Note: Descriptions are shown in the official language in which they were submitted.
CA 02379212 2002-O1-09
WO 01/06013 PCT/US00/19203
METHODS FOR DETERMINING THE SPECIFICITY AND SENSITIVITY OF
OLIGONUCLEOTIDES FOR HYBRIDIZATION
1. FIELD OF THE INVENTION
The field of this invention relates to the field detecting and reporting
polynucleotide"~
sequences, including genomic sequences, genomic transcript sequences (e.g.,
mRNAs from
cells and/or cDNA sequences derived therefrom) copy numbers and single
nucleotide
polymorphisms (SNPs), by nucleic acid hybridization, e.g., on nucleic acid
microarrays. In
Pa~icular, the invention relates to methods for identifying and/or selecting
polynucleotide
sequences, particularly oligonucleotide sequences, which may be used as
hybridization
probes (e.g., on nucleic acid microarrays) that are both sensitivity and
specific to particular
target polynucleotide sequences of interest.
2. BACKGROUND
Within the past decade, several technologies have made it possible to monitor
the
expression level of a large number of genetic transcripts at any one time
(see, e.g., Schena et
al., 1995, Science 270:467-470; Lockhart et al., 1996, Nature Biotechnology
14:1675-1680;
Blanchard et al., 1996, Nature Biotechnology 14:1649; Ashby et al., U.S.
Patent No.
5,569,588, issued October 29, 1996). For example, techniques are known for
preparing
microarrys of cDNA transcripts (see, e.g., DeRisi et al., 1996, Nature
Genetics 14:457-460;
Shalon et al., 1996, Genome Res. 6:689-645; and Schena et al., 1995, Proc.
Natl. Acad. Sci.
U.S.A. 93:10539-11286). Alternatively, high-density arrays containing thousand
of
oligonucleotides complementary to defined sequences, at defined locations on a
surface
using photolithographic techniques for synthesis in situ are described, e.g.,
Fodor et al.,
1991, Science 251:767-773; Pease et al., 1994, Proc. Natl. Acad. Sci. U.S.A.
91:5022-5026;
Lockhart et al., 1996, Nature Biotechnology 14:1675; U.S. Patent Nos.
5,578,832;
5,556,752; and 5,510,270). Methods for generating arrays using inkjet
technology for
oligonucleotide synthesis are also known in the art (see, e.g., Blanchard,
International
Patent Publication WO 98/41531, published September 24, 1998; Blanchard et
al., 1996,
Biosensors and Bioelectronics 11:687-690; Blanchard, 1998, in Synthetic DNA
Arrays in
Genetic Engineering, Vol. 20, J.K. Setlow, Ed., Plenum Press, New York at
pages 111-
123).
Applications of this technology include, for example, identification of genes
which
are up regulated or down regulated in various physiological states,
particularly diseased
states. Additional exemplary uses for transcript arrays include the analyses
of members of
-1-
CA 02379212 2002-O1-09
WO 01/06013 PCT/US00/19203
signaling pathways, and the identification of targets for various drugs. See,
e.g., Friend and
Hartwell, International Publication No. WO 98/38329 (published September 3,
1998);
Stoughton, U.S. Patent Application Serial No. 09/099,722 (filed June 19,
1998); Stoughton
and Friend, U.S. Patent Application Serial No. 09/074, 983 (filed May 8,
1998); Friend and
Stoughton, U.S. Provisional Application Serial Nos. 60/084,742 (filed May 8,
1998),
60/090,004 (filed June 19, 1998), and 60/090,046 (filed June 19, 1998).
Oligonucleotide sequences are particularly useful as probes on microarrays and
in
other applications that involve nucleic acid hybridization. The
oligonucleotides can be
custom synthesized, by techniques known in the art (see, e.g., Froehler et
al., 1986, Nucleic
Acid Res. 14:5399-5407; McBride et al., 1983, Tetrahedron Lett. 24:246-248),
with any
desired DNA sequence. Further, oligonucleotides are small enough that their
thermodynamic properties (e.g., their free binding energies to complementary
and/or
partially complementary sequences) can be at least partially predicted.
However, because
of their small size, oligonucleotide probes frequently correspond to genomic
sequences that
1 S are non-unique and, as a result, may hybridize to more than one
polynucleotide sequence in
a sample. For example, a particular oligonucleotide probe may not only
hybridize to a
particular mRNA transcript of interest in a sample, but may also hybridize to
other
homologs, analogs, splice variants or even marginally related sequence of that
transcript
that are also, often times in greater abundances, in a sample. As a result of
such "cross-
hybridization," many oligonucleotide probes can result in false positive
measurement,
reflecting a lack of specificity. Conversely, an oligonucleotide probe may
also hybridize to
a target polynucleotide sequence of interest more weakly than predicted, e.g.,
from
predicted hybridization binding energies. Such probes can result in false
negative
hybridization measurements, reflecting a lack of sensitivity.
As a result of these limitations, current microarrays require a plurality of
probe
pairs, which are both matched to and intentionally mismatched to a target
sequence, in order
to empirically distinguish signal arising from a target polynucleotide
sequence of interest
(e.g., a particular mRNA sequence of interest) from signal arising from cross-
hybridization
with other polynucleotide sequences. Currently, in situ synthesized microarray
chips
require more than 20 oligonucleotide probe pairs per gene or gene region
reported (Lockhart
et al., supra). However, unless a large number of probes is employed, such a
match-
mismatch scheme can only screen out cross-hybridization from distantly related
sequences.
In particular, the ability of such a match-mismatch scheme to distinguish
between true
hybridization and cross-hybridization to closely related sequences (e.g.,
closely related
homologs and splice variants) is typically limited or even very poor.
Furthermore, the
"reporting density" (i.e., the number of genes detected per unit of surface
area) for a
-2-
CA 02379212 2002-O1-09
WO 01/06013 PCT/US00/19203
microarray is limited, e.g., by the density with which polynucleotide probes
may be laid
down as well as by the number of polynucleotide probes required per gene. The
number of
polynucleotide probes that may be laid down on a microarray chip is therefore
limited by
the technology used to produce the microarray. Photolithographic techniques
discussed
above for producing oligonucleotide microarrays having a high spatial density
of probes are
expensive to synthesize and therefore require a large capital investment.
Oligonucleotide
microarrays produced using the above discussed inkjet technology methods are,
by contrast,
much cheaper and faster to produce both per chip design and per chip. Thus,
such
microarrays are generally preferred for detecting genetic transcripts.in
cells. However,
microarray chips produced by such inkjet technology have a limited probe
density that is
only a fraction of the probe density of chips produced by photolithography
methods. Thus,
because microarrays currently known in the art must use a number of redundant
probes
(e.g., 20) and have limited probe density, the number of genetic transcripts
that may be
effectively detected on a single microarray chip is limited to about 10,000
gene transcripts
using expensive, photolithographic arrays, and only about 750 to 2,500 gene
transcripts
using less expensive, inkjet arrays.
There exists,. therefore, a need for methods which identify particular
oligonucleotide
sequences that may be used as both sensitive and specific probes for target
polynucleotide
sequences. In particular, there is a need for methods that can identify
particular sequences
that hybridize to a particular sequence of interest, such as the sequence of a
particular gene
or gene transcript, with little or no cross-hybridization to other
polynucleotide sequences in
a sample. There is also a need for methods to design nucleic acid arrays which
have less
require fewer polynucleotide probe sequences to detect individual genes of
interest, and
which therefore contain polynucleotide probe sequences to detect more genes of
interest
than do microarrays that a currently available in the art.
Discussion or citation of a reference herein shall not be construed as an
admission
that such reference is prior art to the present invention.
3. SUMMARY OF THE INVENTION
The present invention provides compositions and methods that can be used to
evaluate binding properties of molecules of a first type, which are referred
to herein as
probe molecules, to molecules of a second type, which are referred to herein
as target
molecules. Specifically, the methods and compositions of the invention can be
used to
evaluate both the sensitivity and the specificity with which a probe binds to
a particular
target.
-3-
CA 02379212 2002-O1-09
WO 01/06013 PCT/US00/19203
The sensitivity of a probe, as the term is used herein, is understood to refer
to the
absolute amount or level of a particular target (i.e., the number of molecules
of the
particular target) that binds to the probe under particular binding
conditions. The amount or
level of a particular target that binds to a probe under particular binding
conditions is also
S referred to herein as the amount or level of specific binding to the probe
under the particular
binding conditions.
The specificity of a probe, as the term is used herein, is understood to refer
to the
amount or level of a particular target (i.e., the number of molecules of the
particular target)
that binds to the probe under particular binding conditions relative to the
amount or level of
non-specific binding to the probe under the same binding conditions. Non-
specific binding,
as the term is used herein, is understood to refer to the amount of molecules
other than
molecules of the particular target (i.e., the number of molecules that are not
molecules of
the particular target) that bind to the probe under particular binding
conditions.
The methods of the invention involve comparing the amount or number of
molecules in a first sample that bind to molecules of a probe to the amount or
number of
molecules in a second sample that bind to molecules of the same probe. The
first sample,
which is referred to herein as a "specific binding sample," preferably
comprises molecules
of a particular target that is generally a target of interest to a user.
Preferably, the molecules
of the particular target in the specific binding sample are substantially pure
(e.g., at least
~5% pure, preferably at least 90% pure, more preferably at least 95% pure and
even more
preferably 99% pure).
The second sample, which is referred to herein as a "non-specific binding
sample,"
comprises molecules of a plurality of different (i.e., non-identical) targets
other than the
particular target of interest. Preferably, the molecules of the plurality of
different targets are
of the same type and approximately the same abundances as molecules in a real
sample for
which the probe is intended.
The invention is based, at least in part, on the discovery that a meaningful
measurement of nonspecific binding to molecules of a particular probe may be
obtained by
supplying a distinguishable binding sample of competing molecules in which the
competing
molecules are of the same type and have approximately the same abundances as
competing
molecules in a real sample for which the particular probe is intended. Such
binding samples
may be readily obtained, e.g., according to the methods of the invention
described
hereinbelow, and can be used as non-specific binding samples in the methods of
the
invention to obtain a real measurement of non-specific binding that can be
readily compared
to measurements of specific binding from a specific binding sample. Indeed,
the methods
of the invention both the specific and non-specific binding levels to be
measured
-4-
CA 02379212 2002-O1-09
WO 01/06013 PCT/US00/19203
simultaneously. The specific and non-specific binding samples need not be
physically
separate but need only be distinct from one another so that the binding of
molecules from
the specific binding sample can be distinguished from the binding of molecules
from the
non-specific binding sample. For example, in a preferred embodiment the
specific and non-
specific binding samples are differentially labeled, e.g., with fluorescent
labels that
fluoresce at different wavelengths.
The methods of the invention are particularly useful for evaluating large
numbers of
different probes. For example, the methods of the invention can be used to
evaluate a
plurality of different probes by comparing the amount or number of molecules
from a first
sample (i. e., a specific binding sample) that bind to a each of the plurality
of different
probes to the number or amount of molecules from a second sample (i.e., a non-
specific
binding sample) that bind to each of the plurality of different probes. In
preferred
embodiments, the methods of the invention are used to evaluate a plurality of
different
probes in an array of probes, wherein the array comprises a solid (or, in
certain
embodiments, semi-solid) support or surface to which molecules of the
plurality of different
probes are immobilized. Most preferably, the array is an addressable array,
such as a
positionally addressable array wherein each different probe is located at a
specific, known
location on the support or surface such that the identitiy of a particular
probe can be
determined from its location on the support or surface.
The probes and target molecules that can be evaluated using the compositions
and
methods of the invention can be of any type, although they are preferably
molecules of a
type or class that can specifically bind to one another. For example, in
certain
embodiments, the probes can be molecules of a particular antibody (preferably
a
monoclonal antibody) and the target molecules can be molecules to which
antibodies can
specifically bind such as proteins. However, the compositions and methods of
the invention
are particularly useful for evaluating the hybridization properties of
different polynucleotide
probes to examine both the sensitivity and specificity with which the
polynucleotide probes
hybridize to particular target polynucleotides (i.e., to polynucleotide
molecules having
particular nucleotide sequences). Thus, in preferred embodiments of the
invention both the
probes and the target molecules are polynucleotide molecules.
In such preferred embodiments, the sensitivity of a probe is understood to
refer to
the absolute amount of a particular target polynucleotide (i.e., the number of
polynucleotide
molecules having a particular nucleotide sequence) that hybridizes to the
probe under
particular hybridization conditions. The amount of a particular target
polynucleotide that
hybridizes to a probe under particular hybridization conditions is also
referred to herein as
-5-
CA 02379212 2002-O1-09
WO 01/06013 PCT/US00/19203
the amount of specific hybridization to the probe under the particular
hybridization
conditions.
The specificity of a probe, as the term is used in preferred embodiments of
the
invention, is understood to refer to the amount of a particular target
polynucleotide (i.e., the
number of polynucleotide molecules having a particular nucleotide sequence)
that
hybridizes to the probe under particular hybridization conditions compared to
or relative to
the amount of cross hybridization to the probe under the same hybridization
conditions.
Cross-hybridization or non-specific hybridization, as the terms are used
herein, are
understood to refer to the amount of polynucleotides other than the particular
target
polynucleotide (i.e., the number of polynucleotide molecules having nucleotide
sequences
that are different than the nucleotide sequence of the particular target
polynucleotide) that
hybridize to the probe under particular hybridization conditions.
In particularly preferred embodiments, the methods of the invention involve
comparing the number or amount of polynucleotide molecules from a first sample
that
hybridize to molecules of a polynucleotide probe to the number or amount of
polynucleotide molecules from a second sample that hybridize to molecules of
the
polynucleotide probe. The first sample is a "specific hybridization sample"
comprising
molecules of a particular target polynucleotide (i.e., polynucleotide
molecules having a
particular sequence). The polynucleotide sequence can be, for example, the
sequence of a
p~icular gene or gene transcript of a cell or organism. The second sample is a
"non-
specific hybridization sample" comprising a plurality of different (i.e., non-
identical)
polynucleotide molecules, each different polynucleotide molecule having a
different
nucleotide sequence. In particular, the second or non-specific hybridization
sample should
comprise polynucleotide molecules having nucleotide sequences that are
different from the
nucleotide sequence of the particular target polynucleotide in the first or
specific
hybridization sample. For example, in embodiments wherein the sequence of the
particular
target polynucleotide in the first or specific hybridization sample is the
sequence of a
particular gene or gene transcript of a cell or organism, the nucleotide
sequences of the
polynucleotide molecules in the second, non-specific hybridization sample
preferably
comprise sequences representing the other genes or gene transcripts of the
cell or organism.
For example, the invention provides a first preferred embodiment wherein the
first
sample (i.e., the specific hybridization sample) is a substantially pure
(i.e., at least 75%
pure, preferably at least 90% pure and more preferably at least 95% or 99%
pure) sample of
molecules having a particular target nucleotide sequence and the second sample
(i.e., the
non-specific hybridization sample) comprises a plurality of different
polynucleotide
molecules, with each different polynucleotide molecule is the second sample
having a
-6-
CA 02379212 2002-O1-09
WO 01/06013 PCT/US00/19203
different nucleotide sequence. In a particularly preferred aspect of this
first preferred
embodiment, the target polynucleotide molecules is a gene or gene transcript
(e.g., a mRNA
or cDNA molecule) of a cell or organism and the non-specific hybridization
sample is a
polynucleotide sample from a "deletion mutant" of the cell or organism (i.e.,
a variety or
strain of the cell or organism in which the gene or gene transcript
corresponding to the
target polynucleotide is absent or is silent and not expressed).
The invention also provides a second preferred embodiment wherein the first
sample
(i.e., the specific hybridization sample) is a substantially pure sample of
molecules having a
particular target nucleotide sequence and the second sample (i.e., the non-
specific
hybridization sample) comprises a plurality of different polynucleotide
molecules with each
different polynucleotide molecule in the second sample having a different
nucleotide
sequence. In particular, in the second preferred embodiment of the invention,
the
polynucleotide molecules in the non-specific hybridization sample include
molecules of the
target polynucleotide sequence. In a particularly preferred aspect of this
second preferred
embodiment, the target polynucleotide corresponds to a gene or gene transcript
of a cell or
organism and the non-specific hybridization sample is a polynucleotide sample
from the
"wild type" cell or organism which expresses the gene or gene transcript
corresponding to
the target polynucleotide at normal levels or amounts.
The invention further provides a third preferred embodiment wherein the first
sample (i.e., the specific hybridization sample) comprises molecules having a
particular
target polynucleotide sequence as well as molecules having other non-target
polynucleotide
sequences and the second sample (i.e., the non-specific hybridization sample)
comprises a
plurality of different non-target polynucleotide molecules. Thus, in the third
preferred
embodiment of the invention, the specific hybridization sample is identical to
the non-
specific hybridization sample described above for the second preferred
embodiment of the
invention, and the non-specific hybridization sample is identical to the non-
specific
hybridization sample described above for the first preferred embodiment of the
invention.
The invention still further provides a forth preferred embodiment wherein both
the
first and second sample (i.e., the specific and the non-specific hybridization
samples)
comprise a plurality of different polynucleotide molecules, including
molecules of the target
polynucleotide. However, in the forth preferred embodiment of the invention
the amount or
level of molecules of the target polynucleotide in the first, specific
hybridization sample
differs substantially from the amount or level of molecules of the target
polynucleotide in
the second, non-specific hybridization sample.
Although the probes used in the invention can comprise any type of
polynucleotide,
in preferred embodiments the probes comprise oligonucleotide sequences; i.e.,
CA 02379212 2002-O1-09
WO 01/06013 PCT/US00/19203
polynucleotide sequences that are between about 4 and about 200 bases in
length, and are
most preferably between about 15 and about 150 bases in length. In one
embodiment,
shorter oligonucleotide sequences are used that are less than about 40 bases
in length, and
are more preferably between about 15 and about 30 bases in length. However, a
preferred
embodiment of the invention uses longer oligonucleotide probes that are
between about 40
and about 80 bases in length with oligonucleotide sequences between about 50
and about 70
bases in length (e.g., oligonucleotide sequences of about 50 to about 60 bases
in length)
being particularly preferred.
The compositions and methods of the invention can also be used to evaluate
different binding conditions for a probe or for a plurality of different
probes. For example,
the compositions and methods of the invention can be used to evaluate
hybridization
conditions for a polynucleotide probe or for a plurality of different
polynucleotide probes.
Specifically, the amount of binding of molecules from a first binding sample
(i. e., a specific
binding sample) to one or more different probes under particular binding
conditions can be
compared to the amount of binding of molecules from a second binding sample
(i.e., a non-
specific binding sample) to the one or more different probes under the same
binding
conditions. The sensitivity and specificity of the probe or probes under the
particular
binding conditions can then be readily determined. Thus, by performing the
above
comparison under different binding conditions (e.g., under different
hybridization
conditions for polynucleotides) optimum binding conditions can be readily
ascertained, e.g.,
by determining the binding conditions wherein the sensitivity and specificity
of the probe or
probes to the particular target are optimized.
The invention therefore provides, in a first embodiment, a method for
evaluating a
probe comprising comparing the amount of binding of a first sample to the
probe with the
amount of binding of a second sample to the probe. The first sample comprises
molecules
of a particular target and the second sample comprises molecules of a
plurality of different
targets. In one preferred aspect of this first embodiment, the first sample is
a substantially
pure sample of molecules of the particular target (e.g., at least 75%, at
least 90%, at least
95% or at least 99% pure). In another aspect of this first embodiment, each
different target
in the plurality of different targets of the second sample is different from
the particular
target of the first sample. In still other preferred aspects of this
embodiment, the methods
also determine the specificity of the probe, preferably from the ratio of the
amount of
binding of molecules of the particular target in the first sample to the probe
with the amount
of binding of molecules of the plurality of targets in the second sample to
the probe. The
invention still further provides aspects of this first embodiment wherein the
molecules of
_g_
CA 02379212 2002-O1-09
WO 01/06013 PCT/US00/19203
the first and/or second samples are detectably labeled, e.g., with a
fluorescent molecule. In
particularly preferred aspects, the molecules of the particular target in the
first sample are
detectably labeled with a first label, and the molecules of the plurality of
targets in the
second sample are detectably labeled with a second, different label (e.g.,
with a first and
second fluorescent molecule). In preferred aspects of this embodiment, the
probe is
attached to a surface of a support. In other preferred aspects, the probe is
one of a plurality
of probes, preferably wherein the plurality of probes comprises an array of
probes, said
array having a support with at least one surface and wherein each probe is
attached to the
surface of the support.
Aspects of this first embodiment are also provided wherein the probe is a
polynucleotide probe having a particular nucleotide sequence. In various other
aspects
provided by the invention, the molecules of the particular target in the first
sample are
polynucleotide molecules having a nucleotide sequence. In particularly
preferred aspects,
the polynucleotide probe is attached to a surface of a support. In other
preferred aspects the
polynucleotide probe is one of a plurality of polynucleotide probes and,
preferably, the
plurality of polynucleotide probes comprises an array of polynucleotide
probes, said array
having a support with at least one surface wherein each polynucleotide probe
is attached to
the surface of the support.
The invention also provides, in another embodiment, a method for evaluating a
polynucleotide probe having a particular nucleotide sequence, said method
comprising
comparing the amount of hybridization of a first sample to the polynucleotide
probe with
the amount of hybridization of a second sample to the polynucleotide probe,
wherein: the
first sample comprises molecules of a target polynucleotide having a target
nucleotide
sequence; and the second sample comprises a plurality of different
polynucleotide
molecules wherein each different polynucleotide molecule has a different
nucleotide
sequence. In one preferred aspect of this other embodiment, the first sample
is a
substantially pure sample of molecules of the target polynucleotide (e.g., at
least 75%, 90%,
95% or 99% pure). In one aspect, each different polynucleotide molecule in the
second
sample has a nucleotide sequence different from the target nucleotide
sequence. In another
aspect, the plurality of different polynucleotide molecules in the second
sample comprises:
(a) polynucleotide molecules having a nucleotide sequence that is the same as
the target
nucleotide sequence, and (b) a plurality of different polynucleotide molecules
each having a
different nucleotide sequence that is different from the target nucleotide
sequence. In
another aspect, the first sample further comprises polynucleotide molecules
having a
nucleotide sequence different from the target nucleotide sequence. In one
aspect, each
-9-
CA 02379212 2002-O1-09
WO 01/06013 PCT/US00/19203
different polynucleotide molecule in the second sample has a nucleotide
sequence different
from the target nucleotide sequence. In another aspect, the second sample
comprises: (a)
polynucleotide molecules having a nucleotide sequence that is the same as the
target
nucleotide sequence; and (b) a plurality of different polynucleotide
molecules, each
$ different polynucleotide molecule having a different nucleotide sequence
that is different
from the target nucleotide sequence, wherein the amount of polynucleotide
molecules in the
first sample having the target nucleotide sequence differs substantially from
the amount of
polynucleotide molecules in the second sample having the target nucleotide
sequence (e.g.,
by a factor of at least two, at least four, at least eight, at least twenty,
or at least 100).
The invention also provides, in still other embodiments, a method for
evaluating a
plurality of polynucleotide probes wherein each polynucleotide probe in the
plurality of
polynucleotide probes has a particular nucleotide sequence. The method
comprises
comparing the amount of hybridization of a first sample to each polynucleotide
probe in the
plurality of polynucleotide probes with the amount of hybridization of a
second sample to
each polynucleotide probe in the plurality of polynucleotide probes, wherein:
the first
sample comprises molecules of a target polynucleotide having a target
nucleotide sequence;
and (b) the second sample comprises a plurality of different polynucleotide
molecules,
wherein each different polynucleotide molecule has a different nucleotide
sequence.
The invention further provides, in yet other embodiments, a method for
evaluating
hybridization conditions of one or more polynucleotide probes, each of said
one or more
polynucleotide probes having a particular polynucleotide sequence. The method
comprises
comparing the amount of hybridization of a first sample to each of the one or
more
polynucleotide probes under particular hybridization conditions with the
amount of
hybridization of a second sample to each of the one or more polynucleotide
probes under
the same particular hybridization conditions. The first sample preferably
comprises
molecules of a target polynucleotide having a target nucleotide sequence. The
second
sample preferably comprises a plurality of different polynucleotide molecules
wherein each
different polynucleotide molecule has a different nucleotide sequence.
4. BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows a flow chart illustrating an exemplary embodiment of the
invention
wherein the methods are used to evaluate oligonucleotide probes on a
microarray.
- 10-
CA 02379212 2002-O1-09
WO 01/06013 PCT/US00/19203
FIGS. 2A-2B show fluorescence intensity images from the simultaneous
hybridization of two samples to a microarray comprising oligonucleotide probes
complementary to the gene YER019W of the yeast Saccharomyces cerevisiae and
control
oligonucleotide probes as indicated:
FIG. 2A is the fluorescence intensity from a sample of 2 ~g fragmented cRNA
from
yer019 w/- homozygous disruption yeast labeled with Cy3 ("green channel");
FIG. 2B is the fluorescence intensity from a sample of 1.6 ng fragmented pure
YER019W labeled with Cy5 ("red channel")
FIGS. 3A-3B show fluorescence intensity images from the simultaneous
hybridization of two samples to a microarray identical to the array in FIGS.
2A-2B:
FIG. 3A is the fluorescence intensity from a sample of 1.6 ng fragmented pure
YER019W labeled with CY3 ("green channel");
FIG. 3B is the fluorescence intensity from a sample of 2 ~g fragmented cRNA
from
yer019 w/- homozygous disruption yeast labeled with Cy5 ("red channel").
FIGS. 4A-4B show fluorescence intensity images from the simultaneous
hybridization of two samples to a microarray identical to the array in FIGS.
2A-2B:
FIG. 4A is the fluorescence intensity from a sample of 2 ~g fragmented cRNA
from
yer019w/- homozygous disruption yeast, labeled with Cy3 ("green channel");
FIG. 4B is the fluorescence intensity from a sample of 2 pg fragmented cRNA
from
wild-type yeast, labeled with Cy5 ("red channel").
FIGS. SA-C plots the amount of target and non-target hybridization observed
for
individual probes according to their "tiling positions." FIG. 5A plots the
mean normalized
hybridization intensity from the combined signal of labeled YER019W
hybridization in
FIGS. 2B and 3A; FIG. 5B plots the mean normalized hybridization intensity
from the
combined signal of labeled yer019w/- fragmented cRNA hybridization in FIGS. 2A
and 3B;
FIG. SC plots the ratio of the targeted to non-targeted hybridization
intensities plotted in
FIGS. 5A and SB, respectively.
FIG. 6 is a scatter plot that diagrams relationships between sensitivity ("GS
signal,"
horizontal axis) and specificity ("GNS signal," vertical axis) for each
complementary probe
of YER019W using the data in FIGS. 5A and SC, respectively.
-11-
CA 02379212 2002-O1-09
WO 01/06013 PCT/US00/19203
FIG. 7 is a representation of a computer system which may be used to practice
the
analytical methods of the present invention.
5. DETAILED DESCRIPTION
This section presents a detailed description of the present invention and its
applications. The description is by way of several exemplary illustrations, in
increasing
detail and specificity, of the general methods of the invention. These
examples are non-
limiting, and related variants that will be apparent to one of skill in the
art are intended to be
encompassed by the appended claims.
In particular, the invention relates to methods and compositions that can be
used to
evaluate the properties of different probe molecules and, specifically, to
evaluate both the
sensitivity and the specificity with which a probe binds to a particular
target. In particularly
preferred embodiments, both the probe molecules and the target molecules are
polynucleotides. Accordingly, the methods and compositions of the invention
are described
hereinbelow predominantly in terms of these embodiments (i. e., in terms of
probes and
targets that are polynucleotide molecules).
One skilled in the art can readily appreciate other embodiments, however, in
which
the methods and compositions of the invention are used to evaluate different
types of probe
molecules and target molecules. Indeed, the invention is equally applicable to
any type of
probe molecule and target molecule, although the probe molecules and target
molecules are
preferably of a type or class of molecules that can specifically bind to one
another. For
example, one skilled in the art can readily appreciate that in certain,
alternative
embodiments the probes of the invention can comprise antibodies (preferably
monoclonal
antibodies) while the target molecules can be any type of molecule to which an
antibody
can specifically bind. For example, the target molecules of the invention can
also be protein
or peptide molecules. One skilled in the art can also appreciate other
alternative
embodiments, and can make and use such alternative embodiments without undue
experimentation. It is therefore understood that such alternative embodiments
are also to be
encompassed by the appended claims.
Section 5.1 first provides an introductory overview of the invention and, in
particular, presents and defines certain concepts of the invention such as the
concept of a
probe and a target. A detailed description of the particular methods and
compositions of the
invention is then presented in Section 5.2. Specifically, this overview
includes a description
of the methods by which candidate probes are selected and prepared for
evaluation
(Subsection 5.2.1), a description of the hybridization samples used in the
methods of the
invention (Subsection 5.2.2), and a description of how hybridization levels
can be measured
-12-
CA 02379212 2002-O1-09
WO 01/06013 PCT/US00/19203
for each of the two samples (Subsection 5.2.3). Subsection 5.2.4 also
describes methods by
which such hybridization data may be analyzed, e.g., to evaluate one or more
probes and to
determine the sensitivity and/or specificity of one or more probes.
The methods and compositions of the invention have many useful applications,
e.g.,
in the selection and preparation of probes for microarrays. A few of these
applications are
presented in Section 5.3 below. Finally, an exemplary illustration of the
methods and
compositions of the invention is also provided below in Section 6.
Specifically, this
example demonstrates one particular and non-limiting embodiment of the
invention wherein
the methods and compositions of the invention are used to evaluate candidate
probes for the
gene YER019W of the yeast Saccharomyces cerevisiae.
5.1. INTRODUCTION
The present invention provides methods and compositions that can be used to
evaluate binding properties of molecules of a given type, which are referred
to herein as
probe molecules, to molecules of a second type, which are referred to herein
as target
molecules. A probe or probe molecule, as the term is used herein, is
understood to be any
molecule that can be used to detect another molecule. Likewise, a target or
target molecule,
as the term is used herein, is understood to be any molecule that can be
detected by using a
probe.
Generally, a target molecule is detected by detecting the binding of the
target
molecule to a probe molecule. For example, in preferred embodiments probe
molecules are
immobilized on a solid (or, in certain embodiments a semi-solid) support or
surface. A
sample is then contacted to the solid support or surface under conditions such
that target
molecules that are intended to be detected by the probe molecules can bind
thereto. The
support or surface is subsequently washed under conditions such that molecules
that are not
bound to the probe molecules are removed, while the probe molecules and target
molecules
bound thereto remain. Preferably, the molecules in the sample are detectably
labeled, e.g.,
with a fluorescent label or dye. Thus, binding of the target molecules to the
probe
molecules can be detected, e.g., by detecting the detectable label.
Preferably, molecules of a particular probe specifically detect a particular
target.
For example, in preferred embodiments wherein the probe molecules and the
target
molecules are polynucleotide molecules, the molecules of a particular probe
preferably
detect polynucleotide molecules having a particular polynucleotide sequence;
e.g., a
nucleotide sequence that is complementary to the nucleotide sequence of the
probe
molecules. Further, the polynucleotide sequence to be detected is frequently
one sequence
among hundreds or even thousands or hundreds of thousands in a particular
sample. Thus,
-13-
CA 02379212 2002-O1-09
WO 01/06013 PCT/US00/19203
in order for a probe to specifically detect a particular target, it is
generally preferably that
the probe bind specifically to that target. Specifically, it is generally
preferred that the
specific binding of a particular target to a probe be maximized, while the non-
specific
binding of molecules to the probe is preferably minimized.
As noted above, probes and targets can comprise any type or class of molecule,
although they are preferably of a type or class of molecule that specifically
bind to each
other. For example, antibodies are useful probes for detecting molecules such
as proteins
and peptides to which they specifically bind.
In particularly preferred embodiments of the invention, the probes and targets
both
comprise polynucleotide molecules. Specifically, polynucleotide molecules,
which can
generally be characterized by their nucleotide sequences, can bind or
hybridize to other
polynucleotide molecules by forming non-covalent Watson-Crick base pairs.
Thus, target
polynucleotide molecules having a particular nucleotide sequence can be
readily detected by
means of polynucleotide probe molecules having a complementary sequence.
"Target" polynucleotide, as the term is used herein, refers to molecules of a
particular polynucleotide sequence of interest. Exemplary target
polynucleotides which
may be analyzed by the methods and compositions of the present invention
include, but are
not limited to DNA molecules such as genomic DNA molecules, cDNA molecules and
fragments thereof, including oligonucleotides, expressed sequence tags
("ESTs"), sequence
tag sites ("STSs"), etc. Target polynucleotides which may be analyzed by the
methods and
compositions of the invention also include RNA molecules such as, but by no
means
limited to messenger RNA (mRNA) molecules, ribosomal RNA (rRNA) molecules,
cRNA
(i.e., RNA molecules prepared from cDNA molecules that are transcribed in
vivo) and
fragments thereof.
The target polynucleotides may be from any source. For example, the target
polynucleotide molecules may be naturally occurring nucleic acid molecules
such as
genomic or extragenomic DNA molecules isolated from a cell or organism, or RNA
molecules, such as mRNA molecules, isolated from a cell or organism.
Alternatively, the
polynucleotide molecules may be synthesized, including, e.g., nucleic acid
molecules
synthesized enzymatically in vivo or in vitro, such as cDNA molecules, or
polynucleotide
molecules synthesized by PCR, RNA molecules synthesized by in vitro
transcription, etc.
The sample of target polynucleotides can comprise, e.g., molecules of DNA,
RNA, or
copolymers of DNA and RNA. In preferred embodiments, the target
polynucleotides of the
invention will correspond to particular genes or to particular gene
transcripts (e.g., to
particular mRNA sequences expressed in cells or to particular cDNA sequences
derived
from such mRNA sequences). However, in many embodiments, particularly those
- 14-
CA 02379212 2002-O1-09
WO 01/06013 PCT/US00/19203
embodiments wherein the polynucleotide molecules are derived from mammalian
cells, the
target polynucleotides may correspond to particular fragments of a gene
transcript. For
example, the target polynucleotides may correspond to different exons of the
same gene,
e.g., so that different splice variants of that gene may be detected and/or
analyzed.
Preferably, the polynucleotides analyzed by the compositions and methods of
the
invention are derived from a cell or organism. Specifically, the target
polynucleotides are
preferably derived from and correspond to particular genes or gene transcripts
(e.g., mRNA
sequences or cDNA sequences derived therefrom) of a cell or organism. The
probes
evaluated using the methods and compositions of the invention are therefore
preferably
intended for the detection of particular gene and gene transcripts, e.g., in
samples of
polynucleotide molecules derived from, and therefore expressed by, a cell or
organism.
Cells and organisms that can be manipulated by means of routine techniques,
e.g., of
in vitro homologous recombination and/or sexual genetics are particularly
preferred. More
specifically, preferred cells or organism include those cells or organisms for
which specific
deletion strains or mutants (e.g., strains in which one or more particular
genes or interest are
deleted) are readily available. Such cells and organism include bacterial
cells and
organisms such as Escherichia coli, and yeast cells and organisms such as
Saccharomyces
cerevisiae to name a few. Other cells and organisms for which specific
deletion strains or
mutants can be readily obtained without undue experimentation will also be
apparent to
those skilled in the art.
It is understood, however, that the methods and compositions of the invention
can be
used to evaluate probes for polynucleotides from any cell or organism,
including cells form
higher organisms, e.g., plant cells and animal cells including mammalian cells
such as cells
from a mouse, a rat, or a human organism to name a few. The methods and
compositions of
the invention can be used to evaluate probes for polynucleotides from an
organism even
though specific deletion strains or mutants may not be available.
Although, for simplicity, this disclosure often makes reference to single
cells (e.g.,
"RNA is isolated from a cell"), it is understood that more often any
particular step of the
invention will be carned out using a plurality of genetically and
transcriptionally identical
cells, e.g., from a cultured cell line. Such similar cells are also referred
to herein as a "cell
type." Cells of a particular cell type can be either from naturally single
celled organisms
such as yeast or bacteria, or can be derived from multi-cellular higher
organisms. It is also
understood, however, that the methods of the invention can be practiced using
samples (e.g.,
mRNA samples) that are extracted from a plurality of cells, e.g., in a tissue
sample from an
organism such as a patient. Cells in such samples will, in general, still be
genetically
identical, but they will typically comprise different cell types of an
organism that express at
-15-
CA 02379212 2002-O1-09
WO 01/06013 PCT/US00/19203
least some different genes. In other instances, however, the cells may be
cells, such as
cancer cells, that contain one or more genetic mutations and are not,
therefore, genetically
identical.
In preferred embodiments, the target polynucleotides to be analyzed are
prepared in
vitro from nucleic acids extracted from cells. For example, in one embodiment,
RNA is
extracted from cells (e.g., total cellular RNA) and messenger RNA is purified
from the total
extracted RNA. cDNA is then synthesized from the purified mRNA using, e.g.,
oligo-dT or
random primers. Preferably, the target polynucleotides are short and/or
fragmented
polynucleotide molecules which are representative of the original nucleic acid
population of
the cell.
The target polynucleotides to be analyzed by the methods and compositions of
the
invention are preferably detectably labeled. For example, cDNA can be labeled
directly,
e.g., with nucleotide analogs, or indirectly, e.g., by making a second,
labeled cDNA strand
using the first strand as a template. Alternatively, the double-stranded cDNA
can be
transcribed into cRNA and labeled.
Preferably, the detectable label is a fluorescent label, e.g., by
incorporation of
nucleotide analogs. Other labels suitable for use in the present invention
include, but are
not limited to, biotin, imminobiotin, antigens, cofactors, dinitrophenol,
lipoic acid, olefinic
compounds, detectable polypeptides, electron rich molecules, enzymes capable
of
generating a detectable signal by action upon a substrate, and radioactive
isotopes.
Preferred radioactive isotopes include, 32P, 3s5, 'aC, 'sN and'zsl, to name a
few. Fluorescent
molecules suitable for the present invention include, but are not limited to,
fluorescein and
its derivatives, rhodamine and its derivatives, texas red, S'-carboxy-
fluorescein ("FMA"),
2',7'-dimethoxy-4',5'-dichloro-6-carboxy-fluorescein ("JOE"), N,N,N',N'-
tetramethyl-6-
carboxy-rhodamine ("TAMRA"), 6'-carboxy-X-rhodamine ("ROX"), HEX, TET, IRD40
and IRD41. Fluroescent molecules that are suitable for the invention further
include:
cyamine dyes, including but not limited to Cy2, Cy3, Cy3.5, CyS, Cy5.5, Cy7
and FluorX;
BODIPY dyes, including but not limited to BODIPY-FL, BODIPY-TR, BODIPY-TMR,
BODIPY-630/650, and BODIPY-650/670; and ALEXA dyes, including but not limited
to
~-E~-488, ALEXA-532, ALEXA-546, ALEXA-568, and ALEXA-594; as well as other
fluorescent dyes known to those skilled in the art. Electron rich indicator
molecules
suitable for the present invention include, but are not limited to, aferntin,
hemocyanin, and
colliodal gold. Alternatively, in less preferred embodiments the target
polynucleotides may
be labeled by specifically complexing a first group to the polynucleotide. A
second group,
covalently linked to an indicator molecule and which has an affinity for the
first group, can
-16-
CA 02379212 2002-O1-09
WO 01/06013 PCT/US00/19203
be used to indirectly detect the target polynucleotide. In such an embodiment,
compounds
suitable for use as a first group include, but are not limited to, biotin and
iminobiotin.
The target polynucleotides which are analyzed (e.g., detected) by the methods
and
compositions of the invention are contacted to a probe or to a plurality of
probes under
conditions such that polynucleotide molecules having sequences complementary
to the
probe hybridize thereto. As used herein, a "probe" refers to polynucleotide
molecules of a
particular sequence at to which target polynucleotide molecules having a
particular
sequence (generally a sequence complementary to the probe sequence) are
capable of
hybridizing so that hybridization of the target polynucleotide molecules to
the probe can be
detected. The polynucleotide sequences of the probes may be, e.g., DNA
sequences, RNA
sequences or sequences of a copolymer of DNA and RNA. For example, the
polynucleotide
sequences of the probes may be full or partial sequences of genomic DNA, cDNA,
mRNA
or cRNA sequences extracted from cells. The polynucleotide sequences of the
probes may
also be synthesized, e.g., by oligonucleotide synthesis techniques known to
those skilled in
the art. The probe sequences can also be synthesized enzymatically in vivo,
enzymatically
in vitro (e.g., by PCR) or non-enzymatically in vitro.
Preferably, the probes used in the methods of the present invention are
immobilized
to a solid support or surface such that polynucleotide sequences that are not
hybridized or
bound to the probe or probes may be washed off and removed without removing
the probe
or probes and any polynucleotide sequence bound or hybridized thereto. In one
particular
embodiment, the probes will comprise an array of distinct polynucleotide
sequences bound
to a solid (or semi-solid) support or surface such as a glass surface. Most
preferably, the
array is an addressable array wherein each different probe is located at a
specific known
location on the support or surface such that the identity of a particular
probe can be
determined from its location on the support or surface.
Although the probes used in the invention can comprise any type of
polynculeotide,
in preferred embodiments the probes comprise oligonucleotide sequences (i.e.,
polynucleotide sequences that are between about 4 and about 200 bases in
length, and are
more preferably between about 15 and about 150 bases in length). In one
embodiment,
shorter oligonucleotide sequences are used that are between about 4 and about
40 bases in
length, and are more preferably between about 15 and about 30 bases in length.
However, a
more preferred embodiment of the invention uses longer oligonucleotide probes
that are
between about 40 and about 80 bases in length, with oligonucleotide sequences
between
about 50 and about 70 bases in length (e.g., oligonucleotide sequences of
about 60 bases in
length) being particularly preferred.
-17-
CA 02379212 2002-O1-09
WO 01/06013 PCT/US00/19203
The invention is based, at least in part, on the discovery the nonspecific
hybridization to a particular probe may be assayed by supplying a
distinguishable
hybridization sample of competing molecules. In particular, non-specific
hybridization
samples can be readily obtained which comprise polynucleotide sequences other
than the
particular sequences intended to specifically hybridize to a probe. Rather,
the
polynucleotide sequences of such non-specific hybridization samples are
competing
polynucleotides of the same type and having the same abundances as competing
polynucleotides in experimental samples for which the probe is intended.
5.2. OVERVIEW OF THE METHODS OF THE INVENTION
A flow chart illustrating an exemplary, non-limiting embodiment of the
invention is
shown in FIG. 1. This particular embodiment evaluates the hybridization
properties of a
plurality of different polynucleotide probes on a microarray and, more
specifically, can be
used to evaluate the sensitivity and/or specificity with which each of the
different probes
hybridizes to a particular target polynucleotide.
In the particular embodiment depicted in FIG. 1, a microarray or "chip" of
polynucleotide probes is designed and built by first selecting a plurality of
different
oligonucleotide sequences (101) to evaluate by the methods of the invention,
followed by
synthesis of a microarray of the selected oligonucleotide sequences (102),
e.g., by ink jet
synthesis technology.
Two, differentially labeled hybridization samples are also prepared: a
specific
hybridization sample (103) and a non-specific hybridization sample (104). For
example, in
the particular embodiment shown in FIG. 1 the specific hybridization sample
(103) is a
"gene specific" sample comprising the purified target polynucleotide (e.g.,
polynucleotide
molecules of a purified gene of interest) labeled with a green fluorescent
label (e.g., a
fluorophore such as Cy3 that fluoresces green light when stimulated). The
exemplary non-
specific hybridization sample (104) comprises a sample ofpolynucleotides from
a "deletion
strain" of the cell or organism (i.e., from a strain of a cell or organism
that does not express
the target polynucleotide) labeled with a red fluorescent label (e.g., a
fluorophore such as
Cy5 that fluoresces red light when stimulated). Thus, the non-specific
hybridization sample
is a polynucleotide sample wherein the target polynucleotide has been removed
or deleted.
Both the specific and the non-specific hybridization samples are hybridized to
the
probes (105), preferably simultaneously, and the intensity of their respective
labels is
measured. The signal intensities and/or ratios are then analyzed (106) and
used to evaluate
~e hybridization properties of the different probes.
-18-
CA 02379212 2002-O1-09
WO 01/06013 PCT/US00/19203
Each of the steps depicted in FIG. 1 is described in detail below with respect
to
general aspects of the invention and in terms of specific, exemplary
embodiments. In
particular, subsection 5.2.1, below, describes certain exemplary methods by
which
candidate probes can be selected for evaluation by the methods of the
invention. In
particularly preferred embodiments of the invention, the candidate probes
comprise
microarrays of probes. Accordingly, subsection 5.2.1 also describes and
enables
microarrays as well as methods for preparing the candidate probes for
microarrays.
Subsection 5.2.2 describes and enables both the specific hybridization samples
and
the non-specific hybridization samples which are used in the invention to
evaluate the
candidate probes. The description includes a description of certain
particularly preferred
embodiments of both the specific hybridization samples and the non-specific
hybridization
samples which that can be used in the invention. Exemplary methods and
compositions for
labeling such samples are also described in Section 5.2.2, including the
differential labeling
methods that are preferred in the present invention. Subsection 5.2.3
describes methods of
measuring hybridization of the two samples (i.e., the specific and non-
specific hybridization
samples) to the candidate probes, including descriptions of appropriate
hybridization
conditions. Finally, subsection 5.3 describes methods by which the
hybridization data thus
obtained can be analyzed, e.g., to evaluate the sensitivity and specificity of
individual
probes to the target polynucleotide.
5.2.1. SELECTION OF CANDIDATE PROBES
In the exemplary method depicted in FIG. 1, one or more polynucleotide probes
are
provided or selected for analysis according to the methods of the invention.
In particular,
the polynucleotide probes that are analyzed according to the methods of the
invention
comprise a nucleotide sequence that is capable of hybridizing to molecules of
a target
polynucleotide under appropriate hybridization conditions. Generally, the
target
polynucleotide molecules that hybridize to a probe contain at least one
nucleotide sequence
that is complementary to the nucleotide sequence of the probe and can
therefore hybridize
to the probe by forming non-covalent Watson-Crick base pairs with the
nucleotides of the
probe sequence. Accordingly, polynucleotide probes can be selected or provided
by
selecting or providing polynucleotide probes having different nucleotide
sequences.
Because the preferred target polynucleotide molecules of the invention are
polynucleotide
molecules corresponding to genes or gene transcripts of a cell or organism,
the selected or
provided polynucleotide probes preferably have nucleotide sequences that are
complementary to at least a portion of the nucleotide sequence of a gene or
gene transcript
of interest.
-19-
CA 02379212 2002-O1-09
WO 01/06013 PCT/US00/19203
In preferred embodiments, the polynucleotide probes that are selected or
provided
for analysis are oligonucleotide probes; i.e., the probes comprise
oligonucleotide
sequences. Oligonucleotide sequences are short sequences of polynucleotides
that are
preferably between about 4 and about 200 bases (i.e., nucleotides) in length,
and are more
preferably between about 1 S and about 150 bases in length. In one embodiment,
shorter
oligonucleotide sequences are used that are less than about 40 bases in
length, and are
preferably between about 15 and 30 bases in length. However, a preferred
embodiment of
the invention uses longer oligonucleotide sequences between about 40 and about
80 bases in
length, with oligonucleotide sequences between about 50 and about 70 bases in
length being
preferred, and oligonucleotide sequences between about SO and about 60 bases
in length
being even more preferred.
The compositions and methods of the invention can be used, in general, to
evaluate
the hybridization properties of any probe or probes comprising a
polynucleotide sequence
that are immobilized to a solid support or surface. For example, as described
supra, the
1 S probes can comprise DNA sequences, RNA sequences, or copolymer sequences
of DNA
and RNA. The polynucleotide sequences of the probes can also comprise DNA
and/or
RNA analogs or combinations thereof. For example, the polynucleotide sequences
of the
probes can be full or partial sequences of genomic DNA, cDNA, mRNA or cRNA
sequences extracted from cells. The polynucleotide sequences of the probes can
also be
synthesized nucleotide sequences, such as synthetic oligonucleotide sequences.
The probe
sequences can be synthesized either enzymatically in vivo, enzymatically in
vitro (e.g., by
PCR), or non-enzymatically in vitro.
The probe or probes used in the methods and compositions of the invention are
preferably immobilized to a solid support which can be either porous or non-
porous. For
example, the probes can be polynucleotide sequences that are attached to a
nitrocellulose or
nylon membrane or filter. Such hybridization probes are well known in the art
(see, e.g.,
Sambrook et al., eds., 1989, Molecular Cloning. A Laboratory Manual, 2nd Ed.,
Vols. 1-3,
cold Spring Harbor Laboratory, Cold Spring Harbor, New York). Alternatively,
the solid
support or surface can be a glass or plastic surface, or it can be a semi-
solid support such as
a gel.
Microarrays Generally:
In a particularly preferred embodiment, hybridization levels are measured to
microarrays of probes consisting of a solid phase on the surface of which are
immobilized a
population of polynucleotides, such as a population of DNA or DNA mimics or,
alternatively, a population of RNA or RNA mimics. The solid phase may be a
nonporous
-20-
CA 02379212 2002-O1-09
WO 01/06013 PCT/US00/19203
or, optionally, a porous material such as a gel. Microarrays can be employed,
e.g., for
analyzing the transcriptional state of a cell such as the transcriptional
states of cells exposed
to graded levels of a drug of interest or to graded perturbations to a
biological pathway of
interest. Microarrays are particularly useful in the methods of the instant
invention in that
they can be used to simultaneously screen a plurality of different probes to
evaluate, e.g.,
each probe's sensitivity and specificity for a particular target
polynucleotide.
In preferred embodiments, a microarray comprises a support or surface with
ordered
array of binding (e.g., hybridizing) sites, e.g., for a plurality of different
probes.
Microarrays can be made in a number of ways, of which several are described
hereinbelow.
However produced, microarrays share certain characteristics: The arrays are
reproducible,
allowing multiple copies of a given array to be produced and easily compared
with each
other. Preferably, the microarrays are made from materials that are stable
under binding
(e.g., nucleic acid hybridization) conditions. The microarrays are preferably
small, e.g.,
between about S cm2 and 25 cm2, preferably about 12 to 13 cmz. However, larger
arrays are
also contemplated and may be preferable, e.g., for simultaneously evaluating a
very large
number of different probes.
Preferably, a given binding site or unique set of binding sites in the
microarray will
specifically bind (e.g., hybridize) to the product of a single gene or gene
transcript from a
cell or organism (e.g., to a specific mRNA or to a specific cDNA derived
therefrom).
However, as discussed above, in general other, related or similar sequences
will cross
hybridize to a given binding site.
The microarrays used in the methods and compositions of the present invention
include one or more test probes, each of which has a polynucleotide sequence
that is
complementary to a subsequence of RNA or DNA to be detected. Each probe
preferably
has a different nucleic acid sequence, and the position of each probe on the
solid surface of
the array is preferably known. Indeed, the microarrays are preferably
addressable arrays,
more preferably positionally addressable arrays. More specifically, each probe
of the array
is preferably located at a known, predetermined position on the solid support
such that the
identity (i.e., the sequence) of each probe can be determined from its
position on the array
(i.e., on the support or surface).
Preferably, the density of probes on a microarray is about 100 different
(i.e., non-
identical) probes per 1 cmz or higher. More preferably, a microarray used in
the methods of
the invention will have at least 550 probes per 1 cm2, at least 1,000 probes
per 1 cmz, at
least 1,500 probes per 1 cm2 or at least 2,000 probes per 1 cm2. In a
particularly preferred
embodiment, the microarray is a high density array, preferably having a
density of at least
about 2,500 different probes per 1 cm2. The microarrays used in the invention
therefore
-21
CA 02379212 2002-O1-09
WO 01/06013 PCT/US00/19203
preferably contain at least 2,500, at least 5,000, at least 10,000, at least
15,000, at least
20,000, at least 25,000, at least 50,000 or at least 55,000 different (i.e.,
non-identical)
probes.
In one embodiment, the microarray is an array (i.e., a matrix) in which each
$ positions represents a discrete binding site for a product encoded by a gene
(i.e., for an
mRNA or for a cDNA derived therefrom). For example, the binding site can be a
DNA or
DNA analog to which a particular RNA can specifically hybridize. The DNA or
DNA
analog can be, e.g., a synthetic oligomer, a full length cDNA, a less-than
full length cDNA,
or a gene fragment.
Preferably, the microarrays used in the invention have binding sites (i.e.,
probes) for
one or more genes relevant to the action of a drug of interest or in a
biological pathway of
interest. A "gene" is identified as an open reading frame (ORF) that encodes a
sequence of
preferably at least 50, 75, or 99 amino acid residues from which a messenger
RNA is
transcribed in the organism or in some cell or cells of a multicellular
organism. The number
of genes in a genome can be estimated from the number of mRNAs expressed by
the cell or
organism, or by extrapolation of a well characterized portion of the genome.
When the
genome of the organism of interest has been sequenced, the number of ORFs can
be
determined and mRNA coding regions identified by analysis of the DNA sequence.
For
example, the genome of Saccharomyces cerevisiae has been completely sequenced
and is
reported to have approximately 6275 ORFs encoding sequences longer the 99
amino acid
residues in length. Analysis of these ORFs indicates that there are 5,885 ORFs
that are
likely to encode protein products (Goffeau et al., 1996, Science 274:546-567).
In contrast,
the human genome is estimated to contain approximately 105 genes.
Preparing Probes for Microarra~s:
As noted above, the "probe" to which a particular target polynucleotide
molecule
specifically hybridizes according to the invention is a complementary
polynucleotide
sequence to the target polynucleotide. In one embodiment, the probes of the
microarray
comprises sequences greater than 500 nucleotide bases in length that
correspond to a gene
or gene fragment. For example, such probes can comprise DNA or DNA "mimics"
(e.g.,
derivatives and analogs) corresponding to at least a portion of one or more
genes in an
organism's genome. In another embodiment, such probes are complementary RNA or
RNA
mimics.
DNA mimics are polymers composed of subunits capable of specific, Watson-Crick-
like hybridization with DNA, or of specific hybridization with RNA. For
example, the
DNA mimics can comprise nucleic acids modified at the base moiety, at the
sugar moiety,
-22-
CA 02379212 2002-O1-09
WO 01/06013 PCT/US00/19203
or at the phosphate backbone. For example, one particular DNA mimic includes,
but is not
limited to, phosphorothioates.
Such DNA sequences can be obtained, e.g., by polymerise chain reaction (PCR)
amplification of gene segments from, e.g., genomic DNA, mRNA (e.g., from RT-
PCR) or
from cloned sequences. PCR primers are preferably chosen based on known
sequences of
the genes or cDNA that result in amplification of unique fragments (i.e.,
fragments that do
not share more than 10 bases of contiguous identical sequence with any other
fragment on
the microarray). Computer programs that are well known in the art are useful
in the design
of primers with the required specificity and optimal amplification properties,
such as Oligo
version S.0 (National Biosciences). Typically, each probe on the microarray
will be
between about 20 bases and about 50,000 bases, and usually between about 300
bases and
about 1,000 bases in length. PCR methods are well known in the art and are
described, e.g.,
by Innis et al., eds., 1990, PCR Protocols: A Guide to Methods and
Applications,
Academic Press, Inc., San Diego, California. As will be apparent to one
skilled in the art,
1 S controlled robotic systems are useful for isolating and amplifying nucleic
acids.
An alternative, preferred means for generating the polynucleotide probes for a
microarray used in the methods and compositions of the invention is by
synthesis of
synthetic polynucleotides or oligonucleotides, e.g., using N-phosphonate or
phosphoramidite chemistries (Froehler et al., 1986, Nucleic Acid Res. 14:5399-
5407;
McBride et al., 1983, Tetrahedron Lett. 24:246-248). Synthetic sequences are
typically
between about 4 and about 500 bases in length, more typically between about 4
and about
200 bases in length, and even more preferably between about 15 and about 150
bases in
length. In embodiments wherein shorter oligonucleotide probes are used,
synthetic nucleic
acid sequences less than about 40 bases in length are preferred, more
preferably between
about 15 and about 30 bases in length. In embodiments wherein longer
oligonucleotide
probes are used, synthetic nucleic acid sequences are preferably between about
40 and 80
bases in length, more preferably between about 40 and 70 bases in length and
even more
preferably between about SO and 60 bases in length. In some embodiments,
synthetic
nucleic acids include non-natural bases, such as, but not limited to, inosine.
As noted
above, nucleic acid analogs may be used as binding sites for hybridization. An
example of
a suitable nucleic acid analog is peptide nucleic acid (see, e.g., Egholm et
al., 1993, Nature
363:566-568; U.S. Patent No. 5,539,083).
In other alternative embodiments, the hybridization sites (i.e., the probes)
are made
from plasmid or phage clones of genes, cDNAs (e.g., expressed sequence tags),
or inserts
therefrom (see, e.g., Nguyen et al., 1995, Genomics 29:207-209).
- 23
CA 02379212 2002-O1-09
WO 01/06013 PCT/US00/19203
Attaching Probes to the Solid Surface:
The probes are preferably attached to a solid support or surface which may be
made,
e.g., from glass, plastic (e.g., polypropylene, nylon) polyacrylamide,
nitrocellulose, a gel, or
other porous or nonporous material. A preferred method for attaching the
nucleic acids to
the surface is by printing on glass plates, as is described generally by
Schena et al., 1995,
Science 270:467-470. This method is especially useful for preparing
microarrays of cDNA
(see also DeRisi et al., 1996, Nature Genetics 14:457-460; Shalon et al.,
1996, Genome
Res. 6:639-645; and Schena et al., 1995, Proc. Natl. Acad. Sci. U.S.A.
93:10539-11286).
Another preferred method for making microarrays is by making high-density
oligonucleotide arrays. Techniques are known for producing arrays containing
thousand of
oligonucleotides complementary to defined sequences and at defined locations
on a surface
using photolithographic techniques for synthesis in situ (see Fodor et al.,
1991, Science
251:767-773; Pease et al., 1994, Proc. Natl. Acad. Sci. U.S.A. 91:5022-5026;
Lockhart et
al., 1996, Nature Biotechnology 14:1675; U.S. Patent Nos. 5,578,832;
5,556,752; and
5,510,270) or other methods for rapid synthesis and deposition of defined
oligonucleotides
(Blanchard et al., Biosensors & Bioelectronics 11:687-690). When these methods
are used
oligonucleotides (e.g., 25-mers) of known sequence are synthesized directly on
a surface
such as a derivatized glass slide. Usually, the array produced is redundant
with several
oligonucleotide molecules per RNA. Oligonucleotide probes can also be chosen
to detect
particular alternatively spliced mRNAs.
Other methods for making microarrays, e.g., by masking (Maskos and Southern,
1992, Nucl. Acids. Res. 20:1679-1684) can also be used. In principle and as
noted above
any type of array, for example dot blots on a nylon hybridization membrane
(see Sambrook
et al., supra) can be used. However, as will be recognized by those skilled in
the art, very
small arrays will frequently be preferred because hybridization volumes will
be smaller.
In a particularly preferred embodiment, micorarrays used in the invention are
manufactured by means of an ink jet printing device for oligonucleotide
synthesis, e.g.,
using the methods and systems described by Blanchard in International Patent
Publication
No. WO 98/41531, published on September 24, 1998; Blanchard et al., 1996,
Biosensors
and Bioelectronics 11:687-690; Blanchard, 1998, in Synthetic DNA Arrays in
Genetic
Engineering, Vol. 20, J.K. Setlow, ed., Plenum Press, New York at pages 111-
123.
Specifically, the oligonucleotide probes in such microarrays are preferably
synthesized by
serially depositing individual nucleotides for each probe sequence in an array
of
"microdroplets" of a high tension solvent such a propylene carbonate. The
microdroplets
have small volumes (e.g., 100 pL or less, more preferably 50 pL or less) and
are separated
-24-
CA 02379212 2002-O1-09
WO 01/06013 PCT/US00/19203
from each other on the microarray (e.g., by hydrophobic domains) to form
circular surface
tension wells which define the locations of the array elements (i.e., the
different probes).
Selecting Candidate Probes:
In one preferred embodiment, polynucleotide probes having a nucleotide
sequence
that is complementary to the nucleic acid sequence of a particular target
polynucleotide are
selected or provided by a method that is referred to herein as "tiling."
Specifically,
polynucleotide probes having a nucleotide sequence of length l are selected by
selecting
probes having a nucleotide sequence complementary to a sequence of l
consecutive bases of
the target polynucleotide sequence. For example, a polynucleotide probes can
be selected
or provided by selecting or providing a polynucleotide probe having a
nucleotide sequence
complementary to l consecutive bases of the target polynucleotide sequence
beginning at
the i'th base of the target polynucleotide sequence. In more detail, a first
polynucleotide
probe can be selected or provided by selecting or providing a polynucleotide
probe whose
polynucleotide sequence is complementary to the nucleotide sequence
corresponding to
bases i through i + 1 of the target polynucleotide sequence. A second
polynucleotide probe
sequence can be selected or provided by selecting or providing a
polynucleotide probe
whose nucleotide sequence is complementary to the nucleotide sequence
corresponding to
bases (i + n) through (i + n) + l of the target polynucleotide sequence, etc.
As noted supra, l specifies the length of the probe's polynucleotide sequence.
Therefore, l is a positive integer, preferably having a value between about 4
and about 200,
and more preferably having a value between about 15 and about 150. In
embodiments
wherein probes having shorter oligonucleotide sequences are used, l is
preferably less than
about 40, more preferably between about 15 and about 30. In embodiments
wherein probes
having longer oligonucleotide sequences are used, l is preferably between
about 40 and
about 80, more preferably between about 40 and about 70, more preferably
between about
50 and about 60.
n, the "tiling interval," is a positive integer that preferably has a value
between 1 and
about 10. Particularly preferred values of the tiling interval include n = 1,
2, 3, 4 and 5. i,
~'~ch indicates the starting position within the target polynucleotide
sequence, is also a
positive integer. In certain preferred embodiments, the starting position is
at or near the 5'-
end of the target polynucleotide sequence. Thus, i has preferred values less
than about 50,
and more preferably less than about 10. The first base in the target
polynucleotide sequence
is a particularly preferred starting position in such embodiments.
Accordingly, a
particularly preferred value of the starting position is i = 1. In other
preferred embodiments,
only the 3'-end of the target polynucleotide sequence is tiled. For example,
in certain
- 25 -
CA 02379212 2002-O1-09
WO 01/06013 PCT/US00/19203
embodiments, only the last 2,000, more preferably the last 1,000, more
preferably the last
500, and even more preferably the last 350 bases on the 3'-end of the target
polynucleotide
sequence are tiled. In such embodiments the value of the starting position i
is adjusted
accordingly (e.g., i = L - 2,000; i = L - 1,000; i = L - 500; or i = L - 350;
wherein L is the
S length of the target polynucleotide sequence).
In certain embodiments, the probe or probes to be evaluated may be further
selected,
e.g., by selecting only probes that have or are predicted to have the highest
binding (i.e.,
hybridization) energy dG to their target polynucleotide. Methods for
calculating or
predicting the hybridization energies of polynucleotide molecules are well
known in the art
and include, e.g., the nearest neighbor model (see, e.g., SantaLucia, 1998,
Proc. Natl. Acad.
Sci. U.S.A. 95:1460-1465). One skilled in the art can readily adapt such
models to the
different embodiments of the instant invention including, e.g., embodiments
wherein the
polynucleotide molecules are immobilized on the surface of a solid support
such as in an
array of polynucleotide probes (see, e.g., Provisional Patent Application
Serial No.
60/144,382, filed on July 16, 1999; and U.S. Patent Application Serial No.
09/364,751,
filed on July 30, 1999). Binding energies can be readily evaluated from such
models, e.g.,
using mathematical algorithms and software such as those described, e.g., by
Hyndman et
al., 1996, Biotechniques 20:1090-1096.
In such embodiments, the binding energy can be calculated or predicted for
each of a
plurality of candidate probes for a target polynucleotide, such as for
candidate probes
selected by the above described tiling methods. Those probes predicted to have
the highest
binding energy are then selected for evaluation according to the methods of
the present
invention. Alternatively, those probes predicted to have a binding energy
above a particular
threshold (usually a threshold that is selected by a user) can be selected for
evaluation
according to the methods of the invention.
The above described methods are preferred methods for selecting polynucleotide
probes regardless of the nature of the target polynucleotide sequence for
which the probes
are intended. In particular, the methods are preferred regardless of whether
the target
polynucleotide or target polynucleotides correspond to unique genes (e.g., for
which no
analog or homolog sequences are present or suspected of being present in a
sample) or are
members of one or more families of genes (e.g., for which one or more analogs
or homologs
are known and/or are expected to be present in a sample). The methods are also
preferred
regardless of the expected abundance of the target polynucleotides in a
sample.
Nevertheless, it will be understood by the skilled artisan that the methods
and compositions
of the present invention can be used to evaluate probes that are selected or
provided by any
-26-
CA 02379212 2002-O1-09
WO 01/06013 PCT/US00/19203
method, and are not limited to embodiments wherein the probe or probe
sequences are
selected according to the tiling methods described hereinabove.
Although generally, the probes selected for evaluation according to the
methods of
the invention will be probes for only one particular target, it is understood
that, at least in
certain embodiments of the invention, probes for a plurality of different
targets (e.g., for two
or more different polynucleotide sequences) may be simultaneously selected and
evaluated
using the methods and compositions of the invention. For example, in one
embodiment of
the invention a Basic Local Alignment Search Tool ("BLAST") or PowerBLAST
algorithm
can be used to identify different polynucleotide sequences (e.g., among a
database of
expressed sequences such as the GenBank or dbEST database) that do not contain
sequences that are expected or predicted cross-hybridize with each other's
probes. Such
polynucleotide sequences are referred to herein as "orthogonal" sequences or,
in
embodiments wherein the polynucleotide sequences are sequences of particular
genes, as
"orthogonal genes." Different probes that each hybridize to different
orthogonal sequences
c~ be analyzed simultaneously according to the methods of the present
invention with
minimal artifacts due to cross-hybridization by the gene-specific samples.
Algorithms for comparing polynucleotide sequences, such as the BLAST and
PowerBLAST algorithms, are well known in the art (see, e.g., Altschul et al.,
1990, J. Mol.
Biol. 215:403-410; Altschul, 1997, Nucleic Acids Res. 25:3389-3402; and Zhang
and
Madden, 1997, Genome Res. 7:649-656). One skilled in the relevant arts)
therefore readily
appreciates how to use such algorithms to compare polynucleotide sequences,
e.g., using
standard parameters well known in the art.
5.2.2. HYBRIDIZATION SAMPLES
The methods and compositions of the invention evaluate the properties of one
or
more probes by comparing the amount or level of binding of a first sample,
referred to
herein as a specific binding sample, to each of the one or more probes with
the amount or
level of binding of a second sample, referred to herein as a non-specific
binding sample, to
each of the one or more probes. In particularly preferred embodiments, the
methods and
compositions of the invention evaluate the properties of one or more
polynucleotide probes
by comparing the amount or level of binding (i.e., hybridization) of a a first
sample, referred
to herein as a specific hybridization sample, to each of the one or more
polynucleotide
probes with the amount or level of binding of a second sample, referred to
herein as a non-
specific hybridization sample, to each of the one or more polynucleotide
probes.
The first sample (i.e., the specific hybridization sample) comprises molecules
of a
particular target polynucleotide which, generally, is the intended target
polynucleotide of
-27-
CA 02379212 2002-O1-09
WO 01/06013 PCT/US00/19203
the probe or probes to be evaluated. Alternatively, in those embodiments of
the invention
wherein probes are evaluated for two or more different orthogonal target
polynucleotide
sequences, the specific hybridization sample is preferably a sample comprising
the two or
more different polynucleotide sequences. The target polynucleotide (or the two
or more
orthogonal target polynucleotides) is preferably present in the specific
hybridization sample
in an amount or abundance that is comparable to the amount or abundance of the
target
polynucleotide in a sample for which a probe evaluated by the methods of the
invention is
intended (i.e., in a "real" sample). For example, in preferred embodiments
wherein the
target polynucleotide corresponds to a gene or gene transcript expressed by a
cell or
organism, the target polynucleotide is preferably present in the specific
hybridization
sample in an amount or abundance that is comparable to the amount or abundance
of the
target polynucleotide expressed by the cell or organism.
Most preferably, the target polynucleotide(s) is(are) present in the specific
hybridization sample in an amount or abundance that is equal to its amount or
abundance in
a real sample. However, in many embodiments, the amount or abundance of the
target
polynucleotide(s) in a real sample is not known or is only approximately
known.
Accordingly, in alternative embodiments the target polynucleotide can be
present in the
specific hybridization sample in an amount or abundance that is approximately
equal to its
amount or abundance in a real sample. For example, the target polynucleotide
can be
present in the specific hybridization sample in an amount or abundance that is
of the same
order of magnitude as its amount or abundance in a real sample.
Alternatively, the target polynucleotide can be present in the specific
hybridization
sample in an amount or abundance that is within a minimum and a maximum amount
or
abundance that might be expected for any one polynucleotide sequence in a real
sample.
For example, typically amounts or abundances of a polynucleotide sequence in a
real
sample (i.e., in a sample of polynucleotide molecules extracted from a cell or
organism) can
be as low as about 0.0001% or as high as about 3% of the poly A+ mRNA extraced
from
the cell or organsim. More preferably, the amount or abundance can be as high
as about
2%, more preferably about 1% of the poly A+ mRNA extracted from the cell or
organsim.
The amount or abundance of the polynucleotide sequence is more preferably no
lower than
about 0.0003% of the poly A+ mRNA extracted from the cell or organism. Thus,
for
example, the target polynucleotide can be present in the specific
hybridization sample in an
amount or abundance that is equal to or approximately equal to the average or
mean amount
or abundance of polynucleotides in a real sample. For example, in embodiments
wherein
the target polynucleotide corresponds to a gene or gene transcript of a cell
or organism, the
abundance or amount of the target polynucleotide in the specific hybridization
sample can
-28-
CA 02379212 2002-O1-09
WO 01/06013 PCT/US00/19203
be equal to or approximately equal to the mean or average abundance or amount
of genes or
gene transcripts expressed by the cell or organism. Typical values of the mean
or average
abundance or amount of genes or gene transcripts expressed by a cell or
organism are
known to those skilled in the art, and generally depend on the identity of the
cell or
organism from which the gene or gene transcript is derived. For example,
typical preferred
values may include approximately 0.04% of all polyA+ mRNA extracted from a
cell or
organism.
In other embodiments, although the exact amount or abundance of a target
polynucleotide in a real sample may not be known, its qualitative abundance
will be known.
For example, in embodiments wherein the target polynucleotide corresponds to a
gene or
gene transcript of a cell or organism, such genes or gene transcripts are
often characterized
as being expressed at low levels or abundances, moderate levels or abundances,
or at high
levels or abundances. Accordingly, a target polynucleotide can be present in a
specific
hybridization sample in amounts or abundances typical of such qualitative
abundances.
One skilled in the art readily appreciates values for levels or abundances of
genes or gene
transcripts in a sample that correspond to each of the above described
categories (i.e., low,
moderate and high). Generally, such values will depend on the particular cell
type or
organism from which the gene or gene transcript is derived. For example,
preferred values
can be about 1% or more of all polyA+ mRNA extracted from a cell for high
levels or
abundances, between about 0.01 % and 1 % of all polyA+ mRNA extracted from a
cell for
moderate levels or abundances, and less than about 0.01% of all polyA+ mRNA
extracted
from a cell for low levels or abundances.
The second sample (i.e., the non-specific hybridization sample) preferably
comprises a plurality of different polynucleotide molecules, each different
polynucleotide
molecule having a different polynucleotide sequence. In particular, in those
embodiments
of the invention wherein the sequence of the target polynucleotide is the
sequence of a
particular gene or gene transcript in a cell or organism, the nucleotide
sequences of the
polynucleotide molecules in the second, nonspecific hybridization sample
preferably
comprise sequences representing the other genes or gene transcripts of the
cell or organism.
Many possible different embodiments of the specific and non-specific
hybridization
samples are possible. For example, in a first preferred embodiment, the
specific
hybridization sample is a substantially pure sample of molecules having a
particular
polynucleotide sequence. Preferably, these molecules are molecules of a
particular gene or
gene transcript (e.g., mRNA or cDNA) and, accordingly, have the sequence of
that gene or
gene transcript. The specific hybridization sample in this first embodiment
should be at
-29-
CA 02379212 2002-O1-09
WO 01/06013 PCT/US00/19203
least 75% pure (i.e., no more than 25% of the polynucleotide sequences in the
sample are
different from the sequence of the particular target polynucleotide or target
polynucleotides). Preferably, in this first embodiment the specific
hybridization sample is
at least 90% pure, more preferably at least 95% pure and even more preferably
at least 99%
pure.
It is understood that such specific hybridization samples can be readily
prepared by
one skilled in the art according to routine methods currently known in the art
and without
undue experimentation. For example, polynucleotide molecules corresponding to
'a
particular gene or gene transcript can be obtained, e.g., by polymerise chain
reaction (PCR)
amplification of gene segments from genomic DNA, cDNA, mRNA (e.g., by RT-PCR)
or
cloned sequences. PCR primers are preferably chosen based on known sequences
of the
target polynucleotide (e.g., of the particular gene or its gene transcript)
that result in
amplification of unique fragments. As the term is used herein, such "unique
fragments" are
fragments of a polynucleotide sequence that do not share more than 10 bases of
contiguous
identical sequence with any other fragment in a PCR (or RT-PCR) sample.
Computer
programs that are well known in the art are useful and can be used in the
design of primers
with the required specificity and optimal amplification properties, such as
Oligo version 5.0
(National Biosciences). PCR methods are well known in the art and are
described, for
example, in Innis et al., Eds., 1990, PCR Protocols: A Guide to Methods and
Applications,
Academic Press, Inc., San Diego, CA. It will be apparent to one skilled in the
art the
controlled robotic systems are useful for isolating and amplifying nucleic
acids, including
target polynucleotides of interest to a user.
In alternative embodiments, the target polynucleotide in a specific
hybridization
sample can be prepared from plasmid or phage clones of genes, cDNAs (e.g.,
expressed
sequence tags), or inserts therefrom (see, for example, Nguyen et al., 1995,
Genomics
29:207-209).
In this first embodiment, the second hybridization sample (i.e., the non-
specific
hybridization sample) comprises a plurality of different polynucleotide
molecules, each
different polynucleotide molecule having a different nucleotide sequence. In
particular,
each of the nucleotide sequences of the polynucleotide molecule in the non-
specific
hybridization sample should be different from the nucleotide sequence of the
target
polynucleotide in the specific hybridization sample. For example, in
embodiments wherein
the sequence of the target polynucleotide is the sequence of a particular gene
or gene
transcript of a cell or organism, the nucleotide sequences of the
polynucleotide molecules in
the second, non-specific hybridization sample preferably comprise sequences
representing
the other genes or gene transcripts of the cell or organism.
-30-
CA 02379212 2002-O1-09
WO 01/06013 PCT/US00/19203
Such sequences are also preferably present in the non-specific hybridization
sample
in substantially the same abundances or amounts as their abundances or amounts
in a "real"
sample for which the probe or probes are intends, e.g., in the cell or
organism. In particular,
the amount or abundance or each polynucleotide sequence in such a non-specific
hybridization sample preferably differs from its amount or abundance in a real
sample by no
more than a factor of 100, more preferably by no more than a factor of 10,
even more
preferably by no more than a factor of 2, and even more preferably by no more
than a factor
of 1.5 (i.e., by no more than 50%). It is understood, however, that in certain
instances the
relative amounts or abundances of a few polynucleotide sequences (e.g.,
preferably no more
than about 5%, more preferably no more than about 1%, more preferably no more
than
about 0.1 % of the different polynucleotide sequences in a non-specific
hybridization
sample) may differ by more than the preferred amounts recited above, as is
typically
observed, e.g., in samples from mutant cells or organisms or in samples from
cells or
organisms exposed to one or more drugs. However, the mean abundances of
different
polynucleotide sequences in the non-specific hybridization sample preferably
does not
differ substantially from the mean abundance of different polynucleotide
sequence in most
typical "real" samples. More specifically, the mean abundances preferably
change by no
more than a factor of two, more preferably by no more than 50%, even more
preferably by
no more than 10% and most preferably by no more than 1 %.
Most preferably, the cell or organism is a cell or organism, such as E. coli
or the
yeast Saccharomyces cerevisiae, that can be manipulated according to routine
techniques,
e.g., of in vitro homologous recombination and sexual genetics, that are
currently known in
the art. In such embodiments, the non-specific hybridization sample can be
prepared, e.g.,
from deletion mutants of such cells or organisms wherein the gene
corresponding to the
target polynucleotide sequence has been deleted or is silent (i.e., is not
expressed by those
cells).
Such non-specific hybridization samples can also be prepared from cells and
organisms, including mammalian cells and organisms (e.g., mouse, rat and human
cells or
organisms) for which facile techniques, e.g., of in vitro homologous
recombination and
sexual genetics are not readily available. For example, non-specific
hybridization sample
can also be prepared from, e.g., from obligate diploids, such as from cell
cultures including
cultures of mammalian cells (e.g., mouse, rat or human cells) for which
strains deleted for a
specific gene (specifically, the gene corresponding to the target
polynucleotide) are or can
be made available.
Methods of preparing polynucleotide samples from such deletion mutants are
well
known in the art. For example, methods for preparing total and poly(A)+ from a
cell or
-31 -
CA 02379212 2002-O1-09
WO 01/06013 PCT/US00/19203
organism are well known in the art, and are described generally, e.g., in
Sambrook et al.,
eds., 1989, Molecular Cloning. A Laboratory Manual, 2nd Ed., Cold Spring
Harbor
Laboratory Press, Cold Spring Harbor, New York. In one embodiment, RNA is
extracted
from cells of the various types of interest using guanidinium thiocyanate
lysis followed by
CsCI centrifugation (Chirgwin et al., 1979, Biochemistry 18:5294-5299). In an
alternative
embodiment, which is preferred for S. cerevisiae, RNA is extracted from cells
using phenol
and chloroform, as described in Ausubel et al. (Ausubel et al., eds., 1989,
Current
Protocols in Molecular Biology, Vol. III, Green Publishing Associates, Inc.,
John Wiley &
Sons, Inc., New York at pp. 13.12.1-13.12.5). Poly(A)+ RNA is selected by
selection with
oligo-dT cellulose.
In one embodiment, RNA can be fragmented by methods known in the art, such as
by incubation with ZnCl2, to generate fragments of RNA. In one embodiment,
isolated
mRNA can be converted to antisense RNA synthesized by in vitro transcription
of double-
stranded cDNA in the presence of labeled dNTPs (Lockhart et al., 1996, Nature
Biotechnology 14:1675).
In other embodiments, the polynucleotide molecules of the non-specific
hybridization sample comprise DNA molecules, such as fragmented genomic DNA,
first
strand cDNA which is reverse transcribed from mRNA, or PCR products of
amplified
mRNA or cDNA. Methods for preparing such samples are also well known in the
art, and
one skilled in the art will readily appreciate how to prepare such non-
specific hybridization
samples with undue experimentation.
Alternatively, in those embodiments wherein deletion mutants of the cell or
organism are not readily available, non-specific hybridization samples can
also be prepared
from mixtures of polynucleotides from which the target polynucleotide has been
removed.
For example, non-specific hybridization samples can be prepared from a library
or libraries
of selected clones that do not contain a clone or clones corresponding to the
target
polynucleotide, or from which clones corresponding to the target
polynucleotide have been
removed. Non-specific hybridization samples can also be prepared, e.g., from
DNA or
mRNA samples prepared from cells, as described above, which have been
subtractively
hybridized with the gene or interest (i.e., the gene corresponding to the
target
polynucleotide) to remove it from the sample or, at least, to partially remove
it from the
sample by reducing its abundance.
In a second preferred embodiment of the invention, the specific hybridization
sample is identical to the specific hybridization sample of the first
preferred embodiment
described above. However, the non-specific hybridization sample of this second
embodiment is preferably derived from a source with a normal amount of the
target
-32-
CA 02379212 2002-O1-09
WO 01/06013 PCT/US00/19203
polynucleotide in addition to a plurality of other polynucleotide sequences.
For example, in
those embodiments wherein the sequence of the target polynucleotide is the
sequence of a
particular gene or gene transcript of a cell or organism, the nucleotide
sequences of the
polynucleotide molecules in the second, non-specific hybridization sample
preferably
comprise sequences representing both the gene or gene transcript corresponding
to the
particular target polynucleotide and the other genes or gene transcripts of
the cell or
organism.
Such non-specific hybridization samples can be prepared from normal or "wild
type" cells or organisms that express the gene or gene transcript
corresponding to the target
polynucleotide sequence, as well as other genes or gene transcripts, in normal
amounts,
using methods of extracting polynucleotides from cells or organisms that are
well known in
the art and are described above for the preparation of a non-specific
hybridization sample in
the first particularly preferred embodiment of the invention. Accordingly,
this second
embodiment is particularly preferred in those aspects of the invention wherein
the target
polynucleotide corresponds, e.g., to a gene or gene transcript of a cell or
organism for which
deletion mutants are not readily available. The level of non-specific
hybridization can be
evaluated from such a sample according to the below described methods of the
present
invention; e.g., by subtracting the hybridization signal obtained from the
specific
hybridization sample from the hybridization signal obtained from the non-
specific
hybridization sample.
In a third preferred embodiment of the invention, the specific hybridization
sample
may contain , not only the target polynucleotide sequence, but also other non-
target
polynucleotide sequences. For example, in those embodiments of the invention
wherein the
sequence of the target polynucleotide is the sequence of a particular gene or
gene transcript
of a cell or organism, the nucleotide sequences of the polynucleotide
molecules in the first,
specific hybridization sample may comprise polynucleotide sequences
corresponding to
both the target polynucleotide and to the other genes or gene transcripts of
the cell or
organism. Thus, in such a third embodiment, the specific hybridization sample
is preferably
identical to the non-specific hybridization sample described, above, for the
second preferred
embodiment of the invention. In particular, the specific hybridization sample
in this third
embodiment of the invention is most preferably a polynucleotide sample
obtained from a
normal or wild type cell or organism that expresses the gene or gene
transcript of the target
polynucleotide, as well as other genes or gene transcripts, at normal levels
for the cell or
organism.
The second or non-specific hybridization sample in this third preferred
embodiment
of the invention is preferably identical to the non-specific hybridization
sample described,
-33-
CA 02379212 2002-O1-09
WO 01/06013 PCT/US00/19203
above, for the first preferred embodiment of the invention. In particular, the
non-specific
hybridization in this third preferred embodiment preferably comprises a
plurality of
different nucleotide molecules, with each different polynucleotide molecule
having a
different polynucleotide sequence and with each polynucleotide sequence in the
non-
specific hybridization sample being different from the polynucleotide sequence
of the target
polynucleotide. For example, in particularly preferred aspects of this third
embodiment, the
nonspecific hybridization sample is a polynucleotide sample obtained from a
deletion
mutant of the cell or organism wherein the gene corresponding to the target
polynucleotide
sequence has been deleted or is silent. Such an embodiment is particularly
preferred, e.g.,
in applications wherein it is important to evaluate the probe specificity
and/or sensitivity at
the natural abundance of the target polynucleotide.
In a fourth preferred embodiment of the invention, both the specific
hybridization
sample and the non-specific hybridization sample contain: (a) polynucleotide
molecules
having the polynucleotide sequence of the target polynucleotide; and (b) a
plurality of
different polynucleotide molecules, with each different polynucleotide
molecule having a
different polynucleotide sequence that is also different from the sequence of
the target
polynucleotide. In this fourth embodiment, the amount or level of molecules of
the target
polynucleotide in the first or specific hybridization sample differs
substantially from the
amount or level of the molecules of the target polynucleotide in the second or
non-specific
hybridization sample. Specifically, the amount or level of molecules of the
target
polynucleotide preferably differs by at least a factor of two, and more
preferably by at least
a factor four, more preferably by at least a factor of eight, still more
preferably by at least a
factor of 20, and even more preferably by at least a factor of 100.
Preferably in this fourth embodiment, the different polynucleotide molecules
(i.e.,
the polynucleotide molecules having sequences that are different from the
target
polynucleotide sequence) are substantially identical in both the specific and
non-specific
hybridization samples. In particular, each of the other "non-target"
polynucleotide
sequences is preferably present in substantially the same amount or abundance
in both
samples, and these amounts or abundances are preferably substantially the same
as the
mounts or abundances of the polynucleotide sequences in a "real" sample (e.g.,
in a sample
of polynucleotides from the cell or organism). Specifically, the amount or
abundance of
each non-target polynucleotide molecule preferably differs by no more than a
factor of 100
between the two hybridization samples, more preferably by no more than a
factor of 10,
even more preferably by no more than a factor of two, and still more
preferably by no more
than a factor of 1.5 (i.e., by no more than 50%). It is understood, however,
that larger
changes in the relative abundance of a few polynucleotide sequences (e.g.,
preferably no
-34-
CA 02379212 2002-O1-09
WO 01/06013 PCT/US00/19203
more than about S%, more preferably no more than about 1%, more preferably no
more than
about 0.1 % of the different polynucleotide sequences in a non-specific
hybridization
sample) may occur between the two hybridization samples and/or between the
hybridization
samples and a real sample, as will typically be seen in samples derived, e.g.,
from mutant
cells or organisms or from cells or organisms treated with one or more drugs.
However, the
mean change in the abundance or amount of all non-target polynucleotide
sequences is
preferably no more than a factor of two, more preferably no more than SO%,
even more
preferably no more than 10%, and still more preferably no more than 1%.
The polynucleotide molecules of both the specific hybridization sample and the
non-
specific hybridization sample are preferably detectably labeled. Preferably,
the detectable
label is a fluorescent label, e.g., by incorporation of nucleotide analogs.
Other labels
suitable for use in the present invention include, but are not limited to,
biotin, imminobiotin,
antigens, cofactors, dinitrophenol, lipoic acid, olefinic compounds,
detectable polypeptides,
electron rich molecules, enzymes capable of generating a detectable signal by
action upon a
substrate, and radioactive isotopes. Preferred radioactive isotopes include,
3zP, 3sS,'aC, isN
and'zsl, to name a few. Fluorescent molecules suitable for the present
invention include,
but are not limited to, fluorescein and its derivatives, rhodamine and its
derivatives, texas
red, 5'-carboxy-fluorescein ("FMA"), 2',7'-dimethoxy-4',5'-dichloro-6-carboxy-
fluorescein
("JOE"), N,N,N',N'-tetramethyl-6-carboxy-rhodamine ("TAMR_A"), 6'-carboxy-X-
rhodamine ("ROX"), HEX, TET, IRD40 and IRD41. Fluroescent molecules that are
suitable for the invention further include: cyamine dyes, including but not
limited to Cy2,
Cy3, Cy3.5, CyS, Cy5.5, Cy7 and FluorX; BODIPY dyes, including but not limited
to
BODIPY-FL, BODIPY-TR, BODIfY-TMR, BODIPY-630/650, and BODIPY-650/670;
~d ~EXA dyes, including but not limited to ALEXA-488, ALEXA-532, ALEXA-546,
ALEXA-568, and ALEXA-594; as well as other fluorescent dyes known to those
skilled in
the art. Electron rich indicator molecules suitable for the present invention
include, but are
not limited to, aferritin, hemocyanin, and colliodal gold. Alternatively, in
less preferred
embodiments the target polynucleotides may be labeled by specifically
complexing a first
group to the polynucleotide. A second group, covalently linked to an indicator
molecule
and which has an affinity for the first group, can be used to indirectly
detect the target
polynucleotide. In such an embodiment, compounds suitable for use as a first
group
include, but are not limited to, biotin and iminobiotin.
In particularly preferred embodiments, the two samples (i.e., the specific
hybridization sample and the non-specific hybridization sample) are
differentially labeled.
Specifically, each sample is labeled with a different detectable label (e.g.,
with a different,
-35-
CA 02379212 2002-O1-09
WO 01/06013 PCT/US00/19203
distinct fluorophore) such that the two samples can be simultaneously detected
and
distinguished from each other by detecting each sample's respectively label.
For example,
in one embodiment, the specific hybridization sample can be labeled using
fluorescein-
labeled dNTP, which fluoresces green, while the non-specific hybridization
sample can be
$ labeled using rhodamine-labeled dNTP, which fluoresces red. Each sample can
then be
readily detected and distinguished from the other sample by detecting the
characteristic
green or red fluorescence of each sample's respective label.
Indeed, by using such differential labeling the two hybridization samples can
be
distinguished from each other and separately detected even when they have been
mixed.
Thus, the specific hybridization sample and the non-specific hybridization
sample need not
be kept separate from each other, but can be mixed and hybridize to the
polynucleotide
probe or probes simultaneously, e.g., on the same microarray and in the same
experiment.
It is therefore not a requirement of the present invention that the specific
hybridization
sample and the non-specific hybridization sample be physically separate
samples. The two
samples need only be distinguishable, e.g., by means of the differential
labeling scheme
described above. Alternatively, one skilled in the art can readily appreciate
that, in such
embodiments, the specific hybridization sample and the non-specific
hybridization can be
thought of as the same sample with two different, distinct (e.g.,
differentially labeled)
components: a specific hybridization component (corresponding to the specific
hybridization sample discussed above), and a non-specific hybridization
component
(corresponding to the non-specific hybridization sample discussed above).
5.2.3. MEASURING HYBRIDIZATION LEVELS
The properties of one or more probes are evaluated according to the methods
and
compositions of the present invention by comparing the amount or level of
binding of the
first, specific binding sample to the probe or probes with the amount or level
of the second,
non-specific binding sample to the probe or probes. In preferred embodiments
wherein the
probes and targets comprise polynucleotide molecules, the amount or level of
hybridization
of the first, specific hybridization sample to the polynucleotide probe or
probes is compared
with the amount or level of hybridization of the second, non-specific
hybridization sample
to the polynucleotide probe or probes. Accordingly, in the methods of the
invention
preferably include a step wherein hybridization levels of the specific
hybridization sample
and the non-specific hybridization sample to the polynucleotide probe or
probes are
obtained or provided, e.g., by contacting the two samples to the
polynucleotide probe or
probes under conditions such that polynucleotide molecules in the samples can
bind or
-36-
CA 02379212 2002-O1-09
WO 01/06013 PCT/US00/19203
hybridize to molecules of the probe or probes, and measuring the amount of
polynucleotides
from each of the two samples that bind or hybridize to molecules of the probe
or probes.
Hvbridization Conditions:
The conditions under which the polynucleotides are contacted to the probe or
probes
are known in the art as the "hybridization conditions." Preferably, the
hybridization
conditions are optimized such that specific binding of polynucleotide
molecules to the
probe or probes (e.g., binding of polynucleotide molecules from the specific
hybridization
sample) is high while non-specific binding of polynucleotide molecules to the
probe or
probes (e.g., binding of polynucleotide molecules from the non-specific
hybridization
sample) is low. In some embodiments, however, the optimal hybridization
conditions may
not be known or may only be approximately known. For example, in certain
embodiments
the methods and compositions of the invention can be used to evaluate
particular
hybridization conditions, e.g., to determine whether the hybridization
conditions are
optimal. For example, one or more of the hybridization parameters (e.g., the
temperature
and/or the salt concentration) can be systematically varied, and the methods
of the invention
can be used to determine, e.g., the specificity and/or sensitivity of the
probe or probes for
each hybridization condition. Appropriate or preferred hybridization
conditions are then
identified as the hybridization conditions for which the sensitivity and/or
specificity of the
probe or probes are greatest.
In particular embodiments wherein the probe or probes comprise double-stranded
DNA sequences, the probe or probes, or arrays containing such probes are
preferably
subj ected to denaturing conditions to render the DNA single-stranded prior to
contacting
with the target polynucleotide molecules. Arrays containing single-stranded
probe DNA
(e.g., synthetic oligodeoxyribonucleic acids) may also need to be denatured
prior to
contacting with the target polynucleotide molecules, e.g., to remove hairpins
or dimers
which form due to self complementary sequences.
Optimal hybridization conditions will depend on the length (e.g., oligomer
versus
polynucleotide greater than 200 bases) and type (e.g., RNA or DNA) of probes
and target
nucleic acids. General parameters for specific (i.e., stringency)
hybridization conditions for
nucleic acids are described, e.g., in Sambrook et al., eds., 1989, Molecular
Cloning: A
Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring
Harbor,
New York at pp. 9.47-9.51 and 11.55-11.61; and in Ausubel et al., 1987,
Current Protocols
in Molecular Biology, Greene Publishing and Wiley-Interscience, New York. In
embodiments, wherein cDNA microarrays are used, typical hybridization
conditions are
hybridization in S x SSC plus 0.2% SDS at 65 °C for four hours,
followed by washes at
-37-
CA 02379212 2002-O1-09
WO 01/06013 PCT/US00/19203
25 °C in low stingency wash buffer (1 x SSC plus 0.2% SDS), followed by
10 minutes at
25 °C in higher stringency wash buffer (0.1 x SSC plus 0.2% SDS) (Shena
et al., 1996,
Proc. Natl. Acad. Sci. U.S.A. 93:10614). Useful hybridization conditions are
also provided,
e.g., Tijessen, 1993, Hybridization With Nucleic Acid Probes, Elsevier Science
Publishers;
S B.V. and Kricka, 1992, Nonisotopic DNA Probe Techniques, Academic Press, San
Diego.
In preferred embodiments wherein oligonucleotide probes are used, preferred
hybridization conditions can comprise hybridization at a temperature at or
near the mean
melting temperature of the probes (e.g., within S °C or, more
preferably, within 2 °C) in 1 M
NaCI, 50 mM MES buffer (pH 6.5), 0.5% sodium sarcosine and 32% formamide.
The melting temperature (Tm) of a target polynucleotide from a particular
probe is
known in the art as referring to the temperature at which one-half (i.e., 50%)
of the target
polynucleotide molecules in a sample are bound to molecules of the probe. As
the term is
used herein to describe and enable appropriate hybridization conditions, the
melting
temperature of a probe refers to the melting temperature at which one-half of
the target
polynucleotide molecules in a sample having a nucleotide sequence that is
complementary
to the nucleotide sequence of the probe are hybridized thereto. Methods for
determining the
melting temperature of a particular polynucleotide duplex are well known in
the art and
include, e.g., predicting the melting temperature using well known physical
models adapted
to experimental data (see, e.g., SantaLucia, 1998, Proc. Natl. Acad. Sci.
U.S.A. 95:11460-
11465 and the references cited therein). Mathematical algorithms and software
for
predicting melting temperatures using such models are readily available as
described, e.g.,
by Hyndman et al., 1996, Biotechniques 20:1090-1096. Although the specific
parameter
used in such models are generally derived for polynucleotide duplexes in
solution,
appropriate parameters can readily be obtained to predict melting temperatures
and other
hybridization properties of target polynucleotides hybridizing to
polynucleotide probes that
are immobilized on a solid surface, e.g., in a microarray. Indeed, one skilled
in the art will
readily appreciate how to obtain parameters that are appropriate for the
probes of a specific
microarray, as described, e.g., by Stoughton et al., in U.S. Provisional
Application Serial
No. 60/144,382 filed on July 16, 1999 and U.S. Patent Application Serial No.
09/364,751
filed on July 30, 1999. The melting temperature of an RNA/DNA duplex
approximately 25
base pairs in length in 1 M salt solution is typically between about 60
°C and about 70 °C.
Signal Detection:
The hybridization conditions used in the methods of the invention, including
the
specific hybridization conditions described above, also include washing
conditions. The
wash conditions are preferably such that polynucleotide molecules that are not
bound to the
-38-
CA 02379212 2002-O1-09
WO 01/06013 PCT/US00/19203
probe or probes are removed (e.g., from the microarray of probes) while the
probes and
polynucleotide molecules that are bound thereto remain. The amount of
polynucleotides
hybridized to each probe can then be measured or determined, e.g., by
measuring or
determining the amount of the a detectable label.
As noted above, preferably polynucleotides from the two different samples
(i.e.,
from the specific hybridization sample and the non-specific hybridization
sample) are
hybridized to the probes simultaneously. For example, in preferred embodiments
of the
invention wherein a plurality of different probes on a microarray are
evaluated, the two
samples are simultaneously hybridized to the binding sites on the microarray.
In such
embodiments, the polynucleotide molecules from each of the two samples are
differentially
labeled so that they can be distinguished. For example, cDNA in a specific
hybridization
sample can be labeled using fluorescein-labeled dNTP and cDNA from a non-
specific
hybridization sample can be labeled using rhodamine-labeled dNTP. When the two
cDNAs
are mixed and hybridized to the microarray, the relative intensity of signal
from each cDNA
1 S set is determined for each site on the array, and any relative difference
in abundance of a
particular mRNA is thereby detected.
In the example described above, cDNA from the specific hybridization sample
will
fluoresce green when the fluorophore (i.e., the fluorescein label) is
stimulated, and the
cDNA from the non-specific hybridization sample will fluoresce red. As a
result, when a
p~icular probe (e.g., on the microarray) hybridizes specifically to a
particular target
polynucleotide (i.e., the target polynucleotide of the specific hybridization
sample) the
binding site for that probe on the microarray will emit a wavelength
characteristic of the
fluorescein label (i.e., green). In contrast, when a probe on the microarray
cross-hybridizes
to other polynucleotides (i.e., from the non-specific hybridization sample)
the binding site
for the probe on the microarray will emit a wavelength characteristic of both
labels. A
probe that hybridizes more specifically to the target polynucleotide of the
specific
hybridization sample will fluoresce with a higher ratio of green to red
fluorescence, whereas
a probe that hybridizes less specifically to that target polynucleotide will
fluoresce with a
lower ratio of green to red fluroescence.
The use of such a two-color fluorescence labeling and detection scheme as been
described, e.g., to define alterations in gene expression (see, e.g., Shena et
al., 1995, Science
270:467-470). An advantage of using cDNA labeled with two different
fluorophores is that
a direct and internally controlled compraison of hybridization levels
corresponding to both
specific and non-specific hybridization can be made. Variations due to minor
differences in
experimental conditions (e.g., hybridization conditions) will not affect
subsequent analysis.
However, it is understood that the invention can also be practiced using two
physically
-39-
CA 02379212 2002-O1-09
WO 01/06013 PCT/US00/19203
separate samples and comparing, for example, the absolute amount of cDNA or
mRNA (or
other polynucleotides) from a specific hybridization sample that hybridizes to
a probe and
the absolute amount of cDNA or mRNA from a non-specific hybridization sample
that
hybridizes to the same probe.
When fluorescently labeled targets are used, the fluorescence emission at each
site
of a microarray can be, preferably, detected by scanning confocal laser
microscopy. In one
embodiment, a separate scan, using the appropriate excitation line, is carried
out for each of
the two fluorophores used. Alternatively, a laser can be used that allows
simultaneous
specimen illumination at wavelengths specific to the two fluorophores and
emissions from
the two fluorophores can be analyzed simultaneously (see Shena et al., 1996,
Genome
Res. 6:639-645). In a preferred embodiment, the arrays are scanned with a
laser fluorescent
scanner with a computer controlled X-Y stage and a microscope objective.
Sequential
excitation of the two fluorophores is achieved with a multi-line, mixed gas
laser, and the
emitted light is split by wavelength and detected with two photomultiplier
tubes. Such
fluorescence laser scanning devices are described, e.g., in Schena et al.,
1996, Genome
Res. 6:639-645. Alternatively, the fiber-optic bundle described by Ferguson et
al., 1996,
Nature Biotech. 14:1681-1684, may be used to hybridization levels at a large
number of
binding sites simultaneously.
Signals are recorded and, in a preferred embodiment, analyzed by computer,
e.g.,
using a 12 bit analog to digital board. In one embodiment, the scanned image
is despeckled
using a graphics program (e.g., Hijaak Graphics Suite) and then analyzed using
an image
gridding program that creates a spreadsheet of the average hybridization at
each wavelength
at each site. If necessary, an experimentally determined correction for "cross
talk" (or
overlap) between the channels for the two fluorophores may be made. For any
particular
hybridization site on the microarray, a ratio of the emission of the two
fluorophores can be
calculated. The ratio is independent of the absolute amount or level of
hybridization of
either sample, but is useful, as explained above, for determining the relative
amounts of
specific and cross-hybridization from the two samples.
5.2.4. DATA ANALYSIS
The methods and compositions of the present invention are useful for
evaluating one
or more probes and, more specifically, can be used to evaluate the sensitivity
and/or
specificity with which a probe or probes bind or hybridize to a particular
target. The
sensitivity of a probe, as the term is used herein, is understood to refer to
the absolute
amount or level of a particular target (i.e., the number of molecules of the
particular target)
that binds to the probe under particular binding conditions. The amount or
level of a
-40-
CA 02379212 2002-O1-09
WO 01/06013 PCT/US00/19203
particular target that binds to a probe under particular binding conditions is
also referred to
herein as the amount or level of specific binding to the probe under the
particular binding
conditions. In preferred embodiments of the invention wherein the probes and
targets are
polynucleotide molecules, the sensitivity of a probe (i.e., of a
polynucleotide probe) is
understood to refer to the absolute amount of a particular target
polynucleotide (i.e., the
number of polynucleotide molecules having a nucleotide sequence) that
hybridizes to the
polynucleotide probe under particular hybridization conditions. The amount of
a particular
target polynucleotide that hybridizes to a probe under particular
hybridization conditions is
also referred to herein as the amount of specific hybridization of the probe
under the
pa~icular hybridization conditions.
The specificity of a probe, as the term is used herein, is understood to refer
to the
amount or level of a particular target (i.e., the number of molecules of the
particular target)
that binds to the probe under particular binding conditions relative to the
amount or level of
non-specific binding to the probe under the same binding conditions. Non-
specific binding,
as the term is used herein, is understood to refer to the amount of molecules
other than
molecules of the particular target (i.e., the number of molecules that are not
molecules of
the particular target) that bind to the probe under particular binding
conditions. In preferred
embodiments of the invention wherein the probes and targets are polynucleotide
molecules,
the sensitivity of a probe is understood to refer to the amount of a
particular target
polynucleotide (i.e., the number of polynucleotide molecules having a
particular nucleotide
sequence) that hybridizes to the probe under particular hybridization
conditions compared
to or relative to the amount of cross-hybridization to the probe under the
same hybridization
conditions. Cross-hybridization or non-specific hybridization, as the terms
are used in
preferred embodiments of the invention, are understood to refer to the amount
of
polynucleotides other than the particular target polynucleotide (i.e., the
number of
polynucleotide molecules having nucleotide sequences different that the
nucleotide
sequence of the particular target polynucleotide) that hybridize to the probe
under particular
hybridization conditions.
In the methods and compositions of the present invention, the specific
hybridization
T of a target polynucleotide to the probe p is directly related to the
intensity I of the
hybridization signal from the specific hybridization sample. This relationship
can be
readily expressed, e.g., by the equation:
Tp = s ~ I p (Equation 1 )
wherein Ips is the intensity of the hybridization signal at probe p for the
specific
hybridization sample (i.e., the sample comprising the target polynucleotide
sequence). s
denotes a correction factor, e.g., for detector and label efficiencies.
-41 -
CA 02379212 2002-O1-09
WO 01/06013 PCT/US00/19203
Likewise, the amount of cross-hybridization X to the probe p is directly
related to
the intensity of the hybridization signal from the non-specific hybridization
sample; i.e.,
by:
XP = s ~ I p s (Equation 2)
wherein IpNS 1S the intensity of the hybridization signal at probe p for the
non-specific
hybridization sample (i.e., the sample deleted for the target polynucleotide).
One skilled in the art can therefore readily appreciate that by comparing the
hybridization intensities Ips and IPNS from the two samples the sensitivity
and specificity of a
p~lcular probe can be readily determined. In particularly preferred
embodiments, such a
comparison includes determining or obtaining the ratio of the hybridization
intensity from
the specific hybridization sample to the hybridization from the non-specific
hybridization
sample (i.e., the ratio Ips ~ Ip"'s ). Specifically, as noted above the
specificity of a probe is
can be defined to be the amount of a particular target that binds or
hybridizes to the probe
under particular conditions relative to the amount of non-specific binding or
hybridization
to that probe under the same conditions. Thus, in one embodiment, the
specificity S of the
probep is provided by the equation
T
SP = X (Equation 3)
P
However, from Equations 1 and 2 above, it is readily apparent to one skilled
in the art that
the specificity can also be obtained or provided by the equation
Is
Sp = 1 Ns (Equation 4)
Thus, the ratio of the hybridization intensities of the specific and non-
specific hybridization
samples provide a measure or value for the specificity of the probe.
Likewise, in the methods and compositions of the present invention the
sensitivity
of a probe p is determined or provided from the hybridization intensity of the
specific
hybridization sample to that probe; i.e., from Ips. Generally, the sensitivity
of a probe will
correlate with that probe's specificity. Thus, in preferred embodiments, those
probes that
are more specific for a target polynucleotide will also be more sensitive for
that target
polynucleotide.
Preferably, the analytical methods of the present invention are implemented by
means of a computer system such as those described hereinbelow. FIG. 7
illustrates an
exemplary computer system suitable for implementation of the analytic methods
of this
-42-
CA 02379212 2002-O1-09
WO 01/06013 PCT/US00/19203
invention. A computer system such as the exemplary computer system 701
typically
comprises one or more internal components and is linked to one or more
external
components. The internal components of such computer systems comprise a
processor
element 702 interconnected with a memory 703. For example, the computer system
can be
an Intel Pentium based processor of 200 MHz or greater clock rate and with 32
MB or more
of main memory.
The external components include mas storage 704. The mass storage can be,
e.g.,
one or more hard disks which are typically packaged together with the
processor and the
memory. Such hard disks are typically of 1 GB or greater storage capacity.
Other external
components include one or more user interface devices 705 which can include,
for example,
a monitor and a keyboard together with a pointing device 706 such as a "mouse"
or other
graphical input device. Typically, the computer system is also linked to a
network link 707,
which can be, e.g., part of an Ethernet link to one or more other local
computer systems, to
one or more remote computer systems or to one or more wide area communication
networks
such as the Internet. Such a network link allows the computer system to share
data and
processing tasks with other computer systems.
Loaded into the memory during operation of this system are several software
components which are both standard in the art and special to the instant
invention. These
software components collectively cause the computer system to function
according to the
methods of the invention (i.e., they will cause the processor to implement the
methods of
the invention). The software components are typically encoded and stored on
computer
readable media such as the mass storage component 704. However, one or more of
the
software components can be encoded and stored on other forms of computer
readable
media, including, but not limited to, a floppy disk, a CD-ROM or a DAT tape.
Software
component 710 represents an operating system which is responsible for managing
the
computer system and its network interconnections. The operating system can be,
for
example, of the Microsoft WindowsTM family, such as Windows 95, Windows 98,
Windows
2000 or Windows NT. Alternatively, the operating system can be a Macintosh
operating
system or a Unix operating system such as LINUX. Software component 711
represents
common languages and functions conveniently present in the system to assist
programs
implementing the methods specific to the present invention. Languages that can
be used to
program the analytic methods of the invention include, for example, C, C++
and, less
preferably, FORTRAN and JAVA. Most preferably, the methods of the invention
are
programmed in mathematical software packages which allow symbolic entry of
equations
and high-level specification of processing, including specific algorithms to
be used, thereby
freeing a user of the need to procedurally program individual equations and
algorithms.
- 43 -
CA 02379212 2002-O1-09
WO 01/06013 PCT/US00/19203
Such packages include, e.g., Matlab from Mathworks (Natick, MA), Mathematic
from
Wolfram Research (Chapaign, Illinois) or S-Plus from Math Soft (Seattle,
Washington).
Accordingly, software component 712 represents analytic methods of the present
invention
as programmed in a procedural language or symbolic package. The software
components
$ may also include a component 713 containing data, e.g., in a database, used
in the analytical
methods of the invention. For example, the database component may comprise
data
representing the amount of binding (e.g., hybridization) of molecules in one
or more
samples to a probe or probes. Such computer systems can be used to implement
and
practice the methods of the present invention. In particular, a user can cause
execution of
the analysis software component 712 of the system so that the processor
implements the
methods of the invention and thereby evaluates the binding of one or more
probes to one or
more different target molecules.
The compositions of the invention also include computer program products which
can be used to load one or more of the above-described software components
into the
memory of a computer system and cause the processor of the computer system to
implement the methods of the invention. Such computer program products
generally
comprise one or more computer readable storage media (e.g., floppy disks, CD-
ROMS,
DAT tapes) onto which one or more computer program mechanisms are embedded or
encoded. In particular, the computer program mechanisms comprise, e.g., one or
more of
the above described software components, such that the program mechanisms can
be loaded
into the memory of a computer system (such as the memory of exemplary computer
system
701) and cause the processor of that computer system to execute the analytical
methods of
the present invention.
Alternative systems and methods for implementing the analytic methods of the
present invention will also be recognized by those skilled in the art and are,
therefore,
intended to be comprehended within the scope of the accompanying claims. For
example,
those skilled in the art will recognize alternative program structures which
may be used,
e.g., in a computer system or in a computer program product, for implementing
the methods
of the invention. It is therefore understood that systems and products
encompassing such
alternative program structures are also part of the present invention.
5.3. APPLICATIONS TO PROBE AND MICROARRAY DESIGN
The methods and compositions of the invention are particularly useful for the
design
of microarrays that have many uses, e.g., in the fields of biology and drug
discovery. For
example, the methods and compositions of the invention can be used to prepare
microarrays
of probes that are capable of screening and specifically detecting large
numbers of different
-44-
CA 02379212 2002-O1-09
WO 01/06013 PCT/US00/19203
target polynucleotides such as a large number of different genes or gene
transcripts in a cell
or organism.
For example, such "screening chips," as they are referred to herein, can
comprise
probes capable of differentially hybridizing to and thereby detecting at least
2,000 or at least
4,000 different target polynucleotides. More preferably, such screening chips
have probes
capable of differentially hybridizing to and thereby detecting at least
10,000, at least 15,000,
or at least 20,000 different target polynucleotides. In particularly preferred
embodiments,
screening chips can be prepared that have probes capable of differentially
hybridizing to and
thereby detecting more than 50,000, more than 80,000 or more than 100,000
different target
polynucleotides.
In embodiments wherein the screening chips are used to detect polynucleotides
corresponding to genes or gene transcripts of a cell or organism, such
screening chips will
therefore typically have probes that hybridize specifically and
distinguishably to at least
50% of the genes in the genome of a cell or organism. Screening chips can more
preferably
have probes that hybridize specifically and distinguishably to at least 75%,
at least 80%, at
least 85%, at least 90%, at least 95%, or at least 99% of the genes in the
genome of a cell or
organism. In fact, in particularly preferred embodiments, screening chips
comprise probes
that hybridize specifically and distinguishably to all (i.e., 100%) of the
genes in the genome
of a cell or organism.
The methods and compositions of the invention can also be used to prepare
microarrays of probes that are capable of specifically detecting smaller
number of different
target polynucleotides, but with greater sensitivity and specificity. For
example,
microarrays can be designed, using the methods of the present invention, that
can reliably
and accurately detect changes in certain genes, referred to herein as
"signature genes" that
change, e.g., in response to some perturbation of change to a cell or organism
expressing or
potentially capable of expressing those genes.
Specifically, by using the methods described hereinabove, both the sensitivity
and
the specificity can be determined simultaneously for a plurality of different
probes. The
probes can then be ranked (e.g., according to the methods described in U.S.
Provisional
Application Serial No. 60/144,382 filed on July 16, 1999 and U.S. Patent
Application Serial
No. 09/364,751 filed on July 30, 1999), and then selected to select those
particular probes in
the plurality that have the highest sensitivity and specificity for a
particular target. Such
probes can then be used in microarrays, such as the screening and signature
arrays or
"chips" described above.
In particular, by using probes that are optimized for both sensitivity and
specificity,
the number of different probes required to reliably and distinguishably detect
a particular
- 45 -
CA 02379212 2002-O1-09
WO 01/06013 PCT/US00/19203
polynucleotide can be greatly reduced (e.g., to as few as one probe for each
target
polynucleotide). Thus, microarrays can be prepared that have probes that
specifically and
distinguishably hybridize to a greater number of different polynucleotides.
Alternatively,
by using probes that are optimized for both sensitivity and specificity for a
particular target,
microarrays can be prepared that can more reliably and accurately detect the
amount or level
of certain particular target polynucleotides in a sample, or that can more
reliably and
accurately detect changes in the amount or level of certain particular target
polynucleotides
in two or more different samples. Such microarrays will therefore typically
have binding
sites (i.e., probes) that bind specifically and distinguishably to a small
number of different
polynucleotides.
The methods of the invention can readily be applied by a skilled artisan to
select
probes for a plurality of different target polynucleotides, e.g., by repeating
the above
described methods for each particular target polynucleotide in the plurality.
Alternatively,
in certain embodiments the methods of the invention can be used to evaluate
and select
probes for two or more different polynucleotides wherein the two or more
different
polynucleotides are sufficiently orthogonal to each other that they do not
cross-hybridize to
a common polynucleotide sequence. "Orthogonal" polynucleotides refers to two
or more
polynucleotides that contain no common nucleic acid sequences. Orthogonal
sequences are
not, therefore, expected to cross-hybridize. In particular, a complementary
sequence that
hybridizes to a first polynucleotide sequence is not expected to hybridize to
a second
polynucleotide sequence if the first and sequence polynucleotide sequences are
orthogonal
sequences. Thus, more specifically, none of the two or more different target
polynucleotide
molecules used in such an alternative embodiment will hybridize or cross-
hybridize with a
probe that also hybridizes or cross-hybridizes any of the other different
target
polynucleotide molecules used in the embodiment.
Sequences that are sufficiently orthogonal to use in such embodiments of the
present
invention are understood to be sequences that have no more than 50% sequence
identity to
each other, and more preferably have no more than 20%, no more than 10%, no
more than
5%, no more than 2% or no more than 1% sequence identity to each other. Such
sufficiently orthogonal sequences can be readily identified, e.g., by means of
a sequence
comparison algorithm such as the Basic Local Alignment Search Tool (BLAST) or
Power
BLAST algorithms to identify such sequences within a database of nucleotide
sequences
(e.g., within the GenBank or dbEST databases). Sequence comparison algorithms
such as
BLAST and PowerBLAST are well known in the art (see, e.g., Altschul et al.,
1990, J. Mol.
Biol. 215:403-410; Altschul, 1997, Nucleic Acids Res. 25:3389-3402; and Zhang
and
Madden, 1997, Genome Res. 7:649-656). One skilled in the relevant arts)
therefore readily
-46-
CA 02379212 2002-O1-09
WO 01/06013 PCT/US00/19203
appreciates that such algorithms can be used to compare polynucleotide
sequences, e.g.,
using standard parameters that are well known in the art.
The methods of the invention can also be used to test theoretical models that
predict
properties such as the sensitivity and specificity of polynucleotide probes.
For example,
U.S. Provisional Application Serial No. 60/144,382 filed on July 16, 1999 and
U.S. Patent
Application Serial No. 09/364,751 filed on July 30, 1999 disclose methods
which can be
used to calculate or predict the sensitivity and specificity of a given
oligonucleotide, e.g.,
theoretical models for the thermodynamics of polynucleotide hybridization such
as the
nearest neighbor model (see, e.g., SantaLucia, 1998, Proc. Natl. Acad. Sci.
U.S.A. 95:1460-
1465). Using the methods and compositions of the invention disclosed herein,
hybridization properties of one or more probes such as the sensitivity and
specificity can be
empirically determined and compared, e.g., to the values predicted by such
theoretical
models. Such comparisons can then be used, e.g., to test the reliability
and/or accuracy of
such theoretical models, as well as to refine such models so that they are
more accurate and
reliable. In one preferred embodiment, the methods of the invention can be
used to
establish a database of properties of a plurality of different probes, such as
the specificity
and sensitivity of each probe to a diverse set of target polynucleotides
(e.g., a diverse set of
different genes or gene transcripts). Such a database is well suited to
testing and training
theoretical models of polynucleotide performance (e.g., predicting
polynucleotide
sensitivity and specificity), including the models described above and in U.S.
Provisional
Application Serial No. 60/144,382 filed on July 16, 1999 and in U.S. Patent
Application
Serial No. 09/364,751 filed on July 30, 1999.
6. EXAMPLE
The following example of evaluating different probe molecules is presented as
an
exemplary illustration of the methods and compositions of the previously
described
invention and is not limiting of that invention in any way. Specifically, the
example
presented herein describes the selection of a plurality of oligonucleotide
probes and the
evaluation of their sensitivity and specificity for the gene YER019W of the
yeast
Saccharomyces cerevisiae. The results presented herein demonstrate that probes
which are
both sensitive and specific for a particular target can be readily identified
using the
methods of the invention described hereinabove.
YER019W is a known gene of the yeast Saccharomyces cerevisiae (GenBank
Accession No. U18778) that is about 1.4 kilobases in length (i.e., slightly
longer than the
medium length of yeast ORFs). Although the function of the gene is unknown, it
has
properties that make it an excellent test candidate for probe selection
according to the
-47-
CA 02379212 2002-O1-09
WO 01/06013 PCT/US00/19203
methods of the present invention. First, the sequence of the gene is unique,
with no close
homologs as can be demonstrated by a routine BLAST comparison (Altschul et
al., 1990, J.
Mol. Biol. 215:403-410; Altschul, 1997, Nucleic Acids Res. 25:3389-3402; and
Zhang and
Madden, 1997, Genome Res. 7:649-656) of its sequence with the sequences of the
yeast
genome. This property renders YER019W appropriate for testing subsequence
specificity
against general cross-hybridization. Further, YER019W is expressed at very low
levels in
wild type cells (approximately one copy per cell). Thus, detection of the wild
type levels of
expression by hybridization requires high sensitivity probes. However, its
abundance in
wild type cells is not so low as to be undetectable.
Oligonucleotide probes were selected for evaluation by identifying every other
25-
mer sequence complementary to the YER019W base sequence (GenBank Accession No.
U18778). Thus, the candidate probes consisted of a total of 705 25-mer
sequences
complementary to bases 1-25, 3-27, 5-29, etc. spanning the full length of the
YER019W
sequence.
Three sets of control oligonucleotide probes were also selected. The first set
consisted of SO 25-mer probes complementary to the yeast gene YGR192C (GenBank
Accession No. Z72977), a housekeeping gene that is highly expressed in yeast
(about 200 to
400 copies per cell). Thus, the probes derived from this gene served as
positive controls for
labeling and non-specific hybridization since signal intensity from
hybridization to this
probe should always be high. The second set of control probes consisted of 200
25-mer
probes complementary to the yeast gene YLR040C (GenBank Accession No. Z73212),
a
gene of unknown function that is expressed at extremely low levels (no more
than one copy
per cell) in yeast. These probes thus served as positive sensitivity controls.
The control
probes for both the first and second sets were also selected by tiling every
other position in
a randomly chosen section of the gene YGR192C and YLR040C, respectively.
The third set of probes consisted of 43 20-mer sequences selected from the
yeast
deletion consortium barcodes (see, Shoemaker et al., 1996, Nature Genetics
14:450-456) to
be random nucleotide sequences that are not related to any naturally occurnng
sequences in
yeast and maximally orthogonal to each other. Thus, these probes served as
negative
controls for hybridization of the yeast sequences.
The selected YER019W oligonucleotides, the YGR192C and YLR040C controls
and the negative controls were all printed in duplicate on the top and bottom
half of three
chips, referred to as Chips 978, 979, and 1136, according to the standard
inkjet printing
techniques of Blanchard (see, e.g., International Patent Publication No. WO
98/41531,
published on September 24, 1998; Blanchard et al., 1996, Biosensors and
Bioelectronics
11:687-690; Blanchard, 1998, in Synthetic DNA Arrays in Genetic Engineering,
Vol. 20,
-48-
CA 02379212 2002-O1-09
WO 01/06013 PCT/US00/19203
J.K. Setlow, ed., Plenum Press, New York at pages 111-123). All chips were
hybrdized
ovenight at 66 °C in 200 pL of hybridization solution consisting of 10
mM Tris pH 7.6, 1 M
NaCI, 1 % Triton-X-100; 1 ~g/~L bovine serum albumin, 0.1 ~g/~L sheared hernng
sperm
DNA, SO pM Cy3-labeled gridline oligonucleotide, and 50 pM Cy5-labeled
gridline
oligonucleotide. After hybridization, the chips were washed by shaking for 10
seconds at
room temperature in 6x SSPE, 0.005% Triton-X-100; and for another 10 seconds
at room
temperature in 0.06x SSPE. The chips were dried with pressurized air, and
scanned using a
General Scanning 3000 confocal laser scanner.
Chips 978 and 979 were hybridized simultaneously with differentially labeled
samples. The first sample consisted of fluorescently labeled 1.6 ng fragmented
YER019W
cRNA and served as a specific hybridization sample. This concentration of
YER019W
RNA corresponds to approximately 10 copies of the YER019W transcript per cell
or about
10 times the natural abundances of YER019W. The second sample, which served as
a non-
specific hybridization sample, consisted of 2 ~g fluorescently labeled
fragmented cRNA
from yer019w/- (i.e., a diploid yeast strain specifically deleted for the gene
YER019W)
homozygous disruption yeast mRNA. Specifically, chip 978 was hybridized with
Cy5
labeled YER019W cRNA and Cy3 labeled yer019w/- cRNA. Chip 979 was hybridized
with Cy3 labeled YER019W cRNA and Cy5 labeled yer019w/- cRNA. Chip 1136 was
hybridized with 2 ~g Cy5 labeled fragmented cRNA from wild type yeast and with
2 ~.g
Cy3 labeled fragmented cRNA from yer019w/-.
Combined color images of the three chips 978, 979 and 1136 are shown in FIGS.
2-
4, respectively. Each of the two hybridization samples can be distinguished by
the different
fluorescence color of their respectively labels: Cy3 which fluoresces "green"
(FIGS. 2A,
3A and 4A), and Cy5 which fluoresces "red" (FIGS. 2B, 3B and 4B).
The gene specific signal (i.e., from the specific hybridization sample,
YER019W)
from these images was combined from chips 978 and 979. A plot of the combined
signal
versus the gene tiling position is provided in FIG. 5A. Likewise, the non-
specific signal
(i.e., from the non-specific hybridization sample, yer019w/-) was also
combined from chips
978 and 979 and is plotted versus the gene tiling positions in FIG. 5B.
The signal depicted in FIG. 5A is an indicator of the sensitivity of each
probe from
the gene YER019W. Specifically, peaks in the plot shown in FIG. 5A indicate
probes that
hybridize well (i.e., are sensitive) to YER019W and might therefore be
desirable probes for
detecting that gene in a polynucleotide sample. The signal depicted in FIG. 5B
indicates the
amount of cross-hybridization. Peaks in this plot indicate probes that cross-
hybridize with
other polynucleotide sequences, suggesting that they would be undesirable for
specifically
detecting the YER019W sequence, especially in a sample comprising many
different
-49-
CA 02379212 2002-O1-09
WO 01/06013 PCT/US00/19203
polynucleotide sequences (e.g., a sample of many different yeast
polynucleotide sequences
extracted from a cell or cells). FIG. SC shows a plot of the ratio between the
gene specific
(i.e., YER019W) signal in FIG. 5A and the gene non-specific (i.e., yer019w/-)
signal in
FIG. 5B. The plot therefore indicates the specificity of each probe. Peaks in
this plot
S indicate probes that hybridize well to YER019W while at the same time
exhibiting only
limited cross-hybridization to other polynucleotides. The data in FIGS. 5A and
SC also
indicate that, in general, the sensitivity and specificity may be well-
correlated.
A scatter plot is shown in FIG. 6 that diagrams relationships between the
sensitivity
(GS signal, horizontal axis) and specificity (GS/GNS signal, vertical axis)
for each
complementary probe of YER019W using the data in FIGS. 5A and SC. Those probes
having both high sensitivity and specificity (i.e., in the upper right hand
corner of the scatter
plot) are particularly desirable probes for use, e.g., in a microarray to
detect the YER019W
gene in a sample of many different genes and/or gene transcripts. Such
desirable probes are
also indicated by an (X) in FIG. SC.
Thus, the methods and compositions described hereinbelow allow for the
selection
of the most specific and sensitive probes for detecting a particular
polynucleotide (e.g., a
particular gene).
7. REFERENCES CITED
All references cited herein are incorporated herein by reference in their
entirety and
for all purposes to the same extent as if each individual publication or
patent or patent
application was specifically and individually indicated to be incorporated by
reference in its
entirety for all purposes.
Many modifications and variations of this invention can be made without
departing
from its spirit and scope, as will be apparent to those skilled in the art.
The specific
embodiments described herein are offered by way of example only, and the
invention is to
be limited only by the terms of the appended claims along with the full scope
of equivalents
to which such claims are entitled.
35
-50-
