U.S. patent application number 16/842510 was filed with the patent office on 2020-09-24 for hybridization compositions and methods.
The applicant listed for this patent is Agilent Technologies, Inc.. Invention is credited to Steen Hauge Matthiesen.
Application Number | 20200299769 16/842510 |
Document ID | / |
Family ID | 1000004882120 |
Filed Date | 2020-09-24 |
![](/patent/app/20200299769/US20200299769A1-20200924-D00001.png)
United States Patent
Application |
20200299769 |
Kind Code |
A1 |
Matthiesen; Steen Hauge |
September 24, 2020 |
HYBRIDIZATION COMPOSITIONS AND METHODS
Abstract
The invention provides methods and compositions for hybridizing
at least one molecule to a target. The invention may, for example,
utilize a of cyclic and/or non-cyclic solvent that is non-toxic and
may eliminate or reduce the amount of formamide in the
hybridization composition.
Inventors: |
Matthiesen; Steen Hauge;
(Hillerod, DK) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Agilent Technologies, Inc. |
Santa Clara |
CA |
US |
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|
Family ID: |
1000004882120 |
Appl. No.: |
16/842510 |
Filed: |
April 7, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14352815 |
Apr 18, 2014 |
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PCT/EP2012/070877 |
Oct 22, 2012 |
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16842510 |
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61550016 |
Oct 21, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/6881 20130101;
C12Q 1/6832 20130101; C12Q 1/6841 20130101 |
International
Class: |
C12Q 1/6881 20060101
C12Q001/6881; C12Q 1/6832 20060101 C12Q001/6832; C12Q 1/6841
20060101 C12Q001/6841 |
Claims
1-26. (canceled)
27. A method of hybridizing nucleic acid sequences comprising:
providing a first nucleic acid sequence, providing a second nucleic
acid sequence, providing a hybridization composition comprising at
least one solvent in an amount effective to denature
double-stranded nucleotide sequences, and combining the first and
the second nucleic acid sequence and the hybridization composition
for at least a time period sufficient to hybridize the first and
second nucleic acid sequences, wherein the solvent is chosen from
1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)pyrimidinone,
N,N-dimethyl-acetamide and isobutyramide.
28. A method of hybridizing nucleic acid sequences comprising:
providing a first nucleic acid sequence in an in situ biological
sample, and applying a hybridization composition comprising a
second nucleic acid sequence and at least one solvent in an amount
effective to denature double-stranded nucleotide sequences to said
first nucleic acid sequence for at least a time period sufficient
to hybridize the first and second nucleic acid sequences, wherein
the solvent is chosen from
1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)pyrimidinone, N
,N-dimethyl-acetamide and isobutyramide.
29-32. (canceled)
33. The method according to claim 27, wherein a sufficient amount
of energy to hybridize the first and second nucleic acids is
provided.
34. The method according to claim 33, wherein the energy is
provided by heating the hybridization composition and nucleic acid
sequence.
35. The method according to claim 33, wherein the heating step is
performed by the use of microwaves, hot baths, hot plates, heat
wire, peltier element, induction heating or heat lamps.
36. The method according to claim 27, wherein the first nucleic
acid sequence is double stranded and the second nucleic acid is
single stranded.
37. The method according to claim 27, wherein the denaturation and
hybridization steps occur separately.
38. The method according to claim 27, wherein the step of
hybridizing includes the steps of heating and cooling the
hybridization composition and nucleic acid sequences.
39. The method according to claim 27, wherein the step of
hybridization takes less than 2 hours.
40. The method according to claim 39, wherein the step of
hybridization takes less than 1 hour.
41. The method according to claim 40, wherein the step of
hybridization takes less than 30 minutes.
42. The method according to claim 41, wherein the step of
hybridization takes less than 15 minutes.
43. The method according to claim 42, wherein the step of
hybridization takes less than 5 minutes.
44. The method according to claim 27, wherein the cooling step
takes less than 1 hour.
45. The method according to claim 44, wherein the cooling step
takes less than 30 minutes.
46. The method according to claim 45, wherein the cooling step
takes less than 15 minutes.
47. The method according to claim 46, wherein the cooling step
takes less than 5 minutes.
48. The method according to claim 27, wherein the denaturation step
is performed at 67.degree. C.
49. The method according to claim 27, wherein the first nucleic
acid sequence is in a biological sample.
50. The method according to claim 49, wherein the biological sample
is a cytology or histology sample.
51-57. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a divisional of U.S. patent application
Ser. No. 14/352,815, filed on Apr. 18, 2014, which is a national
stage filing under 35 U.S.C. .sctn. 371 of International
Application No. PCT/EP2012/070877, filed Oct. 22, 2012, which
claims the benefit of U.S. Patent Application No. 61/550,016, filed
Oct. 21, 2011, the contents of all of which are fully incorporated
herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to compositions and methods
for use in hybridization applications. The present invention also
relates to compositions and methods for example, for use in in situ
hybridization (ISH) applications. In one embodiment, the present
invention involves molecular examination of DNA (deoxyribonucleic
acid) and RNA (ribonucleic acid). In particular, the invention can
be used for the molecular examination of DNA and RNA in the fields
of cytology, histology, and molecular biology. In other
embodiments, the present invention relates to the energy (e.g.,
incubation time and heat) required during hybridization between
nucleic acids, e.g., in in situ hybridization targeting DNA and
RNA.
BACKGROUND AND DESCRIPTION
[0003] Double stranded nucleic acid molecules (i.e., DNA, DNA/RNA
and RNA/RNA) associate in a double helical configuration. This
double helix structure is stabilized by hydrogen bonding between
bases on opposite strands when bases are paired in one particular
way (A+T/U or G+C) and hydrophobic bonding among the stacked bases.
Complementary base paring (hybridization) is central to all
processes involving nucleic acid.
[0004] In a basic example of hybridization, nucleic acid probes or
primers are designed to bind, or "hybridize," with a target nucleic
acid, for example, DNA or RNA in a sample. One type of
hybridization application, in situ hybridization (ISH), includes
hybridization to a target in a specimen wherein the specimen may be
in vivo, in vitro, in situ, or for example, fixed or adhered to a
glass slide. The probes may be labeled to make identification of
the probe-target hybrid possible by use of a fluorescence or bright
field microscope/scanner. Such labeled probes can be used, for
example, to detect genetic abnormalities in a target sequence,
providing valuable information about, e.g., prenatal disorders,
cancer, and other genetic or infectious diseases.
[0005] The efficiency and accuracy of nucleic acid hybridization
assays mostly depend on at least one of three major factors: a)
denaturation (i.e., separation of, e.g., two nucleic acid strands)
conditions, b) renaturation (i.e., re-annealing of, e.g., two
nucleic acid strands) conditions, and c) post-hybridization washing
conditions.
[0006] In order for the probes or primers to bind to the target
nucleic acid in the sample, complementary strands of nucleic acid
may be separated. This strand separation step, termed
"denaturation," typically requires aggressive conditions to disrupt
the hydrogen and hydrophobic bonds in the double helix. Once the
complementary strands of nucleic acid have been separated, a
"renaturation" or "reannealing" step allows the primers or probes
to bind to the target nucleic acid in the sample. This step is also
sometimes referred to as the "hybridization" step.
[0007] Traditional hybridization experiments, such as ISH assays,
use high temperatures (e.g., 95.degree. C. to 100.degree. C.)
and/or high concentration formamide-containing solutions (e.g.,
greater than 40%) 10 to denature doubled stranded nucleic acid.
However, these methods have significant drawbacks.
[0008] For example, heat can be destructive to the structure of the
nucleic acid itself because the phosphodiester bonds may be broken
at high temperatures, leading to a collection of broken single
stranded nucleic acids. In addition, heat can lead to complications
when small volumes are used, since evaporation of aqueous buffers
is difficult to control.
[0009] Formamide is a solvent that has a destabilizing effect on
the helical state of, for example, DNA, RNA, and analogs by
displacing loosely and uniformly bound hydrate molecules and by
causing "formamidation" of the Watson-Crick binding sites. Thus,
formamide has a destabilizing effect on double stranded nucleic
acids and analogs, allowing denaturation to occur at lower
temperatures. However, although formamide lowers the melting
temperature (Tm) of double-stranded nucleic acid, when used at high
concentrations, it also significantly prolongs the renaturation
time, as compared to aqueous denaturation solutions without
formamide.
[0010] In addition, using formamide has disadvantages beyond a long
processing time. Formamide is a toxic, hazardous material, subject
to strict regulations for use and waste. Furthermore, the use of of
formamide appears to cause morphological destruction of cellular,
nuclear, and/or chromosomal structure.
[0011] Moreover, the use of formamide, while accepted as the
standard technique for hybridization, is hampered by the long time
required to complete the hybridization, depending on the conditions
and the nucleic acid fragments or sequences used. For example, the
denaturation step is followed by a longer time-consuming
hybridization step, which, e.g., in a traditional fluorescent in
situ hybridization (FISH) protocol takes 14-24 hours, and can even
take up to 72 hours. Examples of traditional hybridization times
are shown in FIGS. 1 and 2.
[0012] The step of re-annealing (i.e., hybridizing) two
complementary strands of nucleic acid chains is by far the most
time-consuming aspect of an assay using hybridization. Until now it
was believed that the use of chaotropic agents, such as formamide,
guanidinium hydrogen, and urea, which interfere with the
Watson-Crick binding sites of nucleic acid bases and thereby
disturb the hydrogen bonds between complementary nucleic acid
bases, was one way to lower the melting temperature (Tm) of the
complementary chains. However, although the use of chaotropic
agents lowers the Tm, these agents appear to significantly prolong
the hybridization time compared to hybridization in an aqueous
solution without a chaotropic agent. Furthermore, besides the
disadvantage of the long processing time, the use of formamide
appears to incur morphological destruction of cellular, nuclear,
and/or chromosomal structure. Finally, formamide is considered a
toxic and hazardous chemical to humans.
[0013] In some embodiments, the present invention provides several
potential advantages over prior art hybridization applications,
such as faster hybridization times, lower hybridization
temperatures, and less toxic hybridization solvents.
SUMMARY OF THE INVENTION
[0014] It is an object of the present invention to provide methods
and compositions which result in hybridization applications having
at least one of the following advantages: highly sensitive,
technically easy, flexible and reliable hybridization procedures,
and fast analyses. In some embodiments, for example, one advantage
may be the ability to tailor the hybridization time by varying the
temperature of the hybridization reaction to a much greater degree
than is available using prior art methods. For example,
hybridization may be possible at room temperature.
[0015] In one embodiment, the compositions and methods of the
invention lower the energy necessary for hybridization. The
compositions and methods of the invention are applicable to any
hybridization technique. The compositions and methods of the
invention are also applicable to any molecular system that
hybridizes or binds using base pairing, such as, for example, DNA,
RNA, a peptide nucleic acid (PNA) or locked nucleic acid (LNA), and
synthetic and natural analogs thereof.
[0016] The nucleic acid hybridization method and compositions of
the present invention may be used for the in vivo or in vitro
analysis of genomic DNA, chromosomes, chromosome fragments, genes,
and chromosome aberrations such as translocations, deletions,
amplifications, insertions, mutations, or inversions associated
with a normal condition or a disease. Further, the methods and
compositions are useful for detection of infectious agents as well
as changes in levels of expression of RNA, e.g., messenger RNA
(mRNA) and its complementary DNA (cDNA).
[0017] Other uses include the in vivo, in vitro, or in situ
analysis of mRNA, viral RNA, viral DNA, small interfering RNA
(siRNA), small nuclear RNA (snRNA), non-coding RNA (ncRNA, e.g.,
tRNA and rRNA), transfer messenger RNA (tmRNA), micro RNA (miRNA),
piwi-interacting RNA (piRNA), long noncoding RNA, small nucleolar
RNA (snoRNA), antisense RNA, double-stranded RNA (dsRNA),
methylations and other base modifications, single nucleotide
polymorphisms (SNPs), copy number variations (CNVs), and nucleic
acids labeled with, e.g., radioisotopes, fluorescent molecules,
biotin, 2,4-dinitrophenol (DNP), digoxigenin (DIG), or antigens,
alone or in combination with unlabeled nucleic acids.
[0018] The nucleic acid hybridization method and compositions of
the present invention are useful for in vivo, in vitro, or in situ
analysis of nucleic acids using techniques such as northern blot,
Southern blot, flow cytometry, autoradiography, fluorescence
microscopy, chemiluminescence, immunohistochemistry, virtual
karyotype, gene assay, DNA microarray (e.g., array comparative
genomic hybridization (array CGH)), gene expression profiling, Gene
ID, Tiling array, gel electrophoresis, capillary electrophoresis,
and in situ hybridizations such as FISH, SISH, CISH.
[0019] In one embodiment, the methods and compositions of the
invention are useful for nucleic acid hybridization applications,
with the proviso that such applications do not include
amplification of the nucleic acid such as, e.g., by polymerase
chain reaction (PCR), in situ PCR, etc.
[0020] The methods and compositions of the invention may be used on
in vitro and in vivo samples 30 such as bone marrow smears, blood
smears, paraffin embedded tissue preparations, enzymatically
dissociated tissue samples, bone marrow, amniocytes, cytospin
preparations, imprints, etc.
[0021] In one embodiment, the invention provides methods and
compositions for hybridizing at least one molecule to a target. The
invention may, for example, reduce the dependence on formamide. For
example, the methods and compositions of the invention may lower
the energy barrier to hybridization using cyclic and/or non-cyclic
solvents. The lower energy barrier may reduce the time and or
temperature necessary for hybridization. Thus, in some aspects, the
present invention overcomes a major time consuming step in
hybridization assays.
[0022] One aspect of the invention is a composition or solution for
use in hybridization applications. Compositions for use in the
invention include an aqueous composition comprising at least one
nucleic acid sequence and at least one solvent in an amount
effective to denature double-stranded nucleotide sequences. In
certain embodiments, the solvent is chosen from
N,N-dimethyl-acetamide, isobutyramide, tetramethylene sulfoxide,
and 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone. In other
embodiments, the solvent is non-cyclic.
[0023] According to yet another aspect, the invention discloses a
method of hybridizing nucleic acid sequences comprising: [0024]
providing a first nucleic acid sequence, [0025] providing a second
nucleic acid sequence, [0026] providing a hybridization composition
comprising at least one solvent in an amount effective to denature
double-stranded nucleotide sequences, and [0027] combining the
first and the second nucleic acid sequence and the hybridization
composition for at least a time period sufficient to hybridize the
first and second nucleic acid sequences, wherein the solvent is
chosen from butadiene sulfone, tetrahydrothiophene 1-oxide
(tetramethylene sulfoxide), d-valerolactam (2-piperidone),
2-pyrrolidone, cyclopentanone, N-methyl-2-pyrrolidone,
1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinonc,
N,N-dimethyl-acetamide and isobutyramide.
[0028] The method may, for example, comprise: [0029] providing a
first nucleic acid sequence, and [0030] applying a hybridization
composition comprising a second nucleic acid sequence and a solvent
in an amount effective to denature double stranded nuleotide
sequences to said first nucleic acid sequence for at least a time
period sufficient to hybridize the first and second nucleic acid
sequences, wherein the solvent is chosen from butadiene sulfone,
tetrahydrothiophene 1-oxide (tetramethylene sulfoxide),
d-valerolactam (2-piperidone), 2-pyrrolidone, cyclopentanone,
N-methyl-2-pyrrolidone, 1,3-dimethyl-3,4,5,6-tetrahydro-2
(1H)-pyrimidinone, N,N-dimethyl-acetamide and isobutyramide.
[0031] In one embodiment, the first nucleic acid sequence is in a
biological sample. In another embodiment, the biological sample is
a cytology or histology sample.
[0032] In one embodiment, the first nucleic acid sequence is a
single stranded sequence and the second nucleic acid sequence is a
double stranded sequence. In another embodiment, the first nucleic
acid sequence is a double stranded sequence in a biological sample
and the second nucleic acid sequence is a single stranded sequence.
In yet another embodiment, both the first and second nucleic acid
sequences are double stranded. In yet another embodiment, both the
first and second nucleic acid sequences are single stranded.
[0033] In one embodiment, a sufficient amount of energy to
hybridize the first and second nucleic acids is provided.
[0034] In one embodiment, the hybridization of the first nucleic
acid sequence to the second nucleic acid sequence occurs in less
than 2 hours, such as, for example, less than 1 hour.
[0035] According to yet another aspect of the present invention,
the hybridization energy is provided by heating the hybridization
composition and nucleic acid sequence. Thus, the step of
hybridizing may include the steps of heating and cooling the
hybridization composition and nucleic acid sequences.
[0036] According to another aspect of the invention, the
denaturation and hybridization steps may occur separately. For
example, the specimen may be denatured with a solution without
probe and thereafter hybridized with probe.
[0037] A further aspect of the invention comprises a method wherein
the step of providing a sufficient amount of energy to hybridize
the nucleic acids involves a heating step performed by the use of
microwaves, hot baths, hot plates, heat wire, peltier element,
induction heating, or heat lamps.
[0038] According to another aspect the present invention relates to
a method wherein the hybridization takes less than 4 hours. In some
embodiments, the hybridization takes less than 2 hours. In other
embodiments, the hybridization takes less than 1 hour. In other
embodiments, the hybridization takes less than 30 minutes. In still
other embodiments, the hybridization takes less than 15 minutes. In
other embodiments, the hybridization takes less than 5 minutes.
[0039] According to a further aspect, the invention relates to the
use of a composition comprising at least one nucleic acid sequence
and at least one solvent in an amount effective to denature
double-stranded nucleotide sequences in hybridization assays. In
certain embodiments, the solvent is chosen from butadiene sulfone,
tetrahydrothiophene 1-oxide (tetramethylene sulfoxide),
d-valerolactam (2-piperidone), 2-pyrrolidone, cyclopentanone,
N-methyl-2-pyrrolidone,
1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone,
N,N-dimethyl-acetamide and isobutyramide.
[0040] In certain embodiments, the solvent is chosen from
N,N-dimethyl-acetamide, isobutyramide, and
1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone.
[0041] In certain embodiments, the solvent is non-cyclic. In other
embodiments, the non-cyclic solvent is chosen from
N,N-dimethyl-acetamide and isobutyramide.
[0042] Non-limiting examples of effective amounts of cyclic and/or
non-cyclic solvents include, e.g., about 1% to about 95% (v/v). In
some embodiments, the concentration of solvent is 5% to 60% (v/v).
In other embodiments, the concentration of solvent is 10% to 60%
(v/v). In still other embodiments, the concentration of solvent is
30% to 50% (v/v). Concentrations of 1% to 5%, 5% to 10%, 10%, 10%
to 20%, 20% to 30%, 30% to 40%, 40% to 50%, or 50% to 60% (v/v) are
also suitable. In some embodiments, the solvent will be present at
a concentration of 0.1%, 0.25%, 0.5%, 1%, 2%, 3%, 4%, or 5% (v/v).
In other embodiments, the solvent will be present at a
concentration of 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%,
11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%,
17%, 17.5%, 18%, 18.5%, 19%, 19.5%, or 20% (v/v).
[0043] According to yet another aspect, the invention relates to
the use of a composition comprising a hybridization composition as
described in this invention for use in hybridization assays.
BRIEF DESCRIPTION OF THE DRAWINGS
[0044] FIG. 1 depicts a typical time-course for single locus
detection with primary labeled FISH probes on formaldehyde fixed
paraffin embedded tissue sections (histological specimens). The
bars represent a hybridization assay performed using a traditional
solution (top) and a typical time-course for a hybridization assay
performed using a composition of the invention (bottom). The first
bar on the left in each time-course represents the deparaffination
step; the second bar represents the heat-pretreatment step; the
third bar represents the digestion step; the fourth bar represents
the denaturation and hybridization steps; the fifth bar represents
the stringency wash step; and the sixth bar represents the mounting
step.
[0045] FIG. 2 depicts a typical time-course for single locus
detection with primary labeled FISH probes on cytological
specimens. The bars represent a hybridization assay performed using
a traditional solution (top) and a typical time-course for a
hybridization assay performed using a composition of the invention
(bottom). The first bar on the left in each time-course represents
the fixation step; the second bar represents the denaturation and
hybridization steps; the third bar represents the stringency wash
step; and the fourth bar represents the mounting step.
DETAILED DESCRIPTION
[0046] A. Definitions
[0047] Unless otherwise defined, scientific and technical terms
used in connection with the present invention shall have the
meanings that are commonly understood by those of ordinary skill in
the art. Further, unless otherwise required by context, singular
terms shall include pluralities and plural terms shall include the
singular.
[0048] In this application, the use of "or" means "and/or" unless
stated otherwise. In the context of a multiple dependent claim, the
use of "or" refers back to more than one preceding independent or
dependent claim in the alternative only.
[0049] Unless the meaning is clearly to the contrary, all ranges
set forth herein are deemed to be inclusive of the endpoints. In
the context of the present invention the following terms are to be
understood as follows:
[0050] "Biological sample" is to be understood as any in vivo, in
vitro, or in situ sample of one or more cells or cell fragments.
This can, for example, be a unicellular or multicellular organism,
tissue section, cytological sample, chromosome spread, purified
nucleic acid sequences, artificially made nucleic acid sequences
made by, e.g., a biologic based system or by chemical synthesis,
microarray, or other form of nucleic acid chip. In one embodiment,
a sample is a mammalian sample, such as, e.g., a human, murine,
rat, feline, or canine sample.
[0051] "Nucleic acid," "nucleic acid chain," and "nucleic acid
sequence" mean anything that binds or hybridizes using base pairing
including, oligomers or polymers having a backbone formed from
naturally occurring nucleotides and/or nucleic acid analogs
comprising nonstandard nucleobases and/or nonstandard backbones
(e.g., PNA or LNA), or any derivatized form of a nucleic acid.
[0052] As used herein, the term "peptide nucleic acid" or "PNA"
means a synthetic polymer having a polyamide backbone with pendant
nucleobases (naturally occurring and modified), including, but not
limited to, any of the oligomer or polymer segments referred to or
claimed as peptide nucleic acids in, e.g., U.S. Pat. Nos.
5,539,082, 5,527,675, 5,623,049, 5,714,331, 5,718,262, 5,736,336,
5,773,571, 5,766,855, 5,786,461, 5,837,459, 5,891,625, 5,972,610,
5,986,053, 6,107,470 6,201,103, 6,228,982 and 6,357,163,
WO96/04000, all of which are herein incorporated by reference, or
any of the references cited therein. The pendant nucleobase, such
as, e.g., a purine or pyrimidine base on PNA may be connected to
the backbone via a linker such as, e.g., one of the linkers taught
in PCT/US02/30573 or any of the references cited therein. In one
embodiment, the PNA has an N-(2-aminoethyl)-glycine) backbone. PNAs
may be synthesized (and optionally labeled) as taught in
PCT/US02/30573 or any of the references cited therein. PNAs
hybridize tightly, and with high sequence specificity, with DNA and
RNA, because the PNA backbone is uncharged. Thus, short PNA probes
may exhibit comparable specificity to longer DNA or RNA probes. PNA
probes may also show greater specificity in binding to
complementary DNA or RNA.
[0053] As used herein, the term "locked nucleic acid" or "LNA"
means an oligomer or polymer comprising at least one or more LNA
subunits. As used herein, the term "LNA subunit" means a
ribonucleotide containing a methylene bridge that connects the
2'-oxygen of the ribose with the 4'-carbon. See generally, Kurreck,
Eur. J. Biochem., 270:1628-44 (2003).
[0054] Examples of nucleic acids and nucleic acid analogs also
include polymers of nucleotide monomers, including double and
single stranded deoxyribonucleotides (DNA), ribonucleotides (RNA),
.alpha.-anomeric forms thereof, synthetic and natural analogs
thereof, and the like. The nucleic acid chain may be composed
entirely of deoxyribonucleotides, ribonucleotides, peptide nucleic
acids (PNA), locked nucleic acids (LNA), synthetic or natural
analogs thereof, or mixtures thereof DNA, RNA, or other nucleic
acids as defined herein can be used in the method and compositions
of the invention.
[0055] "Aqueous solution" is to be understood as a solution
containing water, even small amounts of water. For example, a
solution containing 1% water is to be understood as an aqueous
solution.
[0056] "Hybridization application," "hybridization assay,"
"hybridization experiment," "hybridization procedure,"
"hybridization technique," "hybridization method," etc. are to be
understood as referring to any process that involves hybridization
of nucleic acids. Unless otherwise specified, the terms
"hybridization" and "hybridization step" are to be understood as
referring to the re-annealing step of the hybridization procedure
as well as the denaturation step.
[0057] "Hybridization composition" refers to an aqueous solution of
the invention for performing a hybridization procedure, for
example, to bind a probe to a nucleic acid sequence. Hybridization
compositions may comprise, e.g., a solvent and at least one nucleic
acid sequence. Hybridization compositions do not comprise enzymes
or other components, such as deoxynucleoside triphosphates (dNTPs),
for amplifying nucleic acids in a biological sample.
[0058] "Hybridization solution" refers to an aqueous solution for
use in a hybridization composition of the invention. Hybridization
solutions are discussed in detail below and may comprise, e.g.,
buffering agents, accelerating agents, chelating agents, salts,
detergents, and blocking agents.
[0059] "Repetitive Sequences" is to be understood as referring to
the rapidly reannealing (approximately 25%) and/or intermediately
reannealing (approximately 30%) components of mammalian genomes.
The rapidly reannealing components contain small (a few nucleotides
long) highly repetitive sequences usually found in tandem (e.g.,
satellite DNA), while the intermediately reannealing components
contain interspersed repetitive DNA. Interspersed repeated
sequences are classified as either SINEs (short interspersed repeat
sequences) or LINEs (long interspersed repeated sequences), both of
which are classified as retrotransposons in primates. SINES and
LINEs include, but are not limited to, Alu-repeats, Kpn-repeats,
di-nucleotide repeats, tri-nucleotide repeats, tetra-nucleotide
repeats, penta-nucleotide repeats and hexa-nucleotide repeats. Alu
repeats make up the majority of human SINEs and are characterized
by a consensus sequence of approximately 280 to 300 by that consist
of two similar sequences arranged as a head to tail dimer. In
addition to SINEs and LINEs, repeat sequences also exist in
chromosome telomeres at the termini of chromosomes and chromosome
centromeres, which contain distinct repeat sequences that exist
only in the central region of a chromosome. However, unlike SINEs
and LINEs, which are dispersed randomly throughout the entire
genome, telomere and centromere repeat sequences are localized
within a certain region of the chromosome.
[0060] "Non-toxic" and "reduced toxicity" are defined with respect
to the toxicity labeling of formamide according to "Directive
1999/45/EC of the European Parliament and of the Council of 31 May
1999 concerning the approximation of the laws, regulations and
administrative provisions of the Member States relating to the
classification, packaging, and labelling of dangerous preparations"
(ecb.jrc.it/legislation/1999L0045EC.pdf) ("Directive"). According
to the Directive, toxicity is defined using the following
classification order: T+"very toxic"; T "toxic", C "corrosive", Xn
"harmful", .Xi "irritant." Risk Phrases ("R phrases") describe the
risks of the classified toxicity. Formamide is listed as T (toxic)
and R61 (may cause harm to the unborn child).
[0061] As used herein, the terms "reduced temperature denaturation"
and "low temperature denaturation" refer to denaturations performed
below about 95.degree. C.
[0062] As used herein, the terms "room temperature" and "RT" refer
to about 20.degree. C. to about 25.degree. C., unless otherwise
stated.
[0063] B. Compositions, Buffers, and Solutions
[0064] (1) Hybridization Solutions
[0065] Traditional hybridization solutions are known in the art.
Such solutions may comprise, for example, buffering agents,
accelerating agents, chelating agents, salts, detergents, and
blocking agents.
[0066] For example, the buffering agents may include sodium
chloride/sodium citrate (SSC),
(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), sodium
chloride/sodium phosphate (monobasic)/ethylenediaminetetraacetic
acid (SSPE), piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES),
trimellitic anhydride acid chloride (TMAC), Tris (hydroxymethyl)
aminomethane (TRIS), sodium dodecyl sulfate/Tris (hydroxymethyl)
aminomethane/ethylenediaminetetraacetic (STE), citric acid, a
phosphate buffer, such as, e.g., potassium phosphate or sodium
pyrrophosphate, etc. In some embodiments, the term "phosphate
buffer" refers to a phosphate buffered solution containing
NaH.sub.2PO.sub.4,, 2H.sub.2O (sodium phosphate dibasic dihydrate)
and Na.sub.2HPO.sub.4, H.sub.2O (sodium phosphate monobasic
monohydrate). The buffering agents may be present at concentrations
from 0.01.times. to 50.times., such as, for example, 0.01.times.,
0.1.times., 0.5.times., 1.times., 2.times., 5.times., 10.times.,
15.times., 20.times., 25.times., 30.times., 35.times., 40.times.,
45.times., or 50.times.. Typically, the buffering agents are
present at concentrations from 0.1.times. to 10.times..
[0067] The accelerating agents may include polymers such as
Ficoll.RTM., Polyvinylpyrrolidone (PVP), heparin, dextran sulfate,
proteins such as Bovine serum albumin (BSA), glycols such as
ethylene glycol, glycerol, 1,3 propanediol, propylene glycol, or
diethylene glycol, combinations thereof such as Dernhardt's
solution and Bovine Lacto Transfer Technique Optimizer (BLOTTO),
and organic solvents such as dimethylformamide, Dimethyl sulfoxide
(DMSO), etc. The accelerating agent may be present at
concentrations from 1% to 80% or 0.1.times. to 10.times., such as,
for example, 0.1% (or 0.1.times.), 0.2% (or 0.2.times.), 0.5% (or
0.5.times.), 1% (or 1.times.), 2% (or 2.times.), 5% (or 5.times.),
10% (or 10.times.), 15% (or 15.times.), 20% (or 20.times.), 25% (or
25.times.), 30% (or 30.times.), 40% (or 40.times.), 50% (or
50.times.), 60% (or 60.times.), 70% (or 70.times.), or 80% (or
80.times.). Typically, DMSO, dextran sulfate, and glycol are
present at concentrations from 5% to 10%, such as 5%, 6%, 7%, 8%,
9%, or 10%.
[0068] The chelating agents may include Ethylenediaminetetraacetic
acid (EDTA), ethylene glycol tetraacetic acid (EGTA), etc. The
chelating agents may be present at concentrations from 0.1 mM to 10
mM, such as 0.1 mM, 0.2 mM, 0.5 mM, 1 mNI, 2 mM, 3 mM, 4 mM, 5 mM,
6 mM, 7 mM, 8 mM, 9 mM, or 10 mM. Typically, the chelating agents
are present at concentrations from 0.5 mM to 5 mM, such as 0.5 mM,
1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, or 5
mM.
[0069] The salts may include sodium chloride (NaCl), sodium
phosphate, magnesium phosphate, etc. The salts may be present at
concentrations from 1 mM to 750 mM, such as 1 mNI, 5 mNI, 10 mM, 20
mM, 30 mM, 40 mNI, 50 mM, 100 mM, 200 mM, 300 mM, 400 mM, 500 mM,
600 mM, 700 mM, or 750 mM. In some embodiments, the salts are
present at concentrations from 1 mM to 1000 mM. In other
embodiments, the salts are present at concentrations from 300 mM to
700 mM, 400 mM to 700 mM, or 500 mM to 700 mM.
[0070] The detergents may include TWEEN sodium dodecyl sulfate
(SDS), Triton.TM., CHAPS, deoxycholic acid, etc. The detergent may
be present at concentrations from 0.001% to 10%, such as, for
example, 0.0001, 0.01, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10%.
Typically, the detergents are present at concentrations from 0.01%
to 1%, such as 0.01%, 0.02%, 0.03%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%,
0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%.
[0071] The nucleic acid blocking agents may include, yeast tRNA,
homopolymer DNA, denatured salmon sperm DNA, herring sperm DNA,
total human DNA, COT1 DNA, etc. The blocking nucleic acids may be
present at concentrations of 0.05 mg/mL to 100 mg/mL.
[0072] A great variation exists in the literature regarding
traditional hybridization solutions. For example, a traditional
hybridization solution may comprise 5.times. or 6.times.SSC, 0.01 M
EDTA, 5.times. Dernhardt's solution, 0.5% SDS, and 100 mg/mL
sheared, denatured salmon sperm DNA. Another traditional
hybridization solution may comprise 50 mM HEPES, 0.5 M NaCl, and
0.2 mM EDTA. A typical hybridization solution for FISH on
biological specimens for RNA detection may comprise, e.g.,
2.times.SSC, 10% dextran sulfate, 2 mM vanadyl-ribonucleoside
complex, 50% formamide, 0.02% RNAse-free BSA, and 1 mg/mL E. coli
tRNA. A typical hybridization solution for FISH on biological
specimens for DNA detection may comprise, e.g., 2.times.SSC, 10%
dextran sulfate, 50% formamide, and e.g., 0.3 mg/mL salmon sperm
DNA or 0.1 mg/mL COT1 DNA. Other typical hybridization solutions
may comprise 40% formamide, 10% dextran sulfate, 300 mM NaCl, 5 mM
phosphate buffer, Alu-PNA (blocking PNA) or COT-1 DNA, and in some
cases 0.1 .mu.g/.mu.L total human DNA (THD).
[0073] The compositions of the invention may comprise a
hybridization solution comprising any of the components of
traditional hybridization solutions recited above. The traditional
components may be present at the same concentrations as used in
traditional hybridization solutions, or may be present at higher or
lower concentrations, or may be omitted completely.
[0074] For example, if the compositions of the invention comprise
salts such as NaCl and/or phosphate buffer, the salts may be
present at concentrations that are about twice as high as
traditional concentrations. For example, in some embodiments, the
salts may be present at concentrations of 0-1200 mM NaCl and/or
0-200 mM citrate buffer. In some embodiments, the concentrations of
salts may be, for example, 300 mM NaCl and/or 5 mM citrate buffer,
or 600 mM NaCl and/or 10 mM citrate buffer.
[0075] If the compositions of the invention comprise accelerating
agents such as dextran sulfate, glycol, or DMSO, the dextran
sulfate may be present at concentrations that are about twice as
high as traditional concentrations. For example, in some
embodiments, the dextran sulfate may be present at concentrations
of from 5% to 40%. In some embodiments, the concentration of
dextran sulfate may be 10% or 20%. In some embodiments, the glycol
may be present at concentrations of from 0.1% to 10%, and the DMSO
may be from 0.1% to 10%. In other embodiments, the concentration of
ethylene glycol, 1,3 propanediol, or glycerol may be 1% to 10%. In
some embodiments, the concentration of DMSO may be 1%. In some
embodiments, the aqueous composition does not comprise DMSO.
[0076] If the compositions of the invention comprise citric acid,
the concentrations may range from 25 1 mM to 50 mM and the pH may
range from 5.0 to 8.0. In some embodiments the concentration of
citric acid may be 10 mM and the pH may be 6.2.
[0077] The compositions of the invention may comprise agents that
reduce non-specific binding to, for example, the cell membrane,
such as salmon sperm or small amounts of total human DNA or, for
example, they may comprise blocking agents to block binding of,
e.g., repeat sequences to the target such as larger amounts of
total human DNA or repeat enriched DNA or specific blocking agents
such as PNA or LNA fragments and sequences. These agents may be
present at concentrations of from 0.01-100 .mu.g/.mu.L or 0.01-100
.mu.M. For example, in some embodiments, these agents will be 0.1
.mu.g/.mu.L total human DNA, or 0.1 .mu.g/.mu.L non-human DNA, such
as herring sperm, salmon sperm, or calf thymus DNA, or 5 .mu.M
blocking PNA.
[0078] One aspect of the invention is a composition or solution for
use in hybridization. Compositions for use in the invention include
an aqueous composition comprising a nucleic acid sequence and a
solvent. In some embodiments, the solvent is chosen from
N,N-dimethyl-acetamide, isobutyramide, tetramethylene sulfoxide,
and 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone. In other
embodiments, the solvent is non-cyclic.
[0079] If the compositions of the invention are used in a
hybridization assay, they may further comprise one or more nucleic
acid probes. The probes may be directly or indirectly labeled with
detectable compounds such as enzymes, chromophores, fluorochromes,
and haptens. The DNA probes may be present at concentrations of 0.1
to 100 ng/.mu.L. For example, in some embodiments, the probes may
be present at concentrations of 1 to 10 ng/4. The PNA probes may be
present at concentrations of 0.5 to 5000 nM. For example, in some
embodiments, the probes may be present at concentrations of 5 to
1000 nM.
[0080] In one embodiment, a composition of the invention comprises
a mixture of 15% of N,N-dimethyl-acetamide, isobutyramide,
tetramethylene sulfoxide, or
1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone, 20% dextran
sulfate, 600 mM NaCl, 10 mM citric acid buffer pH 6.2. Another
exemplary composition comprises a mixture of 15% of
N,N-dimethyl-acetamide, isobutyramide, tetramethylene sulfoxide, or
1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone, 20% dextran
sulfate, 600 mM NaCl, 10 mM citric acid buffer pH 6.2, and 0.1
.mu.g/.mu.L herring sperm DNA, or salmon sperm DNA, or calf thymus
DNA, or 0.5% formamide, or 1% ethylene glycol, or 1% 1,3
propanediol.
[0081] (2) Optimization for Particular Applications
[0082] The compositions of the invention can be varied in order to
optimize results for a particular application. For example, the
concentration of cyclic and/or non-cyclic solvents, salt,
accelerating agent, blocking agent, and/or hydrogen ions (i.e. pH)
may be varied in order to improve results for a particular
application. The cyclic and/or non-cyclic solvents is chosen from
butadiene sulfone, tetrahydrothiophene 1-oxide (tetramethylene
sulfoxide), d-valerolactam (2-piperidone), 2-pyrrolidone,
cyclopentanone, N-methyl-2-pyrrolidone,
1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone,
N,N-dimethyl-acetamide and isobutyramide.
[0083] The concentrations of salt and dextran sulfate may also be
varied in order to improve signal intensity and background staining
Generally, as the concentrations of salt and dextran sulfate
increase, the signal intensity may increase while background
decreases. Likewise, signal intensity may increase as dextran
sulfate concentration increases from, for example, 0% to 20%.
[0084] In addition, the types probes used in the compositions of
the invention may be varied to improve results. For example, in
some aspects of the invention, combinations of DNA/DNA probes may
show less background than combinations of DNA/PNA probes in the
compositions of the invention or vice versa. On the other hand, PNA
probes may tend to show stronger signals than DNA probes, for
example, under low salt concentrations.
[0085] C. Applications, Methods, and Uses
[0086] (1) Analytical Samples
[0087] The methods and compositions of the invention may be used
fully or partly in all types of hybridization applications in the
fields of cytology, histology, or molecular biology. According to
one embodiment, the first or the second nucleic acid sequence in
the methods of the invention is present in a biological sample.
Examples of such samples include, e.g., tissue samples, cell
preparations, cell fragment preparations, and isolated or enriched
cell component preparations. The sample may originate from various
tissues such as, e.g., breast (e.g., mammacarcinoma samples), lung,
colorectal, prostate, lung, head & neck, stomach, pancreas,
esophagus, liver, and bladder, or other relevant tissues and
neoplasia thereof, any cell suspension, blood sample, fine needle
aspiration, ascites fluid, sputum, peritoneum wash, lung wash,
urine, feces, cell scrape, cell smear, cytospin or cytoprep
cells.
[0088] The sample may be isolated and processed using standard
protocols. Cell fragment preparations may, e.g., be obtained by
cell homogenizing, freeze-thaw treatment or cell lysing. The
isolated sample may be treated in many different ways depending of
the purpose of obtaining the sample and depending on the routine at
the site. Often the sample is treated with various reagents to
preserve the tissue for later sample analysis, alternatively the
sample may be analyzed directly. Examples of widely used methods
for preserving samples are formalin-fixed followed by
paraffin-embedding and cryo-preservation.
[0089] For metaphase spreads, cell cultures are generally treated
with colcemid, or anther suitable spindle pole disrupting agent, to
stop the cell cycle in metaphase. The cells are then fixed and
spotted onto microscope slides, treated with formaldehyde, washed,
and dehydrated in ethanol. Probes are then added and the samples
are analyzed by any of the techniques discussed below.
[0090] Cytology involves the examination of individual cells and/or
chromosome spreads from a biological sample. Cytological
examination of a sample begins with obtaining a specimen of cells,
which can typically be done by scraping, swabbing or brushing an
area, as in the case of cervical specimens, or by collecting body
fluids, such as those obtained from the chest cavity, bladder, or
spinal column, or by fine needle aspiration or fine needle biopsy,
as in the case of internal tumors. In a conventional manual
cytological preparation, the sample is transferred to a liquid
suspending material and the cells in the fluid are then transferred
directly or by centrifugation-based processing steps onto a glass
microscope slide for viewing. In a typical automated cytological
preparation, a filter assembly is placed in the liquid suspension
and the filter assembly both disperses the cells and captures the
cells on the filter. The filter is then removed and placed in
contact with a microscope slide. The cells are then fixed on the
microscope slide before analysis by any of the techniques discussed
below.
[0091] In a traditional DNA hybridization experiment using a
cytological sample, slides containing the specimen are immersed in
a formaldehyde buffer, washed, and then dehydrated in ethanol. The
probes are then added and the specimen is covered with a coverslip.
The slide is optionally incubated at a temperature sufficient to
denature any double-stranded nucleic acid in the specimen (e.g., 5
minutes at 67.degree. C.) and then incubated at a temperature
sufficient to allow hybridization (e.g., overnight at 45.degree.
C.). After hybridization, the coverslips are removed and the
specimens are subjected to a high-stringency wash (e.g., 10 minutes
at 65.degree. C.) followed by a series of low-stringency washes
(e.g., 2.times.3 minutes at room temperature). The samples are then
dehydrated and mounted for analysis.
[0092] In a traditional RNA hybridization experiment using
cytological samples, cells are equilibrated in 40% formamide,
1.times.SSC, and 10 mM sodium phosphate for 5 min, incubated at
37.degree. C. overnight in hybridization reactions containing 20 ng
of oligonucleotide probe (e.g., a mix of labeled 50 by oligos),
1.times.SSC, 40% formamide, 10% dextran sulfate, 0.4% BSA, 20 mM
ribonucleotide vanadyl complex, salmon testes DNA (10 mg/ml), E.
coli tRNA (10 mg/ml), and 10 mM sodium phosphate. Then washed twice
with 4.times.SSC/40% formamide and again twice with 2.times.SSC/40%
formamide, both at 37.degree. C., and then with 2.times.SSC three
times at room temperature. Digoxigenin-labeled probes can then e.g.
be detected by using a monoclonal antibody to digoxigenin
conjugated to Cy3. Biotin-labeled probes can then e.g. be detected
by using streptavidin-Cy5. Detection can be by fluorescence or
chromogenic, e.g. CISH.
[0093] Histology involves the examination of cells in thin slices
of tissue. To prepare a tissue sample for histological examination,
pieces of the tissue are fixed in a suitable fixative, typically an
aldehyde such as formaldehyde or glutaraldehyde, and then embedded
in melted paraffin wax. The wax block containing the tissue sample
is then cut on a microtome to yield thin slices of paraffin
containing the tissue, typically from 2 to 10 microns thick. The
specimen slice is then applied to a microscope slide, air dried,
and heated to cause the specimen to adhere to the glass slide.
Residual paraffin is then dissolved with a suitable solvent,
typically xylene, toluene, or others. These so-called
deparaffinizing solvents are then removed with a
washing-dehydrating type reagent prior to analysis of the sample by
any of the techniques discussed below. Alternatively, slices may be
prepared from frozen specimens, fixed briefly in 10% formalin or
other suitable fixative, and then infused with dehydrating reagent
prior to analysis of the sample.
[0094] In a traditional DNA hybridization experiment using a
histological sample, formalin-fixed paraffin embedded tissue
specimens are cut into sections of 2-6 ittm and collected on
slides. The paraffin is melted (e.g., 30-60 minutes at 60.degree.
C.) and then removed (deparaffinated) by washing with xylene (or a
xylene substitute), e.g., 2.times.5 minutes. The samples are
rehydrated, washed, and then pre-treated (e.g., 10 minutes at
95-100.degree. C.). The slides are washed and then treated with
pepsin or another suitable permeabilizer, e.g., 3-15 minutes at
37.degree. C. The slides are washed (e.g., 2.times.3 minutes),
dehydrated, and probe is applied. The specimens are covered with a
coverslip and the slide is optionally incubated at a temperature
sufficient to denature any double-stranded nucleic acid in the
specimen (e.g., 5 minutes at 67.degree. C.), followed by incubation
at a temperature sufficient to allow hybridization (e.g., overnight
at 45.degree. C.). After hybridization, the coverslips are removed
and the specimens are subjected to a high-stringency wash (e.g., 10
minutes at 65.degree. C.) followed by a series of low-stringency
washes (e.g., 2.times.3 minutes at room temperature). The samples
are then dehydrated and mounted for analysis.
[0095] In a traditional RNA hybridization experiment using a
histological sample, slides with FFPE tissue sections are
deparaffinized in xylene for 2.times.5 min, immersed in 99% ethanol
2.times.3 min, in 96% ethanol 2.times.3 min, and then in pure water
for 3 min. Slides are placed in a humidity chamber, Proteinase K is
added, and slides are incubated at RT for 5-15 min. Slides are
immersed in pure water for 2.times.3 min, immersed in 96% ethanol
for 10 sec, and air-dried for 5 min. Probes are added to the tissue
section and covered with coverslip. The slides are incubated at
55.degree. C. in humidity chamber for 90 min. After incubation, the
slides are immersed in a stringent wash solution at 55 .degree. C.
for 25 min, and then immersed in TBS for 10 sec. The slides are
incubated in a humidity chamber with antibody for 30 min. The
slides are immersed in TBS for 2.times.3 min, then in pure water
for 2.times.1 min, and then placed in a humidity chamber. The
slides are then incubated with substrate for 60 min, and immersed
in tap water for 5 min.
[0096] In a traditional northern blot procedure, the RNA target
sample is denatured for 10 minutes at 65.degree. C. in RNA loading
buffer and immediately placed on ice. The gels are loaded and
electrophoresed with 1.times. MOPS buffer (10.times. MOPS contains
200 mM morpholinopropansulfonic acid, 50 mNI sodium acetate, 10 mM
EDTA, pH 7.0) at 25 V overnight. The gel is then pre-equilibrated
in 20.times.SSC for 10 min and the RNA is transferred to a nylon
membrane using sterile 20.times.SSC as transfer buffer. The nucleic
acids are then fixed on the membrane using, for example, U V-cross
linking at 120 mJ or baking for 30 min at 120.degree. C. The
membrane is then washed in water and air dried. The membrane is
placed in a sealable plastic bag and prehybridized without probe
for 30 min at 68.degree. C. The probe is denatured for 5 min at
100.degree. C. and immediately placed on ice. Hybridization buffer
(prewarmed to 68.degree. C.) is added and the probe is hybridized
at 68.degree. C. overnight. The membrane is then removed from the
bag and washed twice for 5 min each with shaking in a low
stringency wash buffer (e.g., 2.times.SSC, 0.1% SDS) at room
temperature. The membrane is then washed twice for 15 min each in
prewarmed high stringency wash buffer (e.g., 0.1.times.SSC, 0.1%
SDS) at 68.degree. C. The membrane may then be stored or
immediately developed for detection.
[0097] Additional examples of traditional hybridization techniques
can be found, for example, in Sambrook et al., Molecular Cloning A
Laboratory Manual, 2.sup.nd Ed., Cold Spring Harbor Laboratory
Press, (1989) at sections 1.90-1.104, 2.108-2.117, 4.40-4.41,
7.37-7.57, 8.46-10.38, 11.7-11.8, 11.12-11.19, 11.38, and
11.45-11.57; and in Ausubel et al., Current Protocols in Molecular
Biology, John Wiley & Sons, Inc. (1998) at sections
2.9.1-2.9.6, 2.10.4-2.10.5, 2.10.11-2.10.16, 4.6.5-4.6.9,
4.7.2-4.7.3, 4.9.7-4.9.15, 5.9.18, 6.2-6.5, 6.3, 6.4, 6.3.3-6.4.9,
5.9.12-5.9.13, 7.0.9, 8.1.3, 14.3.1-14.3.4, 14.9, 15.0.3-15.0.4,
15.1.1-15.1.8, and 20.1.24-20.1.25.
[0098] (2) Hybridization Techniques
[0099] The compositions and methods of the present invention can be
used fully or partly in all types of nucleic acid hybridization
techniques known in the art for cytological and histological
samples. Such techniques include, for example, in situ
hybridization (ISH), fluorescent in situ hybridization (FISH;
including multi-color FISH, Fiber-FISH, etc.), chromogenic in situ
hybridization (CISH), silver in situ hybridization (SISH),
comparative genome hybridization (CGH), chromosome paints, and
arrays in situ.
[0100] Molecular probes that are suitable for use in the
hybridizations of the invention are described, e.g., in U.S. Patent
Publication No. 2005/0266459, which is incorporated herein by
reference. In general, probes may be prepared by chemical
synthesis, PCR, or by amplifying a specific DNA sequence by
cloning, inserting the DNA into a vector, and amplifying the vector
an insert in appropriate host cells. Commonly used vectors include
bacterial plasmids, cosmids, bacterial artificial chromosomes
(BACs), PI diverted artificial chromosomes (PACs), or yeast
artificial chromosomes (YACs). The amplified DNA is then extracted
and purified for use as a probe. Methods for preparing and/or
synthesizing probes are known in the art, e.g., as disclosed in
PCT/US02/30573.
[0101] In general, the type of probe determines the type of feature
one may detect in a hybridization assay. For example, total nuclear
or genomic DNA probes can be used as a species-specific 30 probe.
Chromosome paints are collections of DNA sequences derived from a
single chromosome type and can identify that specific chromosome
type in metaphase and interphase nuclei, count the number of a
certain chromosome, show translocations, or identify
extra-chromosomal fragments of chromatin. Different chromosomal
types also have unique repeated sequences that may be targeted for
probe hybridization, to detect and count specific chromosomes.
Large insert probes may be used to target unique single-copy
sequences. With these large probes, the hybridization efficiency is
inversely proportional to the probe size. Smaller probes can also
be used to detect aberrations such as deletions, amplifications,
inversions, duplications, and aneuploidy. For example,
differently-colored locus-specific probes can be used to detect
translocations via split-signal in situ hybridization.
[0102] In general, the ability to discriminate between closely
related sequences is inversely proportional to the length of the
hybridization probe because the difference in thermal stability
decreases between wild type and mutant complexes as probe length
increases. Probes of greater than 10 bp in length are generally
required to obtain the sequence diversity necessary to correctly
identify a unique organism or clinical condition of interest. On
the other hand, sequence differences as subtle as a single base
(point mutation) in very short oligomers (<10 base pairs) can be
sufficient to enable the discrimination of hybridization to
complementary nucleic acid target sequences as compared with
non-target sequences.
[0103] In one embodiment, at least one set of the in situ
hybridization probes may comprise one or more PNA probes, as
defined above and as described in U.S. Pat. No. 7,105,294, which is
incorporated herein by reference. Methods for synthesizing PNA
probes are described in PCT/US02/30573. Alternatively, or in
addition, at least one set of the hybridization probes in any of
the techniques discussed above may comprise one or more locked
nucleic acid (LNA) probes, as described in WO 99/14226, which is
incorporated herein by reference. Due to the additional bridging
bond between the 2' and 4' carbons, the LNA backbone is
pre-organized for hybridization. LNA/DNA and LNA/RNA interactions
are stronger than the corresponding DNA/DNA and DNA/RNA
interactions, as indicated by a higher melting temperature. Thus,
the compositions and methods of the invention, which decrease the
energy required for hybridization, are particularly useful for
hybridizations with LNA probes.
[0104] In one embodiment, the probes may comprise a detectable
label (a molecule that provides an analytically identifiable signal
that allows the detection of the probe-target hybrid), as described
in U.S. Patent Publication No. 2005/0266459, which is incorporated
herein by reference. The probes may be labeled to make
identification of the probe-target hybrid possible by use, for
example, of a fluorescence or bright field microscope/scanner. In
some embodiments, the probe may be labeled using radioactive labels
such as .sup.31P, .sup.33P, or .sup.32S, non-radioactive labels
such as digoxigenin and biotin, or fluorescent labels. The
detectable label may be directly attached to a probe, or indirectly
attached to a probe, e.g., by using a linker. Any labeling method
known to those in the art, including enzymatic and chemical
processes, can be used for labeling probes used in the methods and
compositions of the invention. In other embodiments, the probes are
not labeled.
[0105] In general, in situ hybridization techniques such as CGH,
FISH, CISH, and SISH, employ large, mainly unspecified, nucleic
acid probes that hybridize with varying stringency to genes or gene
fragments in the chromosomes of cells. Using large probes renders
the in situ hybridization technique very sensitive. However, the
successful use of large genomic probes in traditional hybridization
assays depends on blocking the undesired background staining
derived from, e.g., repetitive sequences that are present
throughout the genome. Traditional methods for decreasing
nonspecific probe binding include saturating the binding sites on
proteins and tissue by incubating tissue with prehybridization
solutions containing ficoll, bovine serum albumin (BSA), polyvinyl
pyrrolidone, and nucleic acids. Such blocking steps are
time-consuming and expensive. Advantageously, the methods and
compositions of the invention reduce and/or eliminate the need for
such blocking steps. However, in one embodiment, repetitive
sequences may be suppressed according to the methods known in the
art, e.g., as disclosed in PCT/US02/30573.
[0106] Bound probes may be detected in cytological and histological
samples either directly or indirectly with fluorochromes (e.g.,
FISH), organic chromogens (e.g., CISH), silver particles (e.g.,
SISH), or other metallic particles (e.g., gold-facilitated
fluorescence in situ hybridization, GOLDFISH). Thus, depending on
the method of detection, populations of cells obtained from a
sample to be tested may be visualized via fluorescence microscopy
or conventional brightfield light microscopy.
[0107] Hybridization assays on cytological and histological samples
are important tools for determining the number, size, and/or
location of specific DNA sequences. For example, in CGH, whole
genomes are stained and compared to normal reference genomes for
the detection of regions with aberrant copy number. Typically, DNA
from subject tissue and from normal control tissue is labeled with
different colored probes. The pools of DNA are mixed and added to a
metaphase spread of normal chromosomes (or to a microarray chip,
for array- or matrix-CGH). The ratios of colors are then compared
to identify regions with aberrant copy number.
[0108] FISH is typically used when multiple color imaging is
required and/or when the protocol calls for quantification of
signals. The technique generally entails preparing a cytological
sample, labeling probes, denaturing target chromosomes and the
probe, hybridizing the probe to the target sequence, and detecting
the signal. Typically, the hybridization reaction fluorescently
stains the targeted sequences so that their location, size, or
number can be determined using fluorescence microscopy, flow
cytometry, or other suitable instrumentation. DNA sequences ranging
from whole genomes down to several kilobases can be studied using
FISH. With enhanced fluorescence microscope techniques, such as,
for example, deconvolution, even a single mRNA molecule can be
detected. FISH may also be used on metaphase spreads and interphase
nuclei.
[0109] FISH has been used successfully for mapping repetitive and
single-copy DNA sequences on metaphase chromosomes, interphase
nuclei, chromatin fibers, and naked DNA molecules, and for
chromosome identification and karyotype analysis through the
localization of large repeated families, typically the ribosomal
DNAs and major tandem array families. One of the most important
applications for FISH has been in detecting single-copy DNA
sequences, in particular disease related genes in humans and other
eukaryotic model species, and the detection of infectious agents.
FISH may be used to detect, e.g., chromosomal aneuploidy in
prenatal diagnoses, hematological cancers, and solid tumors; gene
abnormalities such as oncogene amplifications, gene deletions, or
gene fusions; chromosomal structural abnormalities such as
translocations, duplications, insertions, or inversions; contiguous
gene syndromes such as microdeletion syndrome; the genetic effects
of various therapies; viral nucleic acids in somatic cells and
viral integration sites in chromosomes; etc. In multi-color FISH,
each chromosome is stained with a separate color, enabling one to
determine the normal chromosomes from which abnormal chromosomes
are derived. Such techniques include multiplex FISH (m-FISH),
spectral karyotyping (SKY), combined binary ration labeling
(COBRA), color-changing karyotyping, cross-species color banding,
high resolution multicolor banding, telomeric multiplex FISH
(TM-FISH), split-signal FISH (ssFISH), and fusion-signal FISH.
[0110] CISH and SISH may be used for many of the same applications
as FISH, and have the additional advantage of allowing for analysis
of the underlying tissue morphology, for example, in histopathology
applications. If FISH is performed, the hybridization mixture may
contain sets of distinct and balanced pairs of probes, as described
in U.S. Pat. No. 6,730,474, which is incorporated herein by
reference. For CISH, the hybridization mixture may contain at least
one set of probes configured for detection with one or more
conventional organic chromogens, and for SISH, the hybridization
mixture may contain at least one set of probes configured for
detection with silver particles, as described in Powell RD et al.,
"Metallographic in situ hybridization," Hum. Pathol., 38:1145-59
(2007).
[0111] The compositions of the invention may also be used fully or
partly in all types of molecular biology techniques involving
hybridization, including blotting and probing (e.g., Southern,
northern, etc.), and arrays. In some embodiments, the methods and
compositions of the invention are useful for nucleic acid
hybridization applications, with the proviso that such applications
do not include amplification of the nucleic acid such as, e.g., by
PCR, in situ PCR, etc.
[0112] (3) Hybridization Conditions
[0113] The method of the present invention involves the use of
compositions comprising at least one nucleic acid sequence and at
least one solvent in hybridization of nucleic acid chains. The
compositions of the present invention are particularly useful in
said method.
[0114] Hybridization methods using the compositions of the
invention may involve applying the compositions to a sample
comprising a target nucleic acid sequence, most likely in a double
stranded form. Usually, in order to secure access for the probe to
hybridize with the target sequence, the sample and composition arc
heated to denature the target nucleic acids. During denaturation
the solvent interacts with the sequence and facilitates the
denaturation of the target and the re-annealing of the probe to
target.
[0115] Hybridizations using the compositions of the invention may
be performed using the same assay methodology as for hybridizations
performed with traditional compositions. However, 30 the
compositions of the invention allow for shorter hybridization
times. For example, the heat pre-treatment, digestion,
denaturation, hybridization, washing, and mounting steps may use
the same conditions in terms of volumes, temperatures, reagents and
incubation times as for traditional compositions. Additionally, the
compositions of the invention allow for reduction of the
hybridization time in methods comprising longer hybridization
probes or fragments of hybridization probes, for example,
hybridization probes or fragments of hybridization probes
comprising 40 to 500 nucleotides, hybridization probes or fragments
of hybridization probes comprising 50 to 500 nucleotides, or
hybridization probes or fragments of hybridization probes
comprising 50 to 200 nucleotides. A great variation exists in the
traditional hybridization protocols known in the art. For example,
some protocols specify a separate denaturation step of potential
double stranded nucleotides without probe present, before the
following hybridization step, whereas other protocols will denature
the probe and sample together. The compositions of the invention
may be used in any of the traditional hybridization protocols known
in the art.
[0116] Alternatively, assays using the compositions of the
invention can be changed and optimized from traditional
methodologies, for example, by decreasing the hybridization time,
decreasing the hybridization temperatures, and/or decreasing the
hybridization volumes.
[0117] For example, in some embodiments, the denaturation
temperature is 60 to 70.degree. C., 70 to 80.degree. C., 80 to
85.degree. C., 80 to 90.degree. C., or 90 to 100.degree. C. In
other embodiments, the denaturation temperature is 70 to 90.degree.
C., 72 to 92.degree. C., or 75 to 95.degree. C. In other
embodiments, the denaturation temperature is 20 67.degree. C.
[0118] In some embodiments, the compositions of the invention will
produce strong signals when the denaturation temperature is from 60
to 100.degree. C. and the hybridization temperature is from 20 to
60.degree. C. In other embodiments, the compositions of the
invention will produce strong signals when the denaturation
temperature is from 60 to 70.degree. C., 70 to 80.degree. C., 80 to
85.degree. C., 80 to 90.degree. C., or 90 to 100.degree. C., and
the hybridization temperature is from 20 to 30.degree. C., 30 to
40.degree. C., 40 to 50.degree. C., or 50 to 60.degree. C. In other
embodiments, the compositions of the invention will produce strong
signals when the denaturation temperature is 65, 67, 70, or
72.degree. C., and the hybridization temperature is 21, 37, 40, 45,
or 50.degree. C. In some embodiments, the compositions of the
invention will produce strong signals when the denaturation
temperature is 67.degree. C. and the hybridization temperature is
45.degree. C. In other embodiments, the compositions of the
invention will produce strong signals when the denaturation
temperature is 85.degree. C. and the hybridization temperature is
45.degree. C.
[0119] In other embodiments, the compositions of the invention will
produce strong signals when the denaturation time is from 0 to 15
minutes and the hybridization time is from 0 minutes to 24 hours.
In other embodiments, the compositions of the invention will
produce strong signals when the denaturation time is from 0 to 5
minutes and the hybridization time is from 0 minute to 8 hours. In
other embodiments, the compositions of the invention will produce
strong signals when the denaturation time is 0, 1, 2, 3, 4, or 5
minutes, and the hybridization time is 0 minutes, 5 minutes, 15
minutes, 30 minutes, 60 minutes, 180 minutes, or 240 minutes. It
will be understood by those skilled in the art that in some cases,
e.g., RNA detection, a denaturation step is not required with
traditional buffers. The compositions of the invention also
eliminate the need for a denaturation step and/or reduce the
temperature required for denaturation of other types of nucleic
acids such as, for example, DNA. Thus, in one embodiment, the
hybridization time is 0 minutes, i.e., the denaturation step
required with prior art buffers is completely eliminated.
[0120] Accordingly, hybridizations using the compositions of the
invention may be performed in less than 8 hours. In other
embodiments, the hybridization step is performed in less than 6
hours. In still other embodiments, the hybridization step is
performed within 4 hours. In other embodiments, the hybridization
step is performed within 3 hours. In yet other embodiments, the
hybridization step is performed within 2 hours. In other
embodiments, the hybridization step is performed within 1 hour. In
still other embodiments, the hybridization step is performed within
30 minutes. In other embodiments, the hybridization step can take
place within 15 minutes. The hybridization step can even take place
within 10 minutes or in less than 5 minutes. FIGS. 1 and 2
illustrate a typical time-course for hybridization applications
performed on histological and cytological samples, respectively,
using the compositions of the invention compared to hybridization
applications using a traditional compositions.
[0121] Alternatively, assays using the compositions of the
invention can be changed and optimized from traditional
methodologies, for example, by decreasing the stringent wash time
and/or decreasing the stringent wash temperatures.
[0122] After complementary strands of nucleic acid have reannealed
in a hybridization application, the hybridization product will
generally comprise complementary base pairing and non-complementary
base pairing between the probe and the target nucleic acid. Any
non-complementary base pairing is then removed by a series of
post-hybridization washes. Four variables are typically adjusted to
influence the stringency of the post-hybridization washes: [0123]
1. Temperature (as temperature increases, non-perfect matches
between the probe and the target sequence will denature, i.e.,
separate, before more perfectly matched sequences). [0124] 2. Salt
conditions (as salt concentration decreases, non-perfect matches
between the probe and the target sequence will denature, i.e.,
separate, before more perfectly matched sequences). [0125] 3.
Formamide concentration (as the amount of formamide increases,
non-perfect matches between the probe and the target sequence will
denature, i.e., separate, before more perfectly matched sequences).
[0126] 4. Time (as the wash time increases, non-perfect matches
between the probe and the target sequence will denature, i.e.,
separate, before more perfectly matched sequences).
[0127] Stringent wash methods using the compositions of the
invention may involve applying the compositions to a hybridization
product comprising a target nucleic acid sequence hybridized to a
probe. During the stringent wash step, the cyclic and/or non-cyclic
solvent interacts with the hybridization product and facilitates
the denaturation of the mismatched (i.e., non-complementary)
sequences. The cyclic and/or non-cyclic solvents specified in the
present invention may speed up this process, reduce the temperature
required for the stringency wash, and reduce the harshness and
toxicity of the stringency wash conditions compared to
formamide-containing buffers.
[0128] Those skilled in the art will understand that different type
of hybridization assays, different types of samples, different
types of probe targets, different length of probes, different types
of probes, e.g. DNA,/RNA/PNA/LNA oligos, short DNA/RNA probes
(0.5-3 kb), chromosome paint probes, CGH, repetitive probes (e.g.
alpha-satellite repeats), single-locus etc., will effect the
concentrations of e.g. salt and cyclic and/or non-cyclic solvents
required to obtain the most effective post-hybridization washes.
The temperature and incubation time are also important variables
for stringent washes using the compositions of the invention. In
view of the guidance provided herein, one skilled in the art will
understand how to vary these factors to optimize the stringency
washes in hybridization applications.
[0129] Hybridization methods using the compositions of the
invention may also involve applying the compositions to a sample
comprising a target nucleic acid sequence, most likely in a double
stranded form. Usually, in order to secure access for the probe to
hybridize with the target sequence, the probe and sample are heated
together to denature any double stranded nucleic acids. It has been
argued that separate denaturation preserves morphology better,
whereas co-denaturation reduces the number of practical steps. For
these reasons, separate denaturation steps are most often used in
molecular cytogenetics applications, and co-denaturation is most
often used when tissue sections are analyzed.
[0130] Denaturation typically is performed by incubating the target
and probe (either together or separately) in the presence of heat
(e.g., at temperatures from about 70.degree. C. to about 95.degree.
C.) and cyclic and/or non-cyclic solvents. For example, chromosomal
DNA can be denatured by a combination of temperatures above
70.degree. C. (e.g., about 73.degree. C.) and a denaturation buffer
containing 70% cyclic and/or non-cyclic solvent and 2.times.SSC
(0.3M sodium chloride and 0.03M sodium citrate). Denaturation
conditions typically are established such that cell morphology is
preserved.
[0131] Furthermore, the compositions of the invention allow for
fast hybridizations using longer 20 probes or fragments of probes,
for example, probes or fragments of probes comprising 40-500
nucleotides, probes or fragments of probes comprising 50-500
nucleotides, or probes or fragments of probes comprising 50-200
nucleotides.
[0132] As hybridization time changes, the concentration of probe
may also be varied in order to produce strong signals and/or reduce
background. For example, as hybridization time decreases, the
amount of probe may be increased in order to improve signal
intensity. On the other hand, as hybridization time decreases, the
amount of probe may be decreased in order to improve background
staining.
[0133] The compositions of the invention also reduce or eliminate
the need for a blocking step during hybridization applications by
improving signal and background intensity by blocking the binding
of, e.g., repetitive sequences to the target DNA. Thus, there is no
need to use total human DNA, blocking-PNA, COT-1 DNA, or DNA from
any other source as a blocking agent. However, background levels
can be further reduced by adding agents that reduce non-specific
binding, such as to the cell membrane, such as small amounts of
total human DNA or non-human-origin DNA (e.g., salmon sperm DNA) to
a hybridization reaction using the compositions of the
invention.
[0134] The aqueous compositions of the invention furthermore
provide for the possibility to considerably reduce the
concentration of nucleic acid sequences included in the
composition. Generally, the concentration of probes may be reduced
from 2 to 8-fold compared to traditional concentrations. For
example, if HER2 DNA probes and CEN17 PNA probes are used in the
compositions of the invention, their concentrations may be reduced
by 2 to 20-fold compared to their concentrations in traditional
hybridization compositions. In other embodiments, the probe
concentrations are independently reduced by 2-fold, 3-fold, 4-fold,
5-fold, 6-fold 7-fold, 8-fold, 9-fold, or 10-fold. This feature,
along with the absence of any requirement for blocking DNA, such as
blocking-PNA or COT1, allows for an increased probe volume in
automated instrument systems compared to the traditional 10 .mu.L
volume used in traditional systems, which reduces loss due to
evaporation, as discussed in more detail below.
[0135] Reducing probe concentration also reduces background.
However, reducing the probe concentration is inversely related to
the hybridization time, i.e., the lower the concentration, the
higher hybridization time required. Nevertheless, even when
extremely low concentrations of probe are used with the aqueous
compositions of the invention, the hybridization time is still
shorter than with traditional compositions.
[0136] The compositions of the invention often allow for better
signal-to-noise ratios than traditional hybridization compositions.
For example, with certain probes, a one hour hybridization with the
compositions of the invention will produce similar background and
stronger signals than an overnight hybridization in a traditional
compositions. Background is not seen when no probe is added.
[0137] Traditional assay methods may also be changed and optimized
when using the compositions of the invention depending on whether
the system is manual, semi-automated, or automated. For example, a
semi-automated or a fully automated system will benefit from the
short hybridization times obtained with the compositions of the
invention. These changes to traditional hybridization methods may
reduce the difficulties encountered when traditional compositions
are used in such systems. For example, one problem with
semi-automated and fully automated systems is that significant
evaporation of the sample can occur during hybridization, since
such systems require small sample volumes (e.g., 10-150 .mu.L),
elevated denaturation temperatures, and extended hybridization
times (e.g., 14 hours). Thus, proportions of the components in
traditional hybridization compositions arc fairly invariable.
However, since the compositions of the invention allow for faster
hybridizations, evaporation is reduced, allowing for increased
flexibility in the proportions of the components in hybridization
compositions used in semi-automated and fully automated
systems.
[0138] Thus, the compositions and methods of the invention solve
many of the problems associated with traditional hybridization
compositions and methods.
[0139] The disclosure may be understood more clearly with the aid
of the non-limiting examples that follow, which constitute
preferred embodiments of the compositions according to the
disclosure. Other than in the examples, or where otherwise
indicated, all numbers expressing quantities of ingredients,
reaction conditions, and so forth used in the specification and
claims are to be understood as being modified in all instances by
the term "about." Accordingly, unless indicated to the contrary,
the numerical parameters set forth in the following specification
and attached claims are approximations that may vary depending upon
the desired properties sought to be obtained herein. At the very
least, and not as an attempt to limit the application of the
doctrine of equivalents to the scope of the claims, each numerical
parameter should be construed in light of the number of significant
digits and ordinary rounding approaches.
[0140] Notwithstanding that the numerical ranges and parameters
setting forth the broad scope are approximations, the numerical
values set forth in the specific example are reported as precisely
as possible. Any numerical value, however, inherently contains
certain errors necessarily resulting from the standard deviation
found in its respective testing measurements. The examples that
follow illustrate the present invention and should not in any way
be considered as limiting the invention.
EXAMPLES
[0141] Reference will now be made in detail to specific embodiments
of the invention. While the invention will be described in
conjunction with these embodiments, it will be understood that they
are not intended to limit the invention to those embodiments. On
the contrary, the invention is intended to cover alternatives,
modifications, and equivalents, which may be included within the
invention as defined by the appended claims.
[0142] The reagents used in the following examples are from Dako's
Histology FISH Accessory Kit (K5599) (Dako Denmark A/S, Glostrup
Denmark). The kits contain all the key reagents, except for probe,
required to complete a FISH procedure for formalin-fixed,
paraffin-embedded tissue section specimens. All samples were
prepared according to the manufacturer's description. The Dako
Hybridizer (S2451, Dako) was used for the digestion, denaturation,
and hybridization steps.
[0143] Evaluation of FISH slides was performed within a week after
hybridization using a Leica DM6000B fluorescence microscope,
equipped with DAPI, FITC, Texas Red single filters and FITC/Texas
Red double filter under 10.times., 20.times., 40.times., and
100.times. oil objective.
[0144] In the Examples that follow, "dextran sulfate" refers to the
sodium salt of dextran sulfate (D8906, Sigma) having a molecular
weight M.sub.w>500,000. All concentrations of solvents are
provided as v/v percentages. Citrate buffer refers to a citrate
buffered solution containing sodium citrate
(Na.sub.3C.sub.6H.sub.5O.sub.7, 2H.sub.2O; 1.06448, Merck) and
citric acid monohydrate (C.sub.6H.sub.8O.sub.7, H.sub.2O; 1.00244,
Merck).
[0145] General Histology FISH Procedure for Below Examples 1-4
[0146] Slides with cut formalin-fixed paraffin embedded (FFPE)
multiple tissue array sections from humans (tonsils,
mammacarcinoma, kidney, and colon) were baked at 60.degree. C. for
30-60 min, deparaffinated in xylene baths, rehydrated in ethanol
baths, and then transferred to Wash Buffer. The samples were then
pre-treated in Pre-Treatment Solution at a minimum of 95.degree. C.
for 10 min and washed 2.times.3 min. The samples were then digested
with Pepsin RTU at 37.degree. C. for 3 min, washed 2.times.3 min,
dehydrated in a series of ethanol evaporations, and air-dried. The
samples were then incubated with 10 .mu.L FISH probe as described
under the individual experiments. The samples were then washed with
Stringency Wash buffer at 65.degree. C. 10 min, then washed in Wash
Buffer for 2.times.3 min, then dehydrated in a series of ethanol
evaporations, and air-dried. Finally, the slides were mounted with
15 gL Antifade Mounting Medium. When the staining was completed,
observers trained to assess signal intensity, morphology, and
background of the stained slides performed the scoring.
[0147] Scoring Guidelines
[0148] The signal intensities were evaluated on a 0-3 scale with 0
meaning no signal and 3 equating to a strong signal. Between 0 and
3 there are additional grades 0.5 apart from which the observer can
assess signal intensity and background.
[0149] The signal intensity is scored after a graded system on a
0-3 scale.
TABLE-US-00001 0 No signal is seen. 1 The signal intensity is weak.
2 The signal intensity is moderate. 3 The signal intensity is
strong.
[0150] The scoring system allows the use of 1/2 grades.
[0151] The background is scored after a graded system on a 0-3
scale.
TABLE-US-00002 0 Little to no background is seen. 1 Some
background. 2 Moderate background. 3 High Background.
[0152] The scoring system allows the use of 1/2 grades.
EXAMPLE 1
[0153] This example compares the signal intensity and background
from three DNA probes and one PNA probe on FFPE tissue sections
using different solvents, at a denaturation temperature of
67.degree. C. for 10 min or at 82.degree. C. for 5 min.
[0154] FISH Probe Composition I: 3.3 ng/.mu.L HER2 TxRed labeled
DNA probe (1/3 of standard concentration) (size 218 kb) and 1/2 of
the standard concentration (300 nM) of CEN17 FITC labeled PNA
probes (both probes identical with probes from HER2 FISH
pharmDx.TM. kit (K5331, Dako)); 15% ethylene carbonate (E26258,
Sigma-Aldrich); 20% dextran sulfate; 600 mM NaCl; 10 mM citrate
buffer, pH 6.2
[0155] FISH Probe Composition II: 3.3 ng/.mu.L HER2 TxRed labeled
DNA probe (1/3 of standard concentration) (size 218 kb) and 1/2 of
the standard concentration (300 nM) of CEN17 FITC labeled PNA
probes (both probes identical with probes from HER2 FISH
pharmDx.TM. kit (K5331, Dako)); 15% Butadiene Sulfone (B84505
Sigma-Aldrich); 20% dextran sulfate; 600 mM NaCl; 10 mM citrate
buffer, pH 6.2
[0156] FISH Probe Composition III: 3.3 ng/.mu.L HER2 TxRed labeled
DNA probe (1/3 of standard concentration) (size 218 kb) and 1/2 of
the standard concentration (300 nM) of CEN17 FITC labeled PNA
probes (both probes identical with probes from HER2 FISH
pharmDx.TM. kit (K5331, Dako)); 15% Tetra-methylene sulfoxide
(T22403, Sigma-Aldrich); 20% dextran sulfate; 600 mM NaCl; 10 mM
citrate buffer, pH 6.2
[0157] FISH Probe Composition IV: 3.3 ng/.mu.L HER2 TxRed labeled
DNA probe (1/3 of standard concentration) (size 218 kb) and 1/2 of
the standard concentration (300 nM) of CEN17 FITC labeled PNA
probes (both probes identical with probes from HER2 FISH
pharmDx.TM. kit (K5331, Dako)); 15% d-valerolactam (V209,
Sigma-Aldrich); 20% dextran sulfate; 600 mM NaCl; 10 mM citrate
buffer, pH 6.2
[0158] FISH Probe Composition V: 3.3 ng/.mu.L HER2 TxRed labeled
DNA probe (1/3 of standard concentration) (size 218 kb) and 1/2 of
the standard concentration (300 nM) of CEN17 FITC labeled PNA
probes (both probes identical with probes from HER2 FISH
pharmDx.TM. kit (K5331, Dako)); 15% cyclopentanone (08299, Fluka
Analytical/Sigma-Aldrich); 20% dextran sulfate; 600 ml VI NaCl; 10
mM citrate buffer, pH 6.2.
[0159] FISH Probe Composition VI: 3.3 ng/.mu.L HER2 TxRed labeled
DNA probe (1/3 of standard concentration) (size 218 kb) and 1/2 of
the standard concentration (300 nM) of CEN17 FITC labeled PNA
probes (both probes identical with probes from HER2 FISH
pharmDx.TM. kit (K5331, Dako)); 15% 2-pyrrolidone (240338,
Sigma-Aldrich); 20% dextran sulfate; 600 mM NaCl; 10 mM citrate
buffer, pH 6.2.
[0160] FISH Probe Composition VII: 3.3 ng/.mu.L HER2 TxRed labeled
DNA probe (1/3 of standard concentration) (size 218 kb) and 1/2 of
the standard concentration (300 nM) of CEN17 FITC labeled PNA
probes (both probes identical with probes from HER2 FISH
pharmDx.TM. kit (K5331, Dako)); 15% N-methyl-2-pyrrolidone
(806722500, Merck); 20% dextran sulfate; 600 mM NaCl; 10 mM citrate
buffer, pH 6.2.
[0161] The FISH probes were incubated on FFPE tissue sections for
the indicated temperature, for the indicated amount of time, then
at 45.degree. C. for 60 min.
[0162] Results:
TABLE-US-00003 Denaturation Denaturation Signal Intensity
Composition Temperature Time Background Tx Red FITC I 67.degree. C.
10 min +1 3 DNA 3 PNA II 67.degree. C. 10 min +2-21/2 11/2 DNA 21/2
PNA III 67.degree. C. 10 min +0 2 DNA 3 PNA IV 67.degree. C. 10 min
+0 3 DNA 3 PNA V 67.degree. C. 10 min +21/2 2 DNA 3 PNA VI
67.degree. C. 10 min +1/2 3 DNA 3 PNA VII 67.degree. C. 10 min +0 2
DNA 3 PNA I 82.degree. C. 5 min +0-21/2 3 DNA 3 PNA II 82.degree.
C. 5 min +2-21/2 21/2-3 DNA 21/2-3 PNA III 82.degree. C. 5 min +1/2
21/2 DNA 3 PNA IV 82.degree. C. 5 min +11/2 3 DNA 3 PNA V
82.degree. C. 5 min +3 2 DNA 21/2-3 PNA VI 82.degree. C. 5 min +1/2
3 DNA 3 PNA VII 82.degree. C. 5 min +0-1/2 2 DNA 3 PNA
[0163] The scoring was performed on the mamma-carcinoma tissue of a
multi-tissue section. All buffers were not, except for ethylene
carbonate, present in one phase at room temperature (RT) at the
used composition concentrations. Denaturation at 82.degree. C. led
to increased background for the DNA probes of e.g. ethylene
carbonate (composition I), but not for e.g. composition III, IV and
VI.
EXAMPLE 2
[0164] This example compares the signal intensity and background
from DNA probes and PNA probes on FFPE tissue sections using
different solvents, at a denaturation temperature of 67.degree. C.
for 10 min and hybridization at 45.degree. C. for 60 min.
[0165] FISH Probe Composition I: 3.3 ng/.mu.L HER2 TxRed labeled
DNA probe (1/3 of standard concentration) (size 218 kb) and 1/2 of
the standard concentration (300 nM) of CEN17 FITC labeled PNA
probes (both probes identical with probes from HER2 FISH
pharmDx.TM. kit (K5331, Dako)); 15% ethylene carbonate (E26258,
Sigma-Aldrich); 20% dextran sulfate; 600 mM NaCl; 10 mM citrate
buffer, pH 6.2
[0166] FISH Probe Composition II: 3.3 ng/.mu.L HER2 TxRed labeled
DNA probe (1/3 of standard concentration) (size 218 kb) and 1/2 of
the standard concentration (300 nM) of CEN17 FITC labeled PNA
probes (both probes identical with probes from HER2 FISH
pharmDx.TM. kit (K5331, Dako)); 15%
1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone (251569,
Sigma-Aldrich); 20% dextran sulfate; 600 mM NaCl; 10 mM citrate
buffer, pH 6.2
TABLE-US-00004 Signal Intensity Composition Background Tx Red FITC
I +2 3 DNA 3 PNA II +1/2 2 DNA 21/2-3 PNA
[0167] The scoring was performed on the mamma-carcinoma tissue of a
multi-tissue section. Composition II was two-phased at the used
composition concentrations.
EXAMPLE 3
[0168] This example compares the signal intensity and background
from DNA probes and PNA probes on FFPE tissue sections using two
non-cyclic solvents, at a denaturation temperature of 67.degree. C.
for 10 min and hybridization at 45.degree. C. for 60 min.
[0169] FISH Probe Composition I: 3.3 ng/.mu.L HER2 TxRed labeled
DNA probe (1/3 of standard concentration) (size 218 kb) and 1/2 of
the standard concentration (300 nM) of CEN17 FITC labeled PNA
probes (both probes identical with probes from HER2 FISH
pharmDx.TM. kit (K5331, Dako)); 15% N,N-dimethyl-acetamide (72336,
Sigma-Aldrich); 20% dextran sulfate; 600 mM NaCl; 10 mM citrate
buffer, pH 6.2
[0170] FISH Probe Composition II: 3.3 ng/.mu.L HER2 TxRed labeled
DNA probe (1/3 of standard concentration) (size 218 kb) and 1/2 of
the standard concentration (300 nM) of CEN17 FITC labeled PNA
probes (both probes identical with probes from HER2 FISH
pharmDx.TM. kit (K5331, Dako)); 15% isobutyramide (144436,
Sigma-Aldrich); 20% dextran sulfate; 600 mM NaCl; 10 mM citrate
buffer, pH 6.2
TABLE-US-00005 Signal Intensity Composition Background Tx Red FITC
I +1/2 3 DNA 3 PNA II +1/2 3 DNA 3 PNA
[0171] The scoring was performed on the mamma-carcinoma tissue of a
multi-tissue section. Composition I and II were two phased at the
used composition concentrations.
EXAMPLE 4
[0172] This example compares the signal intensity and background
from DNA probes and PNA probes on FFPE tissue sections using DMSO,
at a denaturation temperature of 67.degree. C. for 10 min and
hybridization at 45.degree. C. for 60 min.
[0173] FISH Probe Composition I: 3.3 ng/.mu.L HER2 TxRed labeled
DNA probe (1/3 of standard concentration) (size 218 kb) and 1/2 of
the standard concentration (300 nM) of CEN17 FITC labeled PNA
probes (both probes identical with probes from HER2 FISH
pharmDx.TM. kit (K5331, Dako)); 15% ethylene carbonate (E2625-8,
Sigma-Aldrich); 20% dextran sulfate; 600 mM NaCl; 10 mM citrate
buffer, pH 6.2
[0174] FISH Probe Composition II: 3.3 ng/.mu.L HER2 TxRed labeled
DNA probe (1/3 of standard concentration) (size 218 kb) and 1/2 of
the standard concentration (300 nM) of CEN17 FITC labeled PNA
probes (both probes identical with probes from HER2 FISH
pharmDx.TM. kit (K5331, Dako)); 15% DMSO (Sigma-Aldrich); 20%
dextran sulfate; 600 mM NaCl; 10 mM citrate buffer, pH 6.2
TABLE-US-00006 Signal Intensity Composition Background Tx Red FITC
I +11/2-2 3 DNA 21/2 PNA II +2 1-2 DNA 2-21/2 PNA
[0175] The scoring was performed on the mamma-carcinoma tissue of a
multi-tissue section. Composition II with DMSO was unclear in
appearance (milky white) at the used composition
concentrations.
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