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United States Patent |
6,066,453
|
Pinkel
,   et al.
|
May 23, 2000
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Array-based detection of genetic alterations associated with disease
Abstract
The present invention relates to DNA sequences from regions of copy number
change on chromosome 20. The sequences can be used in hybridization
methods for the identification of chromosomal abnormalities associated
with various diseases.
Inventors:
|
Pinkel; Daniel (Walnut Creek, CA);
Albertson; Donna G. (Lafayette, CA);
Gray; Joe W. (San Francisco, CA)
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Assignee:
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The Regents of the University of California (Oakland, CA)
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Appl. No.:
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908855 |
Filed:
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August 8, 1997 |
Intern'l Class: |
C12Q 001/68; G01N 033/574; G01N 033/53; A01N 055/08 |
Field of Search: |
435/6,7.23,810,975
436/64,813
536/24.31
|
References Cited
Attorney, Agent or Firm: Townsend and Townsend and Crew LLP
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is related to U.S. Ser. No. 08/353,018 filed on Dec. 9,
1994, which is incorporated herein by reference for all purposes.
Claims
What is claimed is:
1. A method of screening for the presence of breast cancer cells in a
sample, the method comprising:
contacting a nucleic acid sample from a human patient with a probe which
binds selectively to a target polynucleotide sequence on a region in which
copy number is altered in breast cancer cells and is selected from the
group consisting of Flpter 0.603, 0.646, 0.675 0.694, 0.772 or 0.867 on
chromosome 20, wherein the probe is contacted with the sample under
conditions in which the probe binds selectively with the target
polynucleotide sequence to form a stable hybridization complex; and
detecting the formation of a hybridization complex.
2. The method of claim 1, wherein the step of detecting the hybridization
complex comprises determining the copy number of the target sequence.
3. The method of claim 1, wherein the probe is labeled with digoxigenin or
biotin.
4. The method of claim 1, wherein the step of detecting the hybridization
complex is carried out by detecting a fluorescent label.
5. The method of claim 4, wherein the fluorescent label is FICT or Texas
red.
6. The method of claim 1, wherein the sample is a tissue section.
7. The method of claim 1, wherein the sample comprises a metaphase cell.
8. The method of claim 1, wherein the sample comprises an interphase cell.
9. The method of claim 1, wherein the sample nucleic acids are from a test
cell and a reference cell.
10. The method of claim 1, wherein the probe is a member of a nucleic acid
array.
11. A kit for the detection of a chromosome abnormality correlated with
breast cancer, the kit comprising a compartment which contains more than
one nucleic acid probe which binds selectively to a target polynucleotide
sequence in a region of a chromosome correlated with breast cancer,
wherein the probes bind selectively with the target polynucleotide
sequence selected from the group consisting of Flpter 0.603, 0.646, 0.675,
0.694, 0.722 and 0.867 on chromosome 20.
Description
FIELD OF THE INVENTION
This invention pertains to the field of cancer genetics. More particularly
this invention pertains to the identification of regions of copy number
increase or decrease associated with cancers and other disease.
BACKGROUND OF THE INVENTION
Chromosome abnormalities are often associated with genetic disorders,
degenerative diseases, and cancer. In particular, the deletion or
multiplication of copies of whole chromosomes or chromosomal segments, and
higher level amplifications of specific regions of the genome are common
occurrences in cancer. See, for example Smith, et al., Breast Cancer Res.
Treat., 18: Suppl. 1: 5-14 (1991, van de Vijer & Nusse, Biochim. Biophys.
Acta. 1072: 33-50 (1991), Sato, et al., Cancer. Res., 50: 7184-7189
(1990). In fact, the amplification and deletion of DNA sequences
containing proto-oncogenes and tumor-suppressor genes, respectively, are
frequently characteristic of tumorigenesis. Dutrillaux, et al., Cancer
Genet. Cytogenet., 49: 203-217 (1990). Clearly, the identification of such
regions and the cloning of the genes involved is crucial both to the study
of tumorigenesis and to the development of cancer diagnostics.
The detection of chromosomal regions of increased or decreased copy number
has traditionally been done by cytogenetics. Because of the complex
packing of DNA into the chromosomes, resolution of cytogenetic techniques
has been limited to regions larger than about 10 Mb; approximately the
width of a band in Giemsa-stained chromosomes. In complex karyotypes with
multiple translocations and other genetic changes, traditional cytogenetic
analysis is of little utility because karyotype information is lacking or
cannot be interpreted. Teyssier, J. R., Cancer Genet. Cytogenet., 37: 103
(1989). Furthermore, conventional cytogenetic banding analysis is time
consuming, labor intensive, and frequently difficult or impossible.
More recently, cloned probes have been used to assess the amount of a given
DNA sequence in a chromosome by Southern blotting. This method is
effective even if the genome is heavily rearranged so as to eliminate
useful karyotype information. However, Southern blotting only gives a
rough estimate of the copy number of a DNA sequence, and does not give any
information about the localization of that sequence within the chromosome.
Comparative genomic hybridization (CGH) is a more recent approach to
identify the presence and localization of amplified/deleted sequences. See
Kallioniemi, et al., Science, 258: 818 (1992). CGH, like Southern
blotting, reveals amplifications and deletions irrespective of genome
rearrangement. Additionally, CGH provides a more quantitative estimate of
copy number than Southern blotting, and moreover also provides information
of the localization of the amplified or deleted sequence in the normal
chromosome.
SUMMARY OF THE INVENTION
The present invention relates to the identification of new regions of copy
number change on chromosome 20. Nucleic acids specific to these regions
are useful as probes or as probe targets for monitoring the relative copy
number of corresponding sequences from a biological sample such as a tumor
cell.
Thus, in one embodiment, this invention provides methods of detecting a
chromosome alteration (e.g., copy number increase or decrease) at about
the following FLpter positions: 0.603, 0.646, and 0.675 (all decrease),
0.694 and 0.722 (both increase), and 0.867 (increase). The methods involve
contacting a nucleic acid sample from a patient with nucleic acid probes
each of which binds selectively to a target regions noted above under
conditions in which the probe forms a stable hybridization complex with
the target sequence; and detecting the hybridization complex. The step of
detecting the hybridization complex can involve determining the copy
number of the target sequence. The probe preferably comprises a nucleic
acid that specifically hybridizes under stringent conditions to a nucleic
acid selected from the probes disclosed here. The probe or the sample
nucleic acid can be labeled, and is more typically fluorescently labeled.
If the sample is labeled, the probes can be attached to a solid surface as
an array.
The probes disclosed here can be used in kits for the detection of a
chromosomal abnormality at the positions on human chromosome 20 noted
above. The kits include a compartment which contains a labeled nucleic
acid probe which binds selectively to a target polynucleotide sequence on
human chromosome 20. The probe preferably includes at least one nucleic
acid that specifically hybridizes under stringent conditions to a nucleic
acid selected from the nucleic acids disclosed here. The kit may further
include a reference probe specific to a sequence in the centromere of
chromosome to 20 or other reference locations.
Definitions A "nucleic acid sample" as used herein refers to a sample
comprising DNA in a form suitable for hybridization to a probes of the
invention. The nucleic acid may be total genomic DNA, total mRNA, genomic
DNA or mRNA from particular chromosomes, or selected sequences (e.g.
particular promoters, genes, amplification or restriction fragments, cDNA,
etc.) within particular amplicons or deletions disclosed here. The nucleic
acid sample may be extracted from particular cells or tissues. The tissue
sample from which the nucleic acid sample is prepared is typically taken
from a patient suspected of having the disease associated with the
amplification or deletion being detected. In some cases, the nucleic acids
may be amplified using standard techniques such as PCR, prior to the
hybridization. The sample may be isolated nucleic acids immobilized on a
solid surface (e.g., nitrocellulose) for use in Southern or dot blot
hybridizations and the like. The sample may also be prepared such that
individual nucleic acids remain substantially intact and comprises
interphase nuclei prepared according to standard techniques. A "nucleic
acid sample" as used herein may also refer to a substantially intact
condensed chromosome (e.g. a metaphase chromosome). Such a condensed
chromosome is suitable for use as a hybridization target in in situ
hybridization techniques (e.g. FISH). The particular usage of the term
"nucleic acid sample" (whether as extracted nucleic acid or intact
metaphase chromosome) will be readily apparent to one of skill in the art
from the context in which the term is used. For instance, the nucleic acid
sample can be a tissue or cell sample prepared for standard in situ
hybridization methods described below. The sample is prepared such that
individual chromosomes remain substantially intact and typically comprises
metaphase spreads or interphase nuclei prepared according to standard
techniques.
A "chromosome sample" as used herein refers to a tissue or cell sample
prepared for standard in situ hybridization methods described below. The
sample is prepared such that individual chromosomes remain substantially
intact and typically comprises metaphase spreads or interphase nuclei
prepared according to standard techniques.
As used herein, a "nucleic acid array" is a plurality of target elements,
each comprising one or more target nucleic acid molecules immobilized on a
solid surface to which probe nucleic acids are hybridized. Target nucleic
acids of some target elements typically are from regions of copy number
change from chromosome 20. The target nucleic acids of a target element
may, for example, contain sequence from specific genes or clones disclosed
here. Other target elements will contain, for instance, reference
sequences. Target elements of various dimensions can be used in the arrays
of the invention. Generally, smaller, target elements are preferred.
Typically, a target element will be less than about 1 cm in diameter.
Generally element sizes are from 1 .mu.m to about 3 mm, preferably between
about 5 .mu.m and about 1 mm.
The target elements of the arrays may be arranged on the solid surface at
different densities. The target element densities will depend upon a
number of factors, such as the nature of the label, the solid support, and
the like. One of skill will recognize that each target element may
comprise a mixture of target nucleic acids of different lengths and
sequences. Thus, for example, a target element may contain more than one
copy of a cloned piece of DNA, and each copy may be broken into fragments
of different lengths. The length and complexity of the target sequences of
the invention is not critical to the invention. One of skill can adjust
these factors to provide optimum hybridization and signal production for a
given hybridization procedure, and to provide the required resolution
among different genes or genomic locations. Typically, the target
sequences will have a complexity between about 1 kb and about 1 Mb,
sometimes 10 kb and about 500 kb, and usually from about 50 kb to about
150 kb.
The term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides
and polymers thereof in either single- or double-stranded form. Unless
specifically limited, the term encompasses nucleic acids containing known
analogues of natural nucleotides which have similar binding properties as
the reference nucleic acid and are metabolized in a manner similar to
naturally occurring nucleotides. Unless otherwise indicated, a particular
nucleic acid sequence also implicitly encompasses conservatively modified
variants thereof (e.g. degenerate codon substitutions) and complementary
sequences and as well as the sequence explicitly indicated
The phrases "hybridizing specifically to" or "specific hybridization" or
"selectively hybridize to", refer to the binding, duplexing, or
hybridizing of a nucleic acid molecule preferentially to a particular
nucleotide sequence under stringent conditions when that sequence is
present in a complex mixture (e.g., total cellular) DNA or RNA.
The term "stringent conditions" refers to conditions under which a probe
will hybridize preferentially to its target subsequence, and to a lesser
extent to, or not at all to, other sequences. A "stringent hybridization"
and "stringent hybridization wash conditions" in the context of nucleic
acid hybridization experiments such as Southern and northern
hybridizations are sequence dependent, and are different under different
environmental parameters. An extensive guide to the hybridization of
nucleic acids is found in Tijssen (1993) Laboratory Techniques in
Biochemistry and Molecular Biology--Hybridization with Nucleic Acid Probes
part I chapter 2. Overview of principles of hybridization and the strategy
of nucleic acid probe assays, Elsevier, N.Y. Generally, highly stringent
hybridization and wash conditions are selected to be about 5.degree. C.
lower than the thermal melting point (T.sub.m) for the specific sequence
at a defined ionic strength and pH. The T.sub.m is the temperature (under
defined ionic strength and pH) at which 50% of the target sequence
hybridizes to a perfectly matched probe. Very stringent conditions are
selected to be equal to the T.sub.m for a particular probe.
An example of stringent hybridization conditions for hybridization of
complementary nucleic acids which have more than 100 complementary
residues on a filter in a Southern or northern blot is 42.degree. C. using
standard hybridization solutions, with the hybridization being carried out
overnight. An example of highly stringent wash conditions is 0.15 M NaCl
at 72.degree. C. for about 15 minutes. An example of stringent wash
conditions is a 0.2.times. SSC wash at 65.degree. C. for 15 minutes (see,
Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2nd ed.)
Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, N.Y.,
(Sambrook et al.) supra for a description of SSC buffer). Often, a high
stringency wash is preceded by a low stringency wash to remove background
probe signal. An example medium stringency wash for a duplex of, e.g.,
more than 100 nucleotides, is 1.times. SSC at 45.degree. C. for 15
minutes. An example low stringency wash for a duplex of, e.g., more than
100 nucleotides, is 4-6.times. SSC at 40.degree. C. for 15 minutes.
An "isolated" polynucleotide is a polynucleotide which is substantially
separated from other contaminants that naturally accompany it, e.g.,
protein, lipids, and other polynucleotide sequences. The term embraces
polynucleotide sequences which have been removed or purified from their
naturally-occurring environment or clone library, and include recombinant
or cloned DNA isolates and chemically synthesized analogues or analogues
biologically synthesized by heterologous systems.
"Subsequence" refers to a sequence of nucleic acids that comprise a part of
a longer sequence of nucleic acids.
A "probe" or a "nucleic acid probe", as used herein, is defined to be a
collection of one or more nucleic acid fragments whose hybridization to a
target can be detected. The probe may be unlabeled or labeled as described
below so that its binding to the target can be detected. The probe is
produced from a source of nucleic acids from one or more particular
(preselected) portions of the genome, for example one or more clones, an
isolated whole chromosome or chromosome fragment, or a collection of
polymerase chain reaction (PCR) amplification products. The probes of the
present invention are produced from nucleic acids found in the regions
described herein. The probe may be processed in some manner, for example,
by blocking or removal of repetitive nucleic acids or enrichment with
unique nucleic acids. Thus the word "probe" may be used herein to refer
not only to the detectable nucleic acids, but to the detectable nucleic
acids in the form in which they are applied to the target, for example,
with the blocking nucleic acids, etc. The blocking nucleic acid may also
be referred to separately. What "probe" refers to specifically is clear
from the context in which the word is used.
The probe may also be isolated nucleic acids immobilized on a solid surface
(e.g., nitrocellulose). In some embodiments, the probe may be a member of
an array of nucleic acids as described, for instance, in WO 96/17958.
Techniques capable of producing high density arrays can also be used for
this purpose (see, e.g., Fodor et al. Science 767-773 (1991) and U.S. Pat.
No. 5,143,854).
"Hybridizing" refers to the binding of two single stranded nucleic acids
via complementary base pairing.
One of skill will recognize that the precise sequence of the particular
probes described herein can be modified to a certain degree to produce
probes that are "substantially identical" to the disclosed probes, but
retain the ability to bind substantially to the target sequences. Such
modifications are specifically covered by reference to the individual
probes herein. The term "substantial identity" of polynucleotide sequences
means that a polynucleotide comprises a sequence that has at least 90%
sequence identity, more preferably at least 95%, compared to a reference
sequence using the methods described below using standard parameters.
Two nucleic acid sequences are said to be "identical" if the sequence of
nucleotides in the two sequences is the same when aligned for maximum
correspondence as described below. The term "complementary to" is used
herein to mean that the complementary sequence is identical to all or a
portion of a reference polynucleotide sequence.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A shows the distribution of the physical map location on chromosome
20 of the clones disclosed here.
FIGS. 1B-E show copy number variation of the clones along chromosome 20 in
a breast cancer cell line (BT474) and 5 breast tumors.
FIG. 2 summarizes the measurements of copy number increase and decrease in
the regions disclosed here.
FIGS. 3A-F show individual tracings along chromosome 20 for BT474 and the 5
breast tumors studied here.
DETAILED DESCRIPTION
The present invention provides new regions of copy number changes on human
chromosome 20. The clones and other information provided herein can be
used to detect the copy number changes in a biological sample and thereby
screen for the presence of disease, such as breast cancer. Generally the
methods involve hybridization of probes that specifically bind one or more
nucleic acid sequences of the target region with nucleic acids present in
a biological sample or derived from a biological sample. The locations of
particular chromosomal regions and/or target regions for particular probes
are typically expressed as the average fractional length from the p
telomere (FLpter).
As used herein, a biological sample is a sample of biological tissue or
fluid containing cells desired to be screened for chromosomal
abnormalities (e.g. amplifications or deletions). In a preferred
embodiment, the biological sample is a cell or tissue suspected of being
cancerous (transformed). Methods of isolating biological samples are well
known to those of skill in the art and include, but are not limited to,
aspirations, tissue sections, needle biopsies, and the like. Frequently
the sample will be a "clinical sample" which is a sample derived from a
patient. Biological samples may also include sections of tissues such as
frozen sections or parafin sections taken for histological purposes. It
will be recognized that the term "sample" also includes supernatant
(containing cells) or the cells themselves from cell cultures, cells from
tissue culture and other media in which it may be desirable to detect
chromosomal abnormalities.
In some embodiments, a chromosome sample is prepared by depositing cells,
either as single cell suspensions or as tissue preparation, on solid
supports such as glass slides and fixed by choosing a fixative which
provides the best spatial resolution of the cells and the optimal
hybridization efficiency. In other embodiments, the sample is contacted
with an array of probes immobilized on a solid surface.
Making Probes
Any probe which hybridizes to regions of altered copy number are suitable
for use in detecting the corresponding regions. Methods of preparing
probes are well known to those of skill in the art (see, e.g. Sambrook et
al, Molecular Cloning: A Laboratory Manual (2nd ed.), Vols. 1-3, Cold
Spring Harbor Laboratory, (1989) or Current Protocols in Molecular
Biology, F. Ausubel et al, ed. Greene Publishing and Wiley-Interscience,
N.Y. (1987)).
Given the strategy for making the nucleic acids of the present invention,
one of skill can construct a variety of vectors and nucleic acid clones
containing functionally equivalent nucleic acids to the particular probes
disclosed here. Cloning methodologies to accomplish these ends, and
sequencing methods to verify the sequence of nucleic acids are well known
in the art. Examples of appropriate cloning and sequencing techniques, and
instructions sufficient to direct persons of skill through many cloning
exercises are found in Berger and Kimmel, Guide to Molecular Cloning
Techniques, Methods in Enzymology volume 152 Academic Press, Inc., San
Diego, Calif. (Berger); Sambrook et al. (1989) Molecular Cloning--A
Laboratory Manual (2nd ed.) Vol. 1-3, Cold Spring Harbor Laboratory, Cold
Spring Harbor Press, N.Y., (Sambrook); and Current Protocols in Molecular
Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture
between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.,
(1994 Supplement) (Ausubel). Product information from manufacturers of
biological reagents and experimental equipment also provide information
useful in known biological methods. Such manufacturers include the SIGMA
chemical company (Saint Louis, Mo.), R&D systems (Minneapolis, Minn.),
Pharmacia LKB Biotechnology (Piscataway, N.J.), CLONTECH Laboratories,
Inc. (Palo Alto, Calif.), Chem Genes Corp., Aldrich Chemical Company
(Milwaukee, Wis.), Glen Research, Inc., GIBCO BRL Life Technologies, Inc.
(Gaithersberg, Md.), Fluka Chemica-Biochemika Analytika (Fluka Chemie AG,
Buchs, Switzerland), Invitrogen, San Diego, Calif., and Applied Biosystems
(Foster City, Calif.), as well as many other commercial sources known to
one of skill.
The nucleic acids provided by this invention, whether RNA, cDNA, genomic
DNA, or a hybrid of the various combinations, are isolated from biological
sources or synthesized in vitro. The nucleic acids and vectors of the
invention are present in transformed or transfected whole cells, in
transformed or transfected cell lysates, or in a partially purified or
substantially pure form.
In vitro amplification techniques suitable for amplifying sequences to
provide a nucleic acid, or for subsequent analysis, sequencing or
subcloning are known. Examples of techniques sufficient to direct persons
of skill through such in vitro amplification methods, including random
priming, the polymerase chain reaction (PCR) the ligase chain reaction
(LCR), Q.beta.-replicase amplification and other RNA polymerase mediated
techniques (e.g., NASBA) are found in Berger, Sambrook, and Ausubel, as
well as Mullis et al., (1987) U.S. Pat. No. 4,683,202; PCR Protocols A
Guide to Methods and Applications (Innis et al. eds) Academic Press Inc.
San Diego, Calif. (1990) (Innis); Arnheim & Levinson (Oct. 1, 1990) C&EN
36-47; The Journal Of NIH Research (1991) 3, 81-94; (Kwoh et al. (1989)
Proc. Natl. Acad. Sci. USA 86, 1173; Guatelli et al. (1990) Proc. Natl.
Acad. Sci. USA 87, 1874; Lomell et al. (1989) J Clin. Chem 35, 1826;
Landegren et al., (1988) Scienice 241, 1077-1080; Van Brunt (1990)
Biotechnology 8, 291-294; Wu and Wallace, (1989) Gene 4, 560; Barringer et
al. (1990) Gene 89, 117, and Sooknanan and Malek (1995) Biotechnology 13:
563-564. Improved methods of cloning in vitro amplified nucleic acids are
described in Wallace et al., U.S. Pat. No. 5,426,039. Improved methods of
amplifying large nucleic acids are summarized in Cheng et al (1994) Nature
369: 684-685 and the references therein.
Nucleic Acids (e.g., oligonucleotides) for in vitro amplification methods
or for use as gene probes, for example, are typically chemically
synthesized according to the solid phase phosphoramidite triester method
described by Beaucage and Caruthers (1981), Tetrahedron Letts.,
22(20):1859-1862, e.g., using an automated synthesizer, as described in
Needham-VanDevanter et al. (1984) Nucleic Acids Res., 12:6159-6168.
Purification of oligonucleotides, where necessary, is typically performed
by either native acrylamide gel electrophoresis or by anion-exchange HPLC
as described in Pearson and Regnier (1983) J. Chrom. 255:137-149. The
sequence of the synthetic oligonucleotides can be verified using the
chemical degradation method of Maxam and Gilbert (1980) in Grossman and
Moldave (eds.) Academic Press, New York, Methods in Enzymology 65:499-560.
The probes are most easily prepared by combining and labeling one or more
of the clones disclosed here. Prior to use, the constructs are fragmented
to provide smaller nucleic acid fragments that easily penetrate the cell
and hybridize to the target nucleic acid. Fragmentation can be by any of a
number of methods well known to hose of skill in the art. Preferred
methods include treatment with a restriction enzyme to selectively cleave
the molecules, or alternatively to briefly heat the nucleic acids in the
presence of Mg.sup.2+. Probes are preferably fragmented to an average
fragment length ranging from about 50 bp to about 2000 bp, more preferably
from about 100 bp to about 1000 bp and most preferably from about 150 bp
to about 500 bp.
One of skill will appreciate that using the clones provided herein, one of
skill in the art can identify or isolate the same or similar probes from
other human genomic libraries using routine methods (e.g. by STS content,
Southern or Northern Blots).
Labeling Nucleic Acids
Methods of labeling nucleic acids (either probes or sample nucleic acids)
are well known to those of skill in the art. Preferred labeled labels are
those that are suitable for use in in situ hybridization. The nucleic acid
probes or samples of the invention may be detectably labeled prior to the
hybridization reaction. Alternatively, a detectable label which binds to
the hybridization product may be used. Such detectable labels include any
material having a detectable physical or chemical property and have been
well-developed in the field of immunoassays.
As used herein, a "label" is any composition detectable by spectroscopic,
photochemical, biochemical, immunochemical, or chemical means. Useful
labels in the present invention include radioactive labels (e.g. .sup.32
P, .sup.125 I, .sup.14 C, .sup.3 H, and .sup.35 S), fluorescent dyes (e.g.
fluorescein, rhodamine, Texas Red, etc.), electron-dense reagents (e.g.
gold), enzymes (as commonly used in an ELISA), colorimetric labels (e.g.
colloidal gold), magnetic labels (e.g. Dynabeads.TM.), and the like.
Examples of labels which are not directly detected but are detected
through the use of directly detectable label include biotin and dioxigenin
as well as haptens and proteins for which labeled antisera or monoclonal
antibodies are available.
The particular label used is not critical to the present invention, so long
as it does not interfere with the in situ hybridization of the probe.
However, probes directly labeled with fluorescent labels (e.g.
fluorescein-12-dUTP, Texas Red-5-dUTP, etc.) are preferred for chromosome
hybridization.
A direct labeled probe, as used herein, is a probe to which a detectable
label is attached. Because the direct label is already attached to the
probe, no subsequent steps are required to associate the probe with the
detectable label. In contrast, an indirect labeled probe is one which
bears a moiety to which a detectable label is subsequently bound,
typically after the probe is hybridized with the target nucleic acid.
In addition the label must be detectible in as low copy number as possible
thereby maximizing the sensitivity of the assay and yet be detectible
above any background signal. Finally, a label must be chosen that provides
a highly localized signal thereby providing a high degree of spatial
resolution when physically mapping the stain against the chromosome.
Particularly preferred fluorescent labels include fluorescein- 12-dUTP and
Texas Red-5-dUTP.
The labels may be coupled to the probes in a variety of means known to
those of skill in the art. In a preferred embodiment the nucleic acid
probes will be labeled using nick translation, PCR, or random primer
extension (Rigby, et al. J. Mol. BioL, 113: 237 (1977) or Sambrook, et
al., Molecular Cloning--A Laboratory Manual, Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y. (1985)).
Detecting the Regions Disclosed Here
As explained above, detection of copy number changes in chromosome 20 is
indicative of the presence and/or prognosis of a large number of cancers.
These include, but are not limited to breast, prostate, cervix, ovary,
bladder, head and neck, and colon.
In a preferred embodiment, the copy number changes are detected through the
hybridization of a probe of this invention to a target nucleic acid (e.g.
a chromosomal sample) in which it is desired to screen for the
amplification or deletion. Suitable hybridization formats are well known
to those of skill in the art and include, but are not limited to,
variations of Southern Blots, in situ hybridization and quantitative
amplification methods such as quantitative PCR (see, e.g. Sambrook,
supra., Kallioniemi et al., Proc. Natl Acad Sci USA, 89: 5321-5325 (1992),
and PCR Protocols, A Guide to Methods and Applications, Innis et al.,
Academic Press, Inc. N.Y., (1990)).
Alternatively, binding to a target nucleic acid can be compared between a
"test" nucleic acid and a "reference" nucleic acid. Preferred sources for
"test" nucleic acids include any organism, organ, tissue, or cell type in
whose DNA it is desirable to identify a chromosomal abnormality. The
"reference" nucleic acid is typically total genomic DNA from a normal cell
and should not include the copy number changes that are the target it is
desired to detect. Hybridization to a particular target sequence is then
compared as described in the Example Section below.
In situ Hybridization.
In some embodiments, the target region is identified using in situ
hybridization. Generally, in situ hybridization comprises the following
major steps: (1) fixation of tissue or biological structure to analyzed;
(2) prehybridization treatment of the biological structure to increase
accessibility of target DNA, and to reduce nonspecific binding; (3)
hybridization of the mixture of nucleic acids to the nucleic acid in the
biological structure or tissue; (4) posthybridization washes to remove
nucleic acid fragments not bound in the hybridization and (5) detection of
the hybridized nucleic acid fragments. The reagent used in each of these
steps and their conditions for use vary depending on the particular
application.
In some applications it is necessary to block the hybridization capacity of
repetitive sequences. In this case, human genomic DNA or Cot-1 DNA, is
used as an agent to block such hybridization. The preferred size range is
from about 200 bp to about 1000 bases, more preferably between about 400
to about 800 bp for double stranded, nick translated nucleic acids.
Hybridization protocols for the particular applications disclosed here are
described in Pinkel et al. Proc. Natl. Acad. Sci. USA, 85: 9138-9142
(1988) and in EPO Pub. No. 430,402. Suitable hybridization protocols can
also be found in Methods o.backslash.in Molecular Biology Vol. 33: In Situ
Hybridization Protocos, K.H.A. Choo, ed., Humana Press, Totowa, N.J.,
(1994). In a particularly preferred embodiment, the hybridization protocol
of Kallioniemi et al., Proc. Natl Acad, Sci USA, 89: 5321-5325 (1992) is
used.
Typically, it is desirable to use dual color FISH, in which two probes are
utilized, each labeled by a different fluorescent dye. A test probe that
hybridizes to the region of interest is labeled with one dye, and a
control probe that hybridizes to a different region (e.g., a centromere)
is labeled with a second dye. A nucleic acid that hybridizes to a stable
portion of the chromosome of interest, or another chromosome, is often
most useful as the control probe. In this way, differences between
efficiency of hybridization from sample to sample can be accounted for.
The FISH methods for detecting chromosomal abnormalities can be performed
on nanogram quantities of the subject nucleic acids. Paraffin embedded
tumor sections can be used, as can fresh or frozen material. Because FISH
can be applied to the limited material, touch preparations prepared from
uncultured primary tumors can also be used (see, e.g., Kallioniemi, A. et
al., Cytogenet. Cell Genet. 60: 190-193 (1992)). For instance, small
biopsy tissue samples from tumors can be used for touch preparations (see,
e.g., Kallioniemi, A. et al., Cytogenet. Cell Genet. 60: 190-193 (1992)).
Small numbers of cells obtained from aspiration biopsy or cells in bodily
fluids (e.g., blood, urine, sputum and the like) can also be analyzed. For
prenatal diagnosis, appropriate samples will include amniotic fluid and
the like.
Arrays
Other formats use arrays of probes or targets to which nucleic acid samples
are hybridized as described below and in WO 96/17958. The array nucleic
acids preferably include nucleic acids selected from a particular region
of copy number change disclosed here. Typically, the array nucleic acids
will include nucleic aid molecules derived from representative locations
along the chromosomal region of interest, a cDNA library, and the like.
These target nucleic acids may be relatively long (typically thousands of
bases) fragments of nucleic acid obtained from, for instance, genomic
clones, inter-Alu PCR products of genomic clones, restriction digests of
genomic clone, cDNA clones and the like. In preferred embodiments the
array nucleic acids are a previously mapped library of clones spanning a
particular region of interest. The arrays can be used with a single
population of sample nucleic acids or can be used with two differentially
labeled collections, as described below.
Many methods for immobilizing nucleic acids on a variety of solid surfaces
are known in the art. For instance, the solid surface may be a membrane,
glass, plastic, or a bead. The desired component may be covalently bound
or noncovalently attached through nonspecific binding. The immobilization
of nucleic acids on solid surfaces is discussed more fully below.
A wide variety of organic and inorganic polymers, as well as other
materials, both natural and synthetic, may be employed as the material for
the solid surface. Illustrative solid surfaces include nitrocellulose,
nylon, glass, diazotized membranes (paper or nylon), silicones,
polyformaldehyde, cellulose, and cellulose acetate. In addition, plastics
such as polyethylene, polypropylene, polystyrene, and the like can be
used. Other materials which may be employed include paper, ceramics,
metals, metalloids, semiconductive materials, cermets or the like. In
addition substances that form gels can be used. Such materials include
proteins (e.g., gelatins), lipopolysaccharides, silicates, agarose and
polyacrylamides. Where the solid surface is porous, various pore sizes may
be employed depending upon the nature of the system.
In preparing the surface, a plurality of different materials may be
employed, particularly as laminates, to obtain various properties. For
example, proteins (e.g., bovine serum albumin) or mixtures of
macromolecules (e.g., Denhardt's solution) can be employed to avoid
non-specific binding, simplify covalent conjugation, enhance signal
detection or the like.
If covalent bonding between a compound and the surface is desired, the
surface will usually be polyfunctional or be capable of being
polyfunctionalized. Functional groups which may be present on the surface
and used for linking can include carboxylic acids, aldehydes, amino
groups, cyano groups, ethylenic groups, hydroxyl groups, mercapto groups
and the like. The manner of linking a wide variety of compounds to various
surfaces is well known and is amply illustrated in the literature. For
example, methods for immobilizing nucleic acids by introduction of various
functional groups to the molecules is known (see, e.g., Bischoffet al.
(1987) Anal. Biochem., 164:336-344; Kremsky et al. (1987) Nucl. Acids Res.
15:2891-2910). Modified nucleotides can be placed on the target using PCR
primers containing the modified nucleotide, or by enzymatic end labeling
with modified nucleotides.
Use of membrane supports (e.g., nitrocellulose, nylon, polypropylene) for
the nucleic acid arrays of the invention is advantageous because of well
developed technology employing manual and robotic methods of arraying
targets at relatively high element densities. Such membranes are generally
available and protocols and equipment for hybridization to membranes is
well known. Many membrane materials, however, have considerable
fluorescence emission, where fluorescent labels are used to detect
hybridization.
To optimize a given assay format one of skill can determine sensitivity of
fluorescence detection for different combinations of membrane type,
fluorochrome, excitation and emission bands, spot size and the like. In
addition, low fluorescence background membranes have been described (see,
e.g., Chu et al. (1992) Electrophoresis 13:105-114).
The sensitivity for detection of spots of various diameters on the
candidate membranes can be readily determined by, for example, spotting a
dilution series of fluorescently end labeled DNA fragments. These spots
are then imaged using conventional fluorescence microscopy. The
sensitivity, linearity, and dynamic range achievable from the various
combinations of fluorochrome and membranes can thus be determined. Serial
dilutions of pairs of fluorochrome in known relative proportions can also
be analyzed to determine the accuracy with which fluorescence ratio
measurements reflect actual fluorochrome ratios over the dynamic range
permitted by the detectors and membrane fluorescence.
Arrays on substrates with much lower fluorescence than membranes, such as
glass, quartz, or small beads, can achieve much better sensitivity. For
example, elements of various sizes, ranging from 1 mm diameter down to 1
.mu.m can be used with these materials. Small array members containing
small amounts of concentrated target DNA are conveniently used for high
complexity comparative hybridizations since the total amount of probe
available for binding to each element will be limited. Thus it is
advantageous to have small array members that contain a small amount of
concentrated target DNA so that the signal that is obtained is highly
localized and bright. Such small array members are typically used in
arrays with densities greater than 10.sup.4 /cm.sup.2. Relatively simple
approaches capable of quantitative fluorescent imaging of 1 cm 2 areas
have been described that permit acquisition of data from a large number of
members in a single image (see, e.g., Wittrup et. al. (1994) Cytometry
16:206-213).
Substrates such as glass or fused silica are advantageous in that they
provide a very low fluorescence substrate, and a highly efficient
hybridization environment. Covalent attachment of the target nucleic acids
to glass or synthetic fused silica can be accomplished according to a
number of known techniques. Nucleic acids can be conveniently coupled to
glass using commercially available reagents. For instance, materials for
preparation of silanized glass with a number of functional groups are
commercially available or can be prepared using standard techniques (see,
e.g., Gait et al. (1984) Oligonucleotide Synthesis: A Practical Approach,
IRL Press, Wash. D.C.). Similarly, quartz cover slips, which have at least
10-fold lower auto fluorescence than glass, can also be silanized.
The targets can also be immobilized on commercially available coated beads
or other surfaces. For surfaces, biotin end-labeled nucleic acid can be
bound to commercially available avidin-coated beads. Streptavidin or
anti-digoxigenin antibody can also be attached to silanized glass slides
by protein-mediated coupling using e.g., protein A following standard
protocols (see, e.g., Smith et al. (1992) Science, 258:1122-1126). Biotin
or digoxigenin end-labeled nucleic acids can be prepared according to
standard techniques.
Hybridization to nucleic acids attached to beads is accomplished by
suspending them in the hybridization mix, and then depositing them on the
glass substrate for analysis after washing. Alternatively, paramagnetic
particles, such as ferric oxide particles, with or without avidin coating,
can be used.
In one particularly preferred embodiment, the target elements are spotted
onto a surface (e.g., a glass or quartz surface). The targets can be made
by dissolving the nucleic acid in a mixture of dimethylsulfoxide (DMSO),
and nitrocellulose and spotting the mixture onto amino-silane coated glass
slides with small capillaries, as described below.
Other Formats
A number of hybridization formats are useful in the invention. For
instance, Southern hybridizations can be used. In a Southern Blot, a
genomic or cDNA (typically fragmented and separated on an electrophoretic
gel) is hybridized to a probe specific for the target region. Comparison
of the intensity of the hybridization signal from the probe for the target
region with the signal from a probe directed to a control (non amplified)
region provides an estimate of the relative copy number of the target
nucleic acid.
Kits Containing Probes.
This invention also provides diagnostic kits for the detection of
chromosomal abnormalities on chromosome 20. In a preferred embodiment, the
kits include one or more probes to the regions disclosed here. The kits
can additionally include blocking probes and instructional materials
describing how to use the kit contents in detecting the target regions.
The kits may also include one or more of the following: various labels or
labeling agents to facilitate the detection of the probes, reagents for
the hybridization including buffers, a metaphase spread, bovine serum
albumin (BSA) and other blocking agents, tRNA, SDS sampling devices
including fine needles, swabs, aspirators and the like, positive and
negative hybridization controls and so forth.
EXAMPLES
The following examples are offered to illustrate, but not to limit the
present invention.
In this example we describe a new implementation of CGH that employs
microarrays of mapped genomic DNA clones in place of metaphase chromosomes
as the hybridization target. This approach improves the resolution by more
that a factor of 100 and references the results to the genetic maps being
produced by the Human Genome Project. We have demonstrated the power of
this approach through a multi-locus analysis of copy number changes on
chromosome 20 in breast cancer. Three new independent regions of copy
number change were resolved in a portion of a chromosome that had
previously been extensively studied, and the boundaries of one region were
mapped to within the length of a clone.
Methods
Arrays: Cloned DNA was isolated from bacterial cultures using standard
procedures. Ten .mu.g of each DNA was ethanol precipitated and dissolved
first in 1 .mu.l of water. Four .mu.l of a solution of nitrocellulose
filter material dissolved in DMSO (0.5 .mu.g/.mu.l) was added and mixed.
The solution was lightly sonicated to reduce the fragment size to several
kb so that it is not too stringy for effective spotting. Sub-nanoliter
amounts of each target solution were deposited using a glass capillary
onto an acid-cleaned, amino propyltrimethoxysilane glass or quartz
surface, and air dried. Final spot diameters were 150-250 .mu.m.
Hybridization: Test and reference genomic DNA were labeled by nick
translation with fluorescein dCTP and Texas red dCTP respectively. Two
hundred to 400 ng of each were mixed with 50 .mu.g of Cot-1 DNA and
ethanol precipitated. The amount of Cot-1DNA was based on fluorimetric
determination since absorbance measurements of some commercial
preparations substantially overestimate the concentration of effective DNA
that they contain. This DNA was dissolved in 10 .mu.l of hybridization mix
to achieve a final composition of 50% formamide/10% dextran
sulfate/2.times.SSC/2% SDS and 100 .mu.g tRNA. The DNA was denatured at
70.degree. C. for 5 minutes, and incubated at 37.degree. C. for several
hours to allow blocking of the repetitive sequences. A well
enclosing.about.1 cm.sup.2 around the perimeter of the array was filled
with the reassociated hybridization mix (10 .mu.l/cm.sup.2 of surface),
and the array was placed in a sealed tube (containing 100 .mu.l of
hybridization solution without probe to prevent evaporation) at 37.degree.
C. for 16-60 hrs on slowly rocking table to actively transport the
hybridization mix over the array. After hybridization the slide was washed
in 50% formamide/2.times.SSC at 45.degree. C. for 10 minutes, followed by
phosphate buffer containing 0.05% NP40 and antifade solution containing 1
.mu.g/ml of DAPI to counter stain the array targets was applied, and a
glass coverslip sealed in place.
Fluorescence imaging and analysis: Fluorescence images of 5 mm.times.7 mm
regions of the array were obtained using a 1.times. magnification imaging
system coupled to a 12 bit CCD camera (Photometrics KAF 1400 chip).
Excitation light, supplied from a mercury arc lamp equipped with a
computer controlled filter wheel, was coupled into the back of the slide
using a quartz prism. After passing through the array elements it
underwent total internal reflection from the outside surface of the cover
slip, passed back through the specimen and into a mirror, and was
reflected back to the specimen by a fixed mirror. A multiband pass filter
(P8100, Chroma Technology, Brattleboro Vt.) was used in the emission light
path. Exposure times were much less than one second for DAPI, and between
0.5 and 2 sec for fluorescein and Texas red. Images were analyzed with
custom software that segmented the array targets based on the DAPI image,
subtracted local background, and calculated several characteristics of the
signals for each target including the total intensity of each
fluorochrome, the fluorescein/Texas red intensity ratio, and the slope of
the scatter plot of the fluorescein and Texas red intensities for each
pixel.
Results
The procedure described here has sufficient sensitivity to allow accurate
analysis of regions as small as 40 kb, approximately 10.sup.-5 of the
genome, using amounts of labeled genomic DNA that are readily available
from most clinical specimens. Control of repetitive sequences is
sufficiently reliable that essentially any clone selected from a library
can be used for a target.
The target DNAs used in our measurements, the STSs or genes they contain,
and their physical (FISH) map locations are listed in Table 1. FIG. 1A
illustrates their distribution along chromosome 20. P1 clones are listed
by number and most are available through the Resource for Molecular
Cytogenetics. In the case of RMC20P154, a clone, RMC20P153 contains the
same STS and is available from RMC. Clones chosen to detect the three
previously known regions of copy number increase were included in the
array (referred to here as A1, A3, and A4), as well as additional clones
designed to provide an approximately 3 Mb resolution scan of the entire
chromosome. DNA for each target was dissolved in DMSO containing a small
amount of nitrocellulose and water, and deposited onto amino-silane coated
quartz or fused silica microscope slides using a glass capillary as
described above. The inclusion of a small amount of nitrocellulose in the
solution substantially increased the amount of hybridizable target DNA
retained in the spots, thereby increasing signal intensities and thus
sensitivity. Quadruplicate 150-300 .mu.m diameter spots were made of each
target DNA. Two hundred to 400 ng each of reference genomic DNAs, labeled
respectively with fluorescein and Texas red, along with 50 .mu.g of Cot-1
DNA to block repetitive sequences, were hybridized for 16 to 72 hours. A
1.times. magnification imaging system was used to acquire CCD camera
images of each of the fluorochromes. Ratios for all of the spots for each
target clone were averaged.
TABLE 1
______________________________________
Target Locus/Gene
FLpter
______________________________________
RMC20C177 0.034
RMC20P107 CDC25B 0.085
RMC20P160 WI-7829 0.158
RMC20P178 D20S186 0.209
RMC20P005 D20S114 0.272
RMC20P099 CST3 0.352
RMC20P090 BcIX 0.526
RMC20P117 G3/N5 0.548
RMC20P037 SRC 0.603
RMC20P154 D20S44 0.646
RMC20P058 TOPO I 0.675
RMC20P100 HUMSEM1 0.694
RMC20P131 D20S178 0.722
RMC20P063 PTP NR1-2 0.755
RMC20P070 D20S120 0.778
B135 0.806
RMC20P127 0.806
B97 0.806
B130 0.806
RMC20P071 D20S100 0.827
RMC20P073 PCK1 0.867
RMC20C033 0.906
RMC20P179 CHRNA+ 0.948
______________________________________
The quantitative capability of our measurements was assessed by hybridizing
artificial test and reference genomes containing 200 ng of total human
genomic DNA spiked with varying amounts of lambda DNA, length 50 kb, to
arrays containing lambda targets. This approximately simulates the
behavior of targets made from human cosmid clones. The ratios were
accurately proportional to copy number ratios over a dynamic range from
below single copy equivalent level, 3 pg, to at least a factor of 10.sup.3
higher. These results indicate that processes such as reassociation of the
double stranded probe fragments and non-specific binding do not
significantly affect the linearity of the assay.
Two studies indicate that quantitative performance is also obtained for
targets made from human genomic clones even though suppression of
repetitive sequences presented additional challenges. In the first, normal
male and female human genomes were compared, FIG. 1B. Ratios in each
comparative hybridization were normalized so that the average of the
targets on chromosome 20 was 1.0. Note that almost all are within 20% of
the average. Thus ratios that fall outside of this range are likely to
indicate significant copy number difference. In this study, the X
chromosome target had a ratio of 0.65.+-.0.05, demonstrating the ability
to detect single copy changes in a diploid genome. The difference in this
result from the expected value of 0.5 is most likely due to incomplete
suppression of the repeat sequences, but the factors mentioned above may
also contribute.
In a second study, array CGH measurements of copy number variation on
chromosome 20 in the breast cancer cell line BT474 compared with
previously published data obtained by FISH (FIG. 1C). While direct
comparisons at identical loci cannot be made because different sets of
clones were used for the two studies, the two sets of measurements are
generally in excellent agreement within the .+-.20% uncertainty expected
for these techniques. For example, the ratio of the highest peak at
fractional length (FLpter) .about.0.8 relative to the p arm was about 10.5
in the array measurements and .about.9 with FISH. Note that the lines
connecting the data points are eye guides only and do not convey
information on copy number at locations between the points where
measurements were made. Thus other independent regions of copy number
change may be revealed, or the locations of the peaks may change somewhat,
if a higher resolution array were to be used.
Analyses of 5 breast tumors (S-50, S-6, S-21, S-59 and S-234), obtained
from the UCSF Breast Cancer SPORE, are shown in FIGS. 1D and 1E and FIG.
2. All of the ratios have been normalized so that the average ratio on
chromosome 20 p is 1.0. The locations of 5 regions of recurrent copy
number increase, A1-A5, and one of decrease, D1, that were present in
these specimens are indicated. Regions A1, A3 and A4 were previously
described in breast cancer. The existence of D1, A2 and A5 was not
detected in the earlier extensive studies employing conventional CGH, FISH
and chromosome microdissection. The tumors in the present study were
selected by FISH to have copy number increase at A4, so our results do not
represent an unbiased analysis of the frequency or amplitude of these
abnormalities in breast cancer. FIG. 1D compares the previously presented
data from cell line BT474 to tumor S-50. The tumor contains levels of copy
number increase as high as BT474 and 4 separate regions of copy number
increase are apparent. A5 represents a newly discovered recurrent region
of copy number change in breast cancer. It has also recently been
identified by FISH in colon cancer.
FIG. 1E shows the remaining 5 tumors, which have lower level copy number
increases. In one, S-21, the only copy number change found was at the
selecting region A4. None have copy number increase at A1 or A2, but three
have an increase at A3. Two of these, S-59 and S-234 demonstrate that A3
is a distinct, separately amplifiable region located very near A4. S6
contains the most distal amplified region, A5, also seen in tumor S-50.
Three of the tumors, S6, S-59, and S-334 contained the copy number
decrease D1 found in BT474. The results of all the measurements are
summarized in FIG. 2 and shown in FIGS. 3A-3F.
The above examples are provided to illustrate the invention but not to
limit its scope. Other variants of the invention will be readily apparent
to one of ordinary skill in the art and are encompassed by the appended
claims. All publications, patents, and patent applications cited herein
are hereby incorporated by reference.
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