U.S. patent application number 13/438749 was filed with the patent office on 2012-10-04 for 5-bromo-2'-deoxy-uridine labeled nucleotide triphosphates and nucleic acid probes and methods of making and using the same.
This patent application is currently assigned to LIFE TECHNOLOGIES CORPORATION. Invention is credited to Zhongting Hu, Anilkumar R. KORE.
Application Number | 20120252030 13/438749 |
Document ID | / |
Family ID | 39951724 |
Filed Date | 2012-10-04 |
United States Patent
Application |
20120252030 |
Kind Code |
A1 |
KORE; Anilkumar R. ; et
al. |
October 4, 2012 |
5-BROMO-2'-DEOXY-URIDINE LABELED NUCLEOTIDE TRIPHOSPHATES AND
NUCLEIC ACID PROBES AND METHODS OF MAKING AND USING THE SAME
Abstract
5-bromo-2'-deoxy-uridine (BrdU) labeled nucleotide triphosphates
and nucleic acid probes are described herein. The BrdU labeled
nucleotide triphosphates include a linker between the nucleotide
triphosphate and the BrdU moiety. The linker can be cleavable or
non-cleavable. The nucleotide triphosphates can be a ribonucleotide
triphosphates, 2'-deoxyribonucleotide triphosphates or
2',3'-dideoxyribonucleotide triphosphates. The nucleic acid probes
can be used for in situ hybridization.
Inventors: |
KORE; Anilkumar R.; (Austin,
TX) ; Hu; Zhongting; (Los Angeles, CA) |
Assignee: |
LIFE TECHNOLOGIES
CORPORATION
Carlsbad
CA
|
Family ID: |
39951724 |
Appl. No.: |
13/438749 |
Filed: |
April 3, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13038184 |
Mar 1, 2011 |
8163897 |
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13438749 |
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12138673 |
Jun 13, 2008 |
7915392 |
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13038184 |
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60929130 |
Jun 14, 2007 |
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Current U.S.
Class: |
435/6.19 ;
536/25.6 |
Current CPC
Class: |
C07H 21/00 20130101 |
Class at
Publication: |
435/6.19 ;
536/25.6 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C07H 19/10 20060101 C07H019/10 |
Claims
1. A compound of the formula: ##STR00038## or a salt thereof,
wherein "Nu" is a nucleotide triphosphate, a 2'-deoxynucleotide
triphosphate or a 2',3'-dideoxynucleotide triphosphate and wherein
L is a linker.
2. The compound of claim 1, wherein L has a length greater than a
C.sub.11 n-alkane carbon chain.
3. The compound of claim 1, wherein "Nu" is uridine triphosphate,
2'-deoxyuridine triphosphate or 2',3'-dideoxytriphosphate.
4. The compound of claim 1, wherein the compound is of the formula:
##STR00039## or a salt thereof, wherein each X is independently H
or OH and "L" is a linker.
5. The compound of claim 1, wherein "L" is a linker having a
chemical structure selected from the group consisting of:
##STR00040## wherein n is an integer of from 6 to 12.
6. A sodium salt of the compound of claim 1.
7. The compound of claim 1, wherein the compound is of the formula:
##STR00041## or a salt thereof, wherein each X is independently H
or OH.
8. A sodium salt of the compound of claim 7.
9. A polynucleotide represented by the formula: ##STR00042##
wherein "Y" is a nucleoside, a nucleotide or a polynucleotide, "Z"
is H, a nucleotide or a polynucleotide, "X" is H or OH, "B" is a
nucleobase and "L" is a linker.
10. The polynucleotide of claim 9, wherein the polynucleotide is
represented by the formula: ##STR00043## wherein "Y" is a
nucleoside, nucleotide or polynucleotide, "Z" is H, a nucleotide or
a polynucleotide, "X" is H or OH and "L" is a linker.
11. The polynucleotide of claim 9, wherein "L" is a linker having a
chemical structure selected from the group consisting of:
##STR00044## wherein n is an integer of from 6 to 12.
12. (canceled)
13. A method for detecting the presence of a nucleic acid analyte
in a tissue sample comprising: incubating the tissue sample with a
composition comprising the polynucleotide of claim 9, wherein the
polynucleotide binds to the nucleic acid analyte; washing the
sample; contacting the sample with an anti-BrdU antibody conjugated
to a detectable moiety or to an enzyme; and detecting the
detectable moiety or enzyme.
14. The method of claim 13, further comprising fixing the sample
prior to incubating the sample.
15. The method of claim 14, wherein fixing comprises contacting the
sample with an acid or a cross-linking agent.
16. A composition comprising: adenosine triphosphate (ATP); uridine
triphosphate (UTP); guanosine triphosphate (GTP); cytidine
triphosphate (CTP); and a compound as set forth in claim 1, wherein
"Nu" is a nucleotide triphosphate.
17-25. (canceled)
Description
[0001] This application claims the benefit of Provisional U.S.
Patent Application No. 60/929,130, filed Jun. 14, 2007, which is
incorporated by reference herein in its entirety.
[0002] The section headings used herein are for organizational
purposes only and should not be construed as limiting the subject
matter described herein in any way.
FIELD
[0003] This application relates generally to nucleic acid probes
for conducting biological assays and to methods of making and using
said probes as well as to compounds related thereto.
INTRODUCTION
[0004] In situ hybridization (ISH) uses a labeled complementary DNA
or RNA strand (i.e., a probe) in the determination of a specific
DNA or RNA sequence in a tissue sample. In contrast to other
methods, ISH enables the morphological demonstration of specific
DNA or RNA sequences in individual cells, tissue sections, single
cells, or chromosome preparations. Hence, ISH can be used to
determine DNA and RNA sequences in a heterogeneous cell population,
and to determine if a gene is expressed in low levels in all of the
cells or in high levels in only a few of the cells.
[0005] Although isotopic in situ hybridization is the most
sensitive method to detect intracellular mRNA distribution,
non-isopotic in situ is a much safer and quicker technique to
localize mRNA. The ISH procedure typically ranges from two to four
days for non-isotopic in situ compared and can be significantly
longer for isotopic in situ hybridization.
SUMMARY
[0006] A compound is provided which is represented by the
formula:
##STR00001##
wherein "Nu" is a nucleotide triphosphate, a 2'-deoxynucleotide
triphosphate or a 2',3'-dideoxynucleotide triphosphate and wherein
L is a linker group. A salt of this compound (e.g., a sodium salt)
is also provided.
[0007] A polynucleotide is also provided which is represented by
the formula:
##STR00002##
wherein "Y" is a nucleoside, a nucleotide or a polynucleotide, "Z"
is H, a nucleotide or a polynucleotide, "X" is H or OH, "B" is a
nucleobase and "L" is a linker group.
[0008] A polynucleotide is also provided which is represented by
the formula:
##STR00003##
wherein "Y" is a nucleoside, a nucleotide or a polynucleotide, "Z"
is H, a nucleotide or a polynucleotide and "X" is H or OH.
[0009] A method for detecting the presence of a nucleic acid
analyte in a tissue sample is also provided which comprises:
[0010] incubating the tissue sample with a composition comprising a
polynucleotide as set forth above, wherein the polynucleotide binds
to the nucleic acid analyte;
[0011] washing the sample;
[0012] contacting the sample with an anti-BrdU antibody conjugated
to a detectable moiety or to an enzyme; and
[0013] detecting the detectable moiety or enzyme.
[0014] A composition is also provided which comprises: adenosine
triphosphate (ATP); uridine triphosphate (UTP); guanosine
triphosphate (GTP); cytidine triphosphate (CTP); and a compound of
the formula:
##STR00004##
or a salt thereof, wherein "Nu" is a ribonucleotide triphosphate
and wherein L is a linker group. A kit is also provided which
comprises a composition as set forth above and an RNA
polymerase.
[0015] A composition is also provided which comprises dATP, dCTP,
dGTP, dTTP and a compound of the formula:
##STR00005##
or a salt thereof, wherein "Nu" is a 2'-deoxyribonucleotide
triphosphate or a 2',3'-dideoxyribonucleotide triphosphate and
wherein L is a linker group. A kit is also provided which comprises
a composition as set forth as set forth above and a DNA polymerase
or terminal transferase.
[0016] These and other features of the present teachings are set
forth herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] The skilled artisan will understand that the drawings,
described below, are for illustration purposes only. The drawings
are not intended to limit the scope of the present teachings in any
way.
[0018] FIG. 1 is an HPLC Profile of a 5-bromo-2'-deoxy-uridine
(BrdU) labeled uridine triphosphate (UTP) probe.
[0019] FIG. 2 is an HPLC Profile of a 5-bromo-2'-deoxy-uridine
(BrdU) labeled 2'-deoxy uridine triphosphate (dUTP) probe.
DESCRIPTION OF THE VARIOUS EMBODIMENTS
[0020] For the purposes of interpreting this specification, the
following definitions will apply and whenever appropriate, terms
used in the singular will also include the plural and vice versa.
In the event that any definition set forth below conflicts with the
usage of that word in any other document, including any document
incorporated herein by reference, the definition set forth below
shall always control for purposes of interpreting this
specification and its associated claims unless a contrary meaning
is clearly intended (for example in interpreting the document where
the term is originally used). The use of "or" herein means "and/or"
unless stated otherwise or where the use of "and/or" is clearly
inappropriate. The use of "a" herein means "one or more" unless
stated otherwise or where the use of "one or more" is clearly
inappropriate. The use of "comprise," "comprises," "comprising,"
"include," "includes," and "including" are interchangeable and not
intended to be limiting. Furthermore, where the description of one
or more embodiments uses the term "comprising," those skilled in
the art would understand that, in some specific instances, the
embodiment or embodiments can be alternatively described using the
language "consisting essentially of" and/or "consisting of."
[0021] As used herein, the term "nucleobase" refers to a nitrogen
containing heterocyclic moiety capable of forming Watson-Crick
hydrogen bonds in pairing with a complementary nucleobase.
Non-limiting examples of suitable nucleobases include: adenine,
cytosine, guanine, thymine, uracil, 5-propynyl-uracil,
2-thio-5-propynyl-uracil, 5-methylcytosine, pseudoisocytosine,
2-thiouracil, 2-thiothymine, 2-aminopurine,
N9-(2-amino-6-chloropurine), N9-(2,6-diaminopurine), hypoxanthine,
N9-(7-deaza-guanine), N9-(7-deaza-8-aza-guanine) and
N8-(8-aza-7-deazaadenine).
[0022] As used herein, the term "nucleoside" refers to a compound
consisting of a nucleobase linked to the C-1' carbon of a ribose
sugar. The ribose may be substituted or unsubstituted. Exemplary
ribose sugars include ribose, 2'-deoxyribose and
2',3'-dideoxyribose (also referred to herein as dideoxyribose).
[0023] As used herein, the term "nucleotide" refers to a phosphate
ester of a nucleoside as a monomer unit or within a
polynucleotide.
[0024] As used herein, the term "nucleotide triphosphate" refers to
a nucleotide with a triphosphate ester group at the 5'
position.
[0025] As used herein, the terms "polynucleotide",
"oligonucleotide" and "nucleic acid` are used interchangeably and
refer to single stranded and double stranded polymers of nucleotide
monomers, including ribonucleotides (RNA) and
2'-deoxyribonucleotides (DNA) linked by internucleotide
phosphodiester bond linkages. A polynucleotide may be composed
entirely of deoxyribonucleotides, entirely of ribonucleotides or
chimeric mixtures thereof.
[0026] As used herein, the term "salt" refers to a salt of a
compound or a mixture of salts of a compound. For example, the salt
can be a sodium salt, or a mixture of the sodium and potassium
salts of the compound. In addition, zwitterionic forms of a
compound are also included in the term "salt." Salts of acidic
functional groups (e.g. a phosphate group) may have a
countercation, such as sodium, potassium, magnesium, calcium,
etc.
[0027] As used herein, the term "hydroxyl protecting group" refers
to any hydroxyl protecting group that is compatible with
oligonucleotide synthesis. For a detailed description of nucleic
acid synthesis methodology, and hydroxyl protecting groups that can
be used therein, please see: Gait, Oligonucleotide Synthesis: A
Practical Approach, IRL Press, Oxford England. Other suitable
hydroxyl protecting groups can be found in: Green et al.,
Protecting Groups In Organic Synthesis, Third Edition, John Wiley
& Sons, Inc. New York, 1999).
[0028] As used herein, the terms "linker", "linker group" and
"linking group" are used interchangeably and refer to any chemical
group (e.g. R.sub.1 and R.sub.2) that connects two or more other
chemical groups. Linking groups can be substituted or unsubstituted
alkyl, alkeneyl or alkynyl groups, which may optionally comprise
one or more heteroatoms (substituted for a carbon atom), such as
oxygen, sulfur, nitrogen or silicon, in the chain as well as one or
more unsaturated carbon-carbon bonds. For example, the linking
group may comprise one or more disulfide, amide and/or ester bonds.
For example, a linking group can be: a) a C.sub.1-10 alkyl group,
b) a C.sub.2-10 alkenyl group, c) a C.sub.2-10 alkynyl group, d) a
C.sub.3-14 cycloalkyl group, e) a C.sub.6-14 aryl group, f) a
C.sub.1-10 heteroalkyl group, g) a C.sub.2-10 heteroalkenyl group,
h) a C.sub.2-10 heteroalkynyl group, i) a 3-14 membered
cycloheteroalkyl group, or j) a 5-14 membered heteroaryl group
wherein each of a)-j) optionally is substituted with one or more
independently selected R.sub.6 groups, wherein R.sub.6 is halogen,
--CN, --NO.sub.2, OXO, --OH, --OR.sub.7, --NH.sub.2, --NHR.sub.7,
--N(R.sub.7).sub.2, or a C.sub.1-10 alkyl group, wherein R.sub.7 is
a substituted or unsubstituted C.sub.1-10 alkyl group. For example,
the linking group can comprise one or more diol and one or more
amide bonds as well as one or more unsaturated carbon-carbon bonds.
In some embodiments, the linker can comprise one or more amide and
one or more ester bonds as well as one or more unsaturated
carbon-carbon bonds.
[0029] Provided herein is a compound of the formula:
##STR00006##
wherein "Nu" is a nucleotide triphosphate, a 2' deoxynucleotide
triphosphate or a dideoxy nucleotide triphosphate and wherein L is
a linker. A salt of the above compound (e.g., a sodium salt) is
also provided.
[0030] According to some embodiments, "Nu" in the above formula is
a uridine triphosphate, a 2'-deoxyuridine triphosphate or a
2',3'-dideoxyuridine triphosphate. In some embodiments, the
compound is of the formula:
##STR00007##
wherein each X in the above formula is independently H or OH and
"L" is a linker group. A salt of the above compound (e.g., a sodium
salt) is also provided.
[0031] According to some embodiments, "L" in either of the above
formulae is a cleavable or a non-cleavable linker. Exemplary
non-cleavable linkers include, but are not limited to, the
following:
##STR00008##
wherein n is an integer greater than 6 (e.g., n=6-12
inclusive).
[0032] Exemplary cleavable linker groups include, but are not
limited to, the following:
##STR00009##
which is cleavable using a mild oxidizing agent;
##STR00010##
which is a thiol cleavable linker;
##STR00011##
which is a thiol cleavable linker with a longer carbon chain;
##STR00012##
which is cleavable by an oxidizing agent;
##STR00013##
which is cleavable under gentle conditions, such as by using a pH
of 8.5 and hydroxyl amine; and
##STR00014##
which is water soluble and cleavable using hydroxyl amine.
Cleavable linkers are disclosed in: Thevenin et al., Biophysics.
J., 59, 358a (1991); Jung et al., Biochim. Biophys. Acta, 761,
152-162 (1983); Farries et al., J. Immunol., 142, 842-847 (1989);
Park et al., J. Biol. Chem., 261, 205-210 (1986); Millar et al., J.
Biol. Chem., 265, 12052-12058 (1990); and Browning et al., J.
Immunol., 143, 1859-1867 (1989).
[0033] In some embodiments, the compound has the formula:
##STR00015##
wherein each X is independently H or OH is also provided. A salt of
the above compound (e.g., a sodium salt) is also provided.
[0034] A BrdU labeled nucleotide as described above can be used to
synthesize a polynucleotide probe. The polynucleotide probe can be
synthesized by polymerase extension in the presence of a template,
an appropriate polymerase and other reagents (e.g. nucleotide
triphosphates, magnesium, etc.) and conditions commonly applied to
polymerase extension reaction. One of skill in the art would be
familiar with the reagents and conditions used to perform a
polymerase extension reaction (see, for example, Ma et al., In
Vitro Mutagenesis of Xanthomonas Campestris .alpha.-Amylase Gene by
Partially Replacing Deoxythymidine Triphosphate with
5-Bromo-2'-Deoxyuridine-5'-Triphosphate Using a PCR technique,
Biotechnology Letters, Vol. 26, No. 2, pp. 171-175(5), (January
2004).
[0035] Accordingly, a polynucleotide is also provided which is
represented by the formula:
##STR00016##
wherein "Y" is a nucleoside, a nucleotide or a polynucleotide, "Z"
is H, a nucleotide or a polynucleotide, "X" is H or OH, "B" is a
nucleobase and "L" is a linker group.
[0036] According to some embodiments, "B" in the above formula is a
uracil group and the polynucleotide is represented by the
formula:
##STR00017##
wherein "X", "Z", "L" and "Y" are defined as set forth above.
[0037] The polynucleotide can be used as a probe for conducting in
situ hybridization assays.
[0038] As set forth above, a 5-bromo-2'-deoxy-uridine (BrdU)
molecule can be linked to a ribonucleotide triphosphate such as
ribo uridine triphosphate (rUTP). The structure of a BrdU-rUTP
(designated BrdU-14-UTP) is set forth below:
##STR00018##
In this probe, 5-BrdU is coupled to a 5-(3-aminoallyl)uridine
5'-triphosphate via a linker group having a 14 atom main chain.
This ribo-derivative can be used for synthesizing BrdU-labeled RNA
probes with RNA polymerases. When BrdU-UTP is used to generate an
antisense probe, BrdU molecules will not anneal to nucleotide A in
the sense probe. Therefore, the BrdU antibody can easily recognize
and bind to BrdU moieties in the probe. The bound BrdU antibodies
can also be easily detected (e.g., by alkaline-phosphatase). The
probes can be used for both section in situ or whole-mount in situ
assays.
[0039] A 5-bromo-2'-deoxyuridine (BrdU) molecule can also be linked
to a 2'-deoxy nucleotide triphosphate such as 2'-deoxy uridine
triphosphate (dUTP). The structure of BrdU-dUTP (designated
BrdU-14-dUTP) is set forth below:
##STR00019##
In the above probe, 5-BrdU is coupled to a 5-[3-aminoallyl]
2'-deoxyuridine 5'-triphosphate via a linker group having a 14 atom
main chain. The 2'-deoxy probe (i.e. dUTP) can be used for to
synthesize BrdU-labeled DNA probes using DNA polymerases.
[0040] A 5-bromo-2'-deoxy-uridine (BrdU) molecule can also be
linked to a 2',3'-dideoxy nucleotide triphosphate such as
2',3'-dideoxy uridine triphosphate (ddUTP). The structure of a
BrdU-ddUTP probe is set forth below:
##STR00020##
The ddUTP-BrdU probe can be used for 3' end labeling of
oligonucleotides with terminal transferase.
[0041] The BrdU labeled probes described herein are non-radioactive
(i.e., non-isotopic) probes. Therefore, the disadvantages of
working with radio isotopes, which include the need for special
laboratories, safety measurements/shielding against exposure to
radioactivity, special training of people working with
radioactivity, expensive waste disposal, and very unstable probes
can be avoided.
[0042] A general reaction scheme for the synthesis of
ribonucleotide-BrdU, 2'-deoxyribonucleotide-BrdU and
2',3'-dideoxy-ribonucleotide-BrdU probes is set forth below.
##STR00021##
[0043] In the above reaction scheme, "AA-UTP" denotes
5-[3-aminoallyl] uridine 5'-triphosphate", AA-dUTP" denotes
5-[3-aminoallyl] 2'-deoxyuridine 5'-triphosphate", AA-ddUTP"
denotes 5-[3-aminoallyl] 2',3'-dideoxyuridine 5'-triphosphate",
"lcaa" denotes a long chain amino acid, "CPG" denotes a controlled
pore glass support, and "DMT" denotes a dimethoxytrityl group. The
designation "H/OH" indicates that either an H or an OH group can be
present. Although a controlled pore glass (CPG) support is depicted
in the above reaction scheme, other supports can also be used (such
supports would be well-known to those skilled in the area of DNA
synthesis). In addition, although a dimethoxytrityl group is
depicted in the above reaction scheme, other hydroxyl protecting
groups could also be used. Such protecting groups would also be
well-known to those skilled in the area of DNA synthesis.
[0044] Moreover, a method is provided which comprises reacting a
compound (1) which is bound to a support:
##STR00022##
wherein "R.sub.1" is a group (i.e. a linking group) linking the
nucleoside moiety to the support (denoted "SUPPORT", which could,
for example, be CPG) and "HPG" is a hydroxyl protecting group, with
the compound (2):
##STR00023##
wherein R.sub.2 is a linking group, and R.sub.3 is H or
SO.sub.3.sup.-, to form a compound (3):
##STR00024##
reacting the compound (3) with the compound (4):
##STR00025##
wherein each X is independently H or OH, to form a compound
(5);
##STR00026##
and removing the compound (5) from the support to form a compound
(6);
##STR00027##
[0045] As set forth in the above method, the BrdU labeled
ribonucleotide, 2'-deoxyribonucleotide or dideoxyribonucleotide
probes can be made by a method comprising reacting the compound
(2):
##STR00028##
with a support bound BrdU moiety. In the compound (2), R.sub.2 is a
linking group, and R.sub.3 is H or SO.sub.3.sup.-. Exemplary
R.sub.2 groups include non-cleavable linking groups and cleavable
linking groups. Exemplary non-cleavable linking groups that can be
used for R.sub.2 include, but are not limited to, the
following:
##STR00029##
wherein n is an integer of at least 6 (e.g., n=6-12).
[0046] As set forth above, a linker which can be cleaved (e.g.,
under mild conditions) can also be used. Exemplary cleavable
linking groups include, but are not limited to:
##STR00030##
which is cleavable using a mild oxidizing agent;
##STR00031##
which is thiol-cleavable;
##STR00032##
which is thiol-cleavable and which has a longer carbon chain;
##STR00033##
which is cleavable by an oxidizing agent;
##STR00034##
which is cleavable under gentle conditions at pH of 8.5 using
hydroxyl amine; and
##STR00035##
which is water soluble and cleavable using hydroxyl amine.
[0047] The length of the linking group between the BrdU label and
the amino allyl modified ribonucleotide, 2'-deoxyribonucleotide or
2',3'-dideoxyribonucleotide (e.g., AA-rUTP, AA-dUTP or AA-ddUTP)
can be varied. According to some embodiments, the length of the
linker group can be at least as long as an 11 carbon atom chain. It
is to be understood that the linker group can include atoms other
than carbon (e.g. sulfur, oxygen, nitrogen or silicon) but without
regard to its composition, will have a length that is at least as
long as an 11 atom carbon chain. The length of the linker group can
be varied to provide a high level of sensitivity during detection.
Moreover, a linker group can be used which allows the BrdU moiety
to project out far enough from the labeled probe to be efficiently
recognized by the antibody during detection.
[0048] A method for detecting the presence of a nucleic acid
analyte in a tissue sample is also described herein. The method
comprises:
[0049] incubating the tissue sample with a composition comprising a
BrdU-labeled polynucleotide as set forth above, wherein the
polynucleotide binds to the nucleic acid analyte;
[0050] washing the sample;
[0051] contacting the sample with an anti-BrdU antibody conjugated
to a detectable moiety or to an enzyme; and
[0052] detecting the detectable moiety or enzyme.
The method as set forth above may further comprise fixing the
sample prior to incubating the sample. Methods for fixing cells for
in-situ analysis are well-known in the art (See, for example,
Sambrook et al., Molecular Cloning, A Laboratory Manual," 3rd Ed.,
Vol. 1, Chapter 7, Protocol 9, sections 7.46 to 7.50 and Ausubel et
al., Current Protocols in Molecular Biology, Vol. 2, Chapter 14, In
Situ Hybridization and Immunohistochemistry). Non-limiting examples
of fixing include, but are not limited to, contacting the sample
with an acid or a cross-linking agent.
[0053] A composition is also provided which comprises: adenosine
triphosphate (ATP); uridine triphosphate (UTP); guanosine
triphosphate (GTP); cytidine triphosphate (CTP); and a compound of
the formula:
##STR00036##
wherein "Nu" is a ribonucleotide triphosphate and wherein L is a
linker group. In some embodiments, a kit is also provided which
comprises a composition as set forth above and an RNA
polymerase.
[0054] In some embodiments, a composition is also provided which
comprises 2' deoxy adenosine triphosphate (dATP); 2' deoxy
guanosine triphosphate (dGTP); 2' deoxy cytidine triphosphate
(dCTP); and thymidine triphosphate (dTTP) and a compound of the
formula:
##STR00037##
wherein "Nu" is a 2'-deoxyribonucleotide triphosphate and wherein L
is a linker group. In some embodiments, a kit is also provided
which comprises a composition as set forth as set forth above and a
DNA polymerase.
Materials and Methods
Reagents
[0055] All the reagents and solvents are used as such without
further purification, unless otherwise stated.
5'-Dimethoxytrityl-5-bromo-2'-deoxy Uridine, 2'-succinoyl-long
chain alkylamine-CPG (5-Br-dU-CPG) and
10-carboxy-decyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite,
N-hydroxysuccinimide ester were purchased from Glen Research, USA.
5-[3-Aminoallyl]uridine 5'-triphosphate, and 5-[3-Aminoallyl]
2'-deoxy uridine 5'-triphosphate were obtained from Ambion. The
detritylation reagent (3% trichloroacetic acid in dichloromethane),
activator (i.e. 5-Ethylthio-1H-tetrazole in anhydrous
acetonitrile), oxidizing solution {i.e. 0.05M I.sub.2 in
THF/H.sub.2O/Pyridine (7:2:1)}, and acetonitrile were obtained from
Glen Research, USA. 50 mM Sodium borate buffer at a pH of 8.5 was
obtained from Ambion. Air tight glass syringes were obtained from
VWR. The probes ware analyzed by Mass Spectroscopy (i.e., LC/MS
(ABI), and by analytical HPLC (Aliance, Water's) using Hypersil SAX
columns, 5 .mu.m, 250.times.4.6 mm (Altech).
Solid Phase Synthesis of BrdU-rUTP and BrdU-dUTP Probes
[0056] BrdU-rUTP and BrdU-dUTP probes were synthesized on long
chain alkylamine controlled pore glass support (80-100 mesh,
500.degree. A) by using a 5' carboxy modifier {i.e.
10-Carboxy-decyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite,
N-hydroxysuccinimide ester} following conventional protocols (see,
for example, Tanaka et al., Nucleic Acid Res., 10, 3240, 1982). 5'
coupling was achieved manually using a syringe technique (see, for
example, Murakami et al., Nucleic Acids Res., 19, 4097, 1991). The
bioconjugation of N-hydroxysuccinimide ester bearing from 5'-BrdU
CPG and 5-[3-aminoallyl]uridine
5'-triphosphate/5-[3-aminoallyl]2'-deoxy uridine 5'-triphosphate
was achieved in 50 mM sodium borate buffer pH 8.5 at room
temperature for 3 hours.
[0057] The 5'-DMT-BrdU loaded CPG, 66 .mu.mol/g (0.5 gm, 33.03
.mu.mol) was poured into a 50 mL gas tight syringe with a glass
wool plug at the inlet. The support was washed initially with
anhydrous CH.sub.3CN (4.times.35 mL). The 5'-dimethoxytrityl (DMT)
protecting group was then removed by washing with 3%
trichloroacetic acid in dichloromethane (5.times.40 mL, 5 min). The
support was then washed with anhydrous CH.sub.3CN (4.times.40 mL).
The coupling of
10-carboxy-decyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite,
N-hydroxysuccinimide ester solution (20 mL, 0.1M solution in
CH.sub.3CN) was drawn into the syringe followed by
5-ethylthio-1H-tetrazole activator in anhydrous acetonitrile
solution (20 mL, 0.3 M in CH.sub.3CN). It was agitated for 20 min
and the solution was ejected. The support was then washed with
anhydrous CH.sub.3CN (4.times.40 mL) and oxidized with a (0.05M, 40
mL) I.sub.2 in THF/H.sub.2O/Pyridine (7:2:1) for 20 min. The
support was then washed with anhydrous acetonitrile (5.times.40
mL), until no more brown color was observed coming from the
support.
[0058] Then support was air-dried and transferred into a vial
containing 5 mL of 50 mM sodium borate buffer pH 8.5. To this, a
solution of 5-[3-aminoallyl]uridine 5'-triphosphate or
5-[3-aminoallyl]2'-deoxy uridine 5'-triphosphate (3 mL, 0.25M) in
sodium borate buffer at a pH of 8.5 was added. The reaction mixture
was slowly agitated for 4 hours at room temperature. After 4 hours,
the supernatant was taken out from the support and washed with
distilled water (6.times.15 mL) in order to remove any traces of
aminoallyl UTP or aminoallyl dUTP. Then the support was washed with
anhydrous acetonitrile (2.times.15 mL) and air-dried. The support
was transferred into a capped vial and treated with concentrated
methanolic NH.sub.4OH 1:4 ratio of (20 mL) for 3 hours at
55.degree. C. temperature in water bath. After removal of ammonia
under nitrogen current, the supernatant was taken from the support.
The support was washed twice with distilled water (2.times.6 mL).
The aqueous layers were combined and lyophilized. The resulting
probe was analyzed by Mass Spectroscopy and analytical HPLC. The
HPLC profile of the BrdU-rUTP probe is shown in FIG. 1. The HPLC
profile of the BrdU-dUTP probe is shown in FIG. 2. The product was
confirmed by mass spectrometry.
Determination of Sensitivity of Probes by Blot Hybridization
[0059] Dot and slot blotting are techniques for immobilizing
several preparations of nucleic acids on the same solid support,
usually a charged nylon membrane or nitrocellulose. Blot
hybridizations with specific nucleic acid probes can provide
information regarding gene expression and genome structure. In
order to find out the sensitivity of the BrdU-14-UTP probe, VIP
(i.e. vasoactive intestinal polypeptide plasmid) DNA was used. VIP
DNA was digested and linearized DNA was used for transcription.
Linearized VIP DNA was transcribed by using normal nucleotide
triphosphates (NTPs) except UTP and BrdU-14-UTP was used in 1:1
ratio in the presence of T3 RNA polymerase, which gave an antisense
strand. In order to obtain a sense strand, the linearized VIP DNA
was transcribed by using T7 RNA Polymerase using normal NTPs. Sense
and antisense probes were spotted on nitrocellulose membrane with
various dilutions. After hybridization, excess probe was washed
away and the hybridized probe was reacted by using alkaline
phosphatase conjugated anti-bromodeoxyuridine antibodies and
detected by using BCIP/NBT color reagents. BCIP refers to
5-Bromo-4-chloro-3-indolylphosphate and NBT refers to nitroblue
tetrazolium chloride. A blot hybridization procedure was carried
out as per standard protocols (Sambrook et al., Molecular Cloning,
A Laboratory Manual, Third Edition, Vol. 1, Chapter 7, protocol 9,
section 7.46 to 7.50; and Ausubel et al., Current Protocols in
Molecular Biology, Vol. 2, Chapter 14, In Situ Hybridization and
Immunohistochemistry). The results indicated that the BrdU-14-UTP
probe had good detection sensitivity in a blot hybridization
format.
[0060] While the foregoing specification teaches the principles of
the present invention, with examples provided for the purpose of
illustration, it will be appreciated by one skilled in the art from
reading this disclosure that various changes in form and detail can
be made without departing from the true scope of the invention.
* * * * *