U.S. patent application number 10/652676 was filed with the patent office on 2004-12-16 for hybridization buffers using low molecular weight dextran sulfate and methods for their use.
This patent application is currently assigned to Ventana Medical Systems, Inc.. Invention is credited to Christensen, Kimberly, Utermohlen, Joseph, Wolf, Catherine.
Application Number | 20040253603 10/652676 |
Document ID | / |
Family ID | 25093987 |
Filed Date | 2004-12-16 |
United States Patent
Application |
20040253603 |
Kind Code |
A1 |
Utermohlen, Joseph ; et
al. |
December 16, 2004 |
Hybridization buffers using low molecular weight dextran sulfate
and methods for their use
Abstract
This invention demonstrates that lower molecular weight dextran
sulfate is an effective volume exclusion agent for hybridization
reactions. The hybridization buffers of this invention utilize
smaller polymers of dextran sulfate, and thus are of a lower
viscosity than conventional hybridization buffers that use higher
molecular weight dextran sulfate polymers as volume exclusion
agents. The lower viscosity of these low molecular weight dextran
sulfate polymer buffers makes them useful for automated
hybridization processes as in dispensing, automated flow, and
mixing on slide.
Inventors: |
Utermohlen, Joseph; (Tucson,
AZ) ; Wolf, Catherine; (Eckbolsheim, FR) ;
Christensen, Kimberly; (Tucson, AZ) |
Correspondence
Address: |
VENTANA MEDICAL SYSTEMS, INC.
1910 INNOVATION PARK DRIVE
TUCSON
AZ
85737
US
|
Assignee: |
Ventana Medical Systems,
Inc.
|
Family ID: |
25093987 |
Appl. No.: |
10/652676 |
Filed: |
August 28, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10652676 |
Aug 28, 2003 |
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09772123 |
Jan 29, 2001 |
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6656685 |
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Current U.S.
Class: |
435/6.16 |
Current CPC
Class: |
C12Q 1/6832 20130101;
C12Q 1/6832 20130101; C12Q 2527/125 20130101 |
Class at
Publication: |
435/006 |
International
Class: |
C12Q 001/68 |
Claims
1-8. (cancelled).
9. A method of automatically hybridizing a polynucleotide probe to
a target, comprising the steps of preparing a section of tissue or
cells to be examined; hybridizing the tissue section or cellular
preparation with a polynucleotide probe composition in the presence
of low molecular weight dextran sulfate wherein said probe
composition contains at least one sequence complementary to a
coding region of the target; removing unhybridized probe from said
tissue section or cellular preparation; and detecting the
hybridized probe-target combination.
10. The method of claim 9 wherein said polynucleotide probe
composition is selected from the group consisting of DNA probes and
RNA probes.
11. The method of claim 9 wherein said tissue section is a
paraffin-embedded tissue section.
12. The method of claim 9 wherein said tissue section is a
fresh-frozen tissue section.
13. The method of claim 9 wherein said polynucleotide probe
composition is labeled with a detectable label.
14. The method of claim 9 wherein said label is selected from the
group consisting essentially of fluorophores, haptens and
chromogens.
15. The method of claim 9 wherein the step of preparing a section
of tissue or cells to be examined comprises a liquid-based
preparation step.
16. The method of claim 9 wherein the step of preparing a section
of tissue or cells to be examined comprises contacting the target
RNA or DNA with blocking DNA to suppress background cross-reactive
signal.
17. The method of claim 9 wherein said hybridization, removal and
detection steps are performed by an automated tissue staining
instrument.
18. The method of claim 9 wherein said probe composition is arrayed
on a solid substrate.
Description
BACKGROUND
[0001] 1. Field of the Invention
[0002] This invention relates to methods for using volume exclusion
agents to enhance in situ hybridization between polynucleotide
probes and their target polynucleotides, particularly in an
automated testing environment. In one aspect, the invention
specifically relates to the use of volume exclusion agents to
facilitate assay and diagnostic procedures for the detection of DNA
and RNA sequences, particularly human papillomavirus (HPV), Epstein
Bar virus (EBV), human immunoglobulin light chain mRNA (Kappa and
Lambda sequences), and Her-2/neu gene.
[0003] 2. Description of Related Art
[0004] Hybridization is a general technique in which the
complementary strands of deoxyribonucleic acid (hereinafter "DNA")
molecules, ribonucleic acid (hereinafter "RNA") molecules, and
combinations of DNA and RNA are separated into single strands and
then allowed to renature or reanneal into base-paired double
helices. At least three major classes of hybridization are
conventionally known and used: solution hybridization which
disrupts the individual cells and extracts the internal nucleic
acids into solution prior to hybridization; filter or blot
hybridization which transfers extracted DNA (or RNA) fragments from
agarose gels to filters or blotters such as cellulose nitrate or
nylon for subsequent hybridization with radioactive DNA or (RNA)
and then detection of hybridization by radioautography or
fluorography; and in situ hybridization ("ISH") which makes
possible the detection and localization of specific nucleic acid or
polynucleotide sequences directly within a structurally intact cell
or cellular component where extraction of nucleic acids from the
cell is undesirable. Although each of these respective
hybridization techniques often employ cells, tissues, and certain
reagents in common, each technique is generally viewed and accepted
within this art as different and completely distinguishable from
any other.
[0005] In situ hybridization is a technique which yields both
molecular and morphological information about intact individual
cells and cellular parts. Rather than requiring the investigator to
laboriously extract DNA and/or RNA from a heterogeneous cell
population, the technique permits detection of DNA and RNA in-situ
within the cellular morphology and allows the investigator to
identify those particular cells or cell parts which contain
specific DNA or RNA sequences of interest. This technique also
allows one to determine simultaneously the biochemical and/or
morphological characteristics of these cells. For this reason, the
in situ hybridization methodology has direct application for many
areas of biomedical and clinical research including developmental
biology, cell biology, genetics, clinical diagnosis, and
pathological evaluation.
[0006] Despite the potential of in-situ hybridization as a
molecular analytical technique, the development of effective
protocols and procedures has been largely haphazard and disjointed.
Since first described in 1969 by Gall et al., P.N.A.S. U.S.A.,
63:378-383 (1969); Methods in Enzymol., 38:370-380 (1971), the in
situ hybridization approach has been directed towards two different
morphological situations: the localization of specific nucleic acid
sequences of interest in the cytoplasm of a cell; and the
identification of specific nucleic acids within the nucleus and/or
chromosomes of a cell.
[0007] Much of the research related to hybridization between target
and probe polynucleotides for assay and diagnostic purposes has
been directed toward optimizing rates of hybridization. In situ
hybridization is particularly problematic due to the inability of
the probes to readily enter into the nucleus or cytoplasm in which
their target polynucleotides are located. To solve this problem,
researchers have attempted, inter alia, to reduce the size of the
probe and to alter cell fixation procedures to facilitate entry of
the probe into the cytoplasm or nucleus, see generally Singer, R.
H., et al., "Optimization of In Situ Hybridization Using Isotopic
and Non-Isotopic Detection Methods," Biotechniques 4(3):230-250,
1986, and Haase, A., et al., "Detection of Viral Nucleic Acids by
In Situ Hybridization," Methods in Virology, Vol. VII, pp. 189-226,
(1984). Amasino, R. M., "Acceleration of Nucleic Acid Rate by
Polyethylene Glycol," Anal. Biochem., 152:304-307 (1986). It has
been reported that the effect of dextran sulfate, the most commonly
used exclusion agent, was most pronounced in mixed phase
hybridizations where the probes exceeded 250 nucleotides. Further,
it has been reported that as the probe size decreases, so would the
enhancing effect of dextran sulfate on the rate of hybridization,
with no effect observed for oligonucleotides of 14 bases. Meinkoth
J. and Wahl J., "Hybridization of Nucleic Acids Immobilized on
Solid Supports" (Review), Anal. Biochem., 138:267-284 at 268
(1984). The use of volume exclusion to enhance in situ
hybridization has also been reported. It was reported that an
average length of 400 nucleotides is optimal for hybridization in
situ in the presence of dextran sulfate. Hasse, A., supra. at
205.
[0008] Early references that disclose the use of dextran sulfate as
a volume exclusion agent include Wahl, G. M., et al., "Efficient
transfer of large DNA fragments from agarose gels to
diazobezyloxymethyl-paper and rapid hybridization using dextran
sulfate," PNAS 76: 3683 (1979); and Ledermann, L. L., et al., "The
rate of nucleic acid annealing to cytological preparations is
increased by the presence of dextran sulfate," Anal. Biochem.,
117(1): 158-163 (1981).
[0009] The in situ localization of HPV DNA using long biotinylated
probes in the presence of dextran sulfate has also been reported by
Beckmann, P. M., et al.; "Detection and Localization of Human
Papillomavirus DNA in Human Genital Condylomas by In Situ
Hybridization with Biotinylated Probes," J. Med. Virol., 16:265-273
(1985); Milde K., Loning, T., "Detection of Papillomavirus DNA in
Oral Papillomas and Carcinomas: Application of In Situ
Hybridization with Biotinylated HPV 16 probes," J. Oral Pathol.,
15:292-296 (1986); and McDougall, J. K., et al., "Methods for
Diagnosing Papillomavirus Infection," in Papillomaviruses, Wiley,
Chicester (CIBA Foundation Symposium 120), pp. 86-103 (1986).
[0010] U.S. Pat. No. 5,985,549 (Singer, et al.) demonstrates the
use of a dextran sulfate hybridization buffer containing formamide
(deionized); dextran sulfate (10%); human DNA or salmon sperm DNA
(100 ug/ml); human tRNA (100 ug/ml); and vanadyl sulfate (10 uM)
for ISH. The molecular weight of dextran sulfate was not disclosed,
and it is assumed that 500,000 average molecular weight was
obtained.
[0011] U.S. Pat. No. 5,750,340 (Kim, I., et al.) disclose a
hybridization solution for performing ISH, the solution consisting
essentially of 8-12% dextran sulfate, 10-30% formamide, and a salt.
No source for the dextran sulfate, or molecular weight, is
specified.
[0012] U.S. Pat. No. 5,116,727 (Brigatti) discloses hybridization
buffers that contain anionic heteropolysaccharides (e.g.
chondroitin A sulfate) as useful volume exclusion agents for
accelerating hybridization reactions. Chondroitin A sulfate
hybridization buffers were of low viscosity which was a useful
property for capillary gap slides and their use in the automated
processing of in situ hybridization reactions. Brigatti teaches
that the low viscosity of this buffer is due to the volume
exclusion agent having an anionic heteropolysaccharide structure;
this was compared to anionic polysaccharides like dextran sulfate.
Brigatti further discloses that anionic homopolysaccharides like
dextran sulfate polymers produce buffers of substantially greater
viscosity based on the their monomeric structure. This high
viscosity makes such hybridization buffers non-ideal for capillary
gap technology since high viscosity inhibits both probe diffusing
in and out from target and during wash steps to wash away excess
probe. Brigatti further teaches that increasing the concentration
of dextran sulfate also increases the viscosity and thus inhibits
the hybridization process.
[0013] U.S. Pat. No. 4,886,741 (Schwartz et al.) describe the use
of dextran sulfate, sodium salt, for use as a volume exclusion
agent for ISH. The average molecular weight is not described, but
by reference to the source (Sigma Chemical, Products for Life
Sciences, St. Louis, Mo.) it has an average molecular weight of
500,000. Schwartz et al. also disclose that dextran sulfate is
typically used at a concentration of about 5-10% (w/v).
[0014] U.S. Pat. No. 4,302,204 (Wahl, G., et al.), describes the
use of dextran sulfate polymers for hybridization buffers used for
in vitro blot hybridization. In this patent however, the preferred
hybridization buffer contained dextran sulfate of 500,000 MW. No
examples were presented that used low molecular weight dextran
sulfate nor was an in situ reaction presented or claimed.
[0015] In the field of nucleic acid hybridization, the need for
rapid assay tests for the accurate and reproducible detection of
nucleic acids has been a long standing problem. Any procedures that
demonstrate a tendency to accelerate the typically multi-hour long
processes are of value, especially for hybridization assays to be
conducted by clinical laboratories.
SUMMARY OF THE INVENTION
[0016] This invention demonstrates that lower molecular weight
dextran sulfate is also an effective volume exclusion agent for
hybridization reactions. The hybridization buffers of this
invention utilize smaller polymers of dextran sulfate, and thus are
of a lower viscosity than conventional hybridization buffers that
use higher molecular weight dextran sulfate polymers as volume
exclusion agents. The lower viscosity of these low molecular weight
dextran sulfate polymer buffers makes them useful for automated
hybridization processes as in dispensing, automated flow, and
mixing on slide.
[0017] The invention is also directed to a method of automatically
hybridizing a nucleic acid probe to a target, comprising the steps
of preparing a section of tissue or cells to be examined;
hybridizing the tissue section or cellular preparation with a
nucleic acid probe composition in the presence of low molecular
weight dextran sulfate wherein said probe composition contains at
least one sequence complementary to a coding region of the target;
removing unhybridized probe from said tissue section or cellular
preparation; and detecting the hybridized probe-target
combination.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIGS. 1A and 1B are color photographs of human spleen tissue
samples hybridized with a DNA probe collection consisting of probes
possessing target gene-specific domains corresponding to EBV EBER 1
and 2 nuclear RNA, wherein the tissue sample was not treated with
ribonuclease A prior to in situ hybridization (A), or was treated
with ribonuclease A prior to in situ hybridization (B).
[0019] FIGS. 2A and 2B are color photographs of lymphoma tissues
using a probe collection consisting of probes possessing target
gene-specific domains corresponding to human immunoglobulin kappa
light chain mRNA. The lymphoma tissue in FIG. 2A overexpresses the
kappa light chain and the tissue in FIG. 2B overexpresses the
lambda light chain.
[0020] FIGS. 3A and 3B are color photographs of human lymphoma
tissues using a probe collection consisting of probes possessing
target gene-specific domains corresponding to human immunoglobulin
lambda light chain mRNA. The tissue in FIG. 3A over expresses the
lambda light chain and the tissue in FIG. 3B over expresses the
kappa light chain.
[0021] FIG. 4 is a color photograph of a slide-based in situ
hybridization image of mouse pS2 gene expression in the mouse
stomach using mouse pS2 antisense riboprobe diluted in Buffer
B.
[0022] FIG. 5 is a scanned color photograph of the ClonTech Human
Atlas DNA microarray probed with Cy3-labelled amplified cDNA probe
from placental RNA.
[0023] FIG. 6 is a color photograph of a slide having the Her-2/neu
hi-amplification control cell line, (Ventana Cat. No. S1003)
hybridized to a Her-2/neu DNA probe.
[0024] FIG. 7 is a color photograph of a slide having the Her-2/neu
low-amplification control cell line, (Ventana Cat. No. S1002)
hybridized to the same Her-2/neu DNA probe.
[0025] FIG. 8 is a color photograph of a slide having the Her-2/neu
non-amplified control cell line, (Ventana Cat. No. S1001)
hybridized to a Her-2/neu DNA probe.
[0026] FIG. 9 is a color photograph of paraffin-embedded cell lines
of Caski cells, having approximately 500 copies of HPV 16
integrated into the cellular nuclei.
[0027] FIG. 10 is a color photograph of paraffin-embedded control
cell lines of HeLa cells, having approximately 20-50 copies of HPV
18 integrated into the cellular nuclei.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0028] The inventors have demonstrated herein that low molecular
weight dextran sulfate polymers of approximately 10,000 MW
(molecular weight) are effective volume exclusion agents for
hybridization reactions between DNA:DNA, DNA:RNA, and RNA:RNA.
Buffers comprising dextran sulfate within the range of 10,000 MW
performed well for both nick translated labeled probes,
oligonucletide DNA probes, and in vitro transcribed labeled RNA
probes. The target type of nucleic acids were chromosomal
(interphase cells), nuclear RNA (EBER), and mRNA targets. Both
colorimetric ISH and FISH probes were tested with these buffers.
The buffers worked with paraffin embedded formalin-fixed tissues
and cell line, sections, cytospin cell preparations, and
ThinPrep.TM. (a liquid-based preparation) cell preparations.
Surprisingly, these buffers behaved as well or better than
comparable buffers made using standard 500,000-2,000,000 MW dextran
sulfate polymer.
[0029] Generally, volume exclusion agents are added to
hybridization buffers to accelerate the re-association of
complementary DNA. The increase in reassociation kinetics
associated with conventional dextran sulfate polymers (500,000 to
2,000,000 MW) are in the range of 8 to 15 times when compared to
buffers lacking this volume exclusion agent (Wetmur, J. G., 1975,
Biopolymers 14:2517-2524; Wahl and Stark, U.S. Pat. No. 4,302,204).
The preferred concentration (weight to volume) for dextran sulfate
polymers (500,000-2,000,000 MW) is between 5% to 10%. The key work
for describing the acceleration affect of dextran sulfate polymers
as volume exclusion was Wetmur, supra. The relative molecular
weight of dextran sulfates disclosed were 500,000 and 2,000,000 MW.
The effectiveness of low molecular weight dextran sulfate polymers
at as volume exclusion agents were not tested.
[0030] The inventors addressed several fundamental issues when they
undertook to develop these low-viscosity ISH hybridization buffers,
such as:
[0031] whether low molecular weight (8,000 to 15,000) range polymer
dextran sulfate would be an effective volume exclusion agent for
hybridization buffers;
[0032] whether buffers with these smaller polymers would be
effective in automated in situ hybridization reactions using the
Ventana Medical Systems, Inc. coverslip technology;
[0033] whether higher viscosity was a property necessary for
dextran sulfate buffers to be effective for hybridization;
[0034] whether these lower molecular weight polymers would function
with oligonucleotide probes because Schwartz (U.S. Pat. No.
4,886,741) example's were of buffers with high molecular weight
dextran sulfate polymers. The inference being that smaller polymers
may not be effective;
[0035] whether these buffers would mix with other solutions on a
slide to form an effective hybridization solution;
[0036] whether smaller size and greater solubility of volume
exclusion agents impacts rinsing performance in removing probe and
hybridization buffer during post hybridization washes, hence
reducing aberrant signal on sample tissues; and
[0037] whether formamide in the buffer would be incompatible. The
effect of formamide in accelerating hybridization is not well
understood. Most studies suggest that formamide actually reduces
the hybridization reaction (J. R. Hutton, "Renaturation kinetics
and thermal stability of DNA in aqueous solutions of formamide and
urea," Nucelic Acid. Res. 4(10):3537-3555, Oct. 1977) at
concentrations greater than 25% by volume. The properties of higher
molecular weight dextran sulfate polymers as volume exclusion
agents may have been due to the property of increased viscosity, or
its higher molecular weight may have been a synergistic result with
formamide or alditols. The polymers described by Brigatti (U.S.
Pat. No. 5,116,727, supra) were 10-fold smaller than dextran
sulfate 500,000, though these were taught to be distinct from
dextran sulfate since they were heteropolysaccharide polymers, a
property unique to Chondroitin A sulfate volume exclusion agents.
So whether the smaller dextran sulfate polymers were effective
volume exclusion agents was not defined nor understood at the
beginning of these studies.
[0038] For automated processes, a hybridization buffer of low
viscosity is desired for hybridization reactions. Automated in situ
hybridization using Ventana Medical Systems' DISCOVERY.TM.,
NexES.RTM. or BENCHMARK.TM. instruments utilizes a LIQUID
COVERSLIP.TM. to prevent evaporative loss of reagent during the
reaction. The compatibility of hybridization buffer with low
molecular weight dextran sulfate with Ventana's liquid coverslip
system was not known. Compatibility of buffer and a wide range of
probe types on Ventana's slide staining systems was an unknown
prior to these studies. These buffers have been found useful with
synthetic oligo-nucleotide probes, nick translated DNA probes from
plasmids or cosmid origin, and in vitro synthesized RNA.
[0039] Automated slide processing on the Ventana DISCOVERY.TM.
automated ISH stainer was the test system used with these buffers.
This staining system relies on mixing through counter-rotation of
the Liquid Coverslip with air jets to stimulate the mixing of
reagent on the slide and the full distribution of reagents across
the surface of the slide. The Ventana process works best if the
reagents are of viscosities similar to or less than water. Reagents
of high viscosities are not easily adapted to the DISCOVERY system.
Many of the conventional hybridization buffers that use high
molecular weight anionic homopolymers of dextran sulfate are not
effective on this staining system.
[0040] To the best of the inventor's knowledge, the utility of low
molecular weight polymers has not been fully explored in the
hybridization literature as to their effectiveness in driving
nucleic acid re-association. The polymeric size of dextran sulfate
that has been demonstrated as effective in driving nucleic acid
reassociation is in the range of 500,000 to 2,000,000 MW. However,
the inventors have found that much lower MW dextrans having lower
viscosities can be used, with the additive result that low
molecular weight, low-viscosity dextran sulfate based hybridization
buffers can be used in the small-volume environment of an automated
instrument. This was a surprising result because at least one
commentator has observed that homopolymers such as dextran sulfate
will be most effective at high average molecular weights as volume
exclusion agents (see Brigatti, U.S. Pat. No. 5,516,727, discussed
supra). The viscosity and solubility characteristics of any polymer
in solution (at a given concentration) is a function of its Stokes
radius, or the molecular volume of the polymer. Thus larger
polymers have a lower solubility and induce solutions of greater
viscosity for any two polymers of the same monomeric unit.
[0041] The greater solubility of smaller average molecular weight
dextran sulfate polymers allows the formulation of hybridization
buffers with dextran sulfate polymers at concentrations equal to or
greater than 10% in the presence of formamide concentrations
ranging from 5% to 80%. This allows for solutions to be formulated
at 2.times. stock solutions. Mixing hybridization buffers from
2.times. stock solution can be mixed on the slide with aqueous
solutions to make the hybridization buffer with optimal
concentrations for all constituents.
Compositions of Hybridization Buffers With Low Molecular Weight
Dextran Sulfate Polymers
[0042] Buffer A:
[0043] 20% wt/vol dextran sulfate at 10K average molecular
weight
[0044] 50% vol/vol formamide
[0045] 10 mM Tris (15:85 of Tris-HCl:Tris-OH)
[0046] 5 mM EDTA
[0047] 300 mM NaCl
[0048] 30 mM trisodium citrate (Na3C6H5O7)
[0049] 0.05% Brij-35
[0050] pH=7.3
[0051] Buffer A can be used directly with no further dilution, in
an automated format using the manually-applied protocol for ISH.
This solution can also be used via an automated protocol as an
approximately 2.times. concentrated solution, that is dispensed
into an equal volume of 2.times.SSC-Triton X100 for most automated
processes. 1.times.SSC comprises 0.15M NaCl; 0.015 M trisodium
citrate. The resultant on-slide hybridization solution is 50% of
the concentration of Buffer A with the exception of NaCl and
trisodium citrate concentrations, which remain the same. The
concentration of pH can range from 6-8, however, the most preferred
is 7.3.
[0052] Modifications to the base low-molecular weight buffer are
useful for specific applications, for instance, in the
hybridization of DNA or RNA probes to mRNA (Buffer B):
[0053] 20% wt/vol dextran sulfate at 10K average molecular
weight
[0054] 80% vol/vol formamide
[0055] 2.times.SSPE
[0056] 0.05% Brij-35
[0057] 1.times.SSPE is a stock solution comprising the following:
150 mM NaCl, 8 mM Na2HPO4, 2 mM NaH2PO4, 1 mM EDTA, in water.
Buffer B can be used via automated protocol as an approximately 2
times concentration solution, that is dispensed into an equal
volume of 2.times.SSPE, 0.025% Triton-X100, and 0.025% Brij-35 for
most automated processes. The resultant hybridization solution
would be: 10% wt/vol dextran sulfate at 10K average molecular
weight, 40% vol/vol formamide, 2.times.SSPE, and 0.025% Brij-35.
The preferred range of concentration of formamide is from about 20%
to about 80%, a more preferred concentration is from about 40% to
80%, and the most preferred concentration is 80% (40% on the
slide). The preferred range of concentration of SSPE is from about
2-4.times., while the most preferred concentration is
2.times.SSPE.
[0058] Another variant of the low molecular weight dextran sulfate
hybridization buffer of Buffer A is used preferably for
automatically hybridizing "DNA chips" built on glass microscope
slides which are typically sold by companies such as Incyte and
ClonTech. The composition of the buffer (Buffer C) is:
6.times.SSPE, 20% dextran sulfate (10K MW), and 10% formamide.
Buffer C is typically run on the instrument in a 1:1 dilution with
2.times.SSPE, 0.0125% Triton-X100, and 0.025% Brij-35. One of the
great advantages of Buffer C is that is that it is formulated to
maximize speadability of the buffer on the slide surface, thus
covering all of the available probe arrays. A preferred
concentration of SSPE ranges from 2-8.times., while more preferred
is 3-7.times. and most preferred is 6.times.. The preferred
concentration range of formamide is 0-20%, more preferred is from
5-15%, and most preferred is 10%.
[0059] The low-molecular weight dextran sulfate used herein was
sourced from Sigma Chemical. It is sold by Sigma as having an
average molecular weight of approximately 10,000. However, the mean
molecular weight (that is by analysis the molecular weight of a
minimum of 75% of the total population of dextran sulfate polymers)
for three lots of Sigma product Dextran Sulfate (catalogue number
D-6924), was, for lot numbers 49H0530, 100K1379, and 070K0848,
12,750 MW, 13,360 MW, and 13,360 MW, respectively. The range of the
molecular weight for this material is within two standard
deviations (8,000 to 16,000 MW). Therefor, the term "low-molecular
weight dextran sulfate" has some variability associated with it,
since it is a polymer that is not well-controlled in terms of its
average size.
[0060] The invention is also directed to a method of automatically
hybridizing a nucleic acid probe to a target, comprising the steps
of preparing a section of tissue or cells to be examined;
hybridizing the tissue section or cellular preparation with a
nucleic acid probe composition in the presence of low molecular
weight dextran sulfate wherein said probe composition contains at
least one sequence complementary to a coding region of the target;
removing unhybridized probe from said tissue section or cellular
preparation; and detecting the hybridized probe-target
combination.
[0061] Automatic hybridization is the term used to describe the
hybridization of a probe to a target by an automated instrument,
such as those sold by Ventana Medical Systems. These include the
models ES.RTM., NexES.RTM., DISCOVERY.TM., and BENCHMARK.TM..
Preparation of the tissue or cell sample is manual, as is loading
of the slides onto the system. The hybridization process is carried
out by the instrument using a pre-loaded protocol that is adapted
specially for use on the automated stainer. One of ordinary skill
is fully capable of operating an automated staining instrument to
perform automated hybridization reactions, once trained for the
specifics of the instruments. Similarly, nucleic acid probe
compositions are well-known in the art, and available commercially
from a number of sources, including Ventana, Novocastra, Zymed,
Vysis, and Enzo. The removal of unhybridized probe from the sample
is performed by the automated stainer instrument by a
pre-programmed washing function which is a a part of the protocol.
The detection function is similarly a part of the protocol.
Standard detection reagents are used, as sold by Ventana for use on
its instrument platforms. The categories of labels used to detect
hybridized probes include fluorophres, haptens, and chromogens. One
of ordinary skill is aware of specific labels and their strengths
and weaknesses in a particular detection setting. Primary detection
schemes may be used where the probe is directly labeled and
visualized with a fluorophore such as fluoroscein or Texas Red
(available from Molecular Probes, Eugene, Oreg.) or an
antibody-mediated secondary detection scheme may be used.
[0062] The invention may be used with either a tissue sample
preparation such as a tissue sample sectioned from a
paraffin-embedded tissue block using a microtome, or a cellular
composition made from a liquid-based prep such as the Thin
Prep.TM., available from Cytyc, Inc., Boxborough, Mass. A
liquid-based preparation is simply a microscope slide having a
mono-layer of cells spread evenly on its surface. The cells are
collected in a collection vial and suspended in a medium that
preserves them for later analysis. An aliquot of the suspension is
filtered through a filter, and the filter is then imprinted upon
the slide, adhering a thin layer of cells tot he glass surface of
the slide.
[0063] Blocking DNA may be used in conjunction with the nucleic
acid probes to reduce the background signal inherent whenever one
is probing chromosomal DNA. U.S. Pat. No. 5,5447,841 (Gray, J., et
al.) describe the method generally, which is hereby incorporated by
reference.
[0064] The preferred embodiments of the hybridization buffers of
the present invention are best understood by referring to the
following Examples. The Examples, which follow, are illustrative of
specific embodiments of the invention, and various uses thereof.
They are set forth for explanatory purposes only, and are not to be
taken as limiting the invention.
EXAMPLES
[0065] A. General Methods
[0066] Samples for ISH analysis were prepared by cutting
formalin-fixed and paraffin-embedded cells or tissue samples into 4
.mu.m sections and placing the sections onto a standard microscope
glass slide. Subsequent processing and ISH of samples was carried
out in an automated device, such as the DISCOVERY.TM. Automated
ISH/IHC Stainer (Ventana Medical Systems, Inc., Tucson, Ariz.)
described in co-owned and co-pending U.S. Patent App. Ser. Nos.
60/076,198 and 09/259,240, both incorporated herein by reference.
To remove paraffin from the samples, the slides were immersed in an
aqueous solution, heated for approximately 20 minutes, and then
rinsed. The automated deparaffinization procedure is more fully
described in U.S. Ser. Nos. 60/099,018, and 09/259,240 both
incorporated herein by reference. The samples were then treated
with Protease 1 and the slides were heated to 85.degree. C. (for
hybridization to RNA target genes) or 90-95.degree. C. (for
hybridization to DNA target genes) for 4 to 10 minutes.
[0067] Hybridization reactions were typically performed in a
hybridization buffer consisting of a dilution of Buffer A into
2.times.SSC-Triton-X100, at a 1:1 ratio, and between 25 to 125
ng/mnL of each individual probe molecule. ISH reactions were
performed at between 37.degree. C. to 54.degree. C. For ISH using
the oligonucleotide probes, as described in U.S. Provisional patent
application 60/233,177, filed Sep. 15, 2000, incorporated by
reference herein, hybridization reactions were carried out for 1 hr
at 47.degree. C. (except for the poly d(T) probe, wherein the
hybridization reaction was optimally carried out at 37.degree. C.
for 1 hr).
[0068] The hybridization of fluorescein-labeled probes to a
particular target gene in the sample was detected by using a
sequential series of binding proteins, i.e., secondary antibody
detection. However, it is equally possible to use direct detection
when visualizing the bound probes. In secondary detection, first,
an anti-fluorescein mouse monoclonal antibody directed against the
fluorescein hapten bound to probe molecule was added to the sample.
Next, a biotin-labeled polyclonal goat antibody directed against
the mouse antibody was added to the sample. Finally, hybridization
reactions were colormietrically detected using a
5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium
(BCIP/NBT) substrate. This technique, termed "secondary antibody
detection," is routine for one of skill in the art. Primary and
secondary antibodies are available from numerous suppliers,
including Ventana Medical Systems, Tucson, Ariz., which are
optimized for use on the Ventana autostaining systems (ES.RTM.,
NexES.RTM., DISCOVERY.TM., and BENCHMARK.TM..)
[0069] B. Examples
Example 1
[0070] In Situ Hybridization Using Buffer A in Comparision With
Standard Dextran Sulfate Buffer.
[0071] A cocktail of two probes complimentary to the Alu human
repetative sequence was used to evaluate the effectiveness of the
lower molecular weight dextran sulfate as a viable volume exclusion
agent for hybridization reactions. The two Alu probes used for the
experiment to compare hybridization buffers containing low
molecular weight dextran sulfate was a cocktail of two
oligonucleotides, each with 3fluorescein haptens attached per
probe. These probes were dissolved at a combined concentration of
500 ng/mL. The two hybridization buffers are Buffer A, and a
standard dextran sulfate buffer (500,000-2,000,000 mol. weight)
buffer of 10% (wt/vol) dextran sulfate, 50% (vol/vol) formamide,
2.times.SSC, and 0.05% Brij-35, at a final pH of 7.3.
[0072] The hybridization was performed with a DISCOVERY.TM.
instrument using the automated dispense protocol for probe and all
other reagents. The sample was a paraffin-embedded cell line, Oncor
INFORM.TM. Her-2/neu Control Slides, Cat. No. S8100, Level 1,
available from Ventana Medical Systems, Inc., Tucson, Ariz. All
slides were processed by removing paraffin by an automated aqueous
deparaffinization method (see co-pending international patent
application number PCT/US99/20353, incorporated herein by
reference) followed by treatment with Ventana Protease 1 for 8
minutes at 50 degrees C. at a 1:2.7 dilution with Ventana's APK
buffer. The sample cells were then equilibrated with 2.times.SSC
(Ventana 2.times.SSC) then rinsed to remove all but a residual
volume of approximately 100 uL on the slide. The probe and
hybridization buffer at a 2.times. concentration were dispensed
(100 ul) onto the slide and mixed with the residual volume of
2.times.SSC using air-jet mixing. After mixing, the slide was
heated to 85 C. within a 10 minute step, then cooled to 37 C. for a
1 hour hybridization reaction. Standard 2.times.SSC at 37 C. washes
followed the hybridization for removing excess probe and probe
non-specifically bound to DNA. Detection of the hybridized probe to
Alu sequences carried out by secondary antibody detection via the
binding of an anti-fluorescein, mouse antibody to fluorescein
haptens attached to the probe followed by Ventana Enhanced Alkaline
Phosphatase Blue Detection (Ventana cat# 760-061) chemistry. Unless
otherwise indicated, all reagents were obtained from Ventana
Medical Systems, Inc., Tucson, Ariz. and all of these reactions on
the slide were performed under a film of LIQUID COVERSLIP.TM., to
prevent evaporative loss of water during processing.
[0073] The results (observed colormetrically) were that the
hybridization buffer containing the 20% concentration dextran
sulfate of low molecular weight was more effective and more
efficient in Ventana Medical Systems, Inc. staining automation. The
reaction based on the low molecular weight dextran
sulfate-containing buffer yielded a strong, intense signal for most
nuclei across the tissue section, the expected result of a probe
against the Alu satellite sequence. The reactions containing the
hybridization buffer with the 500,000 molecular weight dextran
sulfate did not yield as strong a signal and the intensity was not
the same for each cell nucleus. For these reactions, there were
some slides in which regions of the tissue section stained very
poorly and other regions that did not stain at all. Out of 10
slides hybridized with the 500 K dextran sulfate buffer, 8 of those
slides had regions in the tissue sections which had weak nuclear
signal and some regions which had no signal at all. Conversely,
among the 10 slides hybridized with the low molecular weight
dextran sulfate buffer of the present invention, only one of those
slides had a region of the tissue section that exhibited poor
staining. This observation of poor staining was deduced to be due
to the uneven spreading and/or mixing of the hybridization buffer
across the tissue section. For this experiment, that phenomena was
common among the reactions based on the 500 K dextran sulfate
buffer.
[0074] The hybridization reactions with cocktails made with the low
molecular weight dextran sulfate routinely yielded a more intense
signal with the Alu satellite sequence probe compared to the
reactions with 500K dextran sulfate buffer. This observation
suggests (1) that low molecular weight dextran sulfates are
effective volume exclusion agent for hybridization reactions, and
(2) and such buffers are more efficient than corresponding buffers
made with 500K dextran sulfate for automated hybridization
protocols.
Example 2
[0075] ISH on Human Spleen Samples.
[0076] FIGS. 1A-1B illustrate the results obtained for ISH analysis
of human spleen tissue using a probe collection consisting of
labeled oligonucleotides that are complementary to the EBV early
RNA transcripts, EBER 1 and 2. This experiment demonstrated that
dextran sulfate hybridization buffers using 10,000 MW supports the
specific hybridization between DNA oligonucleotide probes and RNA
targets, in this case nuclear RNAs. These two viral transcripts are
nuclear RNA. Target specificity is demonstrated by the loss of
signal with tissues treated with RNase prior to hybridization (B)
versus tissue sample not treated with ribonuclease A prior to in
situ hybridization (A). The decrease in detectable signal in (B)
indicates that this probe specifically hybridizes to RNA
transcripts, EBER 1 and EBER 2.
Example 3
[0077] ISH on Lymphoma Tissue Samples.
[0078] FIGS. 2 and 3 illustrate that the hybridization buffers with
low molecular weight dextran sulfate support specific hybridization
reactions by a comparison of two probe collections, each specific
to mRNA of the two human immunoglobulin light chain genes, kappa
and lambda respectively. The tissues used were plasmacytoid
lymphoma tissues each being monoclonal in origin for one or the
other light chain mRNA. In FIG. 2A, the tumor was kappa monoclonal
as shown by the abnormally high frequency kappa expressing cells,
whereas the tissue in FIG. 2B was a tissue monoclonal for lambda
gene expression, thus a low level of kappa expressing cells were
found. The reverse was found in the tissue in FIG. 3. Using a
lambda probe this monoclonality was discerned as illustrated in
FIGS. 3A and 3B. The tissue in FIG. 3A shows a high frequency of
cells that are over expressing lambda light chain mRNA; tissue in
FIG. 3B had a low frequency of lambda expressing cells. Thus for
probes from related genes, the low molecular weight dextran sulfate
buffer can support specific hybridization.
Example 4
[0079] ISH Using Buffer B.
[0080] FIG. 4 is a color photograph of a slide-based in situ
hybridization image of mouse pS2 gene expression in the mouse
stomach using mouse pS2 antisense riboprobe diluted in Buffer B.
The mouse mPS2 gene, found to be expressed in normal stomach
epithelium (Lefebvre, O., Wolf, C., Kdinger, M., Chenard, M. P.,
Tomasetto, C., Chambon, P., Rio, M. C., "The mouse one P-Domain
(pS2) and two P-Domain (mSP) genes exhibit distinct patterns of
expression," J. Cell Biol., 122:191-198 (1993)), was used as a
model to study the use of the Buffer B hybridization buffer. The
mPS2 cDNA was provided sub-cloned in a pBluescript plasmid
(Promega, Madison), suitable and linearized for in vitro
transcription.
[0081] Anti-sense and sense DIG-labeled riboprobes were synthetized
using the Roche RNA DIG labeling kit (Roche Molecular cat# 1 175
025) and solubilized in a final volume of 200 .mu.l H2O (stock
solution). A 1:1000 dilution of the mPS2 riboprobes into the Buffer
B buffer provided the working dilution.
[0082] Formalin-fixed, paraffin-embedded mouse stomach sections (5
.mu.m) were hybridized on the DISCOVERY instrument platform, after
on-line deparaffinization and digestion steps. Probe and slides
were co-denatured at 70.degree. C. for 6 minutes, and hybridized
for 3 hours at 60.degree. C. Hybridization was followed by three
stringency washes of 6 minutes each, using 0.1.times.SSC at
65.degree. C.
[0083] The probe was detected by immunohistochemistry using a
biotinylated anti-Dig antibody (Sigma, 1:200), followed by a
Streptavidin-Alkaline Phosphatase & NBT/BCIP colorimetric
detection (Ventana Enhanced Blue detection kit).
[0084] Signal was found in the epithelial cells of the human
stomach using the anti-sense probe, while no signal was detected
using the sense probe. Localization of the signal was found to be
appropriate by comparing with previously published radioactive ISH
results (Lefebvre et al., 1993).
Example 5
[0085] ISH on HPV Control Cell Lines in Paraffin-Embedded
Tissue.
[0086] For ISH using HPV probes as a cocktail of cloned DNAs of HPV
high-risk or low-risk strains (described in co-owned international
patent application PCT/US99/25109), hybridization reactions were
carried out at stringencies which allowed discernment between
high-risk and low-risk strains of HPV in paraffin-embedded tissues.
The hybridization reactions were carried out at 57 degrees C. for 2
hours followed by 2.times.SSC washes at 76.degree. C. The final
concentration of hybridization buffer components were 25% formamide
(vol/vol), 2.times.SSC, 5 mM Tris, 2.5 mM EDTA, 0.025% Brij-35,
0.25% Triton X-100 at 0.25% wt vol. The samples were paraffin
embedded cell lines of (a) Caski (approximately 500 copies of HPV
16) (FIG. 9) and (b) HeLa cells with HPV 18 (copy number within a
range of 20 to 50 per cell) (FIG. 10). The diseased tissues were
paraffin-embedded cervical tissues infected with HPV.
[0087] The results were as follows. FIG. 9 shows that the Caski
paraffin-embedded cell lines have good signal in the expected
nuclear pattern with low to no background on the nuclei. FIG. 10
shows that HeLa embedded cells have a smaller nuclear signal spot,
as expected from the lower copy number with low to no
background.
[0088] A proprietary Her-2/neu gene (c-erbB2) DNA probe was tested
using Buffer A as the volume exclusion agent. The probe was tested
in this buffer using paraffin-embedded cell line slides (Ventana
Medical Systems, Inc. cat. no. S8100) and breast carcinoma tissues.
The embedded cell line kit consists of slides each having a cell
line which has different copy numbers of Her-2/neu gene: the level
1 slides (Oncor catalogue number s8100-1) have cells with the
normal copy number of Her-2/neu gene (3 or less); the level 2
slides (Oncor catalogue number s8100-2) have cells with the low
amplified level of Her-2/neu gene, 4 to 10 copies; the level 3
slide (Oncor catalogue number s8100-3) cells have highly amplified
numbers of the Her-2/neu gene, 10 copies or more. The slides were
processed on the DISCOVERY instrument. The slides were
deparaffinized online, pretreated with a detergent solution at 90
C. then further treated with Ventana Protease 1 (catalogue number
760-2018) for 4 minutes at 37.degree. C. for embedded cells and 10
minutes at 50.degree. C. for embedded tissue. The denaturation was
performed at 90.degree. C. for 10 minutes and hybridization was for
12 hours at 50.degree. C. Post-hybridization washes were 6 minutes
at 50.degree. C. in 2.times.SSC followed by 6 minutes at 60.degree.
C. in 2.times.SSC. Hybrids were detected by indirect fluorescence
detection by binding a FITC-labeled anti-Biotin antibody followed
by FITC-labeled anti-Mouse antibody. Slides were counterstained
with propidium iodide and coverslipped after the finish of the
automated processing.
[0089] The results were as follows. With reference to FIGS. 6-8,
the embedded cell lines have strong signal of the expected pattern
with low to no background on the nuclei. The tissue has signal on
the tumor cells with a low level of background. Thus, the
hybridization buffer with 10% dextran sufate of 10,000 MW allows
controlled hybridization stringency and good results with Her-2/neu
probes using a 12 hour hybridization performed under LIQUID
COVERSLIP.TM., on both control cell lines and patient tissue
specimens. FIG. 6 illustrates signal from for high-amplification of
the Her-2/neu gene (greater than 10 copies/cell). FIG. 7
illustrates signal from low-amplification of the her-2/neu gene
(greater than 4, but less than 10, copies/cell). FIG. 8 illustrates
signal from diploid copy number Her-2/neu gene.
Example 6
[0090] In Vivo ISH Hybridization Using Buffer C
[0091] FIG. 5 is a color photograph of a ClonTech Human Atlas DNA
microarray (ClonTech, Inc., Palo Alto, Calif.) that was probed
using Cy3-labelled amplified cDNA probe from placental RNA (Ambion,
Austin, Tex., Cat# 7950). The probes were labeled according to the
method of Zhao, R., Gish, K., Murphy, M., Yin, Y., Notterman, D.,
Hoffman, W. H., Tom, E., Mack, D. H. and Levine, A. J., "Analysis
of p53-regulated gene expression patterns using oligonucleotide
arrays," Genes & Development 14: 981-983 (2000). Hybridization
Buffer C was used to hybridize the probes to the DNA microarray
oligonucleotides using standard instrument protocols.
[0092] It should be understood that the foregoing disclosure
emphasizes certain specific embodiments of the invention and that
all modifications or alternatives equivalent thereto are within the
spirit and scope of the invention as set forth in the following
appended claims. All patents, patent applications, and references
cited herein are incorporated by reference.
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