U.S. patent application number 13/134477 was filed with the patent office on 2012-01-26 for method for detecting transcription factor-protein interactions.
This patent application is currently assigned to Affymetrix, Inc.. Invention is credited to Xianqiang Li.
Application Number | 20120021938 13/134477 |
Document ID | / |
Family ID | 27505967 |
Filed Date | 2012-01-26 |
United States Patent
Application |
20120021938 |
Kind Code |
A1 |
Li; Xianqiang |
January 26, 2012 |
Method for detecting transcription factor-protein interactions
Abstract
A method is provided for identifying complexes between a
transcription factor and another protein, the method comprising:
isolating from a biological sample transcription factor complexes
based on whether the transcription factor complexes comprise a
particular type of transcription factor; and identifying which of a
plurality of different proteins are present in the isolated
transcription factor complexes.
Inventors: |
Li; Xianqiang; (Palo Alto,
CA) |
Assignee: |
Affymetrix, Inc.
Santa Clara
CA
|
Family ID: |
27505967 |
Appl. No.: |
13/134477 |
Filed: |
June 7, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09947274 |
Sep 5, 2001 |
7981842 |
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13134477 |
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09877738 |
Jun 8, 2001 |
6924113 |
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09947274 |
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09877243 |
Jun 8, 2001 |
6696256 |
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09877738 |
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09877403 |
Jun 8, 2001 |
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09877243 |
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09877705 |
Jun 8, 2001 |
6821737 |
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09877403 |
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Current U.S.
Class: |
506/9 ;
436/501 |
Current CPC
Class: |
G01N 33/6845 20130101;
C12Q 1/6881 20130101; G01N 33/6842 20130101; C40B 30/04
20130101 |
Class at
Publication: |
506/9 ;
436/501 |
International
Class: |
C40B 30/04 20060101
C40B030/04; G01N 33/53 20060101 G01N033/53 |
Claims
1-2. (canceled)
3. A method for identifying complexes between a transcription
factor and another protein, the method comprising: isolating from a
biological sample transcription factor complexes based on whether
the transcription factor complexes comprise a particular type of
transcription factor; and identifying which of a plurality of
different proteins are present in the isolated transcription factor
complexes; wherein identifying which of a plurality of different
proteins are present in the isolated transcription factor complexes
comprises identifying which of a plurality of different
non-transcription factor proteins are present in the isolated
transcription factor complexes.
4. A method according to claim 3 wherein identifying which of a
plurality of different proteins are present in the isolated
transcription factor complexes comprises detecting which of a
plurality of different transcription factor and non-transcription
factor proteins are present in the isolated transcription factor
complexes.
5. A method according to claim 3 wherein isolating transcription
factor complexes comprises contacting the biological sample with an
agent that selectively binds to the particular type of
transcription factor; and isolating those transcription factor
complexes to which the agent binds.
6. A method according to claim 3 wherein isolating transcription
factor complexes comprises contacting the biological sample with an
agent that selectively binds to the particular type of
transcription factor; precipitating those transcription factor
complexes to which the agent binds; and isolating the
precipitate.
7. A method according to claim 3 wherein isolating transcription
factor complexes comprises contacting the biological sample with an
agent immobilized on a solid support that selectively binds to the
particular type of transcription factor; and isolating
transcription factor complexes immobilized on the solid support
through binding to the agent.
8. A method according to claim 3 wherein isolating transcription
factor complexes comprises contacting the biological sample with an
antibody that selectively binds to the particular type of
transcription factor; and isolating those transcription factor
complexes to which the antibody binds.
9. A method according to claim 3 wherein identifying which of a
plurality of different types of proteins are present in the
isolated transcription factor complexes comprises simultaneously
identifying multiple different types of proteins in a same
assay.
10. A method according to claim 9, wherein identifying which of a
plurality of different proteins are present in the isolated
transcription factor complexes comprises detecting which of a
plurality of different transcription factor and non-transcription
factor proteins are present in the isolated transcription factor
complexes, and wherein simultaneously identifying multiple
different types of proteins in a same assay is based on a
characterization of which different types of transcription factors
are present in the isolated transcription factor complexes.
11. A method according to claim 9, wherein identifying which of a
plurality of different proteins are present in the isolated
transcription factor complexes comprises detecting which of a
plurality of different transcription factor and non-transcription
factor proteins are present in the isolated transcription factor
complexes, and wherein simultaneously identifying multiple
different types of proteins in a same assay is based on an
identification of transcription factor probes isolated from the
isolated transcription factor complexes.
12. A method according to claim 9 wherein simultaneously
identifying multiple different types of proteins in a same assay
comprises simultaneously identifying at least 5 different types of
proteins in a same assay.
13. (canceled)
14. A method according to claim 9 wherein simultaneously
identifying multiple different types of proteins in a same assay
comprises simultaneously identifying at least 20 different types of
proteins in a same assay.
15. (canceled)
16. A method according to claim 3 wherein identifying which of a
plurality of different types of proteins are present in the
isolated transcription factor complexes comprises simultaneously
identifying multiple different types of non-transcription factor
proteins in a same assay.
17-20. (canceled)
21. A method for identifying complexes between a transcription
factor and another protein, the method comprising: isolating from a
biological sample transcription factor complexes based on whether
the transcription factor complexes comprise a particular type of
transcription factor; and identifying which of a plurality of
different proteins are present in the isolated transcription factor
complexes by taking an array of immobilized agents capable of
binding to the different types of proteins to be identified and
contacting the isolated transcription factor complexes with the
array under conditions suitable for binding between proteins in the
isolated transcription factor complexes and the agents in the
array.
22. A method for identifying complexes between a protein and a
plurality of transcription factors, the method comprising:
isolating from a biological sample transcription factor complexes
based on whether the transcription factor complexes comprise a
particular type of non-transcription factor protein; and
identifying which of a plurality of different transcription factors
are present in the isolated transcription factor complexes.
23. A method according to claim 22 wherein isolating transcription
factor complexes comprises contacting the biological sample with an
agent that selectively binds to the particular type of
non-transcription factor protein; and isolating those transcription
factor complexes to which the agent binds.
24. A method according to claim 22 wherein isolating transcription
factor complexes comprises contacting the biological sample with an
agent that selectively binds to the particular type of
non-transcription factor protein; precipitating those transcription
factor complexes to which the agent binds; and isolating the
precipitate.
25. A method according to claim 22 wherein isolating transcription
factor complexes comprises contacting the biological sample with an
agent immobilized on a solid support that selectively binds to the
particular type of non-transcription factor protein; and isolating
transcription factor complexes immobilized on the solid support
through binding to the agent.
26. A method according to claim 22 wherein isolating transcription
factor complexes comprises contacting the biological sample with an
antibody that selectively binds to the particular type of
non-transcription factor protein; and isolating those transcription
factor complexes to which the antibody binds.
27. A method according to claim 22 wherein identifying which of a
plurality of different types of transcription factors are present
in the isolated transcription factor complexes comprises
simultaneously identifying multiple different types of
transcription factors in a same assay.
28. A method according to claim 27 wherein simultaneously
identifying multiple different types of transcription factors in a
same assay comprises simultaneously identifying at least 5
different types of transcription factors in a same assay.
29. (canceled)
30. A method according to claim 27 wherein simultaneously
identifying multiple different types of transcription factors in a
same assay comprises simultaneously identifying at least 20
different types of transcription factors in a same assay.
31. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation in part of application
Ser. Nos. 09/877,738, 09/877,243, 09/877,403, and 09/877,705, each
filed on Jun. 8, 2001, each of which are incorporated herein by
reference.
FIELD OF THE INVENTION
[0002] The present invention relates to methods for detecting
activated transcription factors in a cell sample. More
specifically, the invention relates to methods for detecting
interactions between activated transcription factors in a cell
sample and compositions, kits, and uses arising there from.
DESCRIPTION OF RELATED ART
[0003] All living organisms use nucleic acids (DNA and RNA) to
encode the genes which make up the genome for that organism. Each
gene encodes a protein that may be produced by the organism through
expression of the gene.
[0004] It is important to note that the mere presence of a gene in
a cell does not communicate the functionality of that gene to the
cell. Rather, it is only when the gene is expressed and a protein
is produced that the functionality of the gene encoding the protein
is conveyed.
[0005] The systems that regulate gene expression respond to a wide
variety of developmental and environmental stimuli, thus allowing
each cell type to express a unique and characteristic subset of its
genes, and to adjust the dosage of particular gene products as
needed. The importance of dosage control is underscored by the fact
that targeted disruption of key regulatory molecules in mice often
results in drastic phenotypic abnormalities [Johnson, R. S., et
al., Cell, 71:577-586 (1992)], just as inherited or acquired
defects in the function of genetic regulatory mechanisms contribute
broadly to human disease.
[0006] The importance of controlled gene expression in human
disease and the information available to date relating to the
mechanisms of gene regulation have fueled efforts aimed at
discovering ways of overriding endogenous regulatory controls or of
creating new signaling circuitry in cells [Belshaw, P. J., et al.,
Proc. Natl. Acad. Sci. USA, 93:4604-4607 (1996); Ho, S. H., et al.,
Nature (London), 382:822-826 (1996); Rivera, V. M., et al., Nat.
Med., 2:1028-1032; Spencer, D. M., et al., Science, 262:1019-1024
(1993)].
[0007] Critical to this research are effective tools for monitoring
gene expression. It is therefore of interest to be able to rapidly
and accurately determine the relative expression of different genes
in different cells, tissues and organisms, over time, and under
various conditions, treatments and regimes. As will be described
herein in greater detail, there are a great many applications that
arise from being able to effectively monitor which genes are being
expressed by a given cell at a given time.
[0008] Standard molecular biology techniques have been used to
analyze the expression of genes in a cell by measuring DNA. These
techniques include PCR, northern blot analysis, or other types of
DNA probe analysis such as in situ hybridization. Each of these
methods allows one to analyze the transcription of only known genes
and/or small numbers of genes at a time. Nucl. Acids Res. 19,
7097-7104 (1991); Nucl. Acids Res. 18, 4833-4842 (1990); Nucl.
Acids Res. 18, 2789-2792 (1989); European J. Neuroscience 2,
1063-1073 (1990); Analytical Biochem. 187, 364-373 (1990); Genet.
Annal Techn. Appl. 7, 64-70 (1990); GATA 8(4), 129-133 (1991);
Proc. Natl. Acad. Sci. USA 85, 1696-1700 (1988); Nucl. Acids Res.
19, 1954 (1991); Proc. Natl. Acad. Sci. USA 88, 1943-1947 (1991);
Nucl. Acids Res. 19, 6123-6127 (1991); Proc. Natl. Acad. Sci. USA
85, 5738-5742 (1988); Nucl. Acids Res. 16, 10937 (1988).
[0009] Gene expression has also been monitored by measuring levels
of mRNA. Since proteins are transcribed from mRNA, it is possible
to detect transcription by measuring the amount of mRNA present.
One common method, called "hybridization subtraction", allows one
to look for changes in gene expression by detecting changes in mRNA
expression. Nucl. Acids Res. 19, 7097-7104 (1991); Nucl. Acids Res.
18, 4833-4842 (1990); Nucl. Acids Res. 18, 2789-2792 (1989);
European J. Neuroscience 2, 1063-1073 (1990); Analytical Biochem.
187, 364-373 (1990); Genet. Annal Techn. Appl. 7, 64-70 (1990);
GATA 8(4), 129-133 (1991); Proc. Natl. Acad. Sci. USA 85, 1696-1700
(1988); Nucl. Acids Res. 19, 1954 (1991); Proc. Natl. Acad. Sci.
USA 88, 1943-1947 (1991); Nucl. Acids Res. 19, 6123-6127 (1991);
Proc. Natl. Acad. Sci. USA 85, 5738-5742 (1988); Nucl. Acids Res.
16, 10937 (1988).
[0010] Gene expression has also been monitored by measuring levels
of gene product, (i.e., the expressed protein), in a cell, tissue,
organ system, or even organism. Measurement of gene expression by
measuring the protein gene product may be performed using
antibodies known to bind to a particular protein to be detected. A
difficulty arises in needing to generate antibodies to each protein
to be detected. Measurement of gene expression via protein
detection may also be performed using 2-dimensional gel
electrophoresis, wherein proteins can be, in principle, identified
and quantified as individual bands, and ultimately reduced to a
discrete signal. In order to positively analyze each band, each
band must be excised from the membrane and subjected to protein
sequence analysis using Edman degradation. Unfortunately, it tends
to be difficult to isolate a sufficient amount of protein to obtain
a reliable sequence. In addition, many of the bands contain more
than one discrete protein.
[0011] A further difficulty associated with quantifying gene
expression by measuring an amount of protein gene product in a cell
is that protein expression is an indirect measure of gene
expression. It is impossible to know from a protein present in a
cell when that protein was expressed by the cell. As a result, it
is hard to determine whether protein expression changes over time
due to cells being exposed to different stimuli.
[0012] Gene expression has also been monitored by measuring the
amount of particular activated transcription factors present in a
cell. Transcription in a cell is controlled by proteins, referred
to herein as "activated transcription factors" which bind to DNA at
sites outside the core promoter for the gene and activate
transcription. Since activated transcription factors activate
transcription, detection of their presence is useful for measuring
gene expression. Transcriptional activators are found in
prokaryotes, viruses, and eukaryotes, including fungi, plants, and
animals, including mammals, providing a wide range of therapeutic
targets.
[0013] The regulatory mechanisms controlling the transcription of
protein-coding genes by RNA polymerase II have been extensively
studied. RNA polymerase II and its host of associated proteins are
recruited to the core promoter through non-covalent contacts with
sequence-specific DNA binding proteins [Tjian, R. and Maniatis, T.,
Cell, 77:5-8 (1994); Stringer, K. F., Nature (London), 345:783-786
(1990)]. An especially prevalent and important subset of such
proteins, known as transcription factors, typically bind DNA at
sites outside the core promoter and activate transcription through
space contacts with components of the transcriptional machinery,
including chromatin remodeling proteins [Tjian, R. and Maniatis,
T., Cell, 77:5-8 (1994); Stringer, K. F., Nature (London),
345:783-786 (1990); Bannister, A. J. and Kouzarides, T., Nature,
384:641-643 (1996); Mizzen, C. A., et al., Cell, 87:1261-1270
(1996)]. The DNA-binding and activation functions of transcription
factors generally reside on separate domains whose operation is
portable to heterologous fusion proteins [Sadowski, I., et al.,
Nature, 335:563-564 (1988)]. Though it is believed that activation
domains are physically associated with a DNA-binding domain to
attain proper function, the linkage between the two need not be
covalent [Belshaw, P. J., et al., Proc. Natl. Acad. Sci. USA,
93:4604-4607 (1996); Ho, S. H., et al., Nature (London),
382:822-826 (1996)]. In many instances, the activation domain does
not appear to contact the transcriptional machinery directly, but
rather through the intermediacy of adapter proteins known as
coactivators [Silverman, N., et al., Proc. Natl. Acad. Sci. USA,
91:11005-11008 ((1994); Arany, Z., et al., Nature (London),
374:81-84 (1995)].
[0014] One of the difficulties associated with measuring gene
expression by measuring transcription factors is that one must
measure the subset of transcription factors which are "activated."
Certain post-transcriptional modifications occur which render
transcription factors "active" in the sense that they are capable
of binding to DNA. It is thus necessary to distinguish between
activated and non-activated transcription factors so that the
"activated transcription factors" can be selectively measured.
[0015] Several different methods have been developed for detecting
activated transcription factors. One method involves using
antibodies selective for activated transcription factors over
inactive forms of the transcription factor. This method is
impractical for detecting multiple different activated
transcription factors due to difficulties associated with
developing numerous different antibodies having the requisite bind
specificities.
[0016] Another method for detecting activated transcription factors
involves measuring DNA--transcription factor complexes through a
gel shift assay. [Ausebel, F. M. et al eds (1993) Current Protocols
in Molecular Biology Vol. 2 Greene Publishing Associates, Inc. and
John Wiley and Sons, Inc., New York]. According to this method, a
sample containing an activated transcription factor is contacted
with a DNA probe that comprises a recognition sequence for the
transcription factor. A complex between the activated transcription
factor and the DNA probe is formed. The DNA-protein complex is
detected by a gel-shift assay. Since individual gel shift assays
must be performed for each activated transcription factor--DNA
complex, this method is currently impractical for measuring
multiple different activated transcription factors at the same
time.
[0017] U.S. Pat. Nos. 6,066,452 and 5,861,246 describe methods for
determining DNA binding sites for DNA-binding proteins. The DNA
binding sites may then be used as probes to isolate DNA-binding
proteins. Similarly, PCT Publication No. WO 00/04196 describes
methods for identifying cis acting nucleic acid elements as well as
methods for isolating nucleic acid binding factors.
SUMMARY OF THE INVENTION
[0018] The present invention relates to methods and kits for
isolating DNA probes that bind to activated transcription
factors.
[0019] In one embodiment, a method is provided which comprises:
contacting a biological sample with a library of double stranded
DNA probes under conditions where DNA probe--transcription factor
complexes are formed between the DNA probes and activated
transcription factors present in the biological sample; separating
DNA probe--transcription factor complexes from non-complexed DNA
probes in the library using an agarose gel separation; excising a
portion of the agarose gel comprising the separated DNA
probe--transcription factor complexes; and isolating the DNA probes
from the excised portion of the agarose gel.
[0020] In another embodiment, a method is provided which comprises:
contacting a biological sample with a library of double stranded
DNA probes under conditions where DNA probe--transcription factor
complexes are formed between the DNA probes and activated
transcription factors present in the biological sample; separating
DNA probe--transcription factor complexes from non-complexed DNA
probes in the library using an agarose gel separation; excising a
portion of the agarose gel comprising the separated DNA
probe--transcription factor complexes; isolating the DNA probes
from the excised portion of the agarose gel; and identifying which
of the DNA probes in the library are isolated.
[0021] In another embodiment, a kit is provided which comprises: a
library of double stranded DNA probes, each probe comprising a
recognition sequence to which an activated transcription factor is
capable of binding and forming a DNA probe--transcription factor
complex, the DNA probes in the library capable of forming DNA
probe--transcription factor complexes with multiple different
activated transcription factors; and instructions for separating
DNA probe--transcription factor complexes from non-complexed DNA
probes in the library by agarose gel separation.
[0022] Kits are also provided for DNA probe libraries for detecting
activated transcription factors.
[0023] In one embodiment, the kit comprises: first and second
libraries of double stranded DNA probes, each probe in the first
and second libraries comprising a recognition sequence to which an
activated transcription factor is capable of binding and forming a
DNA probe--transcription factor complex, the DNA probes in the
library capable of forming DNA probe--transcription factor
complexes with multiple different activated transcription factors;
wherein the probes of the first library further comprise a first
detectable marker and the probes of the second library further
comprise a second detectable marker that is different than the
first detectable marker.
[0024] Methods, arrays and kits are also provided for detecting
activated transcription factors using a hybridization array.
[0025] In one embodiment, a method is provided which comprises:
taking a library of double stranded transcription factor probes,
the transcription factor probes each comprising a recognition
sequence capable of binding to an activated transcription factor,
the recognition sequence varying within the library for binding to
different activated transcription factors; contacting a biological
sample with the library of double stranded DNA probes under
conditions where DNA probe--transcription factor complexes are
formed between the DNA probes and activated transcription factors
present in the biological sample; isolating the transcription
factor probes from the transcription factor probe--transcription
factor complexes formed; and identifying which transcription factor
probes in the library formed complexes by taking an array of
immobilized hybridization probes capable of hybridizing to at least
one of the strands of the different double stranded transcription
factor probes in the library and contacting the isolated
transcription factor probes with the array under conditions
suitable for hybridization of the strands of the different double
stranded transcription factor probes to the hybridization probes in
the array.
[0026] In another embodiment, a hybridization array is provided for
use in identifying which of a plurality of different activated
transcription factors are present in a biological sample by
immobilizing transcription factor probes that form transcription
factor probe--transcription factor complexes with different
activated transcription factors, the array comprising: a substrate;
and a plurality of hybridization probes immobilized on a surface of
the substrate such that different hybridization probes are
positioned in different defined regions on the surface, the
different hybridization probes comprising a different transcription
factor probe binding region capable of immobilizing a different
transcription factor probe to the array, the transcription factor
probe binding region comprising at least two copies of a compliment
to a portion of a recognition sequence comprised on the
transcription factor probe. The hybridization array may optionally
further comprise an internal standard. For example, the array may
further comprise biotinylated DNA which is employed as an internal
standard.
[0027] In another embodiment, a kit is provided for use in
identifying which of a plurality of different activated
transcription factors are present in a biological sample by
isolating and immobilizing transcription factor probes that form
transcription factor probe--transcription factor complexes with
different activated transcription factors, the kit comprising: a
hybridization array comprising a substrate, and a plurality of
hybridization probes immobilized on a surface of the substrate such
that different hybridization probes are positioned in different
defined regions on the surface, the different hybridization probes
comprising a different transcription factor probe binding region
capable of immobilizing a different transcription factor probe to
the array, the transcription factor probe binding region comprising
at least two copies of a compliment to a portion of a recognition
sequence comprised on the transcription factor probe; and
instructions for separating DNA probe--transcription factor
complexes from non-complexed DNA probes in the library by agarose
gel separation.
[0028] Methods, arrays and kits are also provided for identifying
complexes between transcription factors and other proteins
including other transcription factors. It is noted that these
methods, arrays and kits may be applied more broadly for detecting
complexes between proteins.
[0029] In one embodiment, the method comprises identifying
complexes between a transcription factor and another protein, the
method comprising: isolating from a biological sample transcription
factor complexes based on whether the transcription factor
complexes comprise a particular type of transcription factor; and
identifying which of a plurality of different proteins are present
in the isolated transcription factor complexes.
[0030] According to this embodiment, identifying which of a
plurality of different proteins are present in the isolated
transcription factor complexes may comprise identifying which of a
plurality of different transcription factors are present in the
isolated transcription factor complexes. Identifying which of a
plurality of different proteins are present in the isolated
transcription factor complexes may also comprise identifying which
of a plurality of different non-transcription factor proteins are
present in the isolated transcription factor complexes. Identifying
which of a plurality of different proteins are present in the
isolated transcription factor complexes may also comprise detecting
which of a plurality of different transcription factor and
non-transcription factor proteins are present in the isolated
transcription factor complexes. Identifying which of a plurality of
different types of proteins are present in the isolated
transcription factor complexes may also comprise simultaneously
identifying multiple different types of non-transcription factor
proteins in a same assay.
[0031] Isolating transcription factor complexes may optionally
comprise contacting the biological sample with an agent that
selectively binds to the particular type of transcription factor;
and isolating those transcription factor complexes to which the
agent binds.
[0032] Isolating transcription factor complexes may also optionally
comprise contacting the biological sample with an agent that
selectively binds to the particular type of transcription factor;
precipitating those transcription factor complexes to which the
agent binds; and isolating the precipitate.
[0033] Isolating transcription factor complexes may also optionally
comprise contacting the biological sample with an agent immobilized
on a solid support that selectively binds to the particular type of
transcription factor; and isolating transcription factor complexes
immobilized on the solid support through binding to the agent.
[0034] Isolating transcription factor complexes may also optionally
comprise contacting the biological sample with an antibody that
selectively binds to the particular type of transcription factor;
and isolating those transcription factor complexes to which the
antibody binds.
[0035] Identifying which of a plurality of different types of
proteins are present in the isolated transcription factor complexes
may comprise simultaneously identifying multiple different types of
proteins and/or transcription factors in a same assay.
[0036] Simultaneously identifying multiple different types of
proteins in a same assay may comprise simultaneously identifying at
least 5, 10, 15, 20, 50 or more different types of transcription
factors in a same assay.
[0037] Simultaneously identifying multiple different types of
transcription factors in a same assay may optionally be based on a
characterization of which different types of transcription factors
are present in the isolated transcription factor complexes.
[0038] Simultaneously identifying multiple different types of
transcription factors in a same assay may also optionally be based
on an identification of transcription factor probes isolated from
the isolated transcription factor complexes.
[0039] When the method comprises identifying transcription factors,
the method may optionally further comprise, prior to isolating
transcription factor complexes, contacting the biological sample
with a library of transcription factor probes under conditions
where transcription factor probe--transcription factor complexes
are formed between the transcription factor probes and activated
transcription factors present in the biological sample; and
identifying which of a plurality of different types of
transcription factors are present in the isolated transcription
factor complexes comprises identifying which transcription factor
probes are present in the isolated transcription factor
complexes.
[0040] When the method comprises identifying transcription factors,
the method may also optionally comprise, prior to isolating
transcription factor complexes, taking a library of transcription
factor probes, the transcription factor probes each comprising a
recognition sequence capable of binding to an activated
transcription factor, the recognition sequence varying within the
library for binding to different activated transcription factors,
and contacting the biological sample with the library of
transcription factor probes under conditions where transcription
factor probe--transcription factor complexes are formed between the
transcription factor probes and activated transcription factors
present in the biological sample; wherein identifying which of a
plurality of different types of transcription factors are present
in the isolated transcription factor complexes comprises
identifying which transcription factor probes are present in the
isolated transcription factor complexes.
[0041] When the method comprises identifying transcription factors,
the method may optionally comprise, prior to selectively isolating
transcription factor complexes, taking a library of transcription
factor probes, the transcription factor probes each comprising a
recognition sequence capable of binding to an activated
transcription factor, the recognition sequence varying within the
library for binding to different activated transcription factors,
and contacting a biological sample with the library of
transcription factor probes under conditions where transcription
factor probe--transcription factor complexes are formed between the
transcription factor probes and activated transcription factors
present in the biological sample; wherein identifying which of a
plurality of different types of transcription factors are present
in the isolated transcription factor complexes comprises taking an
array of immobilized hybridization probes capable of hybridizing to
the transcription factor probes in the library and contacting the
isolated transcription factor probes with the array under
conditions suitable for hybridization of the different
transcription factor probes to the hybridization probes in the
array.
[0042] According to another embodiment, a method for identifying
complexes between a protein and a plurality of transcription
factors is provided which comprises: isolating from a biological
sample transcription factor complexes based on whether the
transcription factor complexes comprise a particular type of
non-transcription factor protein; and identifying which of a
plurality of different transcription factors are present in the
isolated transcription factor complexes.
[0043] According to this embodiment, isolating transcription factor
complexes may optionally comprise contacting the biological sample
with an agent that selectively binds to the particular type of
non-transcription factor protein; and isolating those transcription
factor complexes to which the agent binds. Isolating transcription
factor complexes may also optionally comprise contacting the
biological sample with an agent that selectively binds to the
particular type of non-transcription factor protein; precipitating
those transcription factor complexes to which the agent binds; and
isolating the precipitate. Isolating transcription factor complexes
may also optionally comprise contacting the biological sample with
an agent immobilized on a solid support that selectively binds to
the particular type of non-transcription factor protein; and
isolating transcription factor complexes immobilized on the solid
support through binding to the agent. Isolating transcription
factor complexes may also optionally comprise contacting the
biological sample with an antibody that selectively binds to the
particular type of non-transcription factor protein; and isolating
those transcription factor complexes to which the antibody
binds.
[0044] Isolating transcription factor complexes may also optionally
comprise simultaneously identifying multiple different types of
transcription factors in a same assay. Simultaneously identifying
multiple different types of transcription factors in a same assay
may comprise simultaneously identifying at least 5, 10, 15, 20, 50
or more different types of transcription factors in a same
assay.
[0045] Methods for characterizing cell types based on which
activated transcription factors are present in a sample are also
provided.
[0046] In one embodiment, a method is provided which comprises:
taking a library of double stranded transcription factor probes,
the transcription factor probes each comprising a recognition
sequence capable of binding to an activated transcription factor,
the recognition sequence varying within the library for binding to
different activated transcription factors native to different cell
types; contacting a biological sample with the library of double
stranded DNA probes under conditions where DNA probe--transcription
factor complexes are formed between the DNA probes and activated
transcription factors present in the biological sample; isolating
the transcription factor probes from the transcription factor
probe--transcription factor complexes formed; identifying which
transcription factor probes in the library formed complexes by
taking an array of immobilized hybridization probes capable of
hybridizing to at least one of the strands of the different double
stranded transcription factor probes in the library and contacting
the isolated transcription factor probes with the array under
conditions suitable for hybridization of the strands of the
different double stranded transcription factor probes to the
hybridization probes in the array; and identifying a cell type of
the biological sample based on which transcription factor probes
are identified.
[0047] Methods for identifying a disease state based on which
activated transcription factors are present in a biological sample
are also provided.
[0048] In one embodiment, the method comprises taking a library of
double stranded transcription factor probes, the transcription
factor probes each comprising a recognition sequence capable of
binding to an activated transcription factor, the recognition
sequence varying within the library for binding to different
activated transcription factors native to different cell types;
identifying which activated transcription factors are present in a
nuclear extract of a test sample of cells by: contacting the
nuclear extract of the test sample with the library of double
stranded DNA probes under conditions where DNA probe--transcription
factor complexes are formed between the DNA probes and activated
transcription factors present in the test sample, isolating the
transcription factor probes from the transcription factor
probe--transcription factor complexes formed, and identifying which
transcription factor probes in the library formed complexes by
taking an array of immobilized hybridization probes capable of
hybridizing to at least one of the strands of the different double
stranded transcription factor probes in the library and contacting
the isolated transcription factor probes with the array under
conditions suitable for hybridization of the strands of the
different double stranded transcription factor probes to the
hybridization probes in the array; identifying which activated
transcription factors are present in a nuclear extract of a control
sample of cells by: contacting the nuclear extract of the control
sample with the library of double stranded DNA probes under
conditions where DNA probe--transcription factor complexes are
formed between the DNA probes and activated transcription factors
present in the control sample, isolating the transcription factor
probes from the transcription factor probe--transcription factor
complexes formed, and identifying which transcription factor probes
in the library formed complexes by taking an array of immobilized
hybridization probes capable of hybridizing to at least one of the
strands of the different double stranded transcription factor
probes in the library and contacting the isolated transcription
factor probes with the array under conditions suitable for
hybridization of the strands of the different double stranded
transcription factor probes to the hybridization probes in the
array; and comparing which activated transcription factors are
present in the test sample and the control sample.
[0049] Methods for screening drug candidates for modulating an
activated transcription factor's activity are also provided.
[0050] In one embodiment, the method comprises: forming a plurality
of test samples by contacting samples of cells with different
agents; and for each test sample, identifying which of a plurality
of different activated transcription factors are present by: taking
a library of double stranded transcription factor probes, the
transcription factor probes each comprising a recognition sequence
capable of binding to an activated transcription factor, the
recognition sequence varying within the library for binding to
different activated transcription factors, contacting the different
test sample with the library of double stranded DNA probes under
conditions where DNA probe--transcription factor complexes are
formed between the DNA probes and activated transcription factors
present in the test samples, isolating the transcription factor
probes from the transcription factor probe--transcription factor
complexes formed, and identifying which transcription factor probes
in the library formed complexes by taking an array of immobilized
hybridization probes capable of hybridizing to at least one of the
strands of the different double stranded transcription factor
probes in the library and contacting the isolated transcription
factor probes with the array under conditions suitable for
hybridization of the strands of the different double stranded
transcription factor probes to the hybridization probes in the
array; and comparing the activated transcription factors present in
the different test samples.
[0051] Methods for determining sequence binding requirements for an
activated transcription factor are also provided.
[0052] In one embodiment, the method comprises: contacting a sample
comprising an activated transcription factor with a library of
double stranded DNA probes under conditions where DNA
probe--transcription factor complexes are formed between the DNA
probes and the activated transcription factor; separating DNA
probe--transcription factor complexes from non-complexed DNA probes
in the library; isolating the DNA probes from the excised portion
of the agarose gel; and determining a consensus sequence for the
DNA probes isolated in order to assess the binding requirements for
the transcription factor.
[0053] In another embodiment, the method comprises contacting a
sample comprising an activated transcription factor with a library
of double stranded DNA probes under conditions where DNA
probe--transcription factor complexes are formed between the DNA
probes and the activated transcription factor;
[0054] separating DNA probe--transcription factor complexes from
non-complexed DNA probes in the library using an agarose gel
separation; excising a portion of the agarose gel comprising the
separated DNA probe--transcription factor complexes; isolating the
DNA probes from the excised portion of the agarose gel; and
determining a consensus sequence for the DNA probes isolated in
order to assess the binding requirements for the transcription
factor.
[0055] In yet another embodiment, the method comprises: contacting
a sample comprising an activated transcription factor with a
library of double stranded DNA probes under conditions where DNA
probe--transcription factor complexes are formed between the DNA
probes and the activated transcription factor;
[0056] separating DNA probe--transcription factor complexes from
non-complexed DNA probes in the library; isolating the DNA probes
from the excised portion of the agarose gel; and quantifying the
amount of each of the isolated DNA probes.
[0057] In yet another embodiment, the method comprises: contacting
a sample comprising an activated transcription factor with a
library of double stranded DNA probes under conditions where DNA
probe--transcription factor complexes are formed between the DNA
probes and the activated transcription factor; separating DNA
probe--transcription factor complexes from non-complexed DNA probes
in the library using an agarose gel separation; excising a portion
of the agarose gel comprising the separated DNA
probe--transcription factor complexes; isolating the DNA probes
from the excised portion of the agarose gel; and quantifying the
amount of each of the isolated DNA probes.
[0058] Methods are also provided for quantifying expression and
activation of multiple different activated transcription factors.
According to one embodiment, the method comprises: contacting a
biological sample with a library of double stranded DNA probes for
detecting active forms of multiple different transcription factors
under conditions where DNA probe--transcription factor complexes
are formed between the DNA probes and activated transcription
factors present in the biological sample; isolating DNA probes from
the DNA probe--transcription factor complexes; identifying which of
the multiple different transcription factors are present in an
activated form in the biological sample based on which DNA probes
are isolated; and quantifying expression of the multiple different
transcription factors from cDNA for the biological sample.
BRIEF DESCRIPTION OF THE DRAWINGS
[0059] FIG. 1 provides a flow diagram for a method for identifying
which of a plurality of transcription factors are activated in a
given sample of cells.
[0060] FIG. 2 illustrates an array of hybridization probes attached
to a solid support where different hybridization probes are
attached to discrete, different regions of the array. A
transcription factor expression signature is shown based on the
distribution of where transcription factor probes hybridize and
their intensity.
[0061] FIG. 3 illustrates an array of hybridization probes attached
to a solid support where probes from a first sample with a first
color dye and probes from a second sample with a second color dye
are both contacted with the array.
[0062] FIG. 4 illustrates a process whereby the minimum DNA
sequence binding requirements for a given transcription factor can
be rapidly determined.
[0063] FIG. 5 illustrates a variation of the method described in
regard to FIG. 4 where an optimal sequence for binding is
identified.
[0064] FIG. 6 provides the sequences for the probes used to form
the transcription factor probe library used in the experiments
described in Sections 12-19 herein.
[0065] FIG. 7 depicts the layout of the array of hybridization
probes employed in the experiments described in Sections 12-19
herein.
[0066] FIG. 8 is an image of the array described in regard to FIG.
7 when the transcription factor probe library described in FIG. 6
is contacted with the array.
[0067] FIG. 9A is an image of an array described in regard to FIG.
7 that is contacted with transcription factor hybridization probes
isolated from transcription factor probe--transcription factor
complexes formed when Brn3 transcription factor hybridization
probes are contacted with a nuclear extract of HeLa cells.
[0068] FIG. 9B is an image of an array described in regard to FIG.
7 that is contacted with transcription factor hybridization probes
isolated from transcription factor probe--transcription factor
complexes formed when c-Myb transcription factor hybridization
probes are contacted with a nuclear extract of HeLa cells.
[0069] FIG. 9C is an image of an array described in regard to FIG.
7 that is contacted with transcription factor hybridization probes
isolated from transcription factor probe--transcription factor
complexes formed when Smad3/4 transcription factor hybridization
probes are contacted with a nuclear extract of HeLa cells.
[0070] FIG. 9D is an image of an array described in regard to FIG.
7 that is contacted with transcription factor hybridization probes
isolated from transcription factor probe--transcription factor
complexes formed when Brn3, c-Myb, and Smad3/4 transcription factor
hybridization probes are contacted with a nuclear extract of HeLa
cells.
[0071] FIG. 10A is an image of an array described in regard to FIG.
7 that is contacted with transcription factor hybridization probes
isolated from transcription factor probe--transcription factor
complexes formed when the entire library of transcription factor
probes described in regard to FIG. 6 are contacted with a control
sample that does not contain any transcription factors.
[0072] FIG. 10B is an image of an array described in regard to FIG.
7 that is contacted with transcription factor hybridization probes
isolated from transcription factor probe--transcription factor
complexes formed when the entire library of transcription factor
probes described in regard to FIG. 6 are contacted with a nuclear
extract of HeLa cells.
[0073] FIG. 11A is an image of an array described in regard to FIG.
7 that is contacted with transcription factor hybridization probes
isolated from transcription factor probe--transcription factor
complexes formed when the entire library of transcription factor
probes described in regard to FIG. 6 are contacted with a nuclear
extract of HeLa cells.
[0074] FIG. 11B is an image of an array described in regard to FIG.
7 that is contacted with transcription factor hybridization probes
isolated from transcription factor probe--transcription factor
complexes formed when the entire library of transcription factor
probes described in regard to FIG. 6 are contacted with a nuclear
extract of PMA-treated HeLa cells.
[0075] FIG. 12A provides a table of the signal intensities of
regions of the array shown in FIG. 11A.
[0076] FIG. 12B provides a table of the signal intensities of
regions of the array shown in FIG. 11B.
[0077] FIG. 12C provides a table with the ratios between the
intensities of the regions of the arrays shown in FIGS. 12A and
12B.
[0078] FIG. 13 provides an image of a gel shift analysis of Est and
NF-E1 performed on HeLa and PMA-treated HeLa cells.
[0079] FIG. 14A is an image of an array described in regard to FIG.
7 that is contacted with transcription factor hybridization probes
isolated from transcription factor probe--transcription factor
complexes formed when the entire library of transcription factor
probes described in regard to FIG. 6 are contacted with a nuclear
extract of A431 cells.
[0080] FIG. 14B is an image of an array described in regard to FIG.
7 that is contacted with transcription factor hybridization probes
isolated from transcription factor probe--transcription factor
complexes formed when the entire library of transcription factor
probes described in regard to FIG. 6 are contacted with a nuclear
extract of PMA-treated A431 cells.
[0081] FIG. 15 provides an image of a gel shift analysis of Ets,
NF-E1, and NF-kB performed on A431 and PMA-treated A431 cells.
[0082] FIG. 16A is an image of an array described in regard to FIG.
7 that is contacted with transcription factor hybridization probes
isolated from transcription factor probe--transcription factor
complexes formed when the entire library of transcription factor
probes described in regard to FIG. 6 are contacted with a nuclear
extract of Jurkat cells.
[0083] FIG. 16B is an image of an array described in regard to FIG.
7 that is contacted with transcription factor hybridization probes
isolated from transcription factor probe--transcription factor
complexes formed when the entire library of transcription factor
probes described in regard to FIG. 6 are contacted with a nuclear
extract of PMA-treated Jurkat cells.
[0084] FIG. 17A is an image of an array described in regard to FIG.
7 that is contacted with transcription factor hybridization probes
isolated from transcription factor probe--transcription factor
complexes formed when the entire library of transcription factor
probes described in regard to FIG. 6 are contacted with a nuclear
extract of HeLa cells.
[0085] FIG. 17B is an image of an array described in regard to FIG.
7 that is contacted with transcription factor hybridization probes
isolated from transcription factor probe--transcription factor
complexes formed when the entire library of transcription factor
probes described in regard to FIG. 6 are contacted with a nuclear
extract of A431 cells.
[0086] FIG. 17C is an image of an array described in regard to FIG.
7 that is contacted with transcription factor hybridization probes
isolated from transcription factor probe--transcription factor
complexes formed when the entire library of transcription factor
probes described in regard to FIG. 6 are contacted with a nuclear
extract of Jurkat cells.
[0087] FIG. 17D is an image of an array described in regard to FIG.
7 that is contacted with transcription factor hybridization probes
isolated from transcription factor probe--transcription factor
complexes formed when the entire library of transcription factor
probes described in regard to FIG. 6 are contacted with a nuclear
extract of K-562 cells.
[0088] FIG. 17E is an image of an array described in regard to FIG.
7 that is contacted with transcription factor hybridization probes
isolated from transcription factor probe--transcription factor
complexes formed when the entire library of transcription factor
probes described in regard to FIG. 6 are contacted with a nuclear
extract of Y79 cells.
[0089] FIG. 18A is an image of a polyacrylamide gel from a gel
shift analysis for multiple different transcription factors.
[0090] FIG. 18B is an image of an agarose gel from a gel shift
analysis for multiple different transcription factors.
[0091] FIG. 19 illustrates an embodiment of a method provided
according to the present invention for detecting transcription
factor--transcription factor complexes.
DETAILED DESCRIPTION OF THE INVENTION
[0092] The present invention relates to rapid and efficient methods
for identifying multiple different activated transcription factors
in a biological sample at the same time. The methods of the present
invention also provide for the quantification of the multiple
different activated transcription factors. As will be described
herein in greater detail, there are a great many applications that
arise from being able to effectively monitor which genes are being
expressed at a given time. With the assistance of the methods of
the present invention, it is thus possible to rapidly and
effectively monitor the levels of expression of multiple different
genes at the same time.
[0093] The present invention also relates to various compositions,
kits, and devices for use in conjunction with the various methods
of the present invention.
[0094] FIG. 1 provides a general overview of how the present
invention detects multiple different activated transcription
factors and thus allows the expression of multiple different genes
to be simultaneously monitored.
[0095] As illustrated, a biological sample is contacted with a
library of probes. The biological sample is typically derived from
a sample of cells and is preferably a nuclear extract of the cell
sample. The sample contains an unknown mixture of activated
transcription factors. Which activated transcription factors are
present in the sample serves to indicate which genes are currently
being expressed.
[0096] Information about the DNA binding specificity of
transcription factors which one wishes to identify is used to
design a library of transcription factor probes. In this regard, it
is noted that the present invention relates to the detection,
monitoring and optionally quantification of known transcription
factors using a library of transcription factor probes whose
sequences are known. The probes in the library are preferably
double stranded. One of the strands is preferably biotin labeled at
the 5' end to facilitate detection.
[0097] Each transcription factor probe in the library comprises a
DNA sequence that is capable of binding to an activated
transcription factor, referred to herein as the probe's recognition
sequence. At least the recognition sequence varies within the
library of probes such that the probes are capable of binding to a
plurality of different activated transcription factors. Due to the
high level binding specificity of the transcription factors, each
transcription factor probe has binding specificity for a single
transcription factor or family of transcription factors.
[0098] Because the present invention is used to identify known
transcription factors, it is practical to use longer recognition
sequences in the probes in the library as compared to what would be
practical if a random library were used. As a result, at least 1%,
2%, 3%, 5%, 10%, 20%, 30%, 50% or more of the probes in the library
may have recognition sequences greater than 35, 40, 45 or more base
pairs in length. By using longer recognition sequences, the probes
have greater binding specificity. In addition, the probes have
greater binding efficiency to the transcription factors which
improves the yield of probe--transcription factor complexes
isolated. As a result, the method of the present invention provides
a high level of sensitivity for isolating probe--transcription
factor complexes, as described further herein, in combination with
a high signal to noise ratio.
[0099] As a result of contacting the sample with the library of
transcription factor probes, complexes are formed between activated
transcription factors present in the sample and transcription
factor probes in the library which have sequences that match the
sequence specificity of the DNA-binding domains of the activated
transcription factors. By isolating the transcription factor
probe--activated transcription factor complexes, those probes from
the library which bind to transcription factors in the sample are
isolated.
[0100] The isolated transcription factor probes are then
identified. Each probe is specific for a different transcription
factor. Since only those probes from the library which form a
complex with an activated transcription factor will be isolated,
identification of which probes are isolated serves to identify
which activated transcription factors are present. Since the
presence of an activated transcription factor evidences gene
expression, the above described method can be used to determine
which genes were being expressed at the time the sample was taken
based on which activated transcription factors are present.
[0101] The design, operation and applications for the present
invention will now be described in greater detail.
1. Libraries of Transcription Factor Probes
[0102] Libraries of transcription factor probes are provided that
may be used to detect activated transcription factors according to
the present invention. A given library comprises a plurality of
double stranded DNA probes where the DNA sequences of the probes
vary within the library. The DNA sequences employed in the probes
of the libraries preferably have a length between about 10 and 100
base pairs, preferably between about 10 and 75 base pairs, more
preferably between about 15 and 50. Probes of longer lengths may
also be used.
[0103] Each probe in the library comprises a recognition sequence
which is capable of forming a probe--transcription factor complex
with an activated transcription factor. Due to the high level of
DNA binding specificity of transcription factors, each
transcription factor will typically bind to a different DNA
sequence. In some instances, a related family of transcription
factors may bind to the same DNA sequence.
[0104] By designing the library of probes such that any given probe
in the library includes a DNA recognition sequence which a
particular activated transcription factor (or a related family of
activated transcription factors) will bind to, and does not also
include a DNA recognition sequence which other activated
transcription factors will bind to, a given probe may be used to
identify a single activated transcription factor (or a related
family of activated transcription factors).
[0105] It is noted that in certain situations, individual probes
may bind to more than one activated transcription factor and may
nonetheless be used in the library. For example, certain probes may
bind to a related family of activated transcription factors. Less
than 1:1 binding specificity between probes and activated
transcription factors can be readily resolved during analysis of
the isolated probes.
[0106] The DNA recognition sequences used in the probes in the
libraries preferably have a length between about 10 and 100 base
pairs, more preferably between about 10 and 75 base pairs, more
preferably between about 15 and 50 base pairs, more preferably
between about 20 and 40 base pairs, and most preferably a length
between about 25 and 35 base pairs.
[0107] The optimal length for the recognition sequence and the
overall probe may vary somewhat depending on the particular
transcription factor. Hence, one may wish to evaluate the optimal
length for the recognition sequence and the probe for a given
transcription factor using a traditional gel shift assay.
[0108] Because the present invention is used to identify known
transcription factors, it is practical to use longer recognition
sequences in the probes in the library as compared to what would be
practical if a random library were used. For example, at least 1%,
2%, 3%, 5%, 10%, 20%, 30%, 50% or more of the probes in the library
may have recognition sequences greater than 35, 40, 45 or more base
pairs in length. By using longer recognition sequences, the probes
have greater binding specificity and greater binding efficiency to
the transcription factors. As a result, the method of the present
invention provides a high level of sensitivity for isolating
probe--transcription factor complexes, as described further herein,
in combination with a high signal to noise ratio.
[0109] Selection of which DNA recognition sequences to use in a
library may be based on the different transcription factors that
one wishes to detect in a sample. This, in turn, may depend on the
type of organism, cell, or disease state one wishes to identify
and/or monitor the gene expression of. It may also depend on the
different functionality that one wishes to identify or monitor.
[0110] A significant feature of the present invention is the
ability to detect multiple different transcription factors at the
same time. This ability arises from the number of different DNA
recognition sequences used in a library, the number of different
DNA recognition sequences relating directly to the number of
different transcription factors that the library can be used to
detect. A given library of transcription factor probes preferably
has at least 2, 3, 5, 10, 20, 50, 100, 250, or more different DNA
recognition sequences. The upper limit on the number of different
DNA recognition sequences that may be incorporated into a library
is limited only by the number of known DNA recognition
sequences.
[0111] A given library of transcription factor probes may be used
to detect gene expression in a single type of cell or organism or
may be used to detect gene expression in multiple different types
of cells or organisms. When the library is designed to detect gene
expression in multiple different types of cells or organisms, the
library has DNA recognition sequences for multiple different types
of cells or organisms. For example, the library may include DNA
recognition sequences for 2, 3, 4, 5 or more different types of
cells or organisms. In one embodiment, the library includes DNA
recognition sequences for 10, 20, 30, 50, or more different types
of cells or organisms.
[0112] If the sample comprises cells that may be from one or more
different organisms, the DNA recognition sequences used in the
library may be for all or some of the different transcription
factors expressed by the one or more different organisms. For
example, if the library is to be used to classify an unknown type
of bacterium, the library may include DNA recognition sequences for
multiple different types of bacteria, thereby allowing the library
to be used to classify the bacterium.
[0113] If the sample comprises cells from a particular organism,
the DNA recognition sequences used in the library may be for all or
some of the different transcription factors expressed by organism.
If the library is to be used to classify an unknown type of cells
(i.e., determine whether a growth is malignant), the library may
include DNA recognition sequences for multiple different types of
cells including the different types of malignant, benign, and
normal cell types present in the organism.
[0114] If the sample comprises cells of a single cell type, the DNA
recognition sequences used in the library may be for all or some of
the different transcription factors expressed by that cell type.
For example, if one wishes to monitor the expression of only a
particular group of genes, such as the genes associated with a
particular pathway, the library may include DNA recognition
sequences for the transcription factors associated with that group
of genes.
[0115] As one can see, a myriad of different libraries of probes
can be assembled depending on the nature of the sample and the
nature of the analysis to be performed. It is noted that different
libraries may also be formed when a particularly large number of
transcription factors are to be detected or when different binding
conditions are needed for different groups of probes.
[0116] The probes in the library may optionally further comprise a
detectable marker which allows the probe to be detected once
isolated from the transcription factor probe--transcription factor
complex. Since a wide variety of detection techniques may be used
to identify the isolated probes, a similarly wide range of
detectable markers may be used in conjunction with those different
detection techniques.
[0117] The detectable marker may be any marker which can be used to
determine the presence or absence of the DNA probes. In a preferred
embodiment, the detectable marker is biotin and is preferably
attached to one of the 5' ends of probes. Biotin probes have been
found to provide a desirable high level of sensitivity.
[0118] The detectable marker may also be a dye which can be seen
under natural light or with the assistance of an excitation light
source to cause fluorescence. In one embodiment, the detectable
marker is a fluorescent dye. Examples of fluorescent dyes that may
be used include, but are not limited to fluorescein and its
derivatives, rhodamine and its derivatives, dansyl, umbeliferone,
acridimium, and chemiluminescent molecules such as luciferin and
2,3-dihydrophthalazinediones. The fluorescent dye may also be an
energy transfer fluorescent dye.
[0119] The detectable marker may also be a molecule which binds to
an analytically detectable counterpart. For example, the detectable
marker may be covalently attached to or incorporated into the
substrate, for example, as taught by Ward, European Patent
Application No. 63,879. In such instances, the substrate is
detected by adding the analytically detectable counterpart which
specifically binds to the substrate, thereby enabling detection of
the substrate. Examples of such detectable markers and their
analytically detectable counterparts include biotin and either
fluorescent or chemiluminescent avidin. Antibodies that bind to an
analytically detectable antigen may also be used as the detectable
marker. The detectable marker may also be a molecule which, when
subjected to chemical or enzymatic modification, becomes detectable
such as those disclosed in Leary, et al., Proc. Natl. Acad. Sci.
(U.S.A.), 80:4045-4049 (1983).
[0120] In certain instances, it may be desirable to employ multiple
different detectable markers. For example, if one wishes to
classify an unknown type of cell or organism, the library may
include DNA probes where different detectable markers are attached
to the probes for the different types of cells. Hence, probes for
transcription factors expressed by malignant cells may include a
first fluorescent dye whereas probes expressed by benign cells may
include a second fluorescent dye. In another example, probes for
transcription factors expressed by a first type of lung cancer may
include a first fluorescent dye whereas probes expressed by of a
second, different type of lung cancer may include a second
fluorescent dye. This allows one to rapidly visually identify the
type of cell based on which detectable markers are present.
[0121] When one wishes to compare the gene expression of different
groups of cells, multiple libraries may be prepared where each
library contains a different detectable marker. For example, a
first library labeled with a first detectable marker may be used
with a first sample of cells and a second library labeled with a
second detectable marker may be used with a second sample of cells.
This way, the isolated probes may be analyzed together. As
described in Section 6A, when an array format is used for detecting
the isolated probes, the use of different detectable markers is
particularly advantageous. For example, as shown in FIG. 3,
transcription factor probes isolated from complexes formed from a
first sample (e.g., a control sample) may have a green dye, and
transcription factor probes isolated from complexes formed from a
second sample (e.g., a test sample) may have a red dye. Regions in
the array which are green represent genes which only the cells in
the control sample are expressing, regions in the array which are
red represent genes which only cells in the test sample are
expressing, and regions in the array which are both green and red
represent genes which cells in both the control and test samples
are expressing.
2. Preparation of Sample
[0122] Nuclear extracts can be prepared from a sample of cells
using the method described by Dignam, J. D., Lebovitz, R. M., and
Roeder, R. G. (1983) Accurate transcription initiation by RNA
polymerase II in a soluble extract from isolated mammalian nuclei.
Nucleic Acids Research 11:1475-1489. Alternatively, a commercially
available kit, such as Sigma's Nu-CLEAR Extraction Kit (cat.
#N-TRACT) can be used.
3. Contacting Cell Sample with Library of Probes
[0123] Once a nuclear extract of a sample of cells is prepared, the
nuclear extract is contacted with a library of probes and incubated
at 15.degree. C. for 30 min.
4. Isolation of Probe--Transcription Factor Complexes
[0124] After contacting the sample with the library of
transcription factor probes, any probe--activated transcription
factor complexes formed are isolated. Any isolation method which
can effectively isolate the complexes may be used. Isolation is
preferably performed by a form of size separation, more preferably
by electrophoresis, more preferably gel electrophoresis and most
preferably gel electrophoresis using an agarose gel.
[0125] One of the problems overcome by the present invention is the
ability to isolate complexes of DNA probes bound to transcription
factors from other probes in the library. Several methods for
performing this type of isolation were attempted. However, these
methods failed to provide a sufficient yield of
probe--transcription factor complexes from the sample.
[0126] For example, Applicant attempted to isolate
probe--transcription factor complexes by performing an ammonia
precipitation. Applicant also attempted to isolate
probe--transcription factor complexes by passing the sample through
a nitrocellulose filter, the filter serving to immobilize proteins
while allowing DNA that is not bound to protein to pass through the
filter. Unfortunately, neither of these approaches provided a
satisfactory yield of complexes for further characterization of the
isolated probes.
[0127] Applicant also attempted to isolate probe--transcription
factor complexes by using acrylamide gel electrophoresis.
Unfortunately, this approach also did not provide a satisfactory
yield of complexes. Without being bound by theory, it is believed
that this method was hampered by the DNA probes being retained by
the acrylamide gel.
[0128] Applicant successfully isolated probe--transcription factor
complexes from the sample using agaraose gel electrophoresis.
Interestingly, despite the fact that agaraose gel electrophoresis
does not provide the same quality separation as other forms of gel
electrophoresis (e.g., acrylamide gel electrophoresis), the
resolution provided using agaraose is more than sufficient to
effectively separate the probe--transcription factor complexes.
Meanwhile, agarose proved to have satisfactorily low retention of
the DNA probes in the complex, thereby allowing the probes to be
further characterized.
[0129] The separated probe--transcription factor complexes were
isolated by removing the band from the gel containing the
probe--transcription factor complexes.
[0130] It is noted that simultaneous isolation of multiple
different probe--transcription factor complexes using a library of
DNA probes is a significant departure from traditional gel-shift
assays involving the use of a single probe to cause the gel shift
of a single transcription factor. [Ausebel, F. M. et al eds (1993)
Current Protocols in Molecular Biology Vol. 2 Greene Publishing
Associates, Inc. and John Wiley and Sons, Inc., New York]. In such
prior gel-shift assays, since only a single transcription factor
was being detected, no efforts were made to isolate the probe used
to cause the gel shift. By contrast, in the present invention,
since multiple probes are used in combination it is necessary to
isolate the complexes in order to isolate and characterize which of
the plurality of probes in the library formed a complex with a
transcription factor and are present in the band.
[0131] It is also noted that simultaneous isolation of multiple
different probe--transcription factor complexes by isolating and
characterizing the DNA probes is also a significant departure from
U.S. Pat. Nos. 6,066,452 and 5,861,246 and PCT Publication No. WO
00/04196 which describe isolation of the nucleic acid binding
factors, not the probes.
[0132] The following is a detailed description of how one may
isolate probe transcription factor complexes using an agarose gel.
It is noted that other separation and isolation methods may be
employed without departing from the present invention.
[0133] According to one embodiment, a 2% agarose gel in
0.5.times.TBE is prepared. Preferably, 8 mm-wide combs are used.
Each sample is mixed with 2 .mu.l of a gel loading buffer. Table 1
provides an embodiment of a gel loading buffer that may be
used.
TABLE-US-00001 TABLE 1 Gel shift loading buffer: 0.25 X TBE buffer:
60% Glycerol: 40% Bromophenol blue: 0.2% (w/v)
[0134] Add 2 ul of gel shift loading buffer and load into a 0.8 cm
width lane of 2% agarose gel in 0.5% TBE. The resulting agarose gel
is then run in 0.5% TBE at 120V for 16 min. The gel area containing
that contains the protein/DNA complex, which will be above the blue
dye and below the gel lane, is excised and transferred to a 1.5 mL
tube.
5. Separation of Transcription Factor Probes from Transcription
Factor--Probe Complexes
[0135] Once the transcription factor probe--transcription factor
complexes are separated from other proteins and DNA in the sample,
for example, as described in Section 4, the transcription factor
probes are separated from the excised portion of the gel.
[0136] The following is a detailed description of how one may
isolate probe--transcription factor complexes from an excised
portion of an agarose gel. It is noted that other isolation
techniques may be employed without departing from the present
invention.
[0137] It is noted that the following steps describing the
isolation of the DNA probes is specifically designed for CLONTECH's
NucleoTrap Kit. If another commercially available gel extraction
kit is employed, the steps should be modified in accordance with
that manufacturer's instructions.
[0138] 1.0 mL of NT1 solution from CLONTECH's NucleoTrap Kit is
added to the excised portion of the gel. The mixture is then
incubated at 50.degree. C. until the gel is totally dissolved. The
tube is preferably periodically gently inverted in order to mix the
contents until the gel is dissolved.
[0139] 6 .mu.l of beads from a commercially available gel
extraction kit is then preferably added and the resulting mixture
is incubated at room temperature for 10 min. The tube is preferably
periodically gently inverted every 2-3 minutes.
[0140] The tube is then microfuged at 10,000 rpm for 30 sec. The
resulting supernatant is carefully removed and the pellet
resuspended in 150 .mu.l of NT2 solution from CLONTECH's NucleoTrap
Kit.
[0141] The pellet is then microfuged at 10,000 rpm for 30 sec. The
resulting supernatant is then carefully removed and the pellet
resuspended in 150 .mu.l of NT3 solution from CLONTECH's NucleoTrap
Kit. The pellet is then microfuged at 10,000 rpm for 30 sec. The
supernatant is again removed and the pellet is allowed to air-dry
for 10 min.
[0142] 50 .mu.l of dH.sub.2O is then added to resuspend the pellet.
The resulting mixture is incubated at room temperature for 5 min.
The mixture is then gently shaken and incubated for another 5
min.
[0143] The resulting mixture is then microfuged at 10,000 rpm for 1
min. The supernatant is transferred to a fresh 1.5 ml tube. The
isolated supernatant contains the DNA probes that are bound to
proteins. The isolated supernatant is preferably stored on ice
until proceeding to a characterization of the DNA probes.
6. Identifying Isolated Transcription Factor Probes
[0144] A variety of different methods may be used to identify which
of the transcription factor probes from the library are present in
the isolated probe--transcription factor complexes. These methods
preferably also allow for the amount of transcription factor probes
isolated to also be quantified. By identifying which transcription
factor probes form complexes, one is able to determine which
transcription factors are present in an activated form in the
sample, the presence of an activated transcription factor
evidencing expression of the gene associated with the transcription
factor. By quantifying the amount of each transcription factor
probe that forms a complex, one is able to determine the amount of
each transcription factor present and hence the level of expression
of the gene associated with that transcription factor.
[0145] One method that may be used to identify which of the
transcription factor probes from the library are present in the
isolated probe--transcription factor complexes is mass
spectroscopy. According to this method, the length and composition
of each probe can be determined. Therefore, the analyzed results
show whether a specific probe is existing in the complexes and the
interactions between transcription factors and binding probes can
be determined.
[0146] Another method that may be used to identify which of the
transcription factor probes from the library are present in the
isolated probe--transcription factor complexes is based on size
separation. According to this method, one varies the length of the
probes in the transcription factor probe library so that it is
possible to resolve the different sized probes based on a
size-based separation. For example, electrophoresis may be
performed in order to separate the probes based on size. Such
size-based DNA separations are traditionally done with high level
specificity for DNA sequencing. By identifying which transcription
factor probes are present based on the size-based separation, one
can determine which activated transcription factors are present and
can also quantify the amount of each activated transcription
factor.
[0147] Yet another method for identifying which of the probes from
the transcription factor probe library formed complexes involves
hybridization of the transcription factor probes with a
hybridization probe comprising a complement to the transcription
factor probes recognition sequence. According to this method,
detection of a particular transcription factor probe is
accomplished by detecting the formation of a duplex between the
transcription factor probe and a hybridization probe comprising a
complement to the transcription factor probe's recognition
sequence.
[0148] A wide variety of assays have been developed for performing
hybridization assays and detecting the formation of duplexes that
may be used in the present invention. For example, hybridization
probes with a fluorescent dye and a quencher where the fluorescent
dye is quenched when the probe is not hybridized to a target and is
not quenched when hybridized to a target oligonucleotide may be
used. Such fluorescer-quencher probes are described in, for
example, U.S. Pat. No. 6,070,787 and S. Tyagi et al., "Molecular
Beacons: Probes that Fluoresce upon Hybridization", Dept. of
Molecular Genetics, Public Health Research Institute, New York,
N.Y., Aug. 25, 1995, each of which are incorporated herein by
reference. By attaching different fluorescent dyes to different
hybridization probes, it is possible to determine which
transcription factor probes from the library formed complexes based
on which fluorescent dyes are present (e.g, fluorescent dye and
quencher on hybridization probe or fluorescent dye on hybridization
probe and quencher on transcription factor probe). Applicant notes
that one may also attach different fluorescent dyes to different
transcription factor probes and use a change in fluorescence due to
hybridization to a hybridization probe to determine which
transcription factor probes are present (e.g, fluorescent dye and
quencher on transcription factor probe or fluorescent dye on
transcription factor probe and quencher on hybridization
probe).
[0149] A difficulty, however, arises when using multiple different
fluorescer to detect multiple different transcription factor
probes. Namely, there is a limited number of different fluorescers
that may be spectrally resolved. As a result, a limited number of
different transcription factors can be detected at the same time,
for example only as many as five to ten.
[0150] A. Hybridization Arrays for Detecting Isolated Transcription
Factor Probes
[0151] A preferred assay for detecting the formation of duplexes
between transcription factor probes and hybridization probes
comprising their complements involves the use of an array of
hybridization probes immobilized on a solid support. The
hybridization probes comprise sequences that are complementary to
at least a portion of the recognition sequences of the
transcription factor probes and thus are able to hybridize to the
different probes in a transcription factor probe library.
[0152] In order to improve enhance the sensitivity of the
hybridization array, the immobilized probes preferably provide at
least 2, 3, 4 or more copies of at least a portion of the
recognition sequence incorporated into the transcription factor
probes.
[0153] According to the present invention, the hybridization probes
immobilized on the array preferably are at least 25 nucleotides in
length, more preferably at least 30, 40 or 50 nucleotides in
length. The immobilized hybridization probes may be 50, 75, 100
nucleotides or longer in length. In one preferred embodiment the
immobilized probes are between 50 and 100 nucleotides in
length.
[0154] By immobilizing hybridization probes on a solid support
which comprise one or more copies of a complement to at least a
portion of the recognition sequences of the transcription factor
probes, the hybridization probes serve as immobilizing agents for
the transcription factor probes, each different hybridization probe
being designed to selectively immobilize a different transcription
factor probe.
[0155] FIG. 2 illustrates an array of hybridization probes attached
to a solid support where different hybridization probes are
attached to discrete, different regions of the array. Each
different region of the array comprises one or more copies of a
same hybridization probe which incorporates a sequence that is
complementary to a recognition sequence of a transcription factor
probe. As a result, the hybridization probes in a given region of
the array can selectively hybridize to and immobilize a different
transcription factor probe based on the transcription factor
probe's recognition sequence.
[0156] By detecting which regions the isolated transcription factor
probes hybridize to on the array, one can determine which activated
transcription factors are present in the sample and can also
quantify the amount of each activated transcription factor.
[0157] These arrays can be designed and used to study transcription
factor activation in a variety of biological processes, including
cell proliferation, differentiation, transformation, apoptosis,
drug treatment, and others described herein.
[0158] Numerous methods have been developed for attaching
hybridization probes to solid supports in order to perform
immobilized hybridization assays and detect target oligonucleotides
in a sample. Numerous methods and devices are also known in the art
for detecting the hybridization of a target oligonucleotide to a
hybridization probe immobilized in a region of the array. Examples
of such methods and device for forming arrays and detecting
hybridization include, but are not limited to those described in
U.S. Pat. Nos. 6,197,506, 6,045,996, 6,040,138, 5,424,186,
5,384,261, each of which are incorporated herein by reference.
[0159] Several modifications may be made to the hybridization
arrays known in the art in order to customize the hybridization
arrays for use in detecting activated transcription factors through
the characterization of isolated transcription factor probes which
form a complex with the activated transcription factors.
[0160] Since the hybridization probe arrays of the present
invention are designed to hybridize to the probes in the
transcription factor probe library by comprising a sequence that is
complementary to the transcription factor recognition sequence, the
composition of the hybridization probes in the array should
complement the recognition sequences of the probes in the
transcription factor probe library. As discussed in Section 1, a
variety of different libraries of transcription factor probes are
provided that may be used to detect activated transcription factors
according to the present invention.
[0161] Selection of the sequences used in the hybridization probes
may be based on the different transcription factors that one wishes
to detect in a sample. This, in turn, may depend on the type of
organism, cell, or disease state one wishes to identify and/or
monitor the gene expression of.
[0162] A significant feature of the present invention is the
ability to detect multiple different transcription factors at the
same time. This ability arises from the number of different DNA
recognition sequences used in a library, the number of different
DNA recognition sequences relating directly to the number of
different transcription factors that the library can be used to
detect. A given array of hybridization probes preferably has
complements for at least 2, 3, 5, 10, 20, 30, 50, 100, 250 or more
different DNA recognition sequences. The upper limit on the number
of different DNA recognition sequences that the array of
hybridization probes may detect is limited only by the number of
known DNA recognition sequences and hence the number of known
complements to the DNA recognition sequences.
[0163] A given array of hybridization probes may be used to detect
gene expression in a single type of cell or organism or may be used
to detect gene expression in multiple different types of cells or
organisms. When the array is designed for use with a library
designed to detect gene expression in multiple different types of
cells or organisms, the array may include complements to DNA
recognition sequences for multiple different types of cells or
organisms. For example, the array may include complements to DNA
recognition sequences for 2, 3, 4, 5 or more different types of
cells or organisms. In one embodiment, the array may include
complements to DNA recognition sequences for 10, 20, 30, 50, or
more different types of cells or organisms.
[0164] If the sample to be analyzed comprises cells that may be
from one or more different organisms, the DNA recognition sequences
used in the library may be for all or some of the different
transcription factors expressed by the one or more different
organisms. For example, if library is to be used to classify an
unknown type of bacterium, the library may include DNA recognition
sequences for multiple different types of bacteria, thereby
allowing the library to be used to classify the bacterium.
Accordingly, the array used in combination with the library would
include complements to DNA recognition sequences for multiple
different types of bacteria.
[0165] If the sample comprises cells from a particular organism,
the DNA recognition sequences used in the library may be for all or
some of the different transcription factors expressed by organism.
Accordingly, the array used in combination with the library would
include complements to DNA recognition sequences for the different
transcription factors expressed by organism.
[0166] If the library is to be used to classify an unknown type of
cells (i.e., determine whether a growth is malignant), the library
may include DNA recognition sequences for multiple different types
of cells including the different types of malignant, benign, and
normal cell types present in the organism. Accordingly, the array
used in combination with the library would include complements to
DNA recognition sequences for the multiple different types of
cells.
[0167] If the sample comprises cells of a single cell type, the DNA
recognition sequences used in the library may be for all or some of
the different transcription factors expressed by that cell type.
Accordingly, the array used in combination with the library would
include complements to the recognition sequences for all or some of
the different transcription factors expressed by that cell
type.
[0168] i. Procedure for Performing Hybridization Using Array
[0169] Provided below is a description of a procedure that may be
used to hybridize isolated transcription factor probes to a
hybridization array. It is noted that the below procedure may be
varied and modified without departing from other aspects of the
invention.
[0170] An array membrane having hybridization probes attached for
the transcription factor probes is first placed into a
hybridization bottle. The membrane is then wet by filling the
bottle with deionized H.sub.2O. After wetting the membrane, the
water is decanted. Membranes that may be used as array membranes
include any membrane to which a hybridization probe may be
attached. Specific examples of membranes that may be used as array
membranes include, but are not limited to NYTRAN membrane
(Schleicher & Schuell), BIODYNE membrane (Pall), and NYLON
membrane (Roche Molecular Biochemicals).
[0171] 5 ml of prewarmed hybridization buffer is then added to each
hybridization bottle containing an array membrane. The bottle is
then placed in a hybridization oven at 42.degree. C. for 2 hr. An
example of a hybridization buffer that may be used is EXPHYP by
Clonetech.
[0172] After incubating the hybridization bottle, a thermal cycler
may be used to denature the hybridization probes by heating the
probes at 90.degree. C. for 3 min, followed by immediately chilling
the hybridization probes on ice.
[0173] The isolated probe--transcription factor complexes are then
added to the hybridization bottle. Hybridization is preferably
performed at 42.degree. C. overnight.
[0174] After hybridization, the hybridization mixture is decanted
from the hybridization bottle. The membrane is then washed
repeatedly.
[0175] In one embodiment, washing includes using 60 ml of a
prewarmed first hybridization wash which preferably comprises
2.times.SSC/0.5% SDS. The membrane is incubated in the presence of
the first hybridization wash at 42.degree. C. for 20 min with
shaking. The first hybridization wash solution is then decanted and
the membrane washed a second time. A second hybridization wash,
preferably comprising 0.1.times.SSC/0.5% SDS is then used to wash
the membrane further. The membrane is incubated in the presence of
the second hybridization wash at 42.degree. C. for 20 min with
shaking. The second hybridization wash solution is then decanted
and the membrane washed a second time.
[0176] ii. Procedure for Detecting Array Hybridization
[0177] The following describes a procedure that may be used to
detect isolated transcription factor probes isolated on the
hybridization array. It is noted that each membrane should be
separately hybridized, washed and detected in separate containers
in order to prevent cross contamination between samples. It is also
noted that it is preferred that the membrane is not allowed to dry
during detection.
[0178] According to the procedure, the membrane is carefully
removed from the hybridization bottle and transferred to a new
container containing 30 ml of 1.times. blocking buffer. The
dimensions of each container is preferably about 4.5''.times.3.5'',
equivalent in size to a 200 .mu.L pipette-tip container. Table 2
provides an embodiment of a blocking buffer that may be used.
TABLE-US-00002 TABLE 2 1X Blocking Buffer: Blocking reagent: 1%
0.1M Maleic acid 0.15M NaCl Adjusted with NaOH to pH 7.5
[0179] It is noted that the array membrane may tend to curl
adjacent its edges. It is desirable to keep the array membrane
flush with the bottom of the container.
[0180] The array membrane is incubated at room temperature for 30
min with gentle shaking. 1 ml of blocking buffer is then
transferred from each membrane container to a fresh 1.5 ml tube. 3
.mu.l of Streptavidin-AP conjugate is then added to the 1.5 ml tube
and is mixed well. The contents of the 1.5 ml tube is then returned
to the container and the container is incubated at room temperature
for 30 min.
[0181] The membrane is then washed three times at room temperature
with 40 ml of 1.times. detection wash buffer, each 10 min. Table 3
provides an embodiment of a 1.times. detection wash buffer that may
be used.
TABLE-US-00003 TABLE 3 1 X Detection wash buffer: 10 mM Tris-HCl,
pH 8.0 150 mM NaCl 0.05% Tween-20
[0182] 30 ml of 1.times. detection equilibrate buffer is then added
to each membrane and the combination is incubated at room
temperature for 5 min. Table 4 provides an embodiment of a 1.times.
detection equilibrate buffer that may be used.
TABLE-US-00004 TABLE 4 1 X Detection equilibrate buffer: 0.1M
Tris-HCl pH 9.5 0.1M NaCl
[0183] The resulting membrane is then transferred onto a
transparency film. 3 ml of CPD-Star substrate, produced by Applera,
Applied Biosystems Division, is then pipetted onto the
membrane.
[0184] A second transparency film is then placed over the first
transparency. It is important to ensure that substrate is evenly
distributed over the membrane with no air bubbles. The sandwich of
transparency films are then incubated at room temperature for 5
min.
[0185] The CPD-Star substrate is then shaken off and the films are
wiped. The membrane is then exposed to Hyperfilm ECL, available
from Amersham-Pharmercia. Alternatively, a chemiluminescence
imaging system may be used such as the ones produced by ALPHA
INNOTECH. It may be desirable to try different exposures of varying
lengths of time (e.g., 2-10 min).
[0186] The hybridization array may be used to obtain a quantitative
analysis of the amount of transcription factor probe present. For
example, if a chemiluminescence imaging system is being used, the
instructions that come with that system's software should be
followed. If Hyperfilm ECL is used, it may be necessary to scan the
film to obtain numerical data for comparison.
[0187] iii. Normalization of Data from Array Hybridization
[0188] One of the advantages provided by array hybridization for
detecting isolated transcription factor probes is the ability to
simultaneously analyze whether multiple different activated
transcription factors are present.
[0189] A further advantage provided is that the system allows one
to compare a quantification of multiple different activated
transcription factors between two or more samples. When two or more
arrays from multiple samples are compared, it is desirable to
normalize them.
[0190] In order to facilitate normalization of the arrays, an
internal standard may be used so that the intensity of detectable
marker signals between arrays can be normalized. In certain
instances, the internal standard may also be used to control the
time used to develop the detectable marker.
[0191] In one embodiment, the internal standard for normalization
is biotinylated DNA which is spotted on a portion of the array,
preferably adjacent one or more sides of the array. For example,
biotin-labeled ubiquitin DNA may be positioned on the bottom line
and last column of the array. In order to normalize two or more
arrays for comparison of results, the exposure time for each array
should be adjusted so that the signal intensity in the region of
the biotinylated DNA is approximately equivalent on both
arrays.
7. Use of Multiple Libraries in Combination to Compare Gene
Expression Between Different Samples
[0192] When an array format is used for detecting the isolated
probes, it may be desirable to use multiple libraries labeled with
different detectable markers in order to facilitate comparison
between samples. For example, as shown in FIG. 3, probes from a
first sample (e.g., a control sample) may have a green dye, and
probes from a second sample (e.g., a test sample) may have a red
dye. Both probes are separated from their bound complexes, mixed,
and hybridized to a single array. Green spots in the array
represent genes which only the cells in the control sample are
expressing, and red spots in the array represent genes which only
cells in the test sample are expressing. When both dyes hybridize
to the same spot in an equal amount, the balanced mixture of green
and red appears as yellow in the array, representing genes which
cells in both the control and test samples are expressing.
[0193] One embodiment of this application of the present invention
thus relates to a method for comparing gene expression between a
test sample and a control sample, the method comprising forming
transcription factor probe--activated transcription factor
complexes using the test sample and a first library of
transcription factor probes having a first detectable marker;
forming transcription factor probe--activated transcription factor
complexes using the control sample and a second library of
transcription factor probes having the same nucleic acid sequences
as the first library but having a second, different detectable
marker; isolating the transcription factor probes from the first
library which formed complexes involving transcription factors from
the test sample; isolating the transcription factor probes from the
second library which formed complexes involving transcription
factors from the control sample; and detecting the isolated
transcription factor probes from the first and second libraries
using a same hybridization array.
[0194] Another embodiment of this application of the present
invention relates to a kit comprises the first and second library
of transcription factor probes. The kit may optionally further
include a hybridization array comprising complements to the
transcription factor probes. The kit may also include instructions
for isolating the transcription factor probe--activated
transcription factor complexes using agarose gel.
8. Applications for Monitoring Gene Expression Via Detection of
Activated Transcription Factors
[0195] By better understanding which cells express which genes and
how different conditions influence gene expression, fundamental
questions of biology can be answered. Thus, by being able to
rapidly and efficiently detect multiple activated transcription
factors at the same time, the present invention avails itself to
numerous valuable applications relating to the monitoring of gene
expression. Some of these applications are described herein. Other
applications will be apparent to those of ordinary skill.
[0196] a. Characterization of Cell Type
[0197] By detecting and optionally quantifying which activated
transcription factors are present in a cell sample, the methods of
the present invention allow one to identify which genes are being
expressed and to what extent each gene is being expressed.
Different types of cells for a particular organism will express
different genes. As a result, the present invention allows one to
rapidly characterize a cell type based on which activated
transcription factors are present and at what levels.
[0198] One embodiment of this application of the present invention
thus relates to a method for characterizing a cell type, the method
comprising forming transcription factor probe--activated
transcription factor complexes using a test sample and a library of
transcription factor probes comprising recognition sequences
characteristic of different types of cells; isolating the
transcription factor probes from the library which formed complexes
involving transcription factors from the test sample; and detecting
the isolated transcription factor probes using a hybridization
array comprising sequences complementary to the transcription
factor probes in the library.
[0199] A further embodiment of this application of the present
invention relates to a library of transcription factor probes and
hybridization array comprising compliments to the library of
transcription factor probes are provided where the transcription
factor probes comprise recognition sequences from multiple
different cell types. A kit is also provided that comprises both
the library of probes and the hybridization array. The kit may also
include instructions for isolating the transcription factor
probe--activated transcription factor complexes using an agarose
gel, either in combination with the library, the hybridization
array, or both.
[0200] It is noted that different organisms will also express
different activated transcription factors. Characterizing the
mixture of different activated transcription factors expressed by a
particular organism (e.g., a culture of bacteria) can be used to
identify the particular organism. This application of the method of
the present invention may be particularly useful for rapidly
characterizing microbes such as bacteria and diseased tissue.
[0201] One embodiment of this application of the present invention
thus relates to a method for characterizing an organism, the method
comprising forming transcription factor probe--activated
transcription factor complexes using a test sample and a library of
transcription factor probes comprising recognition sequences
characteristic of different organisms; isolating the transcription
factor probes from the library which formed complexes involving
transcription factors from the test sample; and detecting the
isolated transcription factor probes using a hybridization array
comprising sequences complementary to the transcription factor
probes in the library.
[0202] A further embodiment of this application of the present
invention relates to a library of transcription factor probes and
hybridization array comprising compliments to the library of
transcription factor probes are provided where the transcription
factor probes comprise recognition sequences from multiple
different organisms. A kit is also provided that comprises both the
library of probes and the hybridization array. The kit may also
include instructions for isolating the transcription factor
probe--activated transcription factor complexes using an agarose
gel, either in combination with the library, the hybridization
array, or both.
[0203] It is noted that the mixture of different activated
transcription factors expressed by different cell types or
organisms may be used according to the present invention as a form
of an expression signature for that cell type. FIG. 2 illustrates
an array detection format. The pattern formed by the detectable
markers [cubes] in FIG. 2 can be used as a visual fingerprint of
the expression signature and can be used to identify a particular
cell type or organism based on that visual fingerprint. In this
regard, it is envisioned that an array may be developed with a
great multiplicity of immobilizing agents for different
transcription factor probes. It is noted that the number of cubes
shown in the figure is employed to reflect signal intensity.
[0204] The array may include immobilizing agents for transcription
factor probes for different cell types and/or for different
organisms. By comparing the array pattern to a standard for a
particular cell type or organism, the cell type or organism can be
rapidly determined.
[0205] b. Determining the Functions of Different Genes
[0206] Despite the fact that each cell in the human body contains
the same set of genes, the human body is comprised of a wide
diversity of different cell types that work in concert to form the
human body. The wide diversity of cell types present in the human
body and other multicellular organisms is due to variations between
cells regarding which genes are expressed, the level at which the
genes are expressed, and the conditions under which the genes are
expressed. The present invention provides the unique ability of
rapidly determining which of a great number of genes are expressed
by numerous different cell types. By being able to determine which
genes are expressed by which cell types, the functions of different
genes can be deduced.
[0207] c. Diagnosis of Disease States
[0208] Certain disease states may be caused and/or characterizable
by certain genes being expressed or not expressed as compared to
normal cells. Other disease states may result from and/or be
characterizable by certain genes being transcribed at different
levels as compared to normal cells.
[0209] By being able to rapidly monitor the expression levels of
multiple different genes, the present invention provides an
accurate method for diagnosing certain disease states known to be
associated with the expression non-expression, reduced expression,
and/or elevated expression of one or more genes. Conversely, by
comparing the expression non-expression, reduced expression, and/or
elevated expression of one or more genes in normal and abnormal
cells, present invention facilitates the association of one or more
genes with certain disease states. By understanding that a
particular disease state is caused by a different expression
(higher or lower) of one or more proteins, it should be possible to
remedy the disease state by increasing or decreasing the expression
of the one or more proteins, by administering the one or more
proteins or, if particular proteins are overexpressed, by
inhibiting the one or more proteins.
[0210] One embodiment of this application of the present invention
thus relates to a method for diagnosing a disease state of a sample
of cells, the method comprising forming transcription factor
probe--activated transcription factor complexes using the sample of
cells and a library of transcription factor probes comprising
different transcription factor recognition sequences; isolating the
transcription factor probes from the library which form complexes
involving transcription factors from the sample; detecting the
isolated transcription factor probes using a hybridization array
comprising sequences complementary to the transcription factor
probes in the library; and diagnosing a presence of a disease state
based on which transcription factors are activated in the cell
sample as identified by which transcription factor probes are
isolated.
[0211] d. Compound Screening for Drug Candidates
[0212] Being able to monitor transcription factor activity for
multiple different transcription factors at the same time is of
great importance to developing a better understanding of different
roles that various transcription factors play. In addition,
monitoring multiple different transcription factors at the same
time allows one to rapidly screen for compounds that influence
transcription factor activity, referred to herein as a
"transcription factor modulator."
[0213] The present invention may thus be used as a high throughput
screening assay for transcription factor modulators that either up-
or down-regulate genes by influencing the synthesis and activation
of transcription factors for those genes.
[0214] By having a further understanding of what compounds modulate
transcription factor activity, such compounds may be more
effectively used for in vitro modification of signal transduction,
transcription, splicing, and the like, e.g., as tools for
recombinant methods, cell culture modulators, etc. More
importantly, such compounds can be used as lead compounds for drug
development for a variety of conditions, including as
antibacterial, antifungal, antiviral, antineoplastic, inflammation
modulatory, or immune system modulatory agents. Accordingly, being
able to monitor transcription factor activity for multiple
different factors has great use for screening compounds to identify
lead compounds for pharmaceutical or other applications.
[0215] Indeed, because gene expression is fundamental in all
biological processes, including cell division, growth, replication,
differentiation, repair, infection of cells, etc., the ability to
monitor transcription factor activity and identify compounds which
modulator their activity can be used to identify drug leads for a
variety of conditions, including neoplasia, inflammation, allergic
hypersensitivity, metabolic disease, genetic disease, viral
infection, bacterial infection, fungal infection, or the like. In
addition, compounds which specifically target transcription factors
in undesired organisms such as viruses, fungi, agricultural pests,
or the like, can serve as fungicides, bactericides, herbicides,
insecticides, and the like. Thus, the range of conditions that are
related to transcription factor activity includes conditions in
humans and other animals, and in plants, e.g., for agricultural
applications.
[0216] As used herein, the term "transcription factor modulator"
refers to any molecule or complex of more than one molecule that
affects the regulatory region. The present invention contemplates
screens for synthetic small molecule agents, chemical compounds,
chemical complexes, and salts thereof as well as screens for
natural products, such as plant extracts or materials obtained from
fermentation broths. Other molecules that can be identified using,
the screens of the invention include proteins and peptide
fragments, peptides, nucleic acids and oligonucleotides
(particularly triple-helix-forming oligonucleotides),
carbohydrates, phospholipids and other lipid derivatives, steroids
and steroid derivatives, prostaglandins and related arachadonic
acid derivatives, etc.
[0217] Existing methods for monitoring gene expression typically
monitor down-stream expression processes by measuring mRNA or the
resulting gene product. However, why a particular mRNA or protein
is expressed at higher or lower levels is not revealed by these
methods. This is because a given compound can influence the
formation of a transcription factor, influence the activation of
the transcription factor, interact with the activated transcription
factor, interact with the regulatory element to which the
transcription factor binds, or interact with the mRNA that is
produced.
[0218] By contrast because the present invention is specific to
detecting activated transcription factors, the present invention
can be effectively used to screen for drugs that have a mechanism
of action directly related to the expression and/or activation of
transcription factors.
[0219] It should be noted that methods exist for measuring a
transcription factor in a sample. However, because such methods
detect the protein itself, they are unable to determine whether the
transcription factor is activated, i.e., it is capable of binding
to a regulatory element. By being able to detect whether multiple
different transcription factors are activated, the present
invention, when used in combination with an assay for detecting the
amount of activated and unactivated transcription factor, allows
one to evaluate specifically how a given compound influences the
activation of different transcription factors.
[0220] The present invention may be used to screen large chemical
libraries for modulator activity for multiple different
transcription factors. For example, by exposing cells to different
members of the chemical libraries, and performing the methods of
the present invention, one is able to screen the different members
of the library relative to multiple different transcription factors
at the same time.
[0221] It will be appreciated that there are many suppliers of
chemical compounds, including Sigma (St. Louis, Mo.), Aldrich (St.
Louis, Mo.), Sigma-Aldrich (St. Louis, Mo.), Fluka
Chemika-Biochemica Analytika (Buchs Switzerland) and the like.
[0222] In one preferred embodiment, high throughput screening
involves testing a combinatorial library containing a large number
of potential modulator compounds. A combinatorial chemical library
may be a collection of diverse chemical compounds generated by
either chemical synthesis or biological synthesis, by combining a
number of chemical "building blocks" such as reagents. For example,
a linear combinatorial chemical library such as a polypeptide
library is formed by combining a set of chemical building blocks
(amino acids) in every possible way for a given compound length
(i.e., the number of amino acids in a polypeptide compound).
Millions of chemical compounds can be synthesized through such
combinatorial mixing of chemical building blocks.
[0223] Such combinatorial libraries are then screened to identify
those library members (particular chemical species or subclasses)
that modulate one or more transcription factors. The compounds thus
identified can serve as conventional "lead compounds" or can
themselves be used as potential or actual therapeutics for the one
or more transcription factors whose activities the compounds
modulate.
[0224] Preparation and screening of combinatorial libraries is well
known to those of skill in the art. Such combinatorial libraries
include, but are not limited to, peptide libraries (e.g., U.S. Pat.
No. 5,010,175, Furka, Int. J. Pept. Prot. Res. 37:487-493 (1991)
and Houghton et al., Nature 354:84-88 (1991)). Other chemistries
for generating chemical diversity libraries can also be used. Such
chemistries include, but are not limited to: peptoids (PCT
Publication No. WO 91/19735), encoded peptides (PCT Publication WO
93/20242), random bio-oligomers (PCT Publication No. WO 92/00091),
benzodiazepines (U.S. Pat. No. 5,288,514), diversomers such as
hydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc.
Nat. Acad. Sci. USA 90:6909-6913 (1993)), vinylogous polypeptides
(Hagihara et al., J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidal
peptidomimetics with .beta.-D-glucose scaffolding (Hirschmann et
al., J. Amer. Chem. Soc. 114:9217-9218 (1992)), analogous organic
syntheses of small compound libraries (Chen et al., J. Amer. Chem.
Soc. 116:2661 (1994)), oligocarbamates (Cho et al., Science
261:1303 (1993)), and/or peptidyl phosphonates (Campbell et al., J.
Org. Chem. 59:658 (1994)), nucleic acid libraries (see, Ausubel,
Berger and Sambrook, all supra), peptide nucleic acid libraries
(see, e.g., U.S. Pat. No. 5,539,083), antibody libraries (see,
e.g., Vaughn et al., Nature Biotechnology, 14(3):309-314 (1996) and
PCT/US96/10287), carbohydrate libraries (see, e.g., Liang et al.,
Science, 274:1520-1522 (1996) and U.S. Pat. No. 5,593,853), small
organic molecule libraries (see, e.g., benzodiazepines, Baum
C&EN, January 18, page 33 (1993); isoprenoids, U.S. Pat. No.
5,569,588; thiazolidinones and metathiazanones, U.S. Pat. No.
5,549,974; pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134;
morpholino compounds, U.S. Pat. No. 5,506,337; benzodiazepines,
5,288,514, and the like).
[0225] Devices for the preparation of combinatorial libraries are
also commercially available (see, e.g., 357 MPS, 390 MPS, Advanced
Chem Tech, Louisville Ky., Symphony, Rainin, Woburn, Mass., 433A
Applied Biosystems, Foster City, Calif., 9050 Plus, Millipore,
Bedford, Mass.). In addition, numerous combinatorial libraries are
themselves commercially available (see, e.g., ComGenex, Princeton,
N.J., Asinex, Moscow, Ru, Tripos, Inc., St. Louis, Mo., ChemStar,
Ltd, Moscow, RU, 3D Pharmaceuticals, Exton, Pa., Martek
Biosciences, Columbia, Md., etc.).
[0226] Control reactions may be performed in combination with the
libraries. Such optional control reactions are appropriate and
increase the reliability of the screening. Accordingly, in a
preferred embodiment, the methods of the invention include such a
control reaction. The control reaction may be a negative control
reaction that measures the transcription factor activity
independent of a transcription modulator. The control reaction may
also be a positive control reaction that measures transcription
factor activity in view of a known transcription modulator.
[0227] By being able to screen multiple different transcription
factors at the same time, not only is it possible to screen a large
number of potential transcription modulators per day, it is also
possible to screen any potential transcription modulator relative
to a large number of different transcription factors. The ability
to screen multiple different transcription factors at the same time
thus greatly enhances the high throughput capabilities of this
screening assay.
[0228] d. Evaluation of Drug Efficacy
[0229] Given that certain disease states may be caused by an
unusual level of transcription of one or more genes, drugs may be
designed to either stimulate or inhibit transcription in order make
gene expression of diseased cells approach the gene expression of
normal cells. A rapid and effective method for monitoring gene
expression is thus highly advantageous for evaluating the
effectiveness of a drug's ability to alter the transcription of one
or more genes. The effectiveness of a drug being delivered to a
site of action as well as the drug's efficacy in vivo can thus be
evaluated with the assistance of the methods of the present
invention.
[0230] Also of great concern when developing new drugs is the side
effects which the drugs might have. One approach for screening drug
candidates for undesirable side effects would be to employ the
present invention to monitor how gene expression is altered in
response to the administration of a drug candidate. By
understanding how a candidate affects gene expression, candidates
likely to have undesirable side affects can be rapidly
identified.
[0231] Because the biological importance of transcription factors,
they are ideal drug targets. Traditional transcription factor
screening assays only detect one transcription factor at a time. As
a result, existing assays are tremendously in efficient for
detecting how a drug effects different gene expression. However,
with the assistance of the present invention, it is now possible to
screen hundreds and even thousands of transcription factors in a
short amount of time in order to monitor how a given drug affects
the expression of wide range of genes. The present invention will
thus dramatically facilitate the screening process of identifying
new drugs, characterizing their mechanism of the action, and
screening for adverse side effects based on the drug's impact on
expression.
9. Determining Sequence Binding Requirements for Transcription
Factors
[0232] In general, a further application of the present invention
is the rapid and efficient determination of the DNA sequence
binding requirements for a given transcription factor. By being
able to efficiently isolate DNA probes from multiple DNA
probe--transcription factor complexes, the present invention makes
it feasible to identify which DNA sequences bind to transcription
factors, quantify the amount of each DNA sequence isolated, and use
that information to determine different DNA sequence binding
requirements for a given transcription factor in a high throughput
manner.
[0233] As a result of the completion of the human genome project,
many new proteins will be discovered. Based on their primary
structure, transcription factors for these proteins can be
identified. However, the identification of DNA sequences to which
these transcription factors will bind is a more difficult problem
which the present invention helps to address.
[0234] When determining the DNA sequences to which transcription
factors bind, there are several different questions that need to be
answered. One question relates to the identification of an optimal
binding sequence for the transcription factor. Another question
relates to the identification of the minimal sequence required for
binding. Yet another question which is related to the prior
question, relates to the identification of a consensus sequence
which is the minimum sequence required for binding. The present
invention can be used to facilitate the answering of each of these
questions.
[0235] The present invention enables one to rapidly determine a set
of DNA sequences to which an activated transcription factor will
bind and a set of DNA sequences to which an activated transcription
factor will not bind. The amount isolated of each member in the set
of DNA sequences to which an activated transcription factor binds
may also be quantified.
[0236] Using this information, the minimum sequence required for
the transcription factor to bind may be determined. Varying degrees
of consensus sequences among the DNA sequences to which an
activated transcription factor binds can also be determined.
Meanwhile, by quantifying the amount of isolated DNA, how different
base substitutions affect transcription factor binding can also be
evaluated. This enables one to determine the sequences to which a
given transcription factor binds most tightly.
[0237] FIG. 4 illustrates a process whereby the minimum DNA
sequence binding requirements for a given transcription factor can
be rapidly determined. As illustrated, a sample containing an
activated transcription factor is contacted with a library of
probes. The probes in the library comprise a DNA sequence and a
detectable marker.
[0238] Since, in this embodiment, the library of probes are used to
determine the minimum DNA sequence binding requirements, the DNA
sequences used in the library may be variations on a sequence which
the transcription factor is known to bind to. Alternatively, the
sequences used in the library may selected without knowledge of the
binding specificity of the activated transcription factor.
[0239] If the DNA probes used to perform this method are known, a
simple hybridization array having compliments to the DNA probes in
the library may be employed, as described above. However, if a
random library of DNA probes is employed, any isolated DNA probes
can be characterized by existing position-fixed DNA array
technology.
[0240] As a result of contacting the sample with the library of
probes, complexes are formed between the activated transcription
factor present in the sample and DNA probes in the library which
have sequences that satisfy the sequence specificity of the
DNA-binding domain of the activated transcription factor. The
resulting probe--transcription factor complexes are preferably
isolated by purification from agarose gel as described previously.
After isolating the DNA probe--activated transcription factor
complexes, those probes from the library which bind to
transcription factors in the sample may be further isolated and
characterized as discussed previously.
[0241] Since only those probes from the library which form a
complex with an activated transcription factor will be isolated,
identification of which probes are isolated serves to identify the
range of sequences to which the activated transcription factor is
capable of binding.
[0242] By constructing a consensus sequence based on the isolated
probes, one is able to more precisely define the minimum binding
requirements for the transcription factor. Furthermore, once a
consensus sequence and a series of binding sequences are known,
this information can be used to locate the occurrence of those
sequences in 5' untranslation regions within a genome. This will
allow researchers to identify which proteins may be regulated by
the transcription factor. Genomes of different organisms may also
be researched to identify proteins that may be functionally
related.
[0243] Depending on the level of diversity of the library used, a
further round of screening may be used to map the binding
requirements of the transcription factor in greater detail. For
example, when one or more binding sequences are identified, further
experimentation may also be conducted to identify more binding
sequences. This may involve creating a library of random mutations
based on one or more DNA sequences shown to bind to the
transcription factor in the prior screen. Binding of the mutants
may be performed in order to identify other mutants to which the
transcription factor binds. Multiple cycles of generating and
screening mutant libraries may be conducted as is necessary and
desirable.
[0244] FIG. 5 illustrates a variation of the method described in
regard to FIG. 4 where an optimal sequence for binding is
identified. As illustrated in FIG. 5, the isolated DNA probes are
quantified as well as identified. By monitoring how changes in the
sequence affect the amount of each probe isolated, one is able to
see how the different sequences compete for binding to the
transcription factor. As a result, an optimal transcription factor
binding sequence can be identified.
[0245] Depending on the level of diversity of the library used to
perform the first screen, when one or more binding sequences are
identified, further experimentation may also be conducted to
identify a better binding sequence. This may involve creating a
library of random mutations based on one or more DNA sequences
shown to bind to the transcription factor in the prior screen.
Quanitification of the binding of the mutants may be performed in
order to identify a stronger binding mutant. Multiple cycles of
generating and screening mutant libraries may be conducted as is
necessary and desirable.
10. Determining Transcription Factor Expression and Activation
[0246] Prior to being activated, a transcription factor must be
expressed. However, not all expressed transcription factors are
activated. By determining whether multiple different transcription
factors are being activated according to the present invention, in
combination with determining whether the different transcription
factors are being expressed, the present invention provides a rapid
and efficient method for monitoring how different transcription
factor expression and transcription factor activation change.
[0247] One application of this method relates to the diagnosis of
disease states. By being able to track both changes in
transcription factor expression and activation, more precise
diagnosis of the cause of genetically related diseases may be
discovered.
[0248] A further application of this method relates to the
evaluation of how different agents or conditions affect both
transcription factor expression and activation. For example,
different agents can be screened for their ability to inhibit
and/or activate the expression of certain proteins or impact
different disease states. By knowing how the agent affects both
transcription factor expression and activation, one is able to
identify the mode of action of the agent. By being able to screen
multiple transcription factors at the same time, one is further
able to screen those agents for otherwise unforeseen adverse
affects on the genetic level.
11. Method for Detecting Interactions between Transcription Factors
and Other Proteins
[0249] Yet a further application of the present invention is the
rapid and efficient determination of interactions that occur
between a given transcription factor and other proteins. In one
particular embodiment, the other proteins are other transcription
factors. It is noted that the present invention may be more broadly
applied to detecting the interaction between a given protein and
other proteins.
[0250] Transcription factors generally have at least two domains: a
DNA binding domain which binds to promoter and enhancer modules;
and an activation domain that allows for interaction between
transcription factors and/or with RNA polymerase II. The role
played by interactions between transcription factors is not yet
well understood but is believed to be significant. The role played
by interactions between transcription factors and other proteins is
also not well understood.
[0251] One of the challenges associated with elucidating the roles
that interactions between transcription factors and other proteins
including other transcription factors play is the identification of
the different complexes that transcription factors form. For
example, a mammal is believed to express over 1000 different
transcription factors. These different transcription factors are
expressed by different cells and under different conditions.
Determining which transcription factors form complexes with other
proteins, including other transcription factors under different
conditions can be very difficult. A need thus exists for a high
throughput method for detecting transcription factor--transcription
factor and other transcription factor--protein complexes.
[0252] FIG. 19 illustrates an embodiment of a method provided
according to the present invention for detecting transcription
factor--transcription factor complexes.
[0253] A biological sample to be analyzed is typically derived from
a sample of cells and is preferably a nuclear extract of the cell
sample. The sample contains an unknown mixture of transcription
factors.
[0254] Optionally, the biological sample may be contacted with a
library of probes. These probes may be used later on as detectable
markers or to identify the presence of a given transciption factor.
Each transcription factor probe in the library comprises a DNA
sequence that is capable of binding to an activated transcription
factor, referred to herein as the probe's recognition sequence. At
least the recognition sequence varies within the library of probes
such that the probes are capable of binding to a plurality of
different activated transcription factors. Due to the high level
binding specificity of the transcription factors, each
transcription factor probe has binding specificity for a single
transcription factor or family of transcription factors. The probes
in the library are preferably double stranded. One of the strands
is preferably biotin labeled at the 5' end to facilitate
detection.
[0255] Because the method may be directed toward identifying known
transcription factors, it is practical to use longer recognition
sequences in the probes in the library as compared to what would be
practical if a random library were used to identify previously
unknown transcription factors. As a result, at least 1%, 2%, 3%,
5%, 10%, 20%, 30%, 50% or more of the probes in the library may
have recognition sequences greater than 35, 40, 45 or more base
pairs in length. By using longer recognition sequences, the probes
have greater binding specificity. In addition, the probes have
greater binding efficiency to the transcription factors which
improves the yield of probe--transcription factor complexes
isolated. As a result, the method of the present invention provides
a high level of sensitivity for isolating transcription
factor--transcription factor complexes in combination with a high
signal to noise ratio.
[0256] As a result of contacting the sample with the library of
transcription factor probes, complexes are formed between activated
transcription factors present in the sample and transcription
factor probes in the library which have sequences that match the
sequence specificity of the DNA-binding domains of the activated
transcription factors.
[0257] Some of the transcription factors in the sample may form
complexes between one or more different transcription factors. In
order to detect and determine the composition of these
transcription factor--transcription factor complexes, complexes
which comprise a single type of transcription factor may be
isolated. Isolation of a single type of transcription factor may be
performed by contacting the transcription factor complexes with an
antibody or other agent that can specifically bind to a particular
type of transcription factor. The agent that specifically binds to
the particular type is then used to separate transcription factor
complexes comprising the particular type of transcription factor.
Alternatively, the agent that specifically binds to the particular
type of transcription factor may be immobilized on a solid support
which thus serves to immobilize transcription factor complexes
comprising the particular type of transcription factor.
[0258] Since the agent used to isolate the transcription factor
complexes is specific for a single type of transcription factor, no
other transcription factors should be isolated unless the
transcription factor is part of a complex comprising the particular
type of transcription factor upon which the isolation is based.
[0259] Identification of the different types of transcription
factors present in the pool of isolated complexes may be determined
either by identifying which transcription factors are present, or
by isolating the transcription factor probes bound to the
transcription factors and characterizing them. Both alternatives
are shown in FIG. 19.
[0260] On the left side of FIG. 19, the transcription factor probes
contained among the pool of isolated complexes are characterized.
The transcription factor probes may be characterized by any
methodology available for characterizing DNA probes. In one
preferred embodiment, the isolated transcription factor probes are
characterized using an array of hybridization probes such as the
array that has already been described. In another preferred
embodiment, the isolated transcription factor probes are
characterized using an array of proteins that selectively bind to
the different isolated transcription factor probes. These proteins
may be antibodies against recognition sequences of the probes or
proteins which recognize the recognition sequences (e.g.,
transcription factors) for the isolated transcription factor
probes.
[0261] Detection of transcription factor probes by an array is
advantageous because it facilitates the detection of multiple
different transcription factor probes in a single assay. For
example, 5, 10, 20, 50 or more different transcription factor
probes may be detected in the same assay.
[0262] Since only those probes from the library which form a
complex with an activated transcription factor will be isolated,
identification of which probes are isolated serves to identify
which activated transcription factors are present. Given that the
isolation process is designed to only isolate a single type of
transcription factor, the presence of probes for other types of
transcription factors indicates that these other types of
transcription factors formed complexes with the type of
transcription factor upon which the isolation was based.
[0263] On the right side of FIG. 19, an alternative process for
characterizing the transcription factors contained within the pool
of isolated complexes is illustrated. As shown, transcription
factors are characterized. The transcription factors may be
characterized by any methodology available for characterizing
proteins. In one preferred embodiment, the isolated transcription
factors are characterized using an array of hybridization probes
which comprise recognition sequences for the different
transcription factors that might be isolated. In another preferred
embodiment, the isolated transcription factors are characterized
using an array of agents, such as antibodies, which selectively
bind to different transcription factors that might be isolated.
[0264] Detection of transcription factors by an array is
advantageous because it facilitates the detection of multiple
different transcription factors in a single assay. For example, 5,
10, 20, 50 or more different transcription factors may be detected
in the same assay.
[0265] By performing the method illustrated in FIG. 19, it is
possible to isolate and then optionally characterize all
transcription factors that form a complex with a given type of
transcription factor. As noted previously, a mammalian cell can
express more than 1000 different types of transcription factors.
Accordingly, the above described process allows one to simultaneous
screen for transcription factor--transcription factor complexes
between a given transcription factor and the different types of
transcription factors that may be present in the cell. The above
described process thus greatly reduces the amount of work that
otherwise would need to be performed in order to monitor all these
potential transcription factor--transcription factor complexes.
[0266] It is noted that the above method is described with regard
to isolating transcription factor complexes based on a single type
of transcription factor at a time. In order to determine
transcription factor--transcription factor complexes involving a
different type of transcription factor, the method may be repeated
where an agent is employed for isolating a second different type of
transcription factor. This process may be repeated for however many
transcription factors as one might wish.
[0267] It is noted that isolating transcription factor complexes
may also be based on isolating more than one type of transcription
factor at a time. However, if isolation is performed based on two
or more transcription factors at a time, there will be inherent
ambiguity regarding which types of transcription factors are
involved in a given complex.
[0268] In another embodiment, a method is provided according to the
present invention for detecting transcription factor--protein
complexes. This method may be used for detecting complexes between
different transcription factors and/or for detecting complexes
between a non-transcription factor protein and a transcription
factor. It may be used more generally to detect complexes between
two or more non-transcription factor proteins.
[0269] As noted above, the biological sample to be analyzed is
typically derived from a sample of cells and is preferably a
nuclear extract of the cell sample. Some of the transcription
factors in the sample may form complexes with other proteins which
may or may not be transcription factors. In order to detect and
determine the composition of these transcription factor--protein
complexes, complexes which comprise a single type of protein may be
isolated.
[0270] Isolation of a single type of protein (whether or not a
transcription factor) may be performed by contacting the protein
with an antibody or other agent that can specifically bind to the
particular type of protein. The agent that specifically binds to
the particular type of protein is then used to separate complexes
comprising the particular type of protein. Alternatively, the agent
that specifically binds to the particular type of protein may be
immobilized on a solid support which thus serves to immobilize
complexes comprising the particular type of protein.
[0271] Since the agent used to isolate the protein complexes is
specific for a single type of protein (e.g., a particular type of
transcription factor or a particular type of protein other than a
transcription factor), no other proteins should be isolated unless
the protein is part of a complex comprising the particular protein
upon which the isolation is based.
[0272] Identification of proteins present in the pool of isolated
complexes may be performed by any method available for detecting
proteins. For example, proteins contained within the pool of
isolated complexes may be characterized using an array of agents,
such as antibodies, which selectively bind to different proteins
that might have been isolated. The agents in the array may be
directed toward transcription factors, non-transcription factor
proteins, and combinations thereof.
[0273] Detection of proteins by an array is advantageous because it
facilitates the detection of multiple different proteins in a
single assay. For example, 5, 10, 20, 50 or more different proteins
may be detected in the same assay.
[0274] According to this embodiment, it is possible to isolate and
then optionally characterize all transcription factors that form a
complex with a given type of protein upon which an isolation is
based. As noted previously, a mammalian cell can express more than
1000 different types of transcription factors. Accordingly, the
above described process allows one to simultaneous screen for
transcription factor--protein complexes between a given protein and
the different types of transcription factors. Alternatively, one
may simultaneously screen for transcription factor--protein
complexes between a given transcription factor and multiple
different types of proteins that may also be present in a cell. The
parallel screening allowed by the present invention greatly reduces
the amount of work that otherwise would need to be performed in
order to monitor all these potential transcription factor--protein
complexes.
12. Kits for Use in the Present Invention
[0275] A wide variety of kits may be designed for use with the
present invention. Various examples of these kits have already been
described and additional kits are further described herein.
[0276] In one particular embodiment, a kit is provided which
includes a library of transcription factor probes comprising
recognition sequences for transcription factors. The kit further
includes an array of hybridization probes where each probe
comprises a sequence that is complementary to at least a portion of
the recognition sequences on the probes in the library. In a
preferred embodiment, the probes in the array of hybridization
probes comprise 2, 3, 4, or more copies of a sequence that is
complementary to at least a portion of the recognition sequences on
the probes in the library.
[0277] In another particular embodiment, a kit is provided which
includes a first library of transcription factor probes comprising
recognition sequences for transcription factors, each probe in the
first library further comprising a first detectable marker. The kit
further includes a second library of transcription factor probes
comprising the same recognition sequences for transcription factors
as the first library, each probe in the second library further
comprising a second detectable marker that is different than the
first detectable marker.
[0278] In another particular embodiment, a kit is provided which
includes a library of transcription factor probes comprising
recognition sequences for transcription factors in combination with
instructions for using agarose gel to isolate transcription factor
probe--transcription factor complexes.
[0279] In yet another embodiment, a kit is provided which includes
an array of hybridization probes where each probe comprises a
sequence that is complementary to at least a portion of recognition
sequences of transcription factors. The kit further includes
instructions for using agarose gel to isolate transcription factor
probe--transcription factor complexes.
[0280] In yet another embodiment, a kit is provided which includes
a hybridization array according to the present invention,
hybridization buffer, detection wash buffer, and detection
equilibrate buffer.
[0281] With regard to any of the kit embodiments, it is noted that
the libraries of transcription factor probes and hybridization
arrays may be varied as has been described herein.
13. Array Detection of Different Activated Transcription
Factors
[0282] This section describes experimental results achieved by
employing a library of probes according to the present invention to
simultaneously screen samples of cells for 54 different activated
transcription factors.
[0283] FIG. 6 provides the sequences for the probes used to form
the transcription factor probe library used in this experiment and
the experiments described in Sections 14-20 herein. It is noted
that the probes used were doubled stranded, FIG. 6 only showing the
strand with biotin labeled at the 5' end. The strands not shown are
the complements to the sequences shown in FIG. 6.
[0284] FIG. 6 also shows the hybridization probes used in the
hybridization probe array used in this experiment. FIG. 7 shows the
layout of the array of hybridization probes employed in the
experiments described herein.
[0285] As can be seen in FIG. 7, each hybridization probe was
placed in multiple different regions, in this case, 4 separate
regions. It is noted that 2, 3, 4 or more multiple separate regions
may be employed. Alternatively, only a single region may also be
employed. As can also be seen in FIG. 7, the concentration of
hybridization probe was varied in the different regions. This
serves both as an internal control as well as a mechanism for
quantifying the amount of immobilized probes.
[0286] Biotinylated oligonucleotides which are used as controls
were positioned in the regions of Row O and Column 17. These
oligonucleotides also serve as a legend for the array, allowing a
person to identify positions of rows and columns in the array.
[0287] The transcription factor probe library described in FIG. 6
was contacted with the array of hybridization probes described in
regard to FIGS. 6 and 7. In this instance, unlike the experiments
to be described herein, no intermediate isolation of transcription
factor probe--transcription factor complexes was performed.
[0288] FIG. 8 is an image of the resulting array. As can be seen,
all of the regions contain immobilized transcription factor probes.
As can also be seen, the regions with higher concentrations of the
same hybridization probe appear brighter (e.g., A1 and A2 vs. B1
and B2).
14. Array Detection of Selected Activated Transcription Factors
[0289] Specific transcription factor probes and combinations of
transcription factor probes were also contacted with a nuclear
extract from HeLa cells and probes from any transcription factor
probe--transcription factor complexes that formed being isolated.
The transcription factor probes and combinations of transcription
factor probes used were Brn3, c-Myb, Smad3/4 individually and the
combination of Brn3, c-Myb, and Smad3/4.
[0290] FIGS. 9A-9D are images of the resulting arrays. As can be
seen, only the hybridization probe regions for Brn3, c-Myb, and
Smad3/4 appear to possess immobilized transcription factor probes
in FIGS. 9A-9C respectively. Meanwhile, FIG. 9D shows immobilized
transcription factor probes in the Brn3, c-Myb, and Smad3/4
hybridization probe regions. The ratios of the spot densities among
Brn3, c-Myb, and Smad3/4 has been found to be very similar to what
is observed in arrays with single probe detection.
[0291] As can also be seen, the higher concentration regions appear
consistently darker than the lower concentration regions. The use
of regions with different concentrations of hybridization probes
allows different concentrations of transcription factor probes to
be isolated. If the higher concentration region is saturated, the
lower concentration one can be used for evaluation.
15. Array Detection of Activated Transcription Factors in HeLa
Cells
[0292] The entire library of probes described in regard to FIG. 6
was also contacted with a nuclear extract from HeLa cells and
probes from any transcription factor probe--transcription factor
complexes that formed being isolated.
[0293] FIG. 10B is an image of the resulting array. As a control,
the entire library of probes described in regard to FIG. 6 was also
contacted with a control sample that did not contain any
transcription factors. Probes from any transcription factor
probe--transcription factor complexes that formed were isolated.
Since no transcription factors are present in the control sample,
it is expected that no complexes are formed and hence no probes are
isolated. FIG. 10A is an image of the resulting array.
[0294] As can be seen in FIG. 10A and as would be expected, no
transcription factor probes are immobilized in the array for the
control sample. Meanwhile, as can be seen in FIG. 10B, a myriad of
transcription factor probes are immobilized in the array for the
HeLa cell sample.
16. Comparison Between Activated Transcription Factors in HeLa
Cells and PMA-activated HeLa Cells
[0295] FIG. 11A is an image of the array resulting from the entire
library of probes described in regard to FIG. 6 being contacted
with a nuclear extract from HeLa cells and any probes from any
transcription factor probe--transcription factor complexes that
formed being isolated.
[0296] In comparison, FIG. 11B is an image of the array resulting
from the entire library of probes described in regard to FIG. 6
being contacted with a nuclear extract from PMA-treated HeLa cells
and probes from any transcription factor probe--transcription
factor complexes that formed being isolated.
[0297] As can be seen by comparing FIGS. 11A and 11B, a number of
transcription factors including Ets and NF-E1 can be seen to have
been activated at higher levels in the PMA-treated HeLa cells.
[0298] The arrays shown in FIGS. 11A and 11B were imaged using a
FluorChem imager (from Alpha Innotech Corp) in order to quantify
the intensity of the spots appearing in the different regions of
the array. FIGS. 12A and 12B provide tables of the signal intensity
for the arrays shown in FIGS. 11A and 11B. FIG. 12C provides the
ratio between the intensities shown in FIGS. 12A and 12B.
[0299] As can be seen by comparing the data shown in FIGS. 12A and
12B, the signal intensities for the regions associated with
transcription factors Egr (C5, C6, D5, D6); Ets (E9, E10, F9, F10);
NF-E1 (G4, G5, H4, H5); and Smad3/4 (I15, I16. J15, J16) are more
intense. Meanwhile, Ets (E9, E10, F9, F10) and Smad3/4 (I15, I16,
J15, J16) show no difference in density.
[0300] The results of the experiment described in regard to FIGS.
11A, 11B, and 12A-12C was confirmed by performing a standard gel
shift assay using Ets and NF-E1 probes. Specifically, nuclear
extracts of HeLa and PMA-treated HeLa cells were incubated with Ets
and NF-E1 probes respectively. As can be seen in FIG. 13, bands
corresponding to Ets appear for both PMA-treated and untreated HeLa
cells. By contrast, a band corresponding to NF-E1 was not present
in untreated HeLa cells but appeared in PMA-treated HeLa cells.
17. Comparison Between Activated Transcription Factors in A431
Cells and PMA-Activated A431 Cells
[0301] The entire library of probes described in regard to FIG. 6
was also contacted with a nuclear extract untreated A431 cells and
PMA-treated A431 cells. FIG. 14A is an image of the array for
untreated A431 cells and FIG. 14B is an image of the array for
PMA-treated A431 cells. As can be seen by comparing FIGS. 14A and
14B, transcription factors NF-E1 and NP-kB were found to be
activated by PMA in A431 cells.
[0302] The results of the experiment described in regard to FIGS.
14A and 14B was confirmed by performing a standard gel shift assay
using Ets, NF-E1, and NF-kB probes. Specifically, nuclear extracts
of A431 and PMA-treated A431 cells were incubated with Ets, NF-E1,
and NF-kB probes respectively. As can be seen in FIG. 15, bands
corresponding to Ets appear for both PMA-treated and untreated A431
cells. By contrast, bands corresponding to NF-E1 and NF-kB were not
present in untreated A431 cells but appeared in PMA-treated A431
cells.
18. Comparison Between Activated Transcription Factors in Jurkat
Cells and PMA-Activated Jurkat Cells
[0303] The entire library of probes described in regard to FIG. 6
was also contacted with a nuclear extract untreated Jurkat cells
and PMA-treated Jurkat cells. FIG. 16A is an image of the array for
untreated Jurkat cells and FIG. 16B is an image of the array for
PMA-treated Jurkat cells. As can be seen by comparing FIGS. 16A and
16B, transcription factor API was found to be activated by PMA.
Interestingly, NF-E1 was not induced by PMA, showing that NF-E1
induction by PMA is cell line dependent.
19. Comparison of Activated Transcription Factors between Multiple
Cell Lines
[0304] The entire library of probes described in regard to FIG. 6
was also contacted with nuclear extracts for HeLa, A431, Jurkat,
K-562, and Y79 cells in order to compare the activated
transcription factors present in these different cell lines. FIGS.
17A-17E show the resulting arrays for HeLa, A431, Jurkat, K-562,
and Y79 cells respectively. As can be seen, the mixture of
transcription factors that are activated varies for the different
cells.
20. Gel Shift Analyses of Different Transcription Factors
[0305] Gel shift analyses were performed for multiple different
transcription factors in order evaluate the sensitivity of gel
shift analysis for detecting different transcription factors.
Specifically, a nuclear extract of HeLa cells was incubated with
transcription factor probes for c-Myb, Ets, Smad3/4, Brn3 and
NF-E2. FIG. 18A is an image of a polyacrylamide gel and FIG. 18B is
an image of an agarose gel. As can be seen in both figures, a band
corresponding to probe--transcription factor complexes is
effectively separated. As can also be seen, c-Myb, Ets, and Smad3/4
can be detected. However, Brn3 and NF-E2 are difficult to detect.
By contrast, c-Myb, Ets. Smad3/4, Brn3 and NF-E2 can all be
detected in the array shown in FIG. 10B.
[0306] It will be apparent to those skilled in the art that various
modifications and variations can be made in the compounds,
compositions, kits, and methods of the present invention without
departing from the spirit or scope of the invention. Thus, it is
intended that the present invention cover the modifications and
variations of this invention provided they come within the scope of
the appended claims and their equivalents.
Sequence CWU 1
1
162121DNAArtificial sequenceTranscription factor probe PP01
1cgcttgatga ctcagccgga a 21221DNAArtificial sequenceTranscription
factor probe PP02 2ttccggctga gtcatcaagc g 21326DNAArtificial
sequenceTranscription factor probe PP03 3gatcgaactg accgcccgcg
gcccgt 26426DNAArtificial sequenceTranscription factor probe PP04
4acgggccgcg ggcggtcagt tcgatc 26523DNAArtificial
sequenceTranscription factor probe PP05 5gtctggtaca gggtgttctt ttt
23623DNAArtificial sequenceTranscription factor probe PP06
6aaaaagaaca ccctgtacca gac 23718DNAArtificial sequenceTranscription
factor probe PP07 7cacagctcat taacgcgc 18818DNAArtificial
sequenceTranscription factor probe PP08 8gcgcgttaat gagctgtg
18920DNAArtificial sequenceTranscription factor probe PP09
9tgcagattgc gcaatctgca 201020DNAArtificial sequenceTranscription
factor probe PP10 10tgcagattgc gcaatctgca 201127DNAArtificial
sequenceTranscription factor probe PP11 11agaccgtacg tgattggtta
atctctt 271227DNAArtificial sequenceTranscription factor probe PP12
12aagagattaa ccaatcacgt acggtct 271327DNAArtificial
sequenceTranscription factor probe PP13 13acccaatgat tattagccaa
tttctga 271427DNAArtificial sequenceTranscription factor probe PP14
14tcagaaattg gctaataatc attgggt 271525DNAArtificial
sequenceTranscription factor probe PP15 15tacaggcata acggttccgt
agtga 251625DNAArtificial sequenceTranscription factor probe PP16
16tcactacgga accgttatgc ctgta 251727DNAArtificial
sequenceTranscription factor probe PP17 17agagattgcc tgacgtcaga
gagctag 271827DNAArtificial sequenceTranscription factor probe PP18
18ctagctctct gacgtcaggc aatctct 271925DNAArtificial
sequenceTranscription factor probe PP19 19atttaagttt cgcgcccttt
ctcaa 252025DNAArtificial sequenceTranscription factor probe PP20
20ttgagaaagg gcgcgaaact taaat 252127DNAArtificial
sequenceTranscription factor probe PP21 21ggatccagcg ggggcgagcg
ggggcca 272227DNAArtificial sequenceTranscription factor probe PP22
22tggcccccgc tcgcccccgc tggatcc 272335DNAArtificial
sequenceTranscription factor probe PP23 23gtccaaagtc aggtcacagt
gacctgatca aagtt 352435DNAArtificial sequenceTranscription factor
probe PP24 24aactttgatc aggtcactgt gacctgactt tggac
352531DNAArtificial sequenceTranscription factor probe PP25
25ggaggagggc tgcttgagga agtataagaa t 312631DNAArtificial
sequenceTranscription factor probe PP26 26attcttatac ttcctcaagc
agccctcctc c 312721DNAArtificial sequenceTranscription factor probe
PP27 27gatctcgagc aggaagttcg a 212821DNAArtificial
sequenceTranscription factor probe PP28 28tcgaacttcc tgctcgagat c
212921DNAArtificial sequenceTranscription factor probe PP29
29cggattgtgt attggctgta c 213021DNAArtificial sequenceTranscription
factor probe PP30 30gtacagccaa tacacaatcc g 213132DNAArtificial
sequenceTranscription factor probe PP31 31cgaagtactt tcagtttcat
attactctac aa 323232DNAArtificial sequenceTranscription factor
probe PP32 32ttgtagagta atatgaaact gaaagtactt cg
323327DNAArtificial sequenceTranscription factor probe PP33
33cacttgataa cagaaagtga taactct 273427DNAArtificial
sequenceTranscription factor probe PP34 34agagttatca ctttctgtta
tcaagtg 273541DNAArtificial sequenceTranscription factor probe PP35
35gaccctagag gatctgtaca ggatgttcta gatccaattc g 413641DNAArtificial
sequenceTranscription factor probe PP36 36cgaattggat ctagaacatc
ctgtacagat cctctagggt c 413725DNAArtificial sequenceTranscription
factor probe PP37 37ctcagcttgt actttggtac aacta 253825DNAArtificial
sequenceTranscription factor probe PP38 38tagttgtacc aaagtacaag
ctgag 253922DNAArtificial sequenceTranscription factor probe PP39
39ggaagcgaaa atgaaattga ct 224022DNAArtificial
sequenceTranscription factor probe PP40 40agtcaatttc attttcgctt cc
224125DNAArtificial sequenceTranscription factor probe PP41
41gatcccccca acacctgctg cctga 254225DNAArtificial
sequenceTranscription factor probe PP42 42tcaggcagca ggtgttgggg
ggatc 254325DNAArtificial sequenceTranscription factor probe PP43
43gatcgctcta aaaataaccc tgtcg 254425DNAArtificial
sequenceTranscription factor probe PP44 44cgacagggtt atttttagag
cgatc 254526DNAArtificial sequenceTranscription factor probe PP45
45ggaagcagac cacgtggtct gcttcc 264626DNAArtificial
sequenceTranscription factor probe PP46 46ggaagcagac cacgtggtct
gcttcc 264725DNAArtificial sequenceTranscription factor probe PP47
47ttttggattg aagccaatat gataa 254825DNAArtificial
sequenceTranscription factor probe PP48 48ttatcatatt ggcttcaatc
caaaa 254930DNAArtificial sequenceTranscription factor probe PP49
49acgcccaaag aggaaaattt gtttcataca 305030DNAArtificial
sequenceTranscription factor probe PP50 50tgtatgaaac aaattttcct
ctttgggcgt 305127DNAArtificial sequenceTranscription factor probe
PP51 51cgctccgcgg ccatcttggc ggctggt 275227DNAArtificial
sequenceTranscription factor probe PP52 52accagccgcc aagatggccg
cggagcg 275327DNAArtificial sequenceTranscription factor probe PP53
53tggggaacct gtgctgagtc actggag 275427DNAArtificial
sequenceTranscription factor probe PP54 54ctccagtgac tcagcacagg
ttcccca 275522DNAArtificial sequenceTranscription factor probe PP55
55agttgagggg actttcccag gc 225622DNAArtificial
sequenceTranscription factor probe PP56 56gcctgggaaa gtcccctcaa ct
225722DNAArtificial sequenceTranscription factor probe PP57
57tgtcgaatgc aaatcactag aa 225822DNAArtificial
sequenceTranscription factor probe PP58 58ttctagtgat ttccattcga ca
225927DNAArtificial sequenceTranscription factor probe PP59
59tacagaacat gtctaagcat gctgggg 276027DNAArtificial
sequenceTranscription factor probe PP60 60ccccagcatg cttagacatg
ttctgta 276127DNAArtificial sequenceTranscription factor probe PP61
61gaatggggca ctgaggcgtg accaccg 276227DNAArtificial
sequenceTranscription factor probe PP62 62cggtggtcac gcctcagtgc
cccattc 276326DNAArtificial sequenceTranscription factor probe PP63
63cgaattgatt gatgcactaa ttggag 266426DNAArtificial
sequenceTranscription factor probe PP64 64ctccaattag tgcatcaatc
aattcg 266528DNAArtificial sequenceTranscription factor probe PP65
65tgtcttcctg aatatgaata agaaataa 286628DNAArtificial
sequenceTranscription factor probe PP66 66ttatttctta ttcatattca
ggaagaca 286720DNAArtificial sequenceTranscription factor probe
PP67 67caaaactagg tcaaaggtca 206820DNAArtificial
sequenceTranscription factor probe PP68 68tgacctttga cctagttttg
206927DNAArtificial sequenceTranscription factor probe PP69
69gatcctgtac aggatgttct agctaca 277027DNAArtificial
sequenceTranscription factor probe PP70 70tgtagctaga acatcctgta
caggatc 277131DNAArtificial sequenceTranscription factor probe PP71
71tcgagggtag ggttcaccga aagttcactc g 317231DNAArtificial
sequenceTranscription factor probe PP72 72cgagtgaact ttcggtgaac
cctaccctcg a 317326DNAArtificial sequenceTranscription factor probe
PP73 73agcttcaggt cagaggtcag agagct 267426DNAArtificial
sequenceTranscription factor probe PP74 74agctctctga cctctgacct
gaagct 267527DNAArtificial sequenceTranscription factor probe PP75
75gtgcatttcc cgtaaatctt gtctaca 277627DNAArtificial
sequenceTranscription factor probe PP76 76tgtagacaag atttacggga
aatgcac 277716DNAArtificial sequenceTranscription factor probe PP77
77agtatgtcta gactga 167816DNAArtificial sequenceTranscription
factor probe PP78 78tcagtctaga catact 167939DNAArtificial
sequenceTranscription factor probe PP79 79tcgagagcca gacaaaaagc
cagacattta gccagacac 398039DNAArtificial sequenceTranscription
factor probe PP80 80gtgtctggct aaatgtctgg ctttttgtct ggctctcga
398121DNAArtificial sequenceTranscription factor probe PP81
81attcgatcgg ggcggggcga g 218221DNAArtificial sequenceTranscription
factor probe PP82 82ctcgccccgc cccgatcgaa t 218322DNAArtificial
sequenceTranscription factor probe PP83 83ggatgtccat attaggacat ct
228422DNAArtificial sequenceTranscription factor probe PP84
84agatgtccta atatggacat cc 228525DNAArtificial
sequenceTranscription factor probe PP85 85catgttatgc atattcctgt
aagtg 258625DNAArtificial sequenceTranscription factor probe PP86
86cacttacagg aatatgcata acatg 258724DNAArtificial
sequenceTranscription factor probe PP87 87gatccttctg ggaattccta
gatc 248824DNAArtificial sequenceTranscription factor probe PP88
88gatctaggaa ttcccagaag gatc 248933DNAArtificial
sequenceTranscription factor probe PP89 89ctagagcctg atttccccga
aatgatgagc tag 339033DNAArtificial sequenceTranscription factor
probe PP90 90ctagctcatc atttcgggga aatcaggctc tag
339121DNAArtificial sequenceTranscription factor probe PP91
91agatttctag gaattcaatc c 219221DNAArtificial sequenceTranscription
factor probe PP92 92ggattgaatt cctagaaatc t 219320DNAArtificial
sequenceTranscription factor probe PP93 93gtatttccca gaaaaggaac
209420DNAArtificial sequenceTranscription factor probe PP94
94gttccttttc tgggaaatac 209525DNAArtificial sequenceTranscription
factor probe PP95 95gcagagcata taaaatgagg tagga 259625DNAArtificial
sequenceTranscription factor probe PP96 96tcctacctca ttttatatgc
tctgc 259732DNAArtificial sequenceTranscription factor probe PP97
97gatcgtaaga ttcaggtcat gacctgagga ga 329832DNAArtificial
sequenceTranscription factor probe PP98 98tctcctcagg tcatgacctg
aatcttacga tc 329929DNAArtificial sequenceTranscription factor
probe PP99 99agcttcaggt cacaggaggt cagagagct 2910029DNAArtificial
sequenceTranscription factor probe PP100 100agctctctga cctcctgtga
cctgaagct 2910123DNAArtificial sequenceTranscription factor probe
PP101 101cacccggtca cgtggcctac acc 2310223DNAArtificial
sequenceTranscription factor probe PP102 102ggtgtaggcc acgtgaccgg
gtg 2310328DNAArtificial sequenceTranscription factor probe PP103
103agcttcaggt caaggaggtc agagagct 2810428DNAArtificial
sequenceTranscription factor probe PP104 104agctctctga cctccttgac
ctgaagct 2810515DNAArtificial sequenceTranscription factor probe
PP105 105ctggaatttt ctaga 1510615DNAArtificial
sequenceTranscription factor probe PP106 106tctagaaaat tccag
1510715DNAArtificial sequenceTranscription factor probe PP107
107ctctgcgccc ggccc 1510815DNAArtificial sequenceTranscription
factor probe PP108 108gggccgggcg cagag 1510963DNAArtificial
sequenceHybridization probe MP02 109ttccggctga gtcatcaagc
gttccggctg agtcatcaag cgttccggct gagtcatcaa 60gcg
6311078DNAArtificial sequenceHybridization probe MP04 110acgggccgcg
ggcggtcagt tcgatcacgg gccgcgggcg gtcagttcga tcacgggccg 60cgggcggtca
gttcgatc 7811169DNAArtificial sequenceHybridization probe MP06-1
111aaaaagaaca ccctgtacca gacaaaaaga acaccctgta ccagacaaaa
agaacaccct 60gtaccagac 6911254DNAArtificial sequenceHybridization
probe MP08 112gcgcgttaat gagctgtggc gcgttaatga gctgtggcgc
gttaatgagc tgtg 5411360DNAArtificial sequenceHybridization probe
MP10 113tgcagattgc gcaatctgca tgcagattgc gcaatctgca tgcagattgc
gcaatctgca 6011481DNAArtificial sequenceHybridization probe MP12
114aagagattaa ccaatcacgt acggtctaag agattaacca atcacgtacg
gtctaagaga 60ttaaccaatc acgtacggtc t 8111581DNAArtificial
sequenceHybridization probe MP14 115tcagaaattg gctaataatc
attgggttca gaaattggct aataatcatt gggttcagaa 60attggctaat aatcattggg
t 8111675DNAArtificial sequenceHybridization probe MP16
116tcactacgga accgttatgc ctgtatcact acggaaccgt tatgcctgta
tcactacgga 60accgttatgc ctgta 7511781DNAArtificial
sequenceHybridization probe MP18 117ctagctctct gacgtcaggc
aatctctcta gctctctgac gtcaggcaat ctctctagct 60ctctgacgtc aggcaatctc
t 8111875DNAArtificial sequenceHybridization probe MP20
118ttgagaaagg gcgcgaaact taaatttgag aaagggcgcg aaacttaaat
ttgagaaagg 60gcgcgaaact taaat 7511981DNAArtificial
sequenceHybridization probe MP22 119tggcccccgc tcgcccccgc
tggatcctgg cccccgctcg cccccgctgg atcctggccc 60ccgctcgccc ccgctggatc
c 8112070DNAArtificial sequenceHybridization probe MP24
120aactttgatc aggtcactgt gacctgactt tggacaactt tgatcaggtc
actgtgacct 60gactttggac 7012193DNAArtificial sequenceHybridization
probe MP26 121attcttatac
ttcctcaagc agccctcctc cattcttata cttcctcaag cagccctcct 60ccattcttat
acttcctcaa gcagccctcc tcc 9312263DNAArtificial
sequenceHybridization probe MP28 122tcgaacttcc tgctcgagat
ctcgaacttc ctgctcgaga tctcgaactt cctgctcgag 60atc
6312363DNAArtificial sequenceHybridization probe MP30 123gtacagccaa
tacacaatcc ggtacagcca atacacaatc cggtacagcc aatacacaat 60ccg
6312496DNAArtificial sequenceHybridization probe MP32 124ttgtagagta
atatgaaact gaaagtactt cgttgtagag taatatgaaa ctgaaagtac 60ttcgttgtag
agtaatatga aactgaaagt acttcg 9612581DNAArtificial
sequenceHybridization probe MP34 125agagttatca ctttctgtta
tcaagtgaga gttatcactt tctgttatca agtgagagtt 60atcactttct gttatcaagt
g 8112682DNAArtificial sequenceHybridization probe MP36
126cgaattggat ctagaacatc ctgtacagat cctctagggt ccgaattgga
tctagaacat 60cctgtacaga tcctctaggg tc 8212775DNAArtificial
sequenceHybridization probe MP38 127tagttgtacc aaagtacaag
ctgagtagtt gtaccaaagt acaagctgag tagttgtacc 60aaagtacaag ctgag
7512866DNAArtificial sequenceHybridization probe MP40 128agtcaatttc
attttcgctt ccagtcaatt tcattttcgc ttccagtcaa tttcattttc 60gcttcc
6612975DNAArtificial sequenceHybridization probe MP42 129tcaggcagca
ggtgttgggg ggatctcagg cagcaggtgt tggggggatc tcaggcagca 60ggtgttgggg
ggatc 7513075DNAArtificial sequenceHybridization probe MP44
130cgacagggtt atttttagac cgatccgaca gggttatttt tagaccgatc
cgacagggtt 60atttttagac cgatc 7513178DNAArtificial
sequenceHybridization probe MP46 131ggaagcagac cacgtggtct
gcttccggaa gcagaccacg tggtctgctt ccggaagcag 60accacgtggt ctgcttcc
7813275DNAArtificial sequenceHybridization probe MP48 132ttatcatatt
ggcttcaatc caaaattatc atattggctt caatccaaaa ttatcatatt 60ggcttcaatc
caaaa 7513390DNAArtificial sequenceHybridization probe MP50
133tgtatgaaac aaattttcct ctttgggcgt tgtatgaaac aaattttcct
ctttgggcgt 60tgtatgaaac aaattttcct ctttgggcgt 9013481DNAArtificial
sequenceHybridization probe MP52 134accagccgcc aagatggccg
cggagcgacc agccgccaag atggccgcgg agcgaccagc 60cgccaagatg gccgcggagc
g 8113581DNAArtificial sequenceHybridization probe MP54
135ctccagtgac tcagcacagg ttccccactc cagtgactca gcacaggttc
cccactccag 60tgactcagca caggttcccc a 8113666DNAArtificial
sequenceHybridization probe MP56 136gcctgggaaa gtcccctcaa
ctgcctggga aagtcccctc aactgcctgg gaaagtcccc 60tcaact
6613766DNAArtificial sequenceHybridization probe MP58 137ttctagtgat
ttccattcga cattctagtg atttccattc gacattctag tgatttccat 60tcgaca
6613881DNAArtificial sequenceHybridization probe MP60 138ccccagcatg
cttagacatg ttctgtaccc cagcatgctt agacatgttc tgtaccccag 60catgcttaga
catgttctgt a 8113981DNAArtificial sequenceHybridization probe MP62
139cggtggtcac gcctcagtgc cccattccgg tggtcacgcc tcagtgcccc
attccggtgg 60tcacgcctca gtgccccatt c 8114078DNAArtificial
sequenceHybridization probe MP64 140ctccaattag tgcatcaatc
aattcgctcc aattagtgca tcaatcaatt cgctccaatt 60agtgcatcaa tcaattcg
7814184DNAArtificial sequenceHybridization probe MP66 141ttatttctta
ttcatattca ggaagacatt atttcttatt catattcagg aagacattat 60ttcttattca
tattcaggaa gaca 8414260DNAArtificial sequenceHybridization probe
MP68 142tgacctttga cctagttttg tgacctttga cctagttttg tgacctttga
cctagttttg 6014381DNAArtificial sequenceHybridization probe MP70
143tgtagctaga acatcctgta caggatctgt agctagaaca tcctgtacag
gatctgtagc 60tagaacatcc tgtacaggat c 8114493DNAArtificial
sequenceHybridization probe MP72 144cgagtgaact ttcggtgaac
cctaccctcg acgagtgaac tttcggtgaa ccctaccctc 60gacgagtgaa ctttcggtga
accctaccct cga 9314578DNAArtificial sequenceHybridization probe
MP74 145agctctctga cctctgacct gaagctagct ctctgacctc tgacctgaag
ctagctctct 60gacctctgac ctgaagct 7814681DNAArtificial
sequenceHybridization probe MP76 146tgtagacaag atttacggga
aatgcactgt agacaagatt tacgggaaat gcactgtaga 60caagatttac gggaaatgca
c 8114764DNAArtificial sequenceHybridization probe MP78
147tcagtctaga catacttcag tctagacata cttcagtcta gacatacttc
agtctagaca 60tact 64148117DNAArtificial sequenceHybridization probe
MP80 148gtgtctggct aaatgtctgg ctttttgtct ggctctcgag tgtctggcta
aatgtctggc 60tttttgtctg gctctcgagt gtctggctaa atgtctggct ttttgtctgg
ctctcga 11714963DNAArtificial sequenceHybridization probe MP82
149ctcgccccgc cccgatcgaa tctcgccccg ccccgatcga atctcgcccc
gccccgatcg 60aat 6315066DNAArtificial sequenceHybridization probe
MP84 150agatgtccta atatggacat ccagatgtcc taatatggac atccagatgt
cctaatatgg 60acatcc 6615175DNAArtificial sequenceHybridization
probe MP86 151cacttacagg aatatgcata acatgcactt acaggaatat
gcataacatg cacttacagg 60aatatgcata acatg 7515272DNAArtificial
sequenceHybridization probe MP88 152gatctaggaa ttcccagaag
gatcgatcta ggaattccca gaaggatcga tctaggaatt 60cccagaagga tc
7215399DNAArtificial sequenceHybridization probe MP90 153ctagctcatc
atttcgggga aatcaggctc tagctagctc atcatttcgg ggaaatcagg 60ctctagctag
ctcatcattt cggggaaatc aggctctag 9915463DNAArtificial
sequenceHybridization probe MP92 154ggattgaatt cctagaaatc
tggattgaat tcctagaaat ctggattgaa ttcctagaaa 60tct
6315560DNAArtificial sequenceHybridization probe MP94 155gttccttttc
tgggaaatac gttccttttc tgggaaatac gttccttttc tgggaaatac
6015675DNAArtificial sequenceHybridization probe MP96 156tcctacctca
ttttatatgc tctgctccta cctcatttta tatgctctgc tcctacctca 60ttttatatgc
tctgc 7515796DNAArtificial sequenceHybridization probe MP98
157tctcctcagg tcatgacctg aatcttacga tctctcctca ggtcatgacc
tgaatcttac 60gatctctcct caggtcatga cctgaatctt acgatc
9615887DNAArtificial sequenceHybridization probe MP100
158agctctctga cctcctgtga cctgaagcta gctctctgac ctcctgtgac
ctgaagctag 60ctctctgacc tcctgtgacc tgaagct 8715969DNAArtificial
sequenceHybridization probe MP102 159ggtgtaggcc acgtgaccgg
gtgggtgtag gccacgtgac cgggtgggtg taggccacgt 60gaccgggtg
6916084DNAArtificial sequenceHybridization probe MP104
160agctctctga cctccttgac ctgaagctag ctctctgacc tccttgacct
gaagctagct 60ctctgacctc cttgacctga agct 8416160DNAArtificial
sequenceHybridization probe MP106 161tctagaaaat tccagtctag
aaaattccag tctagaaaat tccagtctag aaaattccag 6016260DNAArtificial
sequenceHybridization probe MP108 162gggccgggcg cagaggggcc
gggcgcagag gggccgggcg cagaggggcc gggcgcagag 60
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