction of multiple
probes in single features is enabled is an elegant and relatively
simple modification of the existing microarray synthesis methods.
First, on the activated substrate, a light directed de-protection
step is performed which is limited in time duration. The time is
selected to be a selected proportion, such as one-half, of the time
which has been found to be necessary to de-protect the entire
surface area of the features. We have found that if one-half the
minimum exposure time necessary to de-protect the entire feature
area is used, approximately one-half of the surface area of each
feature area will be de-protected. This concept is illustrated in
the schematic of FIG. 1. In FIG. 1, the prepared substrate 10 is
coated with reactive groups, such as silanes with reactive hydroxyl
groups, to which photo-labile protecting groups "P" have been
attached. The layer of photo-labile protecting groups is designated
by the reference number 12. This portion of the microarray is
intended to encompass two features, here designated 14 and 16.
Light, designated at 18, is shined on the entire array for a
sufficient period of time to de-protect about one-half of the
photo-labile protecting groups "P". The de-protected areas of the
features are then capped by a protecting groups that will not be
disturbed by the synthesis of the remaining probes, such as adding
an acid labile protecting group, designated in FIG. 2 by "B". Thus
at this point, about one-half of the area of each feature is
protected by a photo-labile protecting group while the other half
is protected by an acid labile protecting group. Then a light
directed de-protection step is performed to saturation so that the
remaining photo-labile protecting groups are all removed from the
microarray area. Following that, an otherwise conventional a probe
synthesis process is conducted to completion in all the areas which
are not capped by the acid-labile blockers. This is illustrated in
FIG. 3 where an illustrated three nucleotide probe set TAA has been
synthesized in feature 14 while a set of probes of sequence GAC has
been synthesized in feature 16. The three nucleotides are
illustrative only since, in actual practice, of course, the probes
are much longer, typically about 25 nucleotides in length, although
they can be constructed to be up to 100 nucleotides with reasonable
accuracy. After the synthesis of this first set of oligonucleotide
probes is complete, the synthesized probes are capped by a capping
agent that is neither light nor acid labile. Then an acid is used
to remove the acid labile protecting groups "B" from the substrate
to expose the areas of the microarray in which no probes have yet
been synthesized. Then another set of probes are synthesized on the
microarray in exactly the same fashion as before, except that the
sequence of the synthesize probes may now be different. Simply for
purposes of illustration, in the trivial example in the
illustrations in FIG. 4, a second probe of sequence CAT has been
constructed in feature 14 while a second probe set of sequence CAG
has been constructed in feature 16. In actual practice,
synthesizing two probes sets in each feature by the method
described here has turned out to be practical and readily
achievable. The relative amounts of the first and second probe sets
can even be adjusted simply by modifying the time of the partial
light directed de-protection step at the start of the process.
[0014] Note that the direction of probe synthesis depends solely on
the nature of the nucleotides uses. When nucleotides are added to
the building probes set, the added nucleotides are added with a
photo-labile protecting group already attached to them. The
direction of probe synthesis, i.e. whether the probes are
synthesized 3' to 5' or 5' to 3', is determined solely by whether
the added nucleotide has the photo-labile protecting group attached
to its 3' or 5' end. Since nucleotides with suitable photo-labile
protecting groups, including NPPOC, which is the preferred reagent
for use herein, are available with the NPPOC attached at either the
3' or 5' end of the nucleotide, it is readily possible to
synthesize probes in either selected orientation. In fact, the
direction of one set of probes in a given feature does not have to
match the direction of the other set of probes in the same feature.
We have made microarrays in which 3' to 5' and 5' to 3' probes are
synthesized in the same feature areas of the microarrays.
[0015] It is recognized that many different compounds may be used
as binding compounds or protecting groups during the synthesis of
microarrays. Although reference is made below to specific compounds
used during the synthesis process, one of ordinary skill in the art
would recognize that other binding compounds and protecting groups
could also be used in the practice of the present invention. The
below examples merely serve as a discussion of one embodiment of
the present invention and is in no way intended to limit its
scope.
[0016] The present invention allows for two or more different
oligonucleotides to be built in a single feature area. To produce
two oligonucleotides per feature area, one layer of a base
associated with a photosensitive protective group, such as NPPOC
protected bases, is coupled to the array surface. The base is then
partially deprotected with an appropriate light source. The amount
of light dosed on the array will have the effect of removing the
photosensitive protective group from only a percentage of the
feature area, thus controlling the ratio of the different
oligonucleotides synthesized. When the desired percentage of the
original base is removed, a second base carrying a protecting group
that is not sensitive to the light being employed, such as acid
labile di-methoxy-trityl (DMT), is coupled to the free hydroxyls on
the surface of the deprotected portion of the feature area. Once
coupled, the remaining photosensitive protective groups are removed
by dosing the feature area with more of the light source. The
hydroxyl groups that are freed by the second dose of light (and
thus not protected by DMT) are thus free to be used to synthesize
light directed DNA probes in the normal fashion. In absence of any
highly acidic compounds, such as trichloroacetic acid (TCA), the
DMT protected sites within each feature will be unaffected and
saved for future use in building a second group of DNA strands.
[0017] After the first group of DNA strands is synthesized, the DNA
is capped with a capping compound, such as acetic anhydride and
tetrahydrofuran, to inhibit further strand building, and the
synthesis of the second group of DNA strands is begun. First, the
DMT protective group is removed from the original deprotected group
by exposure to a compound effective in removing DMT, such as the
highly acidic compound TCA. Once the DMT is removed, the second
group of DNA strands is synthesized in the normal fashion. Upon
completion, the array is placed in a deprotection solution to
remove the base protecting groups and the cap placed on the first
set of DNA strands, resulting in an array with two different
oligonucleotide strands per feature.
[0018] It is envisioned that even more than two species of probes
can be constructed in a common feature of a microarray. To increase
the number of different oligonucleotides that are present in one
feature, several rounds of partial deprotection by light,
intermixed by coupling with different types of protecting groups
may be employed. Each of these protecting groups must be able to be
independently removed from the surface. When each type of group is
removed, DNA strands are built at those locations. For example,
after deprotection of 33% of the sites with light, a DMT group may
be used as a protecting compound. After deprotection of a second
33% of the sites with light, a base labile FMOC group may be added
as a second protecting compound. The remaining groups may then be
removed with light, and a first group of DNA strands built and
capped. TCA may then be used to remove the DMT group, allowing a
second group of DNA strands to be built and capped. Finally, FMOC
may be removed using a weak base, allowing for a third group of DNA
strands to be built and capped.
[0019] It is envisioned that other types of protecting groups could
also be used, thus allowing for the application of different
chemical treatments or different wavelengths of light. The
direction of synthetic DNA may also be controlled using either 5'
amidites or 3' amidites for synthesis of a given strand. It is also
possible to mix 5' and 3' amidites within one strand. Microarrays
can be constructed in which all the features have more than one
probe in them, or the microarray can have some features with a
single probe and some features with multiple probes in them. This
techniques permits the microarray to be highly customized to
particular unique applications.
[0020] The ability to make microarrays with more than one
oligonucleotide in a feature broadens the range of analyses that
can be conducted with microarrays. For example, one interesting
application enable by this approach is the design of microarrays
intended to permit studies of transcript splicing in the expression
of eukaryotic genes. It is believed, for example, that the number
of genes in the human genome may be insufficient to explain all the
proteins in the human body, and that intermediate mRNA splicing may
explain some of the diversity in proteins. This phenomenon is
difficult to study using conventional microarrays. Illustrated in
FIG. 5 is a method for using microarrays with two probes in a
feature to study mRNA splicing. The two probes constructed in this
illustrative feature are designed to detect whether a covalent
linkage between two exons of a gene occurs in a given mRNA sample,
i.e. to see if an mRNA exists in a cell of tissue which includes
both exons. One of the set or probes in the feature is designed to
be complementary to the mRNA of a particular first target exon of
the gene being studied. The mRNA is hybridized to the microarray,
and the mRNA species hybridizes to the first set of probes only if
the target exon is in the mRNA species. This is illustrated at step
20 in FIG. 5. The mRNA binds only to the probe for the first exon
since the probes for the second exon are of the opposite sense as
the probes for the first exon. Then, reverse transcriptase is used
to extend the probe oligonucleotide, using the bound mRNA as a
template, as illustrated at 22 in FIG. 5. The first exon is
covalently extend to be a longer piece of DNA complementary in
sequence to the mRNA, like a cDNA. The mRNA is then removed from
the micoarray, for example using RNase H to digest the mRNA away,
leaving only the extended probe. Then another hybridization step is
performed, and, as indicated at 24 in FIG. 5, the distant end of
the extended first probe will hybridize to the second probe only if
the second probe set is complementary to far end of the extended
fist probe. The second probe set is thus designed to be
complementary to the DNA (not the mRNA) of another target exon in
the gene being studied. Then a DNA extension reaction is performed,
with a DNA polymerase, such as DNA polymerase I, to add nucleotides
to the single stranded part of the complex, beginning at the
terminus of the second probe. The DNA extension reaction will only
occur if the hybridization to the second set of probes occurred. By
incorporating fluorescently labeled nucleotides in the DNA
extension step, indicated at 26 in FIG. 5, the completed double
stranded DNA molecule can be made fluorescent, or detectable in any
other convenient manner. When the microarray is read, only features
which bound to mRNA which contained sequences complementary to both
of the DNA probes in that feature will fluoresce. With a set of
several features designed to test the various exons in a gene, the
entire gene splicing pattern in a cell or tissue can be determined.
Thus it can be determined which exons are linked by common
transcripts in given cells, and information about gene splicing can
be revealed using microarrays.
EXAMPLES
Example 1
[0021] Using the methods described above and a maskless array
synthesizer instrument, two oligonucleotides were synthesized in
the same feature area using the method of the present invention,
with the ratio of one oligonucleotide to the other varied
horizontally across the array. The array was then hybridized with
two oligonucleotides that were complementary to the array
oligonucleotides. One of these oligonucleotides was labeled with
Cy3, the other with Cy5. The resulting scan of the hybridized
microarray to test samples revealed that the Cy3 oligo has
increasing surface density from left to right, while the Cy5 oligo
is increasing in density from right to left. The result were
readily apparent in fluorescent imaging.
Example 2
[0022] An array was designed having six 10.times.10 feature
sections, with different oligonucleotides synthesized in common
features of the array. Three different oligonucleotides were
combined in all possible permutations in the array with each of the
other oligonucleotides. Test samples of known sequence were then
hybridized to the microarray thus made. Each sample hybridized only
the specific area in which probes complementary to that sample had
been synthesized.
* * * * *