U.S. patent number 6,245,518 [Application Number 09/454,822] was granted by the patent office on 2001-06-12 for polynucleotide arrays and methods of making and using the same.
This patent grant is currently assigned to Hyseq, Inc.. Invention is credited to Joerg Baier.
United States Patent |
6,245,518 |
Baier |
June 12, 2001 |
Polynucleotide arrays and methods of making and using the same
Abstract
The present invention relates to methods of using labeled
tracers to generate spatially addressable arrays of immobilized
molecules, particularly polynucleotides, that can be normalized for
differences in immobilization efficiencies at different addresses
in the array. It also relates to the arrays generated by the method
and to use of these arrays to enhance discrimination in array-based
assays, particularly the discrimination between perfectly matched
hybrids and hybrids containing a single mismatch in nucleic acid
hybridization assays.
Inventors: |
Baier; Joerg (Foster City,
CA) |
Assignee: |
Hyseq, Inc. (Sunnyvale,
CA)
|
Family
ID: |
26809421 |
Appl.
No.: |
09/454,822 |
Filed: |
December 6, 1999 |
Current U.S.
Class: |
435/6.11;
435/6.12; 435/91.2; 536/24.33 |
Current CPC
Class: |
B01J
19/0046 (20130101); C12Q 1/6827 (20130101); C12Q
1/6827 (20130101); B01J 2219/00387 (20130101); B01J
2219/00515 (20130101); B01J 2219/00533 (20130101); B01J
2219/00572 (20130101); B01J 2219/00576 (20130101); B01J
2219/00608 (20130101); B01J 2219/00612 (20130101); B01J
2219/00626 (20130101); B01J 2219/00637 (20130101); B01J
2219/00657 (20130101); B01J 2219/00659 (20130101); B01J
2219/00722 (20130101); C12Q 1/6837 (20130101); C40B
40/06 (20130101); C40B 60/14 (20130101); C12Q
2565/501 (20130101); C12Q 2565/549 (20130101) |
Current International
Class: |
B01J
19/00 (20060101); C12Q 1/68 (20060101); C12Q
001/68 (); C12P 019/34 (); C07H 021/04 () |
Field of
Search: |
;435/6,91.2
;536/23.1,24.33 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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|
|
|
|
|
WO 90/03382 |
|
Apr 1990 |
|
WO |
|
WO 98/31836 |
|
Jul 1998 |
|
WO |
|
Other References
Lockhart et al., "Expression monitoring by hybridization to
high-density oligonucleotide arrays, " Nature Biotechnology, 1996,
vol. 14, pp. 1675-1680.* .
Constantine et al., "Use of GeneChip high-density oligonucleotide
arrays for gene expression monitoring," Life Science News, 1998,
pp. 11-14.* .
Fodor et al. 1991, "Light-Directed, Spatially Addressable Parallel
Chemical Synthesis," Science 251:767-73. .
Pease et al. 1994, "Light-generated oligonucleotide arrays for
rapid DNA sequence analysis," Proc. Natl. Acad. Sci. USA
91:5022-5026..
|
Primary Examiner: Brusca; John S.
Assistant Examiner: Kim; Young
Attorney, Agent or Firm: Pennie & Edmonds LLP
Parent Case Text
This application claims the benefit of the Provisional Patent
Application entitled, "IMPROVED POLYNUCLEOTIDE ARRAYS AND METHODS
OF MAKING AND USING THE SAME," Ser. No. 60/111,961, filed Dec. 11,
1998.
Claims
What is claimed is:
1. A spatially-addressable array of compounds, comprising a
substrate having directly attached thereon, optionally by a linker
at each of a plurality of distinct addresses a compound and a
tracer moiety, wherein the structures of the compounds are
identifiable by their spatial addresses, and wherein the amount of
the tracer moiety attached at each address is proportional to the
amount of compounds attached at that address.
2. The array of claim 1 which is a one-dimensional array.
3. The array of claim 2 in which the substrate is a solid-phase
synthesis support, a glass fiber or a capillary tube.
4. The array of claim 1 which is a two-dimensional array.
5. The array of claim 1 in which the substrate is a glass or
plastic sheet.
6. The array of claim 1, in which the directly attached compounds
are polynucleotides.
7. The array of claim 6 in which the polynucleotides are covalently
attached to the substrate, optionally by way of a linker.
8. The array of claim 7, in which the polynucleotides are
covalently attached to the substrate via their 5' or 3' terminal
nucleotide.
9. The array of claim 7 which comprises 10 to 10.sup.6 different
polynucleotides.
10. The array of claim 7 in which each immobilized polynucleotide
is independently 6 to 20 nucleotides in length.
11. The array of claim 10, in which all of the directly attached
polynucleotides are the same length.
12. The array of claim 10, which comprises a complete set of
polynucleotides 6-10 nucleotides in length.
13. The array of claim 6 in which the polynucleotide directly
attached, optionally by a linker, at one or more address is a
mixture of polynucleotides.
14. The array of claim 13, in which the polynucleotide directly
attached, optionally by a linker, at each address is a mixture of
polynucleotides.
15. The array of claim 13 or 14, in which the mixture is of the
formula: N.sub.x B.sub.y N.sub.z, wherein each N represents any of
the five encoding bases and varies for the polynucleotides in a
given mixtures, B represents any of the five encoding bases and is
the same for each of the polynucleotides in a given mixture, and x,
y, and z are each independently integers.
16. The array of claim 1 in which the tracer moiety comprises a
fluorophore.
17. The array of claim 6 in which the tracer moiety is a
fluorescently-labeled polynucleotide.
18. The array of claim 17 in which the fluorescently-labeled
polynucleotide directly attached, optionally by a linker, at each
address has the same nucleotide sequence as the polynucleotide
directly attached, optionally by a linker at that address.
19. The array of claim 17 in which the fluorescent label is
TAMRA.
20. The array of claim 17, in which the same fluorescently-labeled
polynucleotide is directly attached, optionally by a linker, at
each address.
21. The array of claim 20, in which the fluorescently-labeled
polynucleotide comprises a mixture of fluorescently-labeled
polynucleotides.
22. The array of claim 21, in which the mixture of fluorescently
labeled polynucleotides comprises:
NNNGGCAT-F,
NNNCGGAG-F,
NNNAACTG-F,
NNNATGAA-F,
NNNTGTAC-F,
NNNACTGG-F,
NNNGAACC-F,
NNNTACAG-F,
NNNCTGGA-F, and
NNNCCGGA-F,
wherein each N represents any of the five encoding bases and F is a
fluorophore.
23. The array of claim 22, in which F is TAMRA.
24. A method of making a spatially-addressable array of compounds,
comprising the steps of:
(i) directly attaching, optionally by a linker, at a first address
of a substrate a first compound and a first tracer moiety; and
(ii) directly attaching, optionally by a linker, a second address
of a substrate a second compound and a second tracer moiety.
25. In a method of making spatially addressable array of
polynucleotides by directly attaching, optionally by a linker
pre-synthesized polynucleotides at a discrete spatial address on a
substrate, the improvement comprising directly attaching,
optionally by a linker an amount of a tracer moiety at each spatial
address that is proportional to the amount of polynucleotide
attached at that address.
26. In a method of making a spatially addressable array of
compounds by in situ synthesis, the improvement comprising directly
attaching optionally by a linker at each spatial address of the
array an amount of a tracer moiety that is proportional to the
amount of a product of the in situ synthesis directly attached,
optionally by a linker, at that address.
27. A method of increasing the accuracy of an array-based assay
comprising:
contacting an array according to claim 1 with a an analyte compound
that is capable of generating an assay signal upon interacting with
a compound of the array; and
normalizing the assay signals to account for differences in the
amounts of compounds immobilized at different addresses in the
array, thereby increasing the accuracy of the assay.
28. The method of claim 27 in which the assay signals are
normalized by obtaining the ratio of the assay signal intensity at
an address to the background signal intensity at that address.
29. The method of claim 28 in which the background signal intensity
at an address is obtained by measuring the signal intensity of that
address prior to contacting the array with the analyte
compound.
30. A method of normalizing hybridization signals in an array-based
hybridization experiment, comprising the steps of:
contacting an array of immobilized polynucleotides according to
claim 6 with a target nucleic acid under conditions in which
addresses of the array bearing immobilized polynucleotides that are
complimentary to a region of the target nucleic acid produce a
detectable hybridization signal; and
normalizing the hybridization signal at an address by obtaining the
ratio of the hybridization signal intensity at the address to the
background signal intensity at that address.
31. The method of claim 30 in which the background signal intensity
of the address is obtained by measuring the signal intensity at the
address prior to contacting the array with the target nucleic
acid.
32. The method of claim 30 in which the target nucleic acid is
labeled.
33. The method of claim 30 in which the array is further contacted
with a set of labeled solution-phase polynucleotide probes and
labeled probes and array polynucleotides that hybridize adjacently
to the same target nucleic acid molecule are covalently joined.
Description
1. FIELD OF THE INVENTION
The present invention relates to spatially-addressable arrays of
molecules, particularly biological molecules such as peptides and
oligonucleotide probes, and methods of making and using the
same.
2. BACKGROUND OF THE INVENTION
Recent advances in the ability to construct arrays of biological
molecules has greatly facilitated the ease and speed with which
certain biological assays can be performed. For example, in the
areas of nucleic acid sequencing and analysis, the advent of new
technologies for constructing arrays of immobilized target nucleic
acids or oligonucleotide probes has enabled the rapid screening and
sequencing of nucleic acids. Arrays of peptides and small
biomolecules have also proven useful in binding assays used in
pharmaceutical development. The usefulness of these arrays depends
on the ability to generate arrays with spatially addressable
regions of defined composition or sequence.
Several technologies have been developed for producing these arrays
of biological molecules. Several researchers have devised methods
for in situ synthesis of arrays of biological polymers, such as
nucleic acids, peptides, and carbohydrates. These methods use, for
example, physical barriers to separate regions, devices (such as
inkjet printers) for precise delivery of reagents to regions, or
masking techniques that allow the use of light to determine the
course of synthesis. See, e.g., WO 90/03382; Fodor et al., 1991,
Science 251:767-73; Pease et aL., 1994, Proc. Natl. Acad. Sci.
91:5022-26; U.S. Pat. No. 5,424,186, to Fodor et al. Alternatively,
presynthesized biomolecules or biological polymers may be attached
directly to the substrate at precise positions using a variety of
techniques, ranging from simple spotting to robotic delivery
systems. A variety of different substrates and techniques for
attaching the biomolecules to the substrates are also
available.
As noted above, arrays of nucleic acids have proven particularly
valuable. The ability to perform many previously available
techniques has been greatly enhanced by availability of arrays,
which permit many assays to be performed simultaneously on a single
array rather than having to do each assay individually. Other
techniques that would have been virtually impossible are now
possible using polynucleotide arrays.
One technique that has been particularly enhanced by the
availability of arrays of nucleic acids is sequencing by
hybridization (SBH). SBH is a technique for rapidly sequencing
nucleic acids without using gels. In SBH, polynucleotides having
overlapping sequences are hybridized to a target nucleic acid. The
sequences of the polynucleotides that hybridize are then determined
and the common sequences overlapped to generate the sequence of the
nucleic acid. The use of arrays has allowed the generation of
sufficient hybridization information to make SBH feasible on a
large scale.
SBH is divided into three formats, depending on the nature of the
array and the way in which it is interrogated. In Format I, the
target nucleic acid is immobilized and the labeled polynucleotides
are in solution. In Format II, the polynucleotides are immobilized
and the labeled target nucleic acid is in solution. In Format III,
immobilized polynucleotides are hybridized with an unlabeled target
nucleic acid and labeled oligonucleotide probes. Hybridization is
assayed by ligating the labeled oligonucleotide probes to the
immobilized polynucleotides. All three formats require the ability
to distinguish perfectly matched hybrids from hybrids that contain
a single mismatch at any position. For a more detailed discussion
of SBH and the three formats, see WO 98/31836, particularly at
pages 1-3.
While the demand for biological arrays, and in particular
polynucleotide arrays, is high, current methodologies for
constructing such arrays still suffer from certain difficulties.
The most common difficulty is assaying the quality and integrity of
an array once it has been fabricated. While the chemistries
involved in producing the arrays are relatively well understood,
methods for synthesizing arrays still suffer from lack of
reliability and reproducibility, and even failure. However,
identifying regions of attachment failures is very difficult,
particularly with the small spots found in miniaturized arrays.
Thus, quality control of produced arrays is very difficult to
maintain. Furthermore, even minor variations in attachment
efficiencies can make interpretation of results generated from such
arrays very difficult, as the researcher may not be able to tell
whether a difference in signal is real or merely an artifact of the
attachment process. This problem is particularly acute in
applications such as sequencing by hybridization, which require
extremely accurate differentiation of even minor differences in
hybridization.
3. SUMMARY OF THE INVENTION
These and other shortcomings in the art are overcome by the present
invention, which in one aspect provides spatially addressable
arrays of immobilized molecules in which each spot in the array
contains an amount of a detectable label which is proportional to
the amount of molecule immobilized at that spot. The label can be
any molecule which is capable of producing a detectable,
quantifiable signal, such as a radioisotope, fluorophore,
chromophore, chemiluminescent moiety, etc. The labels at each spot
may be the same or different, but are preferably the same.
In another aspect, the invention provides methods of making arrays
of immobilized molecules in which each spot in the array contains
an amount of a detectable label which is proportional to the amount
of molecule immobilized at that spot. In the method, a molecule to
be immobilized at a particular spot on the array is "spiked" with a
detectable label capable of immobilizing to the substrate with the
same efficiency as the molecule. The molecules to be immobilized at
different spots are each "spiked" with the same proportion of
label. Thus, following immobilization, each spot in the array
contains an amount of label which is proportional to the efficiency
of the immobilization technique. Following synthesis, the array can
be scanned or otherwise analyzed for detectable signal to monitor
the fidelity of the array synthesis.
In a preferred embodiment, the label is attached to, incorporated
within, or otherwise associated with the same type of molecule as
that to be immobilized. Accordingly, in this preferred embodiment
of the methods, the molecule to be immobilized is spiked with a
small amount of labeled molecule of the same type. Again, the
molecules to be immobilized at different locations are each spiked
with the same proportion of labeled molecule.
In another aspect, the invention provides methods of increasing the
accuracy of array-based assays. In the method, background signals
produced from an array of spatially addressable immobilized
molecules according to the invention are quantified and
recorded.
The array is contacted with a target molecule capable of
interacting with at least one of the immobilized molecules. The
target molecule is labeled in some manner to produce an assay
signal, or the interaction between the target and immobilized
molecule is such that only those spots on the array where
interaction has taken place produce a detectable assay signal.
Following contact and optional washing, the array is scanned or
otherwise analyzed for detectable assay signal, and the signal from
each labeled spot quantified. The intensities of the signals from
the respective spots are then normalized, typically by obtaining
the ratio I.sub.a /I.sub.b (where I.sub.a is the assay signal
intensity and I.sub.b is the background signal intensity), to
account for signal differences caused by deviations in the
quantities of immobilized molecules. This normalization process
permits signal intensities from different spots on the array to be
directly compared, regardless of the fidelity of the array
synthesis.
The labels giving rise to the background signals and assay signals,
i.e., the moieties used to label the array spots and target
molecules, respectively, may be the same or different. In instances
where the same label is used, the background signals should be
recorded prior to contacting the array with the target molecule.
The assay signal is then obtained by subtracting the background
signal from a particular spot from the total signal from that spot.
In this embodiment, the assay signals are normalized by obtaining
the ratio (I.sub.a -I.sub.b)/I.sub.b, where I.sub.a and I.sub.b are
as previously defined. When different labels are used, the
background signal can be detected and recorded prior to,
concomitant with, or after detection of assay signals.
The spatially-addressable arrays and methods of the invention
provide myriad advantages over conventional arrays of immobilized
molecules. Due to the presence of the labels, the quality of any
array can be verified prior to use by simply scanning the array for
detectable signal, even in instances where the array is highly
miniaturized. Variability in the quantities of molecules
immobilized at different positions within the array, and more
importantly the complete absence of particular spots from the
array, will be readily apparent. Moreover, because the quantity of
label at each spot in the array correlates with the quantity of
molecule immobilized at that spot, assay signals produced from
different spots in the arrays can be normalized and directly
compared, regardless of the fidelity of the array synthesis. This
feature is particularly important for assays in which signal
intensities are critical to determining whether the signal is
real.
The advantages of the methods and arrays of the invention are
illustrated by way of working examples involving nucleic acid
hybridization assays. In the examples, hybridization assays
performed with polynucleotide arrays according to the invention
were able to discriminate perfectly complementary hybrids from
mismatched hybrids which could not be discriminated using
conventional polynucleotide arrays.
4. BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 shows a scan of a polynucleotide array produced with labeled
tracer polynucleotides in each polynucleotide mixture. The scan
detects the fluorescence at each spot.
FIG. 2 shows a scan of the array of FIG. 1 after hybridization and
ligation with the target DNA and labeled polynucleotides.
5. DETAILED DESCRIPTION OF THE INVENTION
5.1 Definitions
As used herein, the following terms shall have the following
meanings:
"Spatially addressable array" refers to an array in which each
element or component of the array is identifiable by its spatial
address, for example its xyz coordinates. Spatial addressable
arrays according to the invention can be one dimensional, for
example a linear array; two dimensional; or three dimensional.
"Address" or "spot" refers to a particular position in an array.
Each address or spot has unique xyz coordinates. The structure of a
compound immobilized at a particular address or spot is definable
by its coordinates.
"Polynucleotide" refers to a nucleic acid sequence which is
immobilized on a substrate. The polynucleotides of the present
invention can contain as few as four bases or as many as several
hundred or more bases. The polynucleotides can be composed of
natural or modified bases or combinations thereof, and can contain
one or more modified interlinkages.
"Target nucleic acid" refers to a nucleic acid of known or unknown
sequence to be analyzed. The target nucleic acid can be virtually
any number of nucleotides in length, but typically is longer than
the polynucleotides of the array.
"Tracer moiety" refers to a molecule capable of generating a
detectable signal (i.e., a label or labeled molecule) that is or is
capable of being immobilized on the spots of the array, and whose
amount is proportional to the amount of a molecule of interest
immobilized at that spot. The presence of the tracer moiety allows
normalization of the array for differences in immobilization
efficiencies.
5.2 The Invention
The problems in the art discussed in the Background section are
solved by the present invention. The methods of the present
invention enable the quality and integrity of arrays of immobilized
molecules to be simply and reliably assessed. Quality is assessed
by the use of small quantities of labels, either by themselves or
attached to the molecules of interest. The labels or labeled
molecules are immobilized on the substrate along with the molecules
of interest, and their immobilization is proportional to the
immobilization of the molecules of interest. The intensity of the
signal from the immobilized labels at a given location provides an
assessment of the amount of the molecule of interest immobilized on
the substrate at that location, and thus provides a way to verify
the quality and integrity of the array as a whole. This intensity
information is also useful when the array is interrogated, as it
provides a way to distinguish real differences in signal intensity
due to experimental results from artifactual variations due simply
to inconsistent immobilization of the molecules. The invention is
also directed to arrays made by these methods and their use in
various assay techniques.
The present methods are applicable to a wide variety of different
molecules that may be placed in arrays. The methods are
particularly exemplified herein in terms of polynucleotides
immobilized on a substrate, but they are equally applicable to
other types of molecules. For example, one of skill in the art
could easily adapt the present methods to apply to other nucleic
acids (both DNA and RNA), peptides, polypeptides, proteins,
carbohydrates, small biomolecules (e.g. drug candidates), or any
other type of molecule that can be immobilized on a substrate by
any method. Preferably, the molecule is one that can be labeled,
although this is not necessary if a label can be immobilized on the
substrate in a fashion similar to the molecule.
The arrays of the present invention may be of any desired size,
from two spots to 10.sup.6 spots or even more. The upper and lower
limits on the size of the substrate are determined solely by the
practical considerations of working with extremely small or large
substrates.
For a given substrate size, the upper limit is determined only by
the ability to create and detect the spots in the array. The
preferred number of spots on an array generally depends on the
particular use to which the array is to be put. For example,
sequencing by hybridization will generally require large arrays,
while mutation detection may require only a small array. In
general, preferred arrays contain from 2 to about 10.sup.6 spots,
more preferably from about 100 to about 10.sup.5 spots,
particularly preferably from about 400 to about 10.sup.4 spots, and
most preferably between about 500 and about 2000 spots.
Furthermore, not all spots on the array need be unique. Indeed, in
many applications, redundancies in the spots are desirable for the
purposes of acting as internal controls.
A variety of techniques have been described for synthesizing and/or
immobilizing arrays of polynucleotides, including in situ
synthesis, where the polynucleotides are synthesized directly on
the surface of the substrate (see, e.g., U.S. Pat. No. 5,744,305 to
Fodor, et al.,) and attachment of pre-synthesized polynucleotides
to the surface of a substrate at discrete locations (see, e.g., WO
98/31836). Additional methods are described in WO 98/31836 at pages
41-45 and 47-48, among other places. The present invention is
suitable for use with any of these currently available, or later
developed, techniques.
In embodiments involving immobilization of pre-synthesized
polynucleotides, the polynucleotide reagent to be deposited at a
particular spot contains a small quantity, typically 0.01 to 0.15%,
and preferably 0.08%, of a label, typically a labeled
polynucleotide. The polynucleotide reagent is then deposited on the
substrate at a spatially defined region, i.e., at a particular
spot. After immobilization, the spot contains an amount of labeled
polynucleotide which is proportional to the amount of
polynucleotide immobilized at that spot. Depositing a number of
such polynucleotide reagents at different spatial addresses yields
an array of polynucleotides whose sequences are identifiable by
their spatial addresses. Moreover, each spot in the array contains
an amount of labeled polynucleotide that is proportional to the
amount of polynucleotide immobilized at that spot.
In embodiments involving in situ synthesis of polynucleotides, the
polynucleotides are synthesized in their usual manner. At the
synthetic step which adds the last nucleotide, the nucleoside
phosphoramidite reagent to be deposited contains a small quantity,
typically 0.01 to 0.15%, and preferably 0.08%, of a label,
typically a labeled nucleoside phosphoramidite. The synthetic
scheme yields an array of polynucleotides whose sequences are
identifiable by their spatial addresses. Moreover each spot in the
array contains an amount of labeled polynucleotide that is
proportional to the amount of full-length polynucleotide
synthesized at that spot.
While the above method contemplates labeling the last nucleotide of
the polynucleotide, those of skill in the art will appreciate that
other positions, or additional positions, could be similarly
labeled to provide information about the proportions of truncated
polynucleotides synthesized. In these embodiments, the labels used
at the various steps should be distinguishable from one
another.
Moreover, while the in situ synthesis method is described utilizing
phosphoramidite reagents, it will be recognized that other reagents
utilizing other synthesis strategies can also be employed, and in
certain circumstances may be preferable, depending on the stability
of the chosen label to the synthesis conditions. Non-limiting
examples of suitable chemistries and reagents are described, for
example in Oligonucleotide Synthesis: A Practical Approach, M. J.
Gait, Ed., IRL Press, Oxford, England, 1985.
The members of the arrays of the invention are immobilized on a
solid substrate.
The nature and geometry of the solid substrate will depend upon a
variety of factors, including, among others, the type of array
(e.g., one-dimensional, two-dimensional or three-dimensional) and
the mode of attachment (e.g., covalent or non-covalent). Generally,
the substrate can be composed of any material which will permit
immobilization of the polynucleotide and which will not melt or
otherwise substantially degrade under the conditions used to
hybridize and/or denature nucleic acids. In addition, where
covalent immobilization is contemplated, the substrate should be
activatable with reactive groups capable of forming a covalent bond
with the polynucleotide to be immobilized.
A number of materials suitable for use as substrates in the instant
invention have been described in the art. Exemplary suitable
materials include, for example, acrylic, styrene-methyl
methacrylate copolymers, ethylene/acrylic acid,
acrylonitrile-butadienestyrene (ABS), ABS/polycarbonate,
ABS/polysulfone, ABS/polyvinyl chloride, ethylene propylene,
ethylene vinyl acetate (EVA), nitrocellulose, nylons (including
nylon 6, nylon 6/6, nylon 6/6-6, nylon 6/9, nylon 6/10, nylon 6/12,
nylon 11 and nylon 12), polycarylonitrile (PAN), polyacrylate,
polycarbonate, polybutylene terephthalate (PBT), polyethylene
terephthalate (PET), polyethylene (including low density, linear
low density, high density, cross-linked and ultra-high molecular
weight grades), polypropylene homopolymer, polypropylene
copolymers, polystyrene (including general purpose and high impact
grades), polytetrafluoroethylene (PTFE), fluorinated
ethylene-propylene (FEP), ethylene-tetrafluoroethylene (ETFE),
perfluoroalkoxyethylene (PFA), polyvinyl fluoride (PVF),
polyvinylidene fluoride (PVDF), polychlorotrifluoroethylene
(PCTFE), polyethylene-chlorotrifluoroethylene (ECTFE), polyvinyl
alcohol (PVA), silicon styreneacrylonitrile (SAN), styrene maleic
anhydride (SMA), metal oxides and glass.
The substrate may be in the form of beads, particles or sheets, and
may be permeable or impermeable, depending on the type of array.
For example, for linear or three-dimensional arrays the substrate
may consist of bead or particles (such as conventional solid phase
synthesis supports), fibers (such as glass wool or other glass or
plastic fibers) or glass or plastic capillary tubes. For
two-dimensional arrays, the substrate is preferably in the form of
plastic or glass sheets in which at least one surface is
substantially flat. Particularly preferred substrates for use with
two-dimensional arrays are glass slides.
The composition of the immobilized polynucleotides is not critical.
The only requirement is that they be capable of hybridizing to a
target nucleic acid of complementary sequence. For example, the
polynucleotides may be composed of all natural or all synthetic
nucleotide bases, or a combination of both. Non-limiting examples
of modified bases suitable for use with the instant invention are
described, for example, in Practical Handbook of Biochemistry and
Molecular Biology, G. Fasman, Ed., CRC Press, 1989, pp. 385-392.
While in most instances the polynucleotides will be composed
entirely of the natural bases (A, C, G, T or U), in certain
circumstances the use of synthetic bases may be preferred.
Moreover, while the backbones of the polynucleotides will typically
be composed entirely of "native" phosphodiester linkages, they may
contain one or more modified linkages, such as one or more
phosphorothioate, phosphoramidite or other modified linkages. As a
specific example, one or more immobilized polynucleotides may be a
peptide nucleic acid (PNA), which contains amide interlinkages.
Additional examples of modified bases and backbones that can be
used in conjunction with the invention, as well as methods for
their synthesis can be found, for example, in Uhlman & Peyman,
1990, Chemical Review 90(4):544-584; Goodchild, 1990, Bioconjugate
Chem. 1(3):165-186; Egholm et al., 1992, J. Am. Chem. Soc.
114:1895-1897; Gryaznov et al., J. Am. Chem. Soc. 116:3143-3144, as
well as the references cited in all of the above.
While the immobilized polynucleotides will in most instances be a
contiguous stretch of nucleotides, they need not be. Stretches of
nucleotides can be interrupted by one or more linker molecules that
do not participate in sequence-specific base pairing interactions
with a target nucleic acid. The linker molecules may be flexible,
semi-rigid or rigid, depending on the desired application. A
variety of linker molecules useful for spacing one molecule from
another or from a solid surface have been described in the art (and
are described more thoroughly infra); all of these linker molecules
can be used to space regions of immobilized polynucleotides from
one another. In a preferred embodiment of this aspect of the
invention, the linker moiety is from one to ten, preferably one to
six, alkylene glycol moieties, preferably ethylene glycol
moieties.
The immobilized polynucleotides may be as few as four, or as many
as hundreds, or even more, nucleotides in length. Specifically
contemplated as polynucleotides according to the invention are
nucleic acids that are typically referred to in the art as
oligonucleotides and also those referred to as nucleic acids. Thus,
the arrays of the present invention are useful not only in
applications where target nucleic acids are hybridized to
immobilized arrays of relatively short (i.e., 6-20 nucleotide)
probes (such as format II SBH), but also in applications where
relatively short probes are hybridized to arrays of immobilized
nucleic acids.
The polynucleotides of the array can be of any desired sequence. In
a preferred embodiment, they can comprise all possible
polynucleotides of a given length N, which would result in an array
of .sub.4.sup.N unique elements. For all polynucleotides of, for
example, 6 bases in length, the sequences would comprise an array
with 4096 unique elements.
Alternatively, the polynucleotides can make up the "universal set"
for sequencing a nucleic acid, as discussed in WO 98/31836,
particularly pages 27-29.
In an alternative embodiment, the set of polynucleotides may
correspond to particular mutations that are to be identified in a
known sequence. For example, if a particular nucleic acid is known
to contain an unidentified mutation at a particular position, then
the mutated position can be identified with an array of eight
polynucleotides, three corresponding to the three possible
substitutions at that position, one corresponding to the deletion
of the base at that position, and four corresponding to the
insertion of the four possible bases at that position.
Alternatively, for a known gene that may contain any of several
possible identified mutations, the array can comprise
polynucleotides corresponding to the different possible mutations.
This embodiment is particularly useful for genes like oncogenes and
tumor suppressors, which frequently have a variety of known
mutations in different positions. Using arrays facilitates
determining whether or not these genes contain mutations by
allowing simultaneous screening with polynucleotides corresponding
to each of these different positions.
In another alternative embodiment, each spot of the array can
comprise a mixture of polynucleotides of different sequences. These
mixtures may comprise degenerate polynucleotides of the structure
N.sub.x B.sub.y N.sub.z, wherein N represents any of the four bases
and varies for the polynucleotides in a given mixtures, B
represents any of the four bases but is the same for each of the
polynucleotides in a given mixture, and x, y, and z are
integers.
Arrays comprising this type of mixture are useful in, for example,
sequencing by hybridization. Alternatively, the spots may comprise
mixtures of polynucleotides that correspond to different regions of
a known nucleic acid; these regions may be overlapping, adjacent,
or nonadjacent. Arrays comprising these types of mixtures are
useful in, for example, identifying specific nucleic acids,
including those from particular pathogens or other organisms. Both
types of mixtures are discussed in WO 98/31836, particularly at
pages 123-128.
The polynucleotides can be isolated from biological samples,
generated by PCR reactions or other template-specific reactions, or
made synthetically. Methods for isolating polynucleotides from
biological samples and/or PCR reactions are well-known in the art,
as are methods for synthesizing and purifying synthetic
polynucleotides. Probes isolated from biological samples and/or PCR
reactions may, depending on the desired mode of immobilization,
require modification at the 3'- or 5'-terminus, or at one or more
bases, as will be discussed more thoroughly below. Moreover, since
the polynucleotide must be capable of hybridizing to a target
nucleic acid, if not already single stranded, it should preferably
be rendered single stranded, either before or after immobilization
on the substrate.
The polynucleotides can be immobilized on the substrate using a
wide variety of techniques. For example, the polynucleotides can be
adsorbed or otherwise non-covalently associated with the substrate
(for example, immobilization to nylon or nitrocellulose filters
using standard techniques); they may be covalently attached to the
substrate; or their association may be mediated by specific binding
pairs, such as biotin and streptavidin. Of these methods, covalent
attachment is preferred.
In order to effect covalent attachment, the substrate must first be
activated, i.e., treated so as to create reactive groups on or
within the substrate that can react with a reactive group on the
polynucleotide to form a covalent linkage. Those of skill in the
art will recognize that the desired reactive group will depend on
the chemistry used to attach the polynucleotides to the substrate
and the composition of the substrate. Typical reactive groups
useful for effecting covalent attachment of polynucleotides to
substrates include hydroxyl, sulfonyl, amino, epoxy, isothiocyanate
and carboxyl groups; however, other reactive groups as will be
apparent to those having skill may also be used and are also
included within the scope of the invention.
For a review of the myriad techniques that can be used to activate
the substrates with suitable reactive groups, see Wiley
Encyclopedia of Packaging Technology, 2d Ed., Brody & Marsh,
Ed., "Surface Treatment," pp. 867-874, John Wiley & Sons
(1997), and the references cited therein (hereinafter "Surface
Treatment"). Chemical methods suitable for generating amino groups
on silicon oxide substrates are described in Atkinson & Smith,
"Solid Phase Synthesis of Oligodeoxyribonucleotides by the
Phosphite Triester Method," In: Oligonucleotide Synthesis: A
Practical Approach, M J Gait, Ed., 1984, IRL Press, Oxford,
particularly at pp. 45-49 (and the references cited therein);
chemical methods suitable for generating hydroxyl groups on silicon
oxide substrates are described in Pease et al., 1994, Proc. Natl.
Acad. Sci. USA 91:5022-5026 (and the references cited therein);
chemical methods for generating functional groups on polymers such
as polystyrene, polyamides and grafted polystyrenes are described
in Lloyd-Williams et al., 1997, Chemical Approaches to the
Synthesis of Peptides and Proteins, Chapter 2, CRC Press, Boca
Raton, Fla. (and the references cited therein).
Those of skill in the art will recognize that in embodiments
employing covalent attachment, the covalent bond formed between the
polynucleotide and the substrate must be substantially stable to
the various conditions under which the array will be assayed, to
avoid loss of polynucleotide during the assay. One such stable bond
is the phosphodiester bond, which connects the various nucleotides
in a polynucleotide, and which can be conveniently formed using
well-known chemistries (see, e.g., Oligonucleotide Synthesis: A
Practical Approach, 1984, supra). Other stable bonds suitable for
use with hydroxyl-activated substrates include phosphorothioate,
phosphoramidite, or other modified nucleic acid interlinkages. For
substrates modified with amino groups, the bond could be a
phosphoramidate, amide or peptide bond. When substrates are
activated with epoxy functional groups, a stable C--N bond could be
formed. Suitable reagents and conditions for forming such stable
bonds are well known in the art. Other stable bonds suitable for
use with the arrays of the invention will be apparent to those of
skill in the art.
In embodiments in which pre-synthesized polynucleotides are
covalently attached to the substrate, the polynucleotides may be
via their 3'-terminus, 5'-terminus or by way of a reactive group at
one of the bases. Synthesis supports and synthesis reagents useful
for modifying the 3'- and/or 5'-terminus of synthetic
polynucleotides, or for incorporating a base modified with a
reactive group into a synthetic polynucleotide, are well-known in
the art and are also commercially available.
For example, methods for synthesizing 5'-modified polynucleotides
are described in Agarwal et al., 1986, Nucl. Acids Res.
14:6227-6245 and Connelly, 1987, Nucl. Acids Res. 15:3131-3139.
Commercially available products for synthesizing 5'-amino modified
polynucleotides include the N-TFA-C6-AminoModiferm,
N-MMT-C6-AminoModiferm and N-MMT-C12-AminoModifierm reagents
available from Clontech Laboratories, Inc., Palo Alto,
California.
Methods for synthesizing 3 '-modified polynucleotides are described
in Nelson et al., 1989, Nucl. Acids Res. 17:7179-7186 and Nelson et
al., 1989, Nucl. Acids Res. 17:7187-7194. Commercial products for
synthesizing 3'-modified polynucleotides include the
3'-Amino-ON.TM. controlled pore glass and Amino Modifier II.TM.
reagents available from Clontech Laboratories, Inc., Palo Alto,
Calif.
Other methods for modifying the 3' and/or 5' termini of
polynucleotides, as well as for synthesizing polynucleotides
containing appropriately modified bases are provided in Goodchild,
1990, Bioconjugate Chem. 1:165-186, and the references cited
therein. Chemistries for attaching such modified polynucleotides to
substrates activated with appropriate reactive groups are
well-known in the art (see, e.g., Ghosh & Musso, 1987, Nucl.
Acids Res. 15:5353-5372; Lund et al., 1988, Nucl. Acids Res.
16:10861-10880; Rasmussen et al., 1991, Anal. Chem. 198:138-142;
Kato & Ikada, 1996, Biotechnology and Bioengineering
51:581-590; Timofeev et al., 1996, Nucl. Acids Res. 24:3142-3148;
O'Donnell et al., 1997, Anal. Chem. 69:2438-2443).
Methods and reagents for modifying the ends of polynucleotides
isolated from biological samples and/or for incorporating bases
modified with reactive groups into nascent polynucleotides are also
well-known and commercially available. For example, an isolated
polynucleotide can be phosphorylated at the 5'-terminus with
phosphorokinase and this phosphorylated polynucleotide covalently
attached to an amino-activated substrate through a phosphoramidate
or phosphodiester linkage. Other methods will be apparent to those
of skill in the art.
In one convenient embodiment, pre-synthesized polynucleotides,
modified at their 3'- or 5'-termini with a primary amino group, are
conjugated to a carboxy-activated substrate. Chemistries suitable
for forming carboxamide linkages between carboxyl and amino
functional groups are well-known in the art of peptide chemistry
(see, e.g., Atherton & Sheppard, Knorr et al., 1989, Tet. Lett.
30(15):1927-1930; Bannworth & Knorr, 1991, Tet. Lett.
32(9):1157-1160; and Wilchek et al., 1994, Bioconjugate Chem.
5(5):491-492; Solid Phase Peptide Synthesis, 1989, IRL Press,
Oxford, England and Lloyd-Williams et al., Chemical Approaches to
the Synthesis of Peptides and Proteins, 1997, CRC Press, Boca
Raton, FL and the references cited therein). Any of these methods
can be used to conjugate amino-modified polynucleotides to a
carboxy-activated substrate.
In another convenient embodiment, the polynucleotides are
synthesized directly on a hydroxy-activated substrate using
commercially available phosphoramidites synthesis reagents. In this
mode, the polynucleotides are covalently attached to the substrate
via their 3'-termini by way of a phosphodiester linkage.
Alternatively, photoprotected phosphoramidites and the
photolithographic methods described in U.S. Pat. No. 5,744,305 to
Fodor et al. and Pease et al., 1994, supra, can be used.
Whether synthesized directly on the activated substrate or
immobilized on the activated substrate after synthesis or
isolation, the polynucleotides can optionally be spaced away from
the substrate by way of one or more linkers. As will be appreciated
by those having skill in the art, such linkers will be at least
bifunctional, i.e., they will have one functional group or moiety
capable of forming a linkage with the activated substrate and
another functional group or moiety capable of forming a linkage
with another linker molecule or the polynucleotides. The linkers
may be long or short, flexible or rigid, charged or uncharged,
hydrophobic or hydrophilic, depending on the particular
application.
In certain circumstances, such linkers can be used to "convert" one
functional group into another. For example, an amino-activated
substrate can be converted into a hydroxyl-activated substrate by
reaction with, for example, 3-hydroxy-propionic acid. In this way,
substrate materials which cannot be readily activated with a
specified reactive functional group can be conveniently converted
into an appropriately activated substrate. Chemistries and reagents
suitable for "converting" such reactive groups are well-known, and
will be apparent to those having skill in the art.
Linkers can also be used, where necessary, to increase or "amplify"
the number of reactive groups on the activated substrate. For this
embodiment, the linker will have three or more functional groups.
Following attachment to the activated substrate by way of one of
the functional groups, the remaining two or more groups are
available for attachment of polynucleotides. Amplifying the number
of functional groups on the activated substrate in this manner is
particularly convenient when the substrate cannot be readily
activated with a sufficient number of reactive groups.
Reagents for amplifying the number of reactive groups are
well-known and will be apparent to those of skill in the art. A
particularly convenient class of amplifying reagents are the
multifunctional epoxides sold under the trade name DENACOL.TM.
(Nagassi Kasei Kogyo K. K.). These epoxides contain as many as
four, five, or even more epoxy groups, and can be used to amplify
substrates activated with reactive groups that react with epoxides,
including, for example, hydroxyl, amino and sulfonyl activated
substrates. The resulting epoxy-activated substrate can be
conveniently converted to a hydroxyl-activated substrate, a
carboxy-activated substrate, or other activated substrate by
well-known methods.
Linkers suitable for spacing biological molecules such as
polynucleotides from solid surfaces are well-known in the art, and
include, by way of example and not limitation, polypeptides such as
polyproline or polyalanine, saturated or unsaturated bifunctional
hydrocarbons such as 1-amino-hexanoic acid, polymers such as
polyethylene glycol, etc. 1,4-Dimethoxytrityl-polyethylene glycol
phosphoramidites useful for forming phosphodiester linkages with
hydroxyl groups, as well as methods for their use in nucleic acid
synthesis on solid substrates, are described, for example in Zhang
et al., 1991, Nucl. 20 Acids Res. 19:3929-3933 and Durand et al.,
1990, Nucl. Acids Res. 18:6353-6359. Other useful linkers are
commercially available.
A critical feature of the arrays of the invention is the presence
of an amount of a label at each position within the array that is
proportional to the amount of polynucleotide immobilized at that
particular spot. Thus, it is important that the efficiencies of the
coupling reactions which are used to immobilize the label and
polynucleotide are substantially similar. For covalent attachment,
this can be conveniently achieved by using the same immobilization
reactive group on both the label and the polynucleotide.
For embodiments employing immobilization of pre-synthesized
polynucleotides, a preferred label is a labeled polynucleotide. The
primary sequences of the labeled and unlabeled polynucleotides at a
particular spot may be the same or different. In fact, the same
labeled polynucleotide may be used at each spot in the array. The
only requirement is that the polynucleotide reagents deposited at
each spot in the array be "spiked" with substantially the same
proportion of labeled polynucleotide.
In a preferred embodiment, the same mixture of labeled
polynucleotides is used to spike the polynucleotide reagent
deposited at each spot. Using the same mixture of labeled
polynucleotides at each spot ensures that the labels at different
spots do not induce sequence-specific anomalies in hybridization
assays, i.e., it ensures that the labels at each array spot
interact similarly with a target nucleic acid in hybridization
assays. Moreover, use of the same label at each spot reduces the
number of labeled polynucleotides that need to be prepared. A
particularly preferred mixture of ten labeled polynucleotides is
described in the examples section.
Virtually any label that produces a detectable, quantifiable signal
and that is capable of being immobilized on a substrate or attached
to a polynucleotide can be used in conjunction with the arrays of
the invention. Suitable labels include, by way of example and not
limitation, radioisotopes, fluorophores, chromophores,
chemiluminescent moieties, etc. In embodiments where the label is
attached to a polynucleotide, the label can be attached to any part
of the polynucleotide, including the free terminus or one or more
of the bases. Preferably, the position of the label will not
interfere with hybridization, detection or other post-hybridization
modifications of the labeled polynucleotide. Suitable methods of
making labeled polynucleotides are well known in the art.
Due to their ease of detection, polynucleotides labeled with
fluorophores are preferred. Fluorophores suitable for labeling
polynucleotides are described, for example, in the Molecular Probes
catalog (Molecular Probes, Inc., Eugene Oreg. 97402-9144), and the
references cited therein. Methods for attaching fluorophore labels
to polynucleotides are well known, and can be found, for example in
Goodchild, 1990, supra. A preferred fluorophore label is the
carboxylic acid of tetramethyl rhodaimine (TAMRA dye), which is
available from Molecular Probes.
In embodiments employing in situ synthesis, a preferred label is a
fluorescentlylabeled nucleic acid synthesis reagent, such as a
labeled nucleoside phosphoramidite. The position at which the
fluorophore is attached to the nucleoside phosphoramidite will
depend on whether the label will be added at the terminal or
internal nucleotides of the nascent polynucleotides. When a
terminal label is desired, the fluorophore can be conveniently
attached to the 5'-hydroxyl. When internal labels are desired, the
flurophore is preferably attached to the base, optionally by way of
a linker. Methods suitable for making fluorescently-labeled
phosphoramidite synthesis reagents are well-known in the art, and
are described, for example, in Goodchild, 1990, supra.
The amount of label used to "spike" the polynucleotide reagent to
be deposited at a particular spot is not critical for success.
However, the amount used should be sufficient to produce a
detectable signal which does not result in a loss of dynamic range
when the array is used in an assay. For the preferred
polynucleotide arrays of the invention, which are synthesized by
depositing pre-synthesized polynucleotides at discrete spots, it
has been found that spiking the polynucleotide reagent with about
0.01 to 0.15%, preferably about 0.08%, of a fluorescently-labeled
polynucleotide yields good results. When mixtures of
fluorescently-labeled polynucleotides are used, the total quantity
of labeled polynucleotides used to spike the reagent should fall
within the above-described ranges.
The polynucleotide arrays according to the invention can be used in
virtually any array in which hybridization is desirable. For
example, the polynucleotide arrays of the invention are useful for
all three formats of sequencing by hybridization, as well as the
myriad other hybridization arrays performed with arrays of nucleic
acids and/or oligonucleotide probes described in the art.
For use in a hybridization array, the background signals from a
polynucleotide array according to the invention are quantified and
recorded. The mode of detection will depend on the nature of the
label. For flourescent labels, the background signals can be
conveniently quantified by scanning the array with a confocal
camera or with a CCD camera, as is well-known in the art.
Use of the arrays of the present invention contemplates the use of
either probe polynucleotides or target nucleic acids that are
capable of generating a signal when appropriately hybridized to the
array. The probe polynucleotides or target nucleic acids may be
labeled, for example, by the labels and techniques described supra
for labeling the tracer polynucleotide. Alternatively, they may be
labeled by any other technique known in the art. Preferred
techniques include direct chemical labeling methods and enzymatic
labeling methods, such as kinasing and nick-translation.
The array is contacted with a target nucleic acid, which may be
labeled or unlabeled, depending on the particular array (for
example, format II vs. format III SBH), under conditions which
discriminate between perfectly complimentary hybrids and hybrids
containing one or more mismatches. The actual hybridization
conditions used will depend upon, among other factors, the G+C
content of the sequence of interest and the lengths of the
immobilized polynucleotides comprising the array. Hybridization
conditions useful for discriminating between perfect compliments
and mismatches for a variety of hybridization arrays have been
described in the art. For example, hybridization conditions useful
for discriminating complimentary and mismatched hybrids in a
variety of SBH and other applications are described in U.S. Pat.
No. 5,525,464 to Drmanac et al., WO 95/09248 and WO 98/31836. A
particularly detailed discussion of the theoretical and practical
considerations involved in determining hybridization conditions,
and including a discussion of the advantages of low-temperature
washing steps, may be found in WO 98/31836, particularly pages
50-62. Additional guidance may be found in Harmes and Higgins,
Nucleic Acid Hybridization: A Practical Approach, 1985, IRL Press,
Oxford, England.
Following contact, the array is optionally washed, typically under
moderate- to high stringency conditions to remove unhybridized
target. If the target is labeled, the array can be scanned or
otherwise analyzed for detectable assay signal, and the signal from
each labeled spot, or alternatively from all spots, quantified.
Only those spots where hybridization occurred will produce a
detectable assay signal. If each spot in array contains the same
quantity of immobilized polynucleotide, in theory, the intensity of
the assay signal at each spot will be proportional to the extent of
hybridization at that spot. For example, spots containing perfectly
complementary hybrids are expected to produce more intense assay
signals than spots containing mismatched hybrids. In practice,
however, differences in signal intensities between different spots
may instead be due to differences in the amounts of polynucleotide
immobilized at the respective spots.
Because each spot in the arrays of the invention contain an amount
of a label or "tracer" proportional to the amount of polynucleotide
immobilized at the particular spot, the assay signals obtained from
the arrays of the invention can be normalized. As a consequence,
signal intensities from spots within a single array, or across
multiple arrays, can be directly compared, without regard to the
fidelity of the particular array synthesis.
The method by which the signals are normalized will depend upon
whether the tracer or background signals are the same as the assay
signals, such as where the polynucleotides and target nucleic acid
are labeled with the same fluorophore. In this embodiment, a
normalized signal of a particular spot is defined by (I.sub.a
-I.sub.b)/I.sub.b, where I.sub.a is the intensity of the assay
signal of the spot (i.e., intensity of the spot after
hybridization) and I.sub.b is the intensity of the background
signal of the spot (i.e., the intensity of the spot before
hybridization).
In embodiments where the background and assay signals are
different, i.e., where the array spots and target nucleic acid are
labeled with different fluorophores, the normalized signal for a
spot is described by I.sub.a /I.sub.b, where I.sub.a is the
intensity of the assay signal of the spot and I.sub.b is the
intensity of the background signal of the same spot.
While the array is illustrated utilizing a labeled target nucleic
acid, those of skill in the art will recognize that the arrays of
the invention are also useful in assays employing unlabeled target
nucleic acids, such as assays employing the principles of format
III SBH. The only requirement is that some component of the
particular assay generate a detectable signal at spots where
hybridization occurs.
6. EXAMPLE
Tracer Labeling of a Polynucleotide Array
An array of polynucleotides was generated using the surface of a
glass slide as the substrate. The polynucleotides used were 8 bases
long, with an information content corresponding to all possible
5-base sequences. The polynucleotides all had the structure
where:
36C(spacer) is a standard, commercially available 36 carbon spacer
element;
N represents a degenerate position generated by synthesizing the
polynucleotide with an equimolar mixture of all four bases
according to standard methods (i.e., each location on the array
contained a mixture of polynucleotides degenerate at the N
positions); and
B represents any one of the four bases (i.e., each location on the
array contained a mixture of polynucleotides identical at the B
positions).
For the methods of the invention, a mixture of 10 labeled
polynucleotide mixtures was prepared. The polynucleotides in the 10
mixtures had the structure describe above, and the labeled mixture
consisted of the following sequences:
NNNGGCAT NNNCGGAG NNNAACTG NNNATGAA NNNTGTAC
NNNACTGG NNNGAACC NNNTACAG NNNCTGGA NNNCCGGA
Each of these polynucleotide mixtures was labeled at its 3' end
with TAMRA dye (Molecular Probes, Eugene, Oreg.). TAMRA dye has an
absorption maximum at 565 nm and an emission maximum at 580 nm; its
molecular extinction coefficient is 89,000.
The glass slide was prepared for attachment of the polynucleotides
of the array by generating isothiocyanate groups (--N.dbd.C--S) on
the surface of the slide. The slide was derivatized with
isothiocyanate groups according to the following protocol:
(1) Soak glass slide in 1 M HCl for 16 hr. (Alternatively, soak in
1 M nitric acid for 3 hr.) Rinse thoroughly with deionized water,
followed by acetone. Allow to air dry.
(2) Soak slide in hexane, acetone, and methanol, respectively, for
10 min each. Air dry when done. The slide must be completely dry
before proceeding to the next step.
(3) Prepare a solution containing 2% aminopropyltriethoxy silane in
95% acetone:water in a plasic container and let stand 10 min to
activate. Submerge slide in this silane solution for about 2 min
and immediately rinse with acetone. Wash slide with 3 consecutive
acetone washes. Allow to completely air dry.
(4) Cure slides by baking in a dry incubator at 98 C for 45 min.
Remove from incubator and allow to cool for at least 10 min.
(5) Dissolve 1,4-phenylene diisothiocyanate (PDC) in a 10% solution
of pyridine:dimethyl formamide to yield a final concentration of
0.2% PDC. Submerge the slide in the PDC solution and incubate for 2
hr at room temperature. Remove the slide and wash by submerging in
methanol for 5 min, followed by two successive baths of acetone for
5 min each. Allow slide to air dry.
For each spot of the array, small volumes of polynucleotides
mixtures containing 50 .mu.M of the particular degenerate
polynucleotide pool for that spot and 0.04 .mu.M of the labeled
polynucleotide mix (0.08% of the total concentration) were
prepared. These mixtures were then spotted onto the prepared slide
using a robotic pin spotting device. The spotted polynucleotides
covalently bonded to the surface of the slide through a bond
between the cyanate molecule on the slide and the 5' amine of the
polynucleotide.
Before assaying the array, the baseline level of fluorescence at
each location in the array was established by scanning the array to
detect the amount of labeled tracer polynucleotide at each spot
(FIG. 1).
The array was then assayed with a 241 base, single-stranded target
DNA derived from exon 7 of the p53 gene and selected sets of TAMRA
dye-labeled 5-base polynucleotides. After allowing the target DNA,
attached polynucleotides, and labeled polynucleotides to hybridize
under conditions intended to discriminate between perfect matches
and single-base mismatches, ligase was added. The ligase covalently
joined labeled polynucleotides to attached polynucleotides in the
spots of the array at which they bound the target DNA in adjacent
positions. The arrays were then washed to remove the target DNA and
unligated labeled polynucleotides. The array was again scanned to
determine the total fluorescent signal at each position on the
array (FIG. 2).
The intensity signals from each spot on the probed array were then
normalized to the baseline signals from the corresponding spot on
the array, to account for differences in polynucleotide attachment
efficiency. The intensities were normalized according to the
formula (I.sub.a -I.sub.b)/I.sub.b, where I.sub.a is the assay
signal intensity and I.sub.b is the background signal intensity.
The normalized intensities were then used to identify mutations in
the p53 gene by discriminating between perfect complements and
mismatches.
As a control, the same assay was also performed on an array
generated by the same protocol as the labeled array, but without
the use of the labeled polynucleotide tracer mixtures. Because it
lacked the tracer in the spots of the array, the intensities could
not be normalized for the attachment of the polynucleotides.
When the results from the two assays were compared, the signals
from the array with the tracer (which could be normalized for the
efficiency of attachment at each spot) led to the detection of the
mutation in the p53 gene, while the signals from the array without
the tracer (which could not be normalized for the efficiency of
attachment at each spot) could not.
The present invention is not to be limited in scope by the
exemplified embodiments which are intended as illustrations of
single aspects of the invention The foregoing specification and
accompanying drawings is considered to be sufficient to enable one
skilled in the art to broadly practice the invention. Indeed,
various modifications of the above-described means for carrying out
the invention which are obvious to those skilled in the relevant
arts are intended to be within the scope of the following claims.
All patents, patents applications, and publications cited herein
are hereby incorporated by reference in their entireties for all
purposes.
* * * * *