Note: Descriptions are shown in the official language in which they were submitted.
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Arrays, Methods and Kits for Preparation and Use of Syntenic Genomic Arrays
for
Diagnostics and Toxicology
Technical Field
This invention relates to molecular biology, genetic diagnostics and array, or
"chip"
or "biochip" technology. In particular, the invention provides methods for
determining
chromosomal abnormalities in a cell, an organism, or a cell population, such
as in cancer cells
or in embryonic cells, and for comparing chromosome abnormalities of a
plurality of
different species of organisms with respect to defects that affect a
chromosomal syntenic
strand that are homologous in the plurality of organisms. The invention
provides methods,
arrays produced by the methods, and kits for analysis of nucleic acids, for
diagnosis,
prognosis, and toxicology.
Background
Genomic DNA array based chips have the potential to solve many of the
limitations of
traditional whole chromosome analysis methods, which rely on hybridization of
samples to
individual metaphase chromosomes. In contrast to metaphase hybridization, in
which the
immobilized genomic DNA is a metaphase spread, array-based hybridization uses
immobilized nucleic acids arranged as an array on a biochip or an array
platform. The array
hybridization approach can provide DNA sequence copy number information across
the
entire genome in a single, timely, cost-effective and sensitive procedure, the
resolution of
which is primarily dependent upon the number, size and map positions of the
DNA elements
within the array. Typically, bacterial artificial chromosomes, or BACs, which
can each
accommodate on average about 1 SO kilobases (kb) of cloned genomic DNA, are
used in the
production of the array.
While array genome profiling represents a revolutionary progression in genetic
testing, certain aspects of the technique continue to limit performance. In
many cases,
application and immobilization of a nucleic acid probe to a substrate produce
uneven deposit
of the genomic nucleic acid across the surface of the spot, yielding samples
that are not
uniform when viewed under magnification, such as in a microscope. Further,
rare but
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troublesome incomplete removal of non-specifically bound nucleic acid test and
reference
sample to areas of the substrate can lead to complexity of analysis.
Summary of Embodiments of the Invention
A problem in toxicology and in environmental analyses is comparing results
obtained
with an experimental animal to those that might be observed in a human, or
affects a human,
or might affect a human.
A featured embodiment of the present invention is a nucleic acid array that
has a
plurality of immobilized elements in an array, the elements at addressible
locations on a
substrate, the elements being "spots" or patches of nucleic acid deposited on
the substrate.
With the array of the featured invention, the plurality of elements comprise
nucleic acid
sequences from a chromosome syntenic strand from each of a plurality of
organisms, and a
first set of the elements and a second set of the nucleic acid sequences in
the elements are
chosen so that the first set and the second set are from different species of
organism, and the
elements that are from a syntenic chromosome of a first species of organism
have nucleic
acid sequences that are homologous to the nucleic acid sequences that are from
the syntenic
chromosome of a second species of organism.
The term array as used herein implies a plurality which is in this case a very
large
number of elements on a surface, for example, at least 10, or at least 100, or
at least 200, or at
least 1,000. The elements are generally non-identical, however duplicate or
triplicate spots
are used for statistical significance. Each non-identical element contains a
nucleic acid
having a nucleic acid sequence that is a marker for that element, and that
distinguishes it from
other non-identical elements on the same surface. The term array further
implies an orderly
arrangement such that each spot is "addressible", i.e., has a known location
and a known
nucleic acid sequence content.
Calibration spots are included in embodiments of each array herein, as
described in
U.S. patent application serial number 10/112,657 filed March 27, 2002. In one
embodiment,
a calibration spot contains a sample of all or substantially all of the non-
identical sequences
in the other elements of the array. In another embodiment, various
concentrations or
dilutions of a calibration spot are included in each array. In yet another
embodiment, a
calibration spot includes a known quantity of a calibration molecule, which
can be pre-
labeled or it not pre-labeled, e.g., the calibration molecule can be obtained
from a species
other than that of interest in the remainder of the elements of the array,
such as Escherichia
coli DNA or Xenopus laevis DNA used as a calibration spot for syntenic arrays
having
nucleic acid elements of human and mouse DNA; in other embodiments, a
calibration
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molecule can be a synthetic nucleic acid having a naturally occurring or a non-
natural nucleic
acid sequence.
In general, the elements of the syntenic array of the embodiments herein has
nucleic
acid that is cloned genornic DNA. For example, the cloned genomic DNA is
carried on a
vector selected from the group of vectors consisting of yeast artificial
chromosomes (YACs),
bacterial artificial chromosomes (BACs), mammalian artificial chromosomes
(MACs), and
phage P1 artificial chromosomes (PACs).
Further, in general at least one of the species of organism is a mammal.
However
may other embodiments of "syntenic arrays" are envisioned as within the scope
of the
invention, such as those comparing any two different organisms, such as
species of crop
plants, or freshwater fish. In various embodiments, at least one of the
organisms is a mamal,
for example, at least of the organisms is a human. Further, at least one
organism is selected
from the group consisting of rodents, non-human primates, marine mammals,
freshwater
mammals, lagomorphs, porcines, bovines, carnivore, caprines, equines,
amphibia, fish, and
insects. More specifically, at least one organism is selected from the group
consisting of a
gorilla, a chimpanzee, a monkey, a dog, a hamster, a mouse, a rat, a rabbit, a
guinea pig, a
sheep, a goat, a swine, a cow, a horse, a frog, a toad, a zebra fish, and a
fly. I~ an exemplary
array, the species of organism are human and mouse. With a human-mouse array,
effects on
genotoxicity of, for examples, experimental mice or mouse cells can be
compared with
effects on genotoxicity on human cells, for example, in culture. In yet
another exemplary
array, the species of organism are human and a wild animal.
Any of the non-human organisms are, in related embodiments, transgenic, i.e.,
a
transgenic mouse that is used for screening compositions to obtain a
particular novel activity
capable of remediating a particular phenotype is then used as a source of DNA,
for
hybridization to the syntenic arrays ("chips") herein. In this manner, any
screen of organisms
or cells can be further analyzed for genotoxicity, in particular, to identify
compounds that do
not result in chromosomal abnormalities. In addition, the animals herein can
have a "model"
disease, which as used herein means a disease in an animal that is induced or
is present
genetically, and that has symptoms and a phenotype similar to that of a
disease of humans or
a non-human animal, and that is useful for analysis of agents capable of
remediating that
disease.
At least one element of nucleic acid of the first species is at least about
50%
homologous to at least one element of nucleic acid of the second species.
Further, at least
one element of nucleic acid of the first species is at least about 70%
homologous to at least
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one element of nucleic acid of the second species, or about 80%, about 85%,
about 90%,
about 95% or about 99% homologous to at least one element of nucleic acid of
the second
species.
The array elements include nucleic acid sequences that are representative of
at least
one chromosome of at least one species. In a related embodiment, the array
elements include
nucleic acid sequences that are representative of a genome of at least one of
the species.
Representative of at least one chromosome means that at least three, four,
five or more
elements are present in the array that contain sequences from different points
along the
chromosome, such that data obtained from a hybridization of a nucleic acid
sample to the
array can be plotted from the p-terminus to the q-terminus for that
chromosome. Similarly,
representative of at least one genome means that all of the chromosomes within
that genome
are represented, e.g., in a human array, elements are present in the array
that contain
sequences from different points along each of all of the 22 autosomes and the
X and the Y
chromosomes. In a related embodiment, the elements of the array include
nucleic acid
sequences representative of genomes of at least two species.
For all of the above syntenic arrays or the methods below, further embodiments
include providing the arrays as mufti-array surfaces. The mufti-array surfaces
have a
plurality of any of the above arrays on a single substrate. Each of the
plurality of arrays is
printed on the surface in a pattern that is non-contiguous with others, so
that a plurality of
hybridizations can be carried out on the same substrate. For example, two
arrays can be
printed with each one at each end of a glass slide, or three can be printed in
a linear
arrangment with one array at each end and one in the middle. The individual
arrays within
the plurality can further be separated by hydrophobic strips such as a Teflon
strip;
alternatively or in addition, or barriers ("dykes") or raised portions of a
surface such as a slide
can be custom designed to be present prior to printing the array, or added
later. In additional
embodiments of the arrays herein, a viscosity-enhancing solute such as a
dextran or a
polyethyelene glycol can be added to the hybridization buffer, to enhance
separation of the
plurality of hybridizations being performed. Finally, a cover such as a cover
slip can be
separately applied to each hybridization mixture on each array within the
mufti-array surface.
Another featured embodiment of the invention is a method of measuring
genotoxicity
of a composition to a cell of a species of organism, the method comprising
contacting a test
cell or a cell population of a first species with the composition; obtaining a
sample of nucleic
acid from the contacted test cell or population; and analyzing the genome of
the sample
nucleic acid for abnormalities by hybridizing the nucleic acid to an array of
syntenic nucleic
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acid immobilized at addressible locations on a substrate, the syntenic array
having elements
of sequences of nucleic acid from the genome of the first species, and having
elements of
sequences of syntenic nucleic acid from the genome of at least a second
species of organism.
In general, the second species is a human. Further, contacting the test cell
with the
compositions is, in some embodiments, adding the composition to a cell or a
population of
the first species in culture. Use of cell culture makes it possible, for
example, to determine
the effects of a variety of agents on human cells.
In an alternative embodiment, contacting the test cell or population is
treating the cell
of the first species in vivo, i.e., treating the intact organism, generally a
multicellular
organism. Thus. treating the cell in vivo is administering the composition by
a route selected
from the group of achninistering orally, topically, transdermally, and
injecting. Injecting can
be intravenous, subcutaneous, intraperitoneal, and any other standard route.
Treating can also
be, depending on the species, by rectal, intravaginal, intrathecal
administration.
In an embodiment of the method, the first species is a subject exposed to the
composition in a natural environment. The subject can be a "wild" organism
such as a wild
animal or plant, or the subject can be an experimental animal as is used in a
laboratory that
has been placed in the wild in order to measure for a presence of a genotoxic
agent. In a
different embodiment, a subject can be a human or animal patient that has been
inadvertantly
or intentionally exposed to an agent, and the agent was not previously known
to be genotoxic.
Accordingly in the method, the first species which is the test species is
selected from
the group consisting of gorilla, chimpanzee, monkey, dog, hamster, mouse, rat,
rabbit, guinea
pig, sheep, goat, swine, cow, horse, frog, toad, fish, and insect. An example
of the first
species is a non-human transgenic experimental animal. Another example of the
first species
is an animal having a model disease, for example, a mouse having experimental
allergic
encephalomyelitis (EAE), or a non-obese diabetic mouse (NOD), or a mouse
treated with
streptozotocin to induce diabetes. However as the method can be practiced
following a
variety of different circumstances, the first species can be a human cell or a
human subject.
Analyzing the genome of the contacted organism according to the method further
comprises comparing hybridization of nucleic acid from the test cell to
hybridization of
nucleic acid from a reference cell or cell population. The reference cell is
from the first
species; alternatively, the reference cell is from the second species, or from
a third species.
In some embodiments, the reference cell or cell population is not administered
the
composition, and is otherwise identical to the test cell. In general in any of
the embodiments
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of the methods herein, the nucleic acid of the elements immobilized in the
array on the
substrate is cloned DNA.
The method generally includes, prior to hybridization, labeling separately
each of the
test cell nucleic acid and the reference cell with a first detectable label
and a second
detectable label. For example, the first and second labels are fluorescent
dyes, and the dyes
have different emission spectra.
The method further includes, after labeling, preparing a first mixture of the
test cell
nucleic acid labeled with the first label or dye and the reference cell
nucleic acid labeled with
the second label or dye, and preparing a second mixture of the test cell
nucleic acid labeled
with the second label or dye and the reference cell nucleic acid labeled with
the first label or
dye, and separately hybridizing each of the first mixture and the second
mixture to iterations
of the syntenic array.
The method further includes comparing the genome of the test cell by
normalizing a
ratio of extent of hybridization of the first and second labels or dyes to
each element for each
of the ftrst and second mixtures. The method further includes plotting the
resulting set of
ratios as a function of the location of each of the nucleic acids as a
distance along a
chromosome from the p-terminus to the q-terminus. Comparing the genome is
further
identifying a chromosome of the test organism having a chromosomal
abnormality. A
chromosomal abnormality includes an increase or decrease in copy number, such
as a
deletion or an amplification, and also includes a translocation, an inversion,
and an insertion,
including a presence of a nucleic acid sequence not previously characterized
at a location
along the chromosome.
The method further includes identifying a chromosomal location along the
chromosome of the abnormality in the test sample, with respect to the array of
immobilized
elements of the test organism. The method further involves, in cases in which
the first
species is non-human and the syntenic array comprises elements of the human
genome,
determining an homologous chromosome and chromosomal location of the
abnormality in
the human genome. In this context, the term, "homologous chromosome" means a
chromosome of one species having substantial nucleic acid homology with a
chromosome of
another species. Another term often used to describe sequence homology in a
different
organism is "orthologous".
The method further can be used, by comparing an amount of chromosomal
abnormalities in the test sample nucleic acid to chromosomal abnormalities in
the reference
sample nucleic acid, as an indication of an extent of genotoxicity of the
chemical
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composition. Further, comparisons of chromosomal locations of the
abnormalities in the test
species and in another species of organism, for example, in a human, can be
made.
The method is used to analyze genotoxicity of the composition which is
exemplified
by and not limited to: a hazardous occupational compound, a chemical weapon,
airborne dust,
photochemical smog, a natural product, a cosmetic, a food additive, an
agricultural product,
an industrial compound, a new chemical entity, a lead compound, a
pharmaceutical product,
sewage, and an enviromnental sample, and an extract or preparation of any of
these agents or
components of any of these agents.
Another featured embodiment of the invention is a method of identifying a
presence of
a genotoxic agent for a cell of a species of organism in a natural
environment, the method
comprising obtain a test cell or a cell population of a first species of
organism in the
environment; obtaining a sample of nucleic acid from the test cell or
population; and
analyzing the genome of the sample nucleic acid for abnormalities by
hybridizing the nucleic
acid to an array of syntenic nucleic acid immobilized at addressible locations
on a substrate,
the syntenic array having elements of sequences of nucleic acid from the
genome of the first
species, and having elements of sequences of syntenic nucleic acid from the
genome of at
least a second species of organism. The first species may be a feral organism,
i.e., one having
a life cycle in the natural environment, or may be a laboratory strain that
has been placed in
the environment. Alternatively, the first species can be a human exposed to an
agent
inadvertently, such as a human subjected to an ocupational hazard which may
be, for
example, a physical force or a chemical composition in the occupational
environment.
Also among the embodiments of the invention provided herein is a kit for use
of the
method according to any of the above, comprising a syntenic array having
immobilized
nucleic acid elements with nucleotide sequences from genomes of a plurality of
species of
organism, and a container. The kit can further include any of the reagents,
e.g., a plurality of
detectible labels, and/or a polymerase for amplification of nucleic acids, or
a computer
program for obtaining and/or analyzing data obtained from hybridization to the
array, and
instructions for use.
Yet another featured embodiment provided herein is a method of identifying the
presence and location of chromosomal abnormalities in cells of a subject
during progression
of a disease, the method comprising obtaining a nucleic acid sample from the
cells affected
by the disease; and analyzing the sample for chromosomal abnormalities by
hybridizing the
sample to elements of a first syntenic nucleic acid array having nucleic acid
from the genome
of a first species, and further hybridizing the sample to elements of a second
syntenic nucleic
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acid array having nucleic acid from the genome of a second species, the
elements of the first
and second arrays being immobilized on a substrate. Accordingly, the method
involves
obtaining an additional nucleic acid sample of cells from the subject at a
time point
representing a different stage of progression of the disease. Progression of
the disease can be
determined by comparing chromosomal abnormalities in samples from a plurality
of different
time points, i.e., at least two different time points.
For studying progression of a disease by the method herein, a first species or
a second
species is human; in this embodiment or in an alternative embodiment, the
disease is an
animal model of a human disease, i.e., the animal model is a strain of
experimental animal, or
is an experimental animal treated to produce a disease condition that is
useful for study of a
human disease. Diseases such as lung cancer, mesothelioma, adenocarcinoma, and
prostate
cancer have "animal models" well known to one of ordinary skill in the art of
experimental
approaches to study of human disease.
Typically, the disease is a cancer, for example, the disease is a solid tumor,
a blood
proliferative condition. The disease is selected from the group of cancers of
skin, lung,
breast, head and neck, prostate, ovary, brain, leukemia, gastric, stomach,
esophagous,
pancreas, and lymphoma. The disease is a stage I cancer; alternatively, the
cancer is selected
from stage II, III and IV cancers. Accordingly in certain embodiments, the
cancer is
metastatic.
Also provided herein is a method of preparing an array of a plurality of
elements of a
class of biological macromolecules immobilized on a substrate, each element of
the array
having a uniform distribution of the macromolecules, the method comprising
contacting the
substrate with the macromolecules in a composition comprising a buffer of
effective ionic
strength such that a uniform distribution of the macromolecules is obtained
across the surface
of the element. The class of biological macromolecules is selected from the
group consisting
of nucleic acids, proteins, lipids, and carbohydrates. For example, the
nucleic acids are DNA,
for example, the nucleic acids are genomic DNA clones. In a related
embodiment, the clones
comprise an artificial chromosome library of a genome. The artificial
chromosomes are
selected from yeast artificial chromosomes (PACs), bacterial artificial
chromosomes (BACs),
mammalian artificial chromosomes (MACs), and phage P1 artificial chromosomes
(PACs).
In a preferred embodiment, the artificial chromosomes are BACs.
Accordingly, the ionic strength of the buffer for the contacting step is at
least 100
mM, for example, the ionic strength is at least 150 mM. Further, the ionic
strength is less
than 1.0 M, for example, the ionic strength is less than 500 mM. The buffer in
certain
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embodiments comprises an organic ion, for example, the buffer is TRIS N
[tris(hydroxymethyl)methyl]glycine (TRIS); 4-(2-hydroxyethyl)-1-
piperazineethanesulfonic
acid (HEPES); 3-(N-morpholino) propanesulfonic acid (MOPS); or 4-
morpholineethanesulfonic acid (MES). In a preferred embodiment, the buffer is
TRIS.
Alternatively, the buffer comprises an inorganic ion, for example, the buffer
comprises a
phosphate ion. In general, the buffer further comprises EDTA. The buffer has a
pH of at
least about 7, and a pH that is less than about 9. The method further includes
drying the array
of elements on the substrate. For any of the methods or arrays herein, the
substrate is
selected from the group consisting of: glass, paper, ceramics, quartz, metals,
plastics, nylon,
teflon, silicones, and cellulose acetate. Typically, the substrate is a glass
slide.
Embodiments of any of the methods herein can further include providing the
arrays as
a mufti-array surface, and conducting each of the hybridizations together on
the same surface,
as described above. Embodiments of the methods herein further include using
any of the
calibration spots as described herein.
Another embodiment of the invention provided herein is a kit for analysis of
genomic
abnormalities comprising a container and an array prepared by the method
according to any of
methods herein. For example, the kit further comprises buffers for
hybridization of the array
to a sample of nucleic acids. In a method of depositing a plurality of samples
of a biological
material on a substrate in an array of elements having addressible locations,
an improvement
is provided, the improvement comprising depositing the samples in a buffer
having ionic
strength sufficient to produce a uniform distribution of the material
throughout each element.
The biological material is typically DNA although the improvement is
applicable to other
biological materials such as proteins. In particular with the arrays herein,
the array includes
elements of genomic sequences of a plurality of chromosomes from cells from a
plurality of
species of organism, and the sequences are syntenic.
Embodiments of any of the kits herein can further include providing the kits
with
arrays having a mufti-array surface. Embodiments of the kits herein further
include arrays
having elements with any of the calibration spots as described herein.
Brief Description of the Drawings
Fig. 1 a is a ratio plot of comparative genomic hybridization (CGH) of
chromosome 18
of a 12 year old patient having delayed development and central nervous system
demyelination compared to a normal control, showing a deletion of 18 qter of
approximately
7 megabases (Mb). The loss of genetic material is indicated by increase in
Cy3TM labeled test
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sample relative to CySTM labeled reference sample (indicated in red using the
graphics
software software commercially available with the arrays), at the right of the
drawing which
is the chromosome 18 q terminus.
Fig. lb is a ratio plot of CGH of chromosome 4 of the same patient as in Fig.
la,
showing a gain in genetic material at 4q of 3.7 Mb. The gain in genetic
material is indicated
by the increase in CySTM labeled reference sample relative to Cy3TM labeled
test sample
(indicated in blue using the graphics software commercially available with the
arrays) at the
right of the drawing which is the chromosome 4 q terminus.
Fig. 2 is a ratio plot of the X chromosome of a male patient compared to a
normal
male control, showing a distal Xp duplication (at the p terminus of the X
chromosome). The
gain in genetic material is indicated by the increase in CySTM labeled
reference sample
relative to Cy3TM labeled test sample.
Detailed Description of Specific Embodiments
In array hybridization, an effective amount of genomic DNA obtained from cells
of
each of a test sample and a reference sample (e.g., a sample from cells known
to be free of a
chromosomal aberration) are each labeled with a detectable label, such as a
fluorescent dye,
and are each then hybridized to an array of nucleic acids obtained from each
of a collection of
BACs. Hybridization can be performed iteratively, on successive replicates of
the array. The
array contains cloned genomic DNA fragments that collectively cover
substantially the entire
genome of a chosen organism, such as a human. The resulting hybridization
produces a
fluorescently labeled array, the pattern of which reflects hybridization of
sequences in the
samples, i.e., the test genomic DNA and the reference genomic DNA, to
homologous
sequences within the arrayed BACs. For each test sample, a copy number,
including possible
deletions and insertions such as translocations, of every homologous sequence
in each of the
test and reference ~genomic DNA samples should directly affect the pattern of
hybridization,
both quantity and location, for example, as a fluorescent signal at discrete
BACs located at
known spots within the array. The versatility of the approach allows the
detection of both
constitutional variations in DNA copy number in clinical cytogenetic samples
such as
amniotic samples, chorionic villus samples (CVS), blood samples and tissue
biopsies, as well
as somatically acquired changes, for example, that arise during progression of
cancers such as
' those in circulating blood cells, or in solid tumors.
The invention provides array-based methods, arrays and kits for determining
genetic
changes in a sample, such as a cell, a tissue or a cell culture population,
compared to that in a
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reference or normal sample. The methods and arrays of the invention provide
greater levels
of sensitivity, capable of detecting smaller genetic changes than previously
available, and of
detecting clonally distinct cell subpopulations. The methods and arrays of the
invention are
sufficiently sensitive to detect clonally distinct (by karyotypic criteria)
cell populations within
a background cell population. Thus, the methods and arrays of the invention
are particularly
suited for accurate determination and analysis of the complex level of, for
example, genetic
mosaicism observed in many solid tumors and other tumorigenically altered
cells and
samples from individuals with an abnormal genetic make-up.
In one embodiment, the invention provides methods and an array for detecting
genetic
mosaicism. Total genomic DNA is isolated from a cell population, e.g., a
cancer cell
population, with a known or unknown genetic constitution, for example, level
of mosaicism.
A predetermined level of genetic mosaicism can be obtained by conventional G-
band
karyotyping, also referred to as "GTG-banding technique" (see, e.g., Wakui
1999 J. Hum.
Genet. 44:85-90); by fluorescence in situ hybridization ("FISH"; see, e.g.,
Zhao (2000)
Cancer Genet. Cytogenet. 118:108-111); or by spectral karyotyping ("SI~Y";
see, e.g.,
Veldman (1997) Nat. Genet. 15:406-410) or a combination thereof (see, e.g.,
Zhao (2001)
Cancer Genet. Cytogenet. 127:143-147). Array-based genome profiling of the
total genomic
DNA from this cell population is obtained as described herein, and the number
of clonal
subpopulations with distinct karyotypes and their respective percentages in
the total
population are measured. Data from the array-based profile are analyzed as a
function of
position on each chromosome of the test genome, and data from iterations of
hybridization of
both sample and reference nucleic acid, each labeled with each of at least a
first and a second
dye are compared, to determine precise chromosomal sites of a genetic
abnormality.
In another embodiment, pre-isolated total genornic DNA from a homogenous
population of cells with a known genetic aberration or with a suspected
genetic aberration is
tested in comparison with isolated genomic DNA from cells having a "normal" or
reference
karyotype, e.g., cells with no known chromosomal aberrations. For example, the
array
genome profile on total genomic DNA has been established for a female abortus
with a
deletion of Xq and simultaneous trisomy of 16q. An effective amount of test
genomic DNA
with normal 46,XX genomic DNA is used as a reference sample. Genetic
aberrations
detectable herein include those that are not visible by prior methods, i.e.,
those that may not
be detected by conventional hybridization to an intact chromosomal metaphase
spread.
By providing methods, apparatuses and kits having genomic arrays to determine
the
aberrant sites in a genome in a sample, i.e., genetic abnormalities, using the
methods of the
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invention, sites of such genetic abnormalities in each chromosome of a cell
population can be
accurately and efficiently determined.
Definitions
Unless defined otherwise, all technical and scientific terms used herein have
the
meaning commonly understood by a person skilled in the art to which this
invention belongs.
As used herein, the following terms have the meanings ascribed to them unless
specified
otherwise.
The terms "array" or "array" or "DNA array" or "nucleic acid array" or "chip"
or
"biochip" as used herein is a plurality of arrayed elements, each arrayed
element comprising a
defined amount of one or more species of biological molecules, e.g., a
preparation of nucleic
acids, immobilized on a substrate surface at a defined, i.e., at an
addressible known location;
as described in further detail, herein. In certain embodiments, an array of
biological
molecules may be an array of proteins (including peptides and polypeptides),
carbohydrates,
or lipids.
The term "aryl-substituted 4,4-difluoro-4-bora-3a, 4a-diaza-s-indacene dye" as
used
herein includes all "boron dipyrromethene difluoride fluorophore" or "BODIPY"
dyes and
"dipyrrometheneboron difluoride dyes" (see, e.g., U.S. Pat. No. 4,774,339), or
equivalents,
are a class of fluorescent dyes commonly used to label nucleic acids for their
detection when
used in hybridization reactions; see, e.g., Chen (2000) J. Org Chem. 65:2900-
2906: Chen
(2000) J. Biochem. Biophys. Methods 42:137-151. See also U.S. Pat. Nos.
6,060,324;
5,994,063; 5,614,386; 5,248,782; 5,227,487; 5,187,288.
The terms "cyanine 5" or "CySTM" and "cyanine 3" or "Cy3TM" refer to
fluorescent
cyanine dyes produced by Amersham Pharmacia Biotech (Piscataway, N.J.;
Amersham Life
Sciences, Arlington Heights, Ill.), as described in detail, herein, or
equivalents. See U.S. Pat.
Nos. 6,027,709; 5,714,386; 5,268,486; 5,151,507; 5,047,519. These dyes are
typically
incorporated into nucleic acids in the form of 5-amino-propargyl-2'-deoxy-
cytidine 5'-
triphosphate coupled to CySTM or Cy3TM.
The terms "fluorescent dye" and "fluorescent label" as used herein includes
all known
fluors, including rhodamine dyes (e.g., tetramethylrhodamine,
dibenzorhodamine, see, e.g.,
U.S. Pat. No. 6,051,719); fluorescein dyes; "BODIPY" dyes and equivalents
(e.g.,
dipyrrometheneboron difluoride dyes, see, e.g., U.S. Pat. No. 5,274,113);
derivatives of 1-
[isoindolyl]methylene-isoindole (see, e.g., U.S. Pat. No. 5,433,896); and all
equivalents. See
also U.S. Pat. Nos. 6,028,190; 5,188,934.
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The terms "hybridizing specifically to", "specific hybridization" and
"selectively
hybridize to," as used herein refer to formation of a nucleic acid base-paired
duplex as a
result of a high extent of complementary base pairing, of a target sample
nucleic acid
molecule or a target reference molecule, to a probe nucleotide sequence
immobilized on a
substrate surface, under stringent conditions. The term "homologous" means
that two nucleic
acid sequences are sufficiently complementary by Watson-Crick rules of base
pairing to
hybridize under stringent conditions.
The term "stringent conditions" refers to conditions under which one nucleic
acid of a
given sequence will hybridize, i.e., will form a nucleic acid duplex
preferentially with a
second nucleic acid sequence (e.g., a sample genomic nucleic acid hybridizing
to an
immobilized nucleic acid probe in an array), compared to forming a duplex to a
lesser extent
with, or not at all with, other sequences. A "stringent hybridization" and
"stringent
hybridization wash conditions" in the context of nucleic acid hybridization
(e.g., as in array,
Southern or Northern hybridizations) are sequence dependent, and are different
under
different environmental parameters. Generally, more stringent conditions are
found at higher
temperatures, and in the presence of agents that act to reduce the stability
of hydrogen bonds,
such as formamide. Stringent hybridization conditions as used herein can
include, e.g.,
hybridization in a buffer comprising 50% formamide, 5 X SSC, and 1% SDS at
42°C, or
hybridization in a buffer comprising 5 X SSC and 1% SDS at 65°C, both
with a wash of 0.2
X SSC and 0.1% SDS at 65°C. Exemplary stringent hybridization
conditions also include a
hybridization buffer of 40% formamide, 1 M NaCI, and 1 % SDS at 37°C,
and a wash in 1 X
SSC at 45°C. Those of ordinary skill will readily recognize that
alternative but comparable
hybridization and wash conditions can be utilized to provide conditions of
similar stringency.
The precise hybridization format is not critical, since as is known in the
art, it is
stringency of the wash conditions that determine whether a soluble, sample
nucleic acid will
specifically hybridize to an immobilized nucleic acid. Wash conditions can
include, e.g.: a
salt concentration of about 0.02 molar at pH 7 and a temperature of at least
about 50°C or
about 55°C to about 60°C; or, a salt concentration of about 0.15
M NaCI at 72°C for about 15
minutes; or, a salt concentration of about 0.2 X SSC at a temperature of at
least about 50°C or
about 55°C to about 60°C for about 15 to about 20 minutes; or,
the hybridization complex is
washed twice with a solution with a salt concentration of about 2 X SSC
containing 0.1%
SDS at room temperature for 15 minutes and then washed twice by 0.1 X SSC
containing
0.1% SDS at 68°C for 15 minutes; or, equivalent conditions. Stringent
conditions for washing
can also be, e.g., 0.2 X SSC/0.1% SDS at 42°C. See Sambrook, Ausubel,
or Tijssen (cited
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WO 2005/012500 PCT/US2004/025124
herein) for detailed descriptions of equivalent hybridization and wash
conditions and for
reagents and buffers, e.g., SSC buffers and equivalent reagents and
conditions.
The term "karyotype" means the chromosomal aspect of the genome, or chromosome
composition, of a cell or cell population. The term "karyotype" has also been
used to mean
the appearance in a light microscope of a stained, complete chromosome set of
the nucleus of
a cell as the chromosomes appear during mitosis, and the chromosomal
complement of an
individual or sample, including the number of chromosomes and including any
abnormalities
which may be deviations from a normal or euploid set. In various embodiments,
the methods
of the invention can be used to determine deviations from the euploid set such
as any
aneuploid variation in the karyotype of a cell population, whether the cell
population is
consistent in karyotype, or whether the cell population is characterized by
genetic mosaicism,
including the number of karyotype subpopulations in a sample and the percent
of the cell
population having a particular karyotype.
Because specific inherited and acquired diseases and conditions have
characteristic
karyotypes, determination of the chromosomally associated abnormalities of a
cell or cell
population can be used to diagnose, detect or prognose those diseases and
conditions.
Similarly, because levels of genetic abnormalities in a cancer or tumor
population indicate a
medical condition or a physiology, e.g., its tumorigenicity, determination of
the karyotype of
a cancer is useful for diagnosis, prognosis and treatment planning.
The phrase "labeled with a detectable composition" or " detectably labeled" as
used
herein refers to a nucleic acid comprising a detectable composition or moiety,
i.e., a label, as
described herein. The label can be another biological molecule, as a nucleic
acid, e.g., a
nucleic acid in the forni of a stem-loop structure as a "molecular beacon," as
described herein.
The label can be colorimetrically or radioactively labeled bases (or, bases
which can bind to a
detectable label), which can be incorporated into the nucleic acid by, e.g.,
nick translation,
random primer extension, amplification with degenerate primers, and the like.
The label can
be detectable by any means, e.g., visual, spectroscopic, photochemical,
bioluminescent,
chemiluminescent, biochemical, fluorescent, immunochemical, physical or
chemical means.
Examples of suitable fluorescent materials include umbelliferone, fluorescein,
fluorescein
isothiocyanate (FITC), rhodamine, dichlorotriazinylamine fluorescein, dansyl
chloride or
phycoerythrin; an example of a chemiluminescent material is luminol; examples
of
bioluminescent materials are luciferase, luciferin, and aequorin.
The term "nucleic acid" as used herein refers to a deoxyribonucleotide or
ribonucleotide in either single- or double-stranded form. The term encompasses
nucleic acids
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containing known analogues of natural nucleotides. The term also encompasses
nucleic-acid-
like structures with synthetic backbones. DNA backbone analogues provided by
the invention
include phosphodiester, phosphorothioate, phosphorodithioate,
methylphosphonate,
phosphoramidate, alkyl phosphotriester, sulfamate, 3'-thioacetal,
methylene(methylimino), 3'-
N-carbamate, morpholino carbamate, and peptide nucleic acids (PNAs); see
Oligonucleotides
and Analogues, a Practical Approach, edited by F. Eckstein, IRL Press at
Oxford University
Press (1991); Antisense Strategies, Annals of the New York Academy of
Sciences, Volume
600, Eds. Baserga and Denhardt (NYAS 1992); Milligan (1993) J. Med. Chem.
36:1923-
1937; Antisense Research and Applications (1993, CRC Press). PNAs contain
peptide and
peptide-related backbones, such as N-(2-aminoethyl) glycine units.
Phosphorothioate
linkages are described, e.g., by U.S. Pat. Nos. 6,031,092; 6,001,982;
5,684,148; see also, WO
97/03211; WO 96139154; Mata (1997) Toxicol. Appl. Pharmacol. 144:189-197.
Other
synthetic backbones encompassed by the term include methyl-phosphonate
linkages or
alternating methylphosphonate and phosphodiester linkages (see, e.g., U.S.
Pat. No.
5,962,674; Strauss-Soukup (1997) Biochemistry 36:8692-8698), and
benzylphosphonate
linkages (see, e.g., U.S. Pat. No. 5,532,226; Samstag (1996) Antisense Nucleic
Acid Drug
Dev 6:153-156). The term "nucleic acid" as used herein structurally includes
the terms gene,
DNA, RNA, cDNA, mRNA, and chemically or enzymatically obtained derivatives and
copies, including the terms oligonucleotide primer, probe and amplification
product.
The term "genomic DNA" or "genomic nucleic acid" means nucleic acid isolated
from a nucleus of one or more cells, and, includes nucleic acid derived from
(e.g., isolated
from, amplified from, cloned from, synthetic versions of) the total cellular
or genomic DNA.
The genomic DNA can be from any organismal source, including eukaryotic
species, or from
microorganisms which are prokaryotic, such as bacteria and blue-green algae,
or acellular,
such as viruses.
The term "a sample comprising a nucleic acid" or "sample of nucleic acid" as
used
herein refers to a sample comprising a DNA, an RNA, or nucleic acid
representative of DNA
or RNA isolated from a natural source, in a form suitable for hybridization
(e.g., as a soluble
aqueous solution) to another nucleic acid or combination thereof (e.g.,
hybrization to
immobilized probes or targets). The nucleic acid may be obtained as a
plurality of isolated,
cloned or amplified portions of a genome or gene; it may be, e.g., a genomic
DNA, mRNA,
or cDNA from substantially an entire genome, substantially all or part of a
particular
chromosome, or selected sequences (e.g. particular promoters, genes,
amplification or
restriction fragments, cDNA library, etc.). The nucleic acid sample may be
extracted from
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particular cells, tissues or body fluids, or, can be from cell cultures,
including cell lines, or
from preserved tissue sample, as described herein.
As used herein, the terms "computer" and "processor" are used in their
broadest
general contexts and incorporate all such devices. The methods of the
invention can be
practiced using any computer/processor and in conjunction with any known
software or
methodology. For example, a computer/processor can be a conventional general-
purpose
digital computer, e.g., a personal "workstation" computer, including
conventional elements
such as microprocessor and data transfer bus. The computer/processor can
further include any
form of memory elements, such as dynamic random access memory, flash memory or
the
like, or mass storage such as magnetic disc optional storage.
Generating and Manipulating Nucleic Acids
Practicing the methods of the invention involves isolation, synthesis,
cloning,
amplification, labeling and hybridization (e.g., hybridization) of nucleic
acids. As described
herein, nucleic acid for analysis and the inunobilized nucleic acid on the
array can be
representative of genomic DNA, including defined parts of, or entire,
chromosomes, or entire
genomes. Comparative genomic hybridization (hybridization) reactions, see,
e.g., U.S. patent
numbers 5,830,645, and 5,976,790. Nucleic acid samples are labeled with a
detectable
moiety, e.g., a fluorescent dye, for example, a first sample can labeled with
a first dye and a
second sample labeled with a second dye (e.g., Cy3TM and CySTM). In one
embodiment, the
test sample nucleic acid is labeled with at least one detectable moiety, e.g.,
a fluorescent dye
that is different than is used to label a second or reference sample of
nucleic acids, for
example, for use in a first iteration of the hybridization. In a second
iteration, the dyes used
for labeling of the test sample and the reference sample are reversed, and
data obtained from
the first iteration is compared to that of the second iteration, as a control
for any variables
such as efficiencies of labeling, detection of emission, and random non-
specific binding.
In certain embodiments, the nucleic acids may be amplified using standard
techniques
such as PCR. Amplification can also be used to subclone or label the nucleic
acid prior to the
hybridization. The sample and/or the immobilized nucleic acid can be
detectably labeled as
described herein. The sample or the probe on the array can be produced from
and collectively
can be representative of a source of nucleic acids from one or more particular
(pre-selected)
portions of a genome, e.g., a collection of polymerase chain reaction (PCR)
amplification
products, substantially an entire set of chromosomes, a selected chromosome or
a
chromosome fragment, or substantially an entire genome, e.g., as a collection
of clones, e.g.,
BACs, PACs, YACs, and the like. The array-immobilized nucleic acid or genomic
nucleic
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acid sample may be processed in some manner prior to splitting or printing on
the substrate,
e.g., by blocking or removal of repetitive nucleic acids or by enrichment with
selected nucleic
acids.
Samples are applied to the immobilized probes (e.g., spotted or printed on the
substrate to form the array) and, after hybridization and washing, the
addressible location
(e.g., spot on the array) and amount of each dye at each spot are read. The
array-immobilized
nucleic acid can be in the form of cloned DNA, e.g., YACs, BACs, PACs, and the
like, as
described herein. In one embodiment, each "spot" or probe element on the array
has a known
sequence, e.g., a known segment of the genome, and/or a known position on each
of the
chromosomes of the genome, or other sequence.
General Techniques
Nucleic acids used to practice this invention, whether RNA, cDNA, genomic DNA,
vectors, viruses or hybrids thereof, are isolated from any of a variety of
sources, genetically
engineered, amplified, and/or expressed/generated recombinantly. Any
recombinant
expression system can be used, including, in addition to bacterial cells,
e.g., mammalian,
yeast, insect or plant cell expression systems.
Nucleic acids can be synthesized in vitro by well-known chemical synthesis
techniques, as described in, e.g., Carruthers (1982) Cold Spring Harbor Symp.
Quant. Biol.
47:411-418; Adams (1983) J. Am. Chem. Soc. 105:661; Belousov (1997) Nucleic
Acids Res.
25:3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19:373-380; Blommers
(1994)
Biochemistry 33:7886-7896; Narang (1979) Meth. Enzymol. 68:90; Brown (1979)
Meth.
Enzymol. 68:109; Beaucage (1981) Tetra. Lett. 22:1859; U.S. Pat. No.
4,458,066. Double
stranded DNA fragments may then be obtained either by chemically synthesizing
the
complementary strand and annealing the strands together under appropriate
conditions, or by
using the single strand as a template for enzymatically synthesizing the
complementary strand
using a DNA polymerase with a primer sequence.
Techniques for the manipulation of nucleic acids, such as, e.g., subcloning,
labeling
probes (e.g., random-primer labeling using Klenow polymerase, nick
translation,
amplification), sequencing, hybridization, G-banding, SKY, FISH and the like
are well
described in the scientific and patent literature, see, e.g., Sambrook, ed.,
MOLECULAR
CLONING: A LABORATORY MANUAL (2ND ED.), Vols. 1-3, Cold Spring Harbor
Laboratory, (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel, ed.
John Wiley & Sons, Inc., New York (1997); LABORATORY TECHNIQUES IN
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BIOCHEMISTRY AND MOLECULAR BIOLOGY: hybridization WITH NUCLEIC ACID
PROBES, Part I. Theory and Nucleic Acid Preparation, Tijssen, ed. Elsevier,
N.Y. (1993).
Clones of Genomic Nucleic Acids
Genomic nucleic acids used in the methods, kits, and apparatus, of the
invention, e.g.,
those immobilized onto arrays or used as samples, can be obtained and
manipulated by
cloning into various vehicles. If necessary, genomic nucleic acid samples can
be screened and
re-cloned or amplified from any source of genomic DNA. Thus, in various
aspects, forms of
genomic nucleic acid used in the methods of the invention (including arrays
and samples)
include genomic DNA, e.g., genomic libraries, which are contained in mammalian
such as
human artificial chromosomes, satellite artificial chromosomes, yeast
artificial chromosomes,
bacterial artificial chromosomes, Pl artificial chromosomes, and the like.
Mammalian artificial chromosomes (MACS) and human artificial chromosomes
(HAC) are, e.g., described in Ascenzioni (1997) Cancer Lett. 118:135-142;
Kuroiwa (2000)
Nat. Biotechnol. 18:1086-1090; andU.S. patent numbers. 5,288,625; 5,721,118;
6,025,155;
and 6,077,697. MACs can contain inserts larger than 400 kilobase (Kb), see,
e.g., Mejia
(2001) Am. J. Hum. Genet. 69:315-326. Auriche (2001) EMBO Rep. 2:102-107, has
built
human minichromosornes having a size of 5.5 kilobase.
Satellite artificial chromosomes, or, satellite DNA-based artificial
chromosomes
(SATACs), are, e.g., described in Warburton (1997) Nature 386:553-555; Roush
(1997)
Science 276:38-39; Rosenfeld (1997) Nat. Genet. 15:333-335). SATACs can be
made by
induced de novo chromosome formation in cells of different mammalian species;
see, e.g.,
Hadlaczky (2001) Curr. Opin. Mol. Ther. 3:125-132; Csonka (2000) J. Cell Sci.
113 (Pt
18):3207-3216.
Yeast artificial chromosomes (YACs) can also be used and typically contain
inserts
ranging in size from 80 to 700 kb. YACs have been used for stable propagation
of genomic
fragments of up to one million base pairs in size; see, e.g., U.S. patent
numbers 5,776,745;
and 5,981,175; Feingold (1990) Proc. Natl. Acad. Sci. USA 87:8637-8641; Tucker
(1997)
Gene 199:25-30; Adam (1997) Plant J.11:1349-1358; and Zeschnigk (1999) Nucleic
Acids
Res. 27:21.
Bacterial artificial chromosomes (BACs) are vectors that can contain inserts
of size
120 Kb or greater, see, e.g., U.S. patent numbers 5,874,259; 6,277,621; and
6,183,957. BACs
are based on the E. coli F factor plasmid system, and DNA cloned into BACs can
be purified
in microgram quantities. Because BAC plasmids are maintained in the bacterial
cell at a copy
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number of at one to two, unwanted genetic rearrangement observed with YACs are
reduced
or eliminated; see e.g., Cao (1999) Genome Res. 9:763-774.
P1 artificial chromosomes (PACs), bacteriophage P1-derived vectors are
described in
Woon (1998) Genomics 50:306-316; Reid (1997) Genomics 43:366-375; Nothwang
(1997)
Genomics 41:370-378; and Kent (1997) Bioteclmiques 23:120-124). P1 is a
bacteriophage
that infects E. coli that can contain 75 to 100 Kb DNA inserts. PACs are
screened in much
the same way as lambda libraries.
Other cloning vehicles can also be used, for example, recombinant viruses;
cosmids,
plasmids or cDNAs; see, e.g., U.S. patent numbers 5,501,979; 5,288,641; and
5,266,489.
These vectors can include marker genes, such as, e.g., luciferase and green
fluorescent
protein genes (see, e.g., Baker (1997) Nucleic Acids Res 25:1950-1956).
Sequences, inserts,
clones, vectors and the like can be isolated from natural sources, or can be
obtained from
such sources as ATCC or GenBank libraries or commercial sources, or prepared
by synthetic
or recombinant methods.
Amplification of Nucleic Acids
Amplification using oligonucleotide primers can be used to generate or
manipulate,
e.g., subclone, genomic nucleic acids used in the methods of the invention, to
incorporate
label into immobilized or sample nucleic acids, to detect or measure levels of
nucleic acids
hybridized to an array, and the like. Amplification, typically with degenerate
primers, is also
useful for incorporating detectable probes (e.g., CySTM or Cy3TM-cytosine
conjugates) into
nucleic acids representative of test or control genomic DNA to be used to
hybridize to
immobilized genomic DNA. Amplification can be used to quantify the amount of
nucleic
acid is in a sample, see, e.g., U.S. patent number 6,294,338. Amplification
methods are also
well known in the art, and include, e.g., polymerise chain reaction (PCR; see,
e.g., PCR
PROTOCOLS, A GUIDE TO METHODS AND APPLICATIONS, ed. Innis, Academic
Press, N.Y. 1990, and PCR STRATEGIES (1995), ed. Innis, Academic Press, Inc.,
N.Y.);
ligase chain reaction (LCR; see , e.g., Barringer 1990, Gene 89:117);
transcription
amplification (see, e.g., Kwoh 1989, Proc. Natl. Acid. Sci. USA 86:1173); self
sustained
sequence replication (see, e.g., Guatelli 1990, Proc. Natl. Acid. Sci. USA
87:1874); Q Beta
replicase amplification (see, e.g., Smith 1997, J. Clin. Microbiol. 35:1477-
1491); automated
Q-beta replicase amplification assay (see, e.g., Burg 1996, Mol. Cell. Probes
10:257-271);
and other RNA polymerise mediated techniques, e.g., nucleic acid sequence
based
amplification, or, "NASBA" (see, e.g., Birch 2001, Lett. Appl. Microbiol.
33:296-301;
Greijer 2001, J. Virol. Methods 96:133-147).
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Hybridizing Nucleic Acids
In practicing the methods of the invention, samples of nucleic acid, e.g.,
isolated,
cloned or amplified genomic nucleic acid, are hybridized to immobilized
nucleic acids. In
various embodiments, the hybridization and/or wash conditions are carried out
under
moderate to stringent conditions. An extensive guide to the hybridization of
nucleic acids is
found in, e.g., Sambrook Ausubel, Tijssen. Generally, highly stringent
hybridization and
wash conditions are selected to be about 5°C lower than the thermal
melting point (TM) for
the specific sequence at a defined ionic strength and pH. The TM is the
temperature (under
defined ionic strength and pH) at which 50% of the target reference sample
sequence labeled
molecules hybridize to a perfectly matched probe. Very stringent conditions
are selected to be
equal to the TM for a particular probe. Exemplary stringent hybridization
conditions for
hybridization of complementary nucleic acids which have more than 100
complementary
residues on an array comprise 42°C using standard hybridization
solutions (see, e.g.,
Sarnbrook), with the hybridization being carried out overnight. Exemplary
highly stringent
wash conditions can comprise 0.15 M NaCI at 72°C for about 15 minutes.
Exemplary
stringent wash conditions can also comprise a 0.2 X SSC wash at 65°C
for 15 minutes (see,
e.g., Sambrook). In one aspect, a high stringency wash is preceded by a medium
or low
stringency wash to remove background probe signal. An exemplary medium
stringency wash
for a duplex of, e.g., more than 100 nucleotides, comprises 1 X SSC at
45°C for 15 minutes.
An exemplary low stringency wash for a duplex of, e.g., more than 100
nucleotides, can
comprise 4 x to 6 X SSC at 40°C for 15 minutes.
In various embodiments, in practicing the array-based comparative
hybridization
(hybridization) reactions of the invention, the fluorescent dyes Cy3TM. and
CySTM. are used to
differentially label nucleic acid fragments from two samples, e.g., nucleic
acid from a
reference or normal control is compared to a test sample nucleic acid from
cell or tissue.
Many commercial instruments are designed to accommodate to detection of two
dyes. To
increase the stability of CySTM, or fluors or other oxidation-sensitive
compounds, antioxidants
and free radical scavengers can be used in hybridization mixes, the
hybridization and/or the
wash solutions. Thus, CySTM/signals are dramatically increased and longer
hybridization
times are possible. See co-pending LT.S. patent application serial number
09/839,658, filed
Apr. 19, 2001.
To further increase hybridization sensitivity, hybridization can be carned out
in a
controlled, unsaturated humidity environment; thus, hybridization efficiency
is significantly
improved if the humidity is not saturated. The hybridization efficiency can be
improved if
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the humidity is dynamically controlled, i.e., if the humidity changes during
hybridization.
Array devices comprising housings and controls that allow the operator to
control the
humidity during pre-hybridization, hybridization, wash and/or detection stages
can be used.
The device can have detection, control and memory components to allow pre-
programming
of the humidity (and temperature and other parameters) during the entire
procedural cycle,
including pre-hybridization, hybridization, wash and detection steps.
The methods of the invention can incorporate hybridization conditions
comprising
temperature fluctuation. Hybridization has much better efficiency in a
changing temperature
environment as compared to conditions where the temperature is set precisely
or at relatively
constant level (e.g., plus or minus a couple of degrees, as with most
commercial ovens).
Reaction chamber temperatures can be fluctuatingly modified by, e.g., an oven,
or other
device capable of creating changing temperatures.
The methods of the invention can comprise hybridization conditions comprising
osmotic fluctuation. Hybridization efficiency (i.e., time to equilibrium) can
also be enhanced
by a hybridization environment that comprises changing hyper-/hypo-tonicity,
e.g., a solute
gradient. A solute gradient is created in the device. For example, a low salt
hybridization
solution is placed on one side of the array hybridization chamber and a higher
salt buffer is
placed on the other side to generate a solute gradient in the chamber.
Fragmentation and Digestion of Nucleic Acid
In practicing the methods of the invention, immobilized and sample nucleic
acids can
be cloned, labeled or immobilized in a variety of lengths. For example, in one
aspect, the
genomic nucleic acid can have a length smaller than about 200 bases. Use of
labeled genomic
DNA limited to this small size significantly improves the resolution of the
molecular profile
analysis, e.g., in array-based hybridization. For example, use of such small
fragments allows
for significant suppression of repetitive sequences and other unwanted,
"background" cross-
hybridization on the immobilized nucleic acid. Suppression of repetitive
sequence
hybridization greatly increases the reliability of the detection of copy
number differences
(e.g., amplifications or deletions) or detection of unique sequences.
The resultant fragment lengths can be modified by, e.g., treatment with DNase.
Adjusting the ratio of DNase to DNA polymerase in a nick translation reaction
changes the
length of the digestion product. Standard nick translation kits typically
generate 300 to 600
base pair fragments. If desired, the labeled nucleic acid can be further
fragmented to
segments of 200 bases, down to as low as about 25 to 30 bases. Random
enzymatic digestion
of the DNA is carried out, using, e.g., a DNA endonuclease, e.g., DNase (see,
e.g., Herrera
21
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(1994) J. Mol. Biol. 236:405-411; Suck (1994) J. Mol. Recognit. 7:65-70), or,
the two-base
restriction endonuclease CviJI (see, e.g., Fitzgerald (1992) Nucleic Acids
Res. 20:3753-3762)
and standard protocols, see, e.g., Sambrook, Ausubel, with or without other
fragmentation
procedures.
Other procedures can also be used to fragment genomic DNA, e.g. mechanical
shearing, sonication (see, e.g., Deininger (1983) Anal. Biochem. 129:216-223),
and the like
(see, e.g., Sambrook, Ausubel, Tijssen). For example, one mechanical technique
is based on
point-sink hydrodynamics that result when a DNA sample is forced through a
small hole by a
syringe pump, see, e.g., Thorstenson (1998) Genome Res. 8:848-855. Fragment
size can be
evaluated by a variety of techniques, including, e.g., sizing electrophoresis,
as by Siles (1997)
J. Chromatogr. A. 771:319-329, that shows analysis of DNA fragmentation using
a dynamic
size-sieving polymer solution in a capillary electrophoresis. Fragment sizes
can also be
determined by, e.g., matrix-assisted laser desorption/ionization time-of
flight mass
spectrometry, see, e.g., Chiu (2000) Nucleic Acids Res. 28:E31.
Comparative Genomic Hybridization (hybridization)
The methods of the invention are used in array-based comparative genomic
hybridization reactions to detect a chromosomal abnormality in the sample, or
detect genetic
mosaicism in cell populations, such as tissue, e.g., biopsy or body fluid
samples.
Hybridization is a molecular cytogenetics approach that can be used to detect
regions in a
genome undergoing changes, e.g., gains or losses of a sequence or of a change
in copy
numbers of a sequence. Analysis of genomes of tumor cells can detect a region
or regions of
anomaly and to monitor the ongoing process.
Hybridization reactions compare the genetic composition of test versus
controls
samples; e.g., whether a test sample of genomic DNA (e.g., from a cell
population suspected
of having one or more genetic defects) has amplified or deleted or mutated
segments, as
compared to a reference sample which is a "negative" control, e.g., "normal"
or "wild type"
genotype, or to a "positive" control, e.g., a known cancer cell or a cell with
a known defect,
e.g., a known translocation or deletion or amplification or the like.
The methods of the invention can be practiced with all known methods and means
and
variations thereof for carrying out comparative genomic hybridization, see,
e.g., U.S. patent
numbers 6,197,501; 6,159,685; 5,976,790; 5,965,362; 5,856,097; 5,830,645;
5,721,098;
5,665,549; and 5,635,351; and, Diago (2001) American J. of Pathol.
May;158(5):1623-
1631;(Theillet (2001) Bull. Cancer 88:261-268; Werner (2001) Pharmacogenomics
2:25-36;
Jain (2000) Pharmacogenomics 1:289-307.
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Arrays, or "BioChips"
The present invention in one embodiment provides arrays and methods of
producing
them. In an alternative embodiment, the methods herein can be practiced with
any known
"array," also referred to as a "array" or "DNA array" or "nucleic acid array"
or "biochip," or
variation thereof. An array is generically a plurality of "probe elements," or
"spots," each
probe element comprising a defined amount of one or more biological molecules,
e.g.,
polypeptides, nucleic acid molecules, or probes, immobilized on a known or
defined
(addressible) location on a substrate surface. Typically, the immobilized
biological molecules
are contacted with a sample for specific binding, e.g., hybridization, between
molecules in the
sample and the array. Immobilized nucleic acids can contain sequences from
specific
messages (e.g., as cDNA libraries) or genes (e.g., genomic libraries),
including, e.g.,
substantially all or a subsection of a chromosome or substantially all of a
genome, including a
human genome. Other elements or spots can contain reference sequences, such as
positive
and negative controls, and the like. The elements of the arrays may be
arranged on the
substrate surface at different sizes and different densities. Different probe
elements of the
arrays can have the same molecular species, e.g., in different amounts,
densities, sizes,
labeled or unlabeled, and the like.
The sizes and densities of the spots or elements sizes depend upon a number of
factors, such as the nature of the label, the substrate support (which is
solid, semi-solid,
fibrous, capillary or porous), and the like. Each spot or element may comprise
substantially
the same nucleic acid sequences, or alternatively can be a mixture of nucleic
acids of
different lengths and/or sequences. Thus, for example, a spot or element
contains more than
one copy of a cloned piece of DNA, and each copy may be broken into fragments
of different
lengths, by methods described herein.
In one embodiment, the array can contain spots encoding all or substantially
all of
syntenic sequences from highly homologous sequences of chromosomes of two or
more
different species. Syntenic sequences are those that are located on the same
chromosome, for
example, a large chromosome such as human chromosome 1, or a small chromosome
such as
human X chromosome, whether or not these are linked by classical genetic
analysis. In one
embodiment, a syntenic an array contains elements having syntenic sequences
from one or
more chromosomes that contain homologous sequences from each of a human and
another
vertebrate, such as a mammal, e.g., a human and a rodent such as a mouse, or a
human and a
chimpanzee. Alternatively, the vertebrate can be a non-mammal, such as a frog
(RaTaa) or an
African clawed toad (~enopus ) or a zebra Bsh. In another embodiment, a
syntenic array
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contains elements having syntenic sequences from one or more chromosomes from
a human
and an invertebrate species of interest, for example, a human and a
Df°osophila. The
immobilized sequences, obtained for example from cloned BAC libraries using
clones known
to be syntenic from each species, are placed in the array at various
addressible locations.
A syntenic array having arrays of spots of nucleic acids with, for example,
identifiable
regions of chromosomes from a human and from another species such as a mouse
can be
prepared using libraries such as BAC libraries comprising the entire genome,
that are known
to one of skill in the art. These can then be used to analyze samples obtained
from human
subjects having diseases or disease conditions, and from animal subjects for
which animal
models are known that are models for the human disease. Examples of diseases
for which
animal models are known include lung cancer, mesothelioma, adenocarcinoma, and
prostate
cancer.
Alternatively, these arrays can be used to test genotoxicity, i.e.,
mutagenicity, of
chemical compositions towards genomes of test animals, by analysis of the
genome of cells,
organisms, or cell populations exposed to or admininstered the chemical
composition. Cell
populations can be obtained from organisms exposed to the chemical, or can be
cultured in
vitro or ex vivo and then exposed to the chemical composition. The arrays of
the invention,
by testing the effect of the chemical on the entire genome, can replace or
supplement present
toxological analyses that require larger numbers of organisms or longer
periods of analysis,
while yielding results showing effect of the chemical on the entire genome,
and relating those
analyses to the human genome.
Syntenic arrays can be prepared having homologous sequences in a plurality of
organisms, such that samples taken from mouse cells exposed to a chemical, or
from mouse
cells having a particular cancer, can, for example, be analyzed with syntenic
immobilized
arrayed probe sequences from a human, a mouse, a dog, and a frog,
simultaneously and in a
time efficient manner.
The array can comprise nucleic acids immobilized on any substrate, e.g., a
solid
surface (e.g., nitrocellulose, glass, quartz, fused silica, plastics and the
like). See, e.g., U.S.
patent number 6,063,338 describing mufti-well platforms comprising cycloole~n
polymers if
fluorescence is to be measured. Arrays used in the methods of the invention
can include
housing having components for controlling humidity and temperature during the
hybridization and wash reactions.
In practicing the methods of the invention, known arrays and methods of making
and
using arrays can be incorporated in whole or in part, or variations thereof,
as described, for
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example, in U.S. patent numbers 6,277,628; 6,277,489; 6,261,776; 6,258,606;
6,054,270;
6,048,695; 6,045,996; 6,022,963; 6,013,440; 5,965,452; 5,959,098; 5,856,174;
5,830,645;
5,770,456; 5,632,957; 5,556,752; 5,143,854; 5,807,522; 5,800,992; 5,744,305;
5,700,637;
5,556,752; and 5,434,049; see also, e.g., WO 99/51773; WO 99/09217; WO
97/46313; WO
96/17958; see also, e.g., Johnston (1998) Curr. Biol. 8:8171-8174; Schummer
(1997)
Biotechniques 23:1087-1092; Fern (1997) Biotechniques 23:120-124; Solinas-
Toldo (1997)
Genes, Chromosomes & Cancer 20:399-407; Bowtell (1999) Nature Genetics Supp.
21:25-
32. See also published U.S. patent applications Nos. 20010018642; 20010019827;
20010016322; 20010014449; 20010014448; 20010012537; 20010008765. The present
invention in various embodiments can use any known array, e.g., GeneChipsTM,
Affymetrix,
Santa Clara, CA; SpectralChipTM Mouse BAC Arrays, SpectralChipTM Human BAC
Arrays
and Custom Arrays of Spectral Genomics, Houston, Texas, and their accompanying
manufacturer's instructions.
Substrate Surfaces
The arrays used to practice the invention can have substrate surfaces of a
rigid, semi-
rigid or flexible material. The substrate surface can be flat or planar, be
shaped as wells,
raised regions, etched trenches, pores, beads, filaments, or the like.
Substrates can be of any
material upon which a "capture probe" can be directly or indirectly bound. For
example,
suitable materials can include paper, glass (see, e.g., US patent number
5,843,767), ceramics,
quartz or other crystalline substrates (e.g. gallium arsenide), metals,
metalloids,
polacryloylmorpholide, various plastics and plastic copolymers, Nylon.TM.,
Teflon.TM.,
polyethylene, polypropylene, poly(4-methylbutene), polystyrene,
polystyrene/latex,
polymethacrylate, polyethylene terephthalate), rayon, nylon, polyvinyl
butyrate),
polyvinylidene difluoride (PVDF); (see, e.g., U.S. patent number 6,024,872),
silicones (see,
e.g., U.S. patent number 6,096,817), polyformaldehyde (see, e.g., U.S patent
numbers
4,355,153; and 4,652,613), cellulose (see, e.g., U.S. patent number
5,068,269), cellulose
acetate (see, e.g., U.S. patent number 6,048,457), nitrocellulose, various
membranes and gels
(e.g., silica aerogels, see, e.g., U.S. patent number 5,795,557), paramagnetic
or
superparamagnetic microparticles (see, e.g., U.S. patent number 5,939,261) and
the like.
Reactive functional groups can be, e.g., hydroxyl, carboxyl, amino groups or
the like. Silane
(e.g., mono- and dihydroxyalkylsilanes, aminoalkyltrialkoxysilanes, 3-
aminopropyl-
triethoxysilane, 3-aminopropyltrimethoxysilane) can provide a hydroxyl
functional group for
reaction with an amine functional group.
Nucleic Acids and Detectable Moieties: Incorporating Labels and Scanning
Arrays
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The methods of the invention use nucleic acids associated with a detectable
label, e.g.,
have incorporated or have been conjugated to a detectable moiety. Any
detectable moiety can
be used. The association with the detectable moiety can be covalent or non-
covalent. In
another embodiment, the array-immobilized nucleic acids and sample nucleic
acids are
differentially detectable, e.g., they have different labels and emit
difference signals. In yet
another embodiment, the array-immobilized nucleic acids are unloaded, and the
test sample
nucleic acid and the reference nucleic acid are differentially blended, and in
another iteration
of the hybridization, the differential labels are changed.
Useful detectible labels or tags include, e.g., radioactive labels, e.g., 32P,
35S,3H, 14C,
lash 1311; fluorescent dyes (e.g., CySTM, Cy3TM, FITC, rhodamine, lanthanide
phosphors,
Texas red), electron-dense reagents (e.g. gold), enzymes, e.g., as commonly
used in an
ELISA (e.g., horseradish peroxidase, (3-galactosidase, luciferase, alkaline
phosphatase),
colorimetric labels (e.g. colloidal gold), magnetic labels (e.g. DynabeadsTM),
biotin,
dioxigenin, or any.haptens and proteins for which antisera or monoclonal
antibodies are
available. The label can be directly incorporated into the nucleic acid to be
detected, or it can
be attached to a probe or antibody having a linked moiety that hybridizes or
binds to the
nucleic acid. A peptide can be made detectable by incorporating (e.g., into a
nucleoside base)
a predeternzined polypeptide epitope recognized by a secondary reporter (e.g.,
leucine zipper
pair sequences, binding sites for secondary antibodies, transcriptional
activator polypeptide,
metal binding domains, epitope tags). Label can be attached by spacer arms of
various
lengths to reduce potential steric hindrance or impact on other useful or
desired properties.
See, e.g., Mansfield (1995) Mol Cell Probes 9:145-156. In array-based
hybridization, fluors
can be paired together; for example, one fluor labeling the control sample
(e.g., the nucleic
acid of known, or normal, karyotype) and another fluor the test sample nucleic
acid (e.g.,
from a CVS or from a cancer cell sample). Exemplary pairs are: rhodamine and
fluorescein
(see, e.g., DeRisi (1996) Nature Genetics 14:458-460); lissamine-conjugated
nucleic acid
analogs and fluorescein-conjugated nucleotide analogs (see, e.g., Shalon
(1996) supra);
Spectrum Red.TM. and Spectrum Green.TM. (Vysis, Downers Grove, Ill.); Cy3TM
and CySTM
Cy3 TM and CySTM can be used together; both are fluorescent cyanine dyes
produced by
Amersham Life Sciences (Arlington Heights, Ill.). Cyanine and related dyes,
such as
merocyanine, styryl and oxonol dyes, are particularly strongly light-absorbing
and highly
luminescent, see, e.g., U.S. patent numbers 4,337,063; 4,404,289; and
6,048,982.
Other fluorescent nucleotide analogs can be used, see, e.g., Jameson (1997)
Methods
Enzymol. 278:363-390; Zhu (1994) Nucleic Acids Res. 22:3418-3422. U.S. patent
numbers
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5,652,099 and 6,268,132 also describe nucleoside analogs for incorporation
into nucleic
acids, e.g., DNA and/or RNA, or oligonucleotides, via either enzymatic or
chemical synthesis
to produce fluorescent oligonucleotides. U.S. patent number 5,135,717
describes
phthalocyanine and tetrabenztriazaporphyrin reagents for use as fluorescent
labels.
Detectable moieties can be incorporated into genomic nucleic acid of the test
or
reference sample, and, if desired, into "target" nucleic acid, by covalent or
non-covalent
means, e.g., by transcription, such as by random-primer labeling using Klenow
polymerase,
or "nick translation," or, amplification, or equivalent. For example, in one
aspect, a
nucleoside base is conjugated to a detectable moiety, such as a fluorescent
dye, e.g., Cy3TM or
CySTM, and then incorporated into a sample genomic nucleic acid. Samples of
genomic DNA
can be incorporated with a Cy3TM- or a CySTM-dCTP conjugate mixed with
unlabeled dCTP.
CySTM is typically excited by the 633 nm line of HeNe laser, and emission is
collected at 680
nm. See also, e.g., Bartosiewicz (2000) Archives of Biochem. Biophysics 376:66-
73; Schena
(1996) Proc. Natl. Acad. Sci. USA 93:10614-10619; Pinkel (1998) Nature
Genetics 20:207-
211; Pollack (1999) Nature Genetics 23:41-46.
In another embodiment, for performing PCR or nick translation to label nucleic
acids,
modified nucleotides synthesized by coupling allylamine-dUTP to the
succinimidyl-ester
derivatives of the fluorescent dyes or haptenes (such as biotin or
digoxigenin) are used; this
method allows custom preparation of most common fluorescent nucleotides, see,
e.g.,
Henegariu (2000) Nat. Biotechnol. 18:345-348.
In certain embodiments of the methods of the invention, labeling with a
detectable
composition (labeling with a detectable moiety) also can include a nucleic
acid attached to
another biological molecule, such as a nucleic acid, e.g., a nucleic acid in
the form of a stem-
loop structure as a "molecular beacon" or an "aptamer beacon." Molecular
beacons as
detectable moieties are well known in the art; for example, Sokol (1998) Proc.
Natl. Acad.
Sci. USA 95:11538-11543, synthesized "molecular beacon" reporter
oligodeoxynucleotides
with matched fluorescent donor and acceptor chromophores on their 5' and 3'
ends. In the
absence of a complementary nucleic acid strand, the molecular beacon remains
in a stem-loop
conformation where fluorescence resonance energy transfer prevents signal
emission. On
hybridization with a complementary sequence, the stem-loop structure opens
increasing the
physical distance between the donor and acceptor moieties thereby reducing
fluorescence
resonance energy transfer and allowing a detectable signal to be emitted when
the beacon is
excited by light of the appropriate wavelength. See also, e.g., Antony (2001)
Biochemistry
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40:9387-9395, describing a molecular beacon comprised of a G-rich 18-mer
triplex forming
oligodeoxyribonucleotide. See also U.S. patent numbers 6,277,581 and
6,235,504.
Aptamer beacons are similar to molecular beacons; see, e.g., Harnaguchi (2001)
Anal.
Biochem. 294:126-131; Poddar (2001) Mol. Cell. Probes 15:161-167; Kaboev
(2000) Nucleic
Acids Res. 28:E94. Aptamer beacons can adopt two or more conformations, one of
which
allows ligand binding. A fluorescence-quenching pair is used to report changes
in
conformation induced by ligand binding. See also, e.g., Yamamoto (2000) Genes
Cells
5:389-396; and Smirnov (2000) Biochemistry 39:1462-1468.
In addition to methods for labeling nucleic acids with fluorescent dyes,
methods for
the simultaneous detection of multiple fluorophores are well known in the art,
see, e.g., U.S.
patent numbers 5,539,517; 6,049,380; 6,054,279; and 6,055,325. For example a
spectrograph
can image an emission spectrum onto a two-dimensional array of light
detectors; a full
spectrally resolved image of the array is thus obtained. Photophysics of the
fluorophore, e.g.,
fluorescence quantum yield and photodestruction yield, and the sensitivity of
the detector are
read time parameters for an oligonucleotide array. With sufficient laser power
and use of
CySTM and/or Cy3TM, which have lower photodestruction yields, an array can be
read in less
than S seconds.
It is desirable for detection and analysis of a mixture of two or more fluors
or
fluorescent dyes such as Cy3TM and CySTM, to create a composite image showing
the amount
of each of the plurality of fluors. To acquire the two or more images, the
array can be scanned
either simultaneously or sequentially. Charge-coupled devices, or CCDs, are
used in array
scanning systems, including practicing the methods of the invention. Thus,
CCDs used in the
methods of the invention can scan and analyze multicolor fluorescence images.
Devices and methods can be used or adapted to practice the methods of the
invention,
including array reading or "scanning" devices, for scanning the array
following hybridization,
and for further analyzing multicolor fluorescence images; see, e.g., U.S.
patent numbers
6,294,331; 6,261,776; 6,252,664; 6,191,425; 6,143,495; 6,140,044; 6,066,459;
5,943,129;
5,922,617; 5,880,473; 5,846,708; and 5,790,727; and, the patents cited in the
discussion of
arrays herein. See also published U.S. patent applications having numbers
20010018514; and
20010007747.
The methods of the invention further comprise data analysis, which can include
the
steps of determining, e.g., fluorescent intensity as a function of substrate
position, removing
"outliers" (data deviating from a predetermined statistical distribution), or
calculating the
relative binding affinity of the immobilized array targets from the remaining
data. The
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resulting data can be displayed as an image with color in each region varying
according to the
light emission or binding affinity between targets and probes. See, e.g., U.S.
patent numbers
5,324,633; 5,863,504; and 6,045,996. The invention can also incorporate a
device for
detecting a labeled marker on a sample located on a support, see, e.g., U.S.
patent number
5,578,832.
In one embodiment, the data are displayed as a ratio plot of normalized data
from two
independent arrays, for example, in which Cy3TM -labeled test sample data
normalized to
CySTM-labeled reference sample are shown in red, and CySTM-labeled test sample
data
normalized to Cy3TM-labeled reference data, are shown in blue. The normalized
ratio,
displayed on the ordinate, from each of the individual clones, is displayed
linerearly ordered
according to position on a chromosome along the abscissa, such that the p-ann
terminus
clone is displayed on the left, and the q-arm terminus is displayed on the
right, for each
chromosome. Reciprocal values (each normalized to a reference control) are
used for red and
blue plots, so that the obtained red and blue functions deviate in opposite
directions (one
positive and one negative) if a genetic abnormality that is a deletion of one
or more BAC
clones, is significant. Similarly, the two functions deviate in the same
direction (both
positive) if a genetic abnormality that is an insertion compared to one or
more BAC clones, is
observed. Non-significant deviations that are due, e.g., to non-specific
binding or other
random effects, appear in only one of the two functions.
Sources of Genomic Nucleic Acid for Sample Preparation
The invention provides methods of detecting a chromosomal abnormality in a
sample
comprising nucleic acid, such as a cell population or a tissue or fluid
sample, by performing
an array-based comparative genomic hybridization. The nucleic acid can be
isolated from or,
amplified from or, cloned from genomic DNA. The genomic DNA can be from any
source,
for example, the cell, tissue or fluid sample from which the nucleic acid
sample is prepared is
taken from a subject or a cell exposed to a chemical composition or a physical
force, to
determine whether the composition or force is associated with a genetic
defect, and to
compare the abnormality with that of a patient having or suspected of having a
pathology or a
condition. A causality relationship may be established between the composition
or force and
the chromosomal abnormality, or the diagnosis or prognosis of the pathology or
condition can
be associated with a genetic defect, e.g., a cancer or tumor comprising cells
with genomic
nucleic acid base substitutions, amplifications, deletions and/or
translocations. The test
sample (and a control reference sample) can be a cell sample, such as tissue
or fluid from,
e.g., amniotic samples, CVS, serum, blood, chord, blood or urine samples,
central nervous
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system (CNS) or bone marrow aspirations, fecal samples, saliva, tears, tissue
and surgical
biopsies, needle or nucleic acids from punch biopsies, and the like. The
reference sample may
be a standard that is used for each analysis, i.e., a uniform standard as a
negative control for
chromosomal abnormalities, or a positive control for a particular known
syndrome, defect, or
disease.
Methods of isolating cell, tissue or fluid samples are well known to those of
skill in
the art sample sources include, but are not limited to, aspirations, tissue
sections, drawing of
blood or other biological fluids, surgical or needle biopsies, and the like. A
"clinical sample"
derived from a patient includes frozen sections or paraffin sections taken for
histological
purposes. The sample can also be derived from supernatants (of cell cultures),
lysates of cells,
cells from tissue culture in which it may be desirable to detect a genetic
abnormality,
including chromosomal abnormalities and changes in gene or chromosomal copy
numbers.
Chromosomal Abnormalities
The methods, arrays and kits of the invention can be used for detecting
genotoxic
effects of a chemical composition or preparation, or a physical force, and can
also be used for
diagnosing diseases and conditions, formulating appropriate treatment plans
and preparing a
prognosis, using one or more models from a heterologous species, to
extrapolate to the
species of interest, i.e., a human. The methods and arrays herein provide a
surrogate for use
of human cells, by analyzing syntenic strands of chromosomes from more than
one species or
genus on a single surface. The syntenic arrays can be arranged, by selection
of clones that
carry nucleic acid sequences from numbers and locations at selected portions
of an
organisms' genome, to focus on chromosomes or parts of chromosomes of interest
in two or
more species of organism, or can include an entire genome of at least one of a
plurality of
species of organisms. Causality of chromosome defects or abnormalities can be
associated
with one or more compositions or forces, and further associated with one or
more known
genetic defects. Further, methods, apparatuses and kits of the invention can
be used for
analyzing progression of a disease, e.g., a cancer or tumor comprising cells
with genomic
nucleic acid base substitutions, amplifications, deletions and/or
translocations, or, an
inherited condition. In some situations, the amount or degree of different
subpopulations
comprising different genetic makeups (karyotypes) in a tumor or other cancer
cell population
from a patient can be helpful in classifying the cancer or formulating a
treatment plan or
prognosis.
Chromosome abnormalities are also common causes of congenital malformations
and
spontaneous abortions. They include structural abnormalities such as
deletions, insertions,
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translocations and amplifications of various portions of chromosome;
polyploidy;
monosomy; trisomy; and mosaicism.
Methods of the invention can also be used to detect aneuploidy (a deviation
from
euploidy, commonly complete diploidy) of all or parts of one or more
chromosomes, for
example, chromosomes 13, 18, 21, X, and Y from genomic DNA from newborn
uncultured
blood samples (see, e.g., Jalal (1997) Mayo Clin. Proc. 72:705-710). Mosaicism
has been
found in approximately 1%-2% of viable pregnancies as determined by CVS at 9-
11 weeks of
gestation. It has been detected in pregnancies with both diploid and trisomic
fetuses and
appears to have an important effect on the intrauterine fetal survival, see,
e.g., Harrison
(1993) Hum. Genet. 92:353-358. Experimental cells of human origin or from
another species
are used to analyze for aneuploidy produced by a chemical composition or a
physical force,
and arrays are designed to report abnormalities along a syntenic set of clones
of a
chromosome and its hornolog in another species.
Preimplantation genetic diagnosis of oocytes and embryos has become the
technique
of choice to select against abnormal embryos before embryo transfer in vitro
fertilization
(IVF) programs. Thus, in another embodiment, the methods of the invention are
used for
preimplantation genetic diagnosis and the diagnosis of structural
abnormalities in oocytes and
embryos. See, e.g., Fung (2001) J. Histochem. Cytochem. 49:797-798. The
methods,
apparatuses and kits of the invention are useful in conjunction with CVS and
fetal
karyotyping. See, e.g., Sanz (2001) Fetal Diagn. Ther. 16:95-97.
Genetic mosaicism is frequent among transgenic animals produced by pronuclear
microinjection. A successful method for the screening of founder animals for
germline
mosaicism prior to mating would greatly reduce the costs associated with the
propagation of
the transgenic lines, and improve the efficiency of transgenic livestock
production. A
syntenic array using the methods herein enables analysis of animals of a
variety of species on
a single surface, such as a glass slide. In each analysis, two mixtures of
test and reference
nucleic acids are made: the test sample separately labeled with a each of a
first detectable
label and a different and distinguishable second is mixed with a reference
sample labeled
with each of the second detectable label and the first detectable label,
respectively. The two
mixtures are hybridized to iterations of the syntenic array on a surface
having a plurality of
arrays, i.e., on a mufti-array surface. Each of the iterations of the array
has elements having
nucleic acids from the syntenic strand of the chromosome from each of the
species. Results
obtained for each species are compared directly for results obtained from a
second or more
species. Thereafter, one of the species can be used as a surrogate for
analysis of the chemical
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composition, physical force, or progression of the condition. The methods of
the invention
are useful in the production of transgenic animals, particularly, the
screening of founder
animals for germline mosaicism prior to mating. See, e.g., Ibanez (2001) Mol.
Reprod. Dev.
58:166-172.
Toxicology Using Whole Organisms and Cells in Cultures
Methods herein are suitable for analysis of abnormalities in cell samples
obtained
from organisms (species of animals, plants, fungi, etc.) and from samples of
cells in culture.
Cells may be primary cultures or established cell lines. Primary cultures may
be obtained cell
samples taken from whole metazoan animals or from multi-cellular plants, for
example, that
have been exposed (test samples) to a chemical or physical agent in an
environment, or from
unexposed control organisms which may be used as reference samples. Organisms,
primary
cultures, and established cell lines are exposed to an agent under rigorous
controlled
laboratory conditions, or are placed in a natural environment to monitor that
environment for
the presence of a deleterious agent, or are feral organism obtained from the
environment as a
means of monitoring a possible past history of genotoxic agents in that
environment.
Primary cell cultures are obtained from tissues and organs by culture methods
using
media known to one of ordinary skill in the art of cell cultures. Suitable
animal cell types are
ascites, epithelial (including epidermal, tracheal/bronchial, renal tubule,
hematopoietic)
endothelial, meural cells, lymphocytes and the like. Cells may be obtained
from wild-type
outbred strains such as BN (Brown Norway) rat, or from inbred isogenic strains
such as F344
rat, some of which exhibit substantially greater sensitivity to chemical
agents than outbred
strains, for example, are tens to hundreds fold more sensitive as indicated by
presenting
similar effects at tens to hundreds fold decreased dose of the agent. Suitable
rodent strains
are obtained from Charles River Laboratory, Wilmington, MA or other suppliers.
Toxicology studies using the syntenic arrays herein are also performed with
genetically engineered organisms or with mutant strains, any of which may be
heterozygous
or homozygous for one or more transgenes or mutant alleles (see Dashwood, R.,
J. Biochem.
Mol. Biol. 36(1)35-42, 2003). Mutant animals such as the non-obese diabetic
mouse (NOD),
or animals treated to obtain a model system of a human disease, such as
streptozotocin-
treated diabetic mice, or myelin-treated mice having experimental allergic
encephalomyelitis
(EAE, a model for multiple sclerosis) may be used. Further, mouse model
strains carrying
engineered reporter genes such as lacZ, lacl, c-mycllacZ, YpsL, and gptd
transgenic animals,
and knock-out strains such as p53+~-, XPA-~-, XPL-~-, and the like have been
used to detect
frequency and nature of point mutations or deletions spanning one or a few
base pairs within
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the gene. Mouse mutant strains are available from Jackson Laboratories, Bar
Harbor, ME
and other suppliers. While none of these strains, however, is engineered to be
capable of
detecting large chromosomal abnormalities such as deletions, amplifications,
or other major
chromosomal changes in response to an agent in an environment, any of these
cell lines or
strains, or rodents in vivo, can be analyzed by the methods herein for such
chromosomal
abnormalities. DNA is prepared from a cell sample and labelled according to
Example 2
herein, and tested on the syntenic arrays designed for use both with human and
non-human
species, e.g., rodent species such as rat or mouse, as described in Example 4.
Data obtained is
complementary to that obtained by the use of such reporter genes, and both
data can be
obtained from the same groups of animals.
Advantageously, data obtained using the syntenic arrays herein indicate both
the
presence of a chromosomal abnormality in the test samples, and its location on
the
chromosome of the test organism, e.g., rat, mouse, guinea pig, further
indicates its location on
a homologous chromosome of another species, such as a human. The test organism
therefore
is used herein as a surrogate as prospective designed toxicological tests on
humans cannot be
performed.
Mufti-array Surfaces
Mufti-array surfaces provided herein have on each surface a plurality of
copies of the
array, i.e., arrays of biological molecules, for example, nucleic acids. The
term "mufti-array
surface" or "surface" as used herein means an article of manufacture having a
plurality of
micro-arrays applied to a side or a face of a substrate. In general the micro-
arrays are printed
or spotted or otherwise deposited on the face of the substrate, in an
arrangement such that the
arrays are non-contiguous, i.e., the arrays are distal from each other on the
surface, or are not
in contact, compared to the size of each array and the spacings of the spots
within each array.
A mufti-array surface having a plurality of arrays is desirable for the
following
procedures: hybridizations are conducted in duplicate or triplicate on a
single surface.
Previous to the present invention, duplicate or triplicate or even a greater
number of
replicated spots have been described that are present on a single surface,
however all spots
were exposed to hybridization of a single hybridization mixture prepared from
a biological
sample. The hybridization mixture is a solution that typically contains a
nucleic acid sample
from a test subject or a reference subject, and is labeled with a fluorescent
dye, or is a mixture
of two different samples of nucleic acids of different origins, each labeled
with the same or a
different dye. The hybridization mixture is formed prior to hybridization with
the spots or
elements of the array on the surface, for example, the mixture includes
nucleic acids from test
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subject labeled with a first fluorescent dye and nucleic acids from a
reference sample labeled
with a different and distinguishable dye. The reference sample can be nucleic
acids from a
normal individual of the same species as the test subject, or can be nucleic
acids of a different
species, or nucleic acids from a single BAC clone or from a mixture of BAC
clones. For
BAC clones, NCBI maintains a human BAC resource, which provides genome-wide
inforniation concerning large-insert clones that integrate cytogenetic,
radiation-hybrid,
linkage, and sequence maps of the genome. See
www.ncbi.nlm.nih.gov/genome/cyto/hrbc.shtml.
It is desirable, in analyzing such data, to perform the hybridization in two
different
formats that reverse the fluorescent labels, what is commonly described as a
"label reversal",
"label swap" or "dye swap" analysis. In a dye swap analysis, at least two
nucleic acid
samples are to be compared, and at least two mixtures are made. In the first
mixture, a first
label such as a first fluorescent dye is used to identify the reference
nucleic acid probe, and a
second label such as a second fluorescent dye is used to identify the test
sample, and after
labeling each, the mixture is made. Then the labels are reversed, i.e., a
second mixture is
made in which the reference nucleic acid probe carries the second dye and the
test sample
carries the first dye. Each of the two mixtures provides a reference for the
purpose of plotting
amounts of hybridization of each solution nucleic acid, reference and test
sample, to each of
the immobilized cloned nucleic acids. The results are plotted as a function of
the linear
position of each of the cloned immobilized nucleic acids on a chromosome. Then
a
representation is made of a portion or of an entire chromosome, or of a
plurality of
chromosomes, or of a complete set of chromosomes (autosomes with or without
sex
chromosomes), i.e., of the entire genome. Results obtained from analyzing both
sets of data
are combined to reveal changes that would otherwise be undetectable if label
reversal was not
used. This is because small fluctuations from a ratio of 1.0 become
statistically significant
when the dye swap data are plotted together, which might not be significant if
only a single
mixture was used.
Further, it is desirable to compare multiple test subjects with the same
reference
sample. In any of these uses, multiple iterations of identical arrays on a
single surface are
highly advantageous or necessary.
Prior to the methods and surfaces as described herein, it has been necessary
to
conduct such analyses using a plurality or a multiplicity of replicas of the
array of elements
printed on each of several different surfaces, each replica having one
complete iteration of all
of the elements within the array, or possibly having two iterations with
elements interspersed,
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or possibly having two iterations closely aligned to eachother, the two
iterations serving as
statistical replicas for improved accuracy of a single hybridization mixture.
It has not been
possible to contact a single surface successfully with two different mixtures
and maintaining
a separate integrity of each of the different mixtures. For example prior to
the present
methods, a dye swap analysis was performed with two mixtures, the first being
a mixture of
the test nucleic acid labeled with the first dye and mixed with the reference
nucleic acid
labeled with the second dye, and the second being the test nucleic acid
labeled with the
second dye mixed with the reference nucleic acid labeled with the first dye,
the two mixtures
were then analyzed using separate arrays on each of two different surfaces.
The use of a multiplicity of different surfaces for separate hybridizations of
each of
different nucleic acid mixtures can be a source of variability, e.g., in
efficiency of binding of
spots to each surface, hybridization due to variability in conditions, minor
variations in
concentration of each nucleic acid, variation in concentrations, different
efficiencies in
elution of non-specifically bound materials due to minor variations in washing
procedures or
solutions, at the time of hybridization to each separate surface, or
variations in
photomultiplier settings in a scanner used to visualize and evaluate the
array, after
hybridization to each separate surface. Accordingly, the present surfaces
provided herein
address this problem in the prior common usage by having mufti-arrays, which
are a plurality
of the arrays present together on a single surface.
In a non-limiting example, two arrays are located at distal ends of a planar
substrate
such as a standard glass microscope slide, however alternative shapes and
sizes of substrates,
and shapes and sizes of arrays, are within the scope of surfaces, kits and
methods envisioned
herein. For example, a substrate may be a one inch by 3 inch microscope slide,
and may have
a plurality of arrays such as two arrays, one at either end, or four arrays in
a linear
arrangement. A larger substrate such as a square slide may have four arrays,
one in each
corner, or nine arrays with three arrays on each side and one in the middle.
Further, barriers to maintain separation of fluids deposited on each array
during
hybridization may be used, the barners being placed between each of the
arrays, in addition
to embodiments of the surface in the absence of barriers, as described herein.
The barriers
are physical "dykes" or "dams" having a height above the plane of the
substrate face or
surface, and such barriers include raised portions of the substrate as
manufactured, or as
added subsequently. Alternatively, the barriers may be hydrophobic materials
that are printed
on the substrate to produce a "strip" which can prevent the flow of an aqueous
solution from
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one array to another. The barriers can be added before or after printing or
depositing the
micro-arrays, to produce the mufti-array surfaces.
The barriers are comprised of a material that is not soluble in aqueous
solution, and
the material hydrophobic. Exemplary hydrophobic materials for barrier
construction or
printing include: polyethylene, silicone, paraffin, and Teflon~.
Hybridization using the "mufti-array surfaces" having multiple arrays on a
single
surface of a single substrate, is conducted by adding each of the
hybridization mixtures to an
iteration of the array and protecting the hybridization mixture with a cover
to prevent loss of
volume of solution by evaporation. The cover further acts to confine each
hybridization of a
particular sample or mixture of samples, labeled with one or more dyes as
described above, to
the appropriate micro-array. A pre-determined amount of hybridization mixture
is deposited
above each of the arrays, such that addition of a cover, for example placed
directly on the
fluid, yields a resulting thin layer of fluid above the array in which the
sample nucleic acids
can hybridize to complementary sequences within the array. Hybridization for
each array on
the surface is conducted under a separate cover.
Conditions for hybridization can be modified, for example, the hybridization
solution
can be altered, to improve and assure fluid separation of the multiple
hybridizations on the
surface. For example, viscosity of the hybridization fluid may be increased to
reduce fluidity
by adding one or more solutes that do not interact with the nucleic acids
during the
hybridization. Exemplary solutes include small molecules that are viscous
liquids such as
glycerol; polymers of small molecules such as sugars, exemplified by but not
limited to
dextrans; and starches such as corn starch; polymers of amino acids which are
synthetic
polypeptides or naturally occurring proteins such as albumins and gelatins;
and synthetic
polymers, for example, polyethylene glycol, or polyacrylamide or agarose, each
solute at a
concentration sufficient to increase viscosity without significantly affecting
mobility of the
dissolved nucleic acids for interaction and hybridization (annealing to form a
double stranded
complex) to the immobilized nucleic acids. The viscosity increasing solute may
be
chemically modified to improve its properties, for example, to render it
resistant to digestion
by extracellular enzymes of bacteria and fungi. Solutions for hybridization
may be stored
with antibiotic or growth inhibiting materials to retard spoilage during
storage; alternatively,
solutions may be frozen or lyophilized for convenient storage for later use.
The mufti-array surfaces and methods herein are not limited to performance of
dye
swap analyses. For example, a mufti-array surface having two or more, e.g.,
four, five, six or
even nine iterations of an array can be used to analyze multiple samples, for
example, a
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plurality of members of a nuclear family, or multiple siblings and a proband
carrying a
chromosomal disorder, which can now be analyzed together on a single substrate
having
multiple micro-arrays, using separate hybridizations. Further, any multiple
number of
subjects can be analyzed simultaneously on a single substrate, or any one
subject can be
analyzed in mixtures of different reference samples. Different reference
samples can be
prepared in advance from each of relevant different species for extensive
repeated. use as a
standard of comparison with multiple different test samples, or from specific
animal strains
having one or more of several different known transgenes or mutations, or
different
predetermined single BAC clone nucleic acid or mixtures of nucleic acids from
two or more
BAC clones.
Chromosomal analysis using calibration spots and disease-negative clones
Calibration spots that act as positive controls for hybridization of a sample,
and that
are located within an array have been described (see, U.S. patent application
2003-0186250-
Al, published Oct. 2, 2003, and incorporated herein in its entirety by
reference). In
embodiments of the surfaces and methods provided herein, calibration spots may
include a
subset of cloned nucleic acids, for example, those clones of the human genome
carrying
sequences not known by any published references to be associated with a
chromosomal
disorder or disease. The term, "non-reactive" as applied to a specific cloned
sequence of
nucleic acid of known chromosomal location, means that the nucleic acid
generally
hybridizes to a full extent to a genomic nucleic acid from any test subject,
i.e., and is "non-
reactive" because it does not give a false "positive" diagnosis of a
chromosomal disorder.
Because the non-reactive or backbone clones are positive controls for
hybridization, they are
therefore expected to be non-reactive with a test sample for detection of a
chromosomal
disorder.
A calibration spot may be a mixture of nucleic acids from any combination of
other
elements present in the array, or can be a mixture of a subset of such
elements, or can be a
nucleic acid not so represented in the array. For example, a calibration spot
can be a mixture
of backbone clones as defined above, for any one syntenic set of clones
representing the
syntenic chromosome as chosen by the user, or for all of the chromosomes in
the human
genome or in a genome of any other organism. An exemplary calibration spot may
comprise
a mixture of nucleic acids, for example, from backbone clones, for example,
from about 10,
from about 20, from about 40, or from about 80 clones such as backbone or non-
reactive
clones. An exemplary but non-limiting calibration spot contains 72 non-
reactive backbone
clones, selected to represent nucleic acid from each of the set of human
autosomes and sex
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chromosomes. An alternative calibration spot contains nucleic acid from an
unrelated
heterologous species, such as a fish or amphibian, for purposes of
standardizing
hybridization, in which case an internal control carrying a recognizable label
can be added or
"spiked" into each hybridization mixture of a test sample and a reference
sample.
Representation of each chromosome is made by calculating ratios of labels in
each of
the two double dye-labeled hybridizations (dye swap) and relative amounts are
plotted
graphically as a function of distance of each cloned chromosomal portion from
the p terminus
conventionally shown on the left. By convention, one of the two double labeled
materials is
plotted in a consistent color (e.g., red), and the other in a different color
(e.g., blue), such that
deletion of a portion of nucleic acid in a test subject is displayed in red
above the 1.0 ratio
line (see Figs. 1 and 2), and an insertion such as an amplification is plotted
as blue above the
1.0 ratio line.
In addition, the arrays provided herein as shown in drawings and examples
herein,
include cloned nucleic acids from portions of each chromosome that are not
associated with
any known chromosomal disorders, so that representations of a chromosome of a
test
subject's DNA is facilitated, and a chromosomal disorder on a given chromosome
is more
readily distinguished from normal portions of that chromosome.
EXAMPLES
The following examples are offered to illustrate embodiments of the invention,
and
are not to be construed as further limiting. The methods are used throughout
the examples.
Example 1. Making BAC Clone Nucleic Acid Array
BAC clones (Tart of et al., 1987, CA Cetheda Res. Lab Focus 86:184; available
from
the Bio Laboratories, Carlsbud CA) containing inserts of greater than thirty
kilobases (30 kb),
and up to about 300 kb, are grown up in Terrific Broth medium (commercially
available from
numerous suppliers). Large inserts, e.g., clones >300 kb, and small inserts,
about 1 to 20 kb,
can also be used. DNA is prepared by a modified alkaline lysis protocol (see,
e.g.,
Sambrook). A genomic DNA sample used in labeling experiments is prepared by
protocol to
be substantially free of RNA and proteins. Any general protocol for this
purpose can be used,
and the following procedure is not to be construed as limiting.
Following lysis of cells and removal of cell debris, ribonuclease (DNase-free,
10
mg/ml) is added to the DNA sample to a final concentration of 20-100
microg/ml, and the
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mixture is incubated at 37°C for 30 min. Proteinase K is added at a
final concentration of 100
microg/ml, and the sample is incubated at 50°C for one hour. The sample
is cooled to room
temperature, and an equal volume ofphenol:chloroform:isoamyl alcohol (25:24:1)
is added.
The two phases are gently mixed by rotation on a wheel or end-to-end turning,
for 10 min,
and the phases are separated by centrifugation at 10,000g for 3 min.
The aqueous layer is removed, and is similarly re-extracted until no interface
material
is observed. Chloroform is used to remove remaining phenol, the mixture is
again
centrifuged, the aqueous layer is removed to a clean tube, and DNA is
precipated by adding a
one-twelfth volume of SM NaCl. The solutions are mixed by slow end-to-end
turning,
followed by addition of 2.5 volumes of ice cold 100% ethanol (or a 0.75 volume
of room
temperature isopropanol). After addition of ice-cold ethanol, the sample is
incubated at -
20°C for 30 min to one hour (or about 15-30 min at room temperature if
isopropanol is used).
The DNA precipitate is collected by micro-centrifiguration at maximum speed
for 10
min. The ethanol (or isopropanol) supernatant is carefully removed, and lml of
70% ethanol
is added to the pellet to remove precipated salt. The 70% ethanol is gently
removed, and a
second rinsing of the pellet with 70% ethanol is performed. The second 70%
ethanol is
removed, and the pellet is dissolved in sterile distilled water. A DNA
concentration of 100-
200 nanograms/microliter is obtained, the DNA having an average molecular
weight greater
than the 8,454 base pair lambda DNA-BstE II digested marker, and substantially
free of
RNA. The DNA is labeled as described herein.
The DNA is then chemically modified as described by U.S. patent number
6,048,695.
The modified DNA is then dissolved in proper buffer and printed directly on
clean glass
surfaces as described by U.S. patent number. 6,048,695. Usually multiple spots
are printed
for each clone. Two or more iterations or sets of each complete array are
printed on the
surface, each complete array separated by a barner, or separated by a space
having no spots,
i.e., a plurality of non-contiguous arrays.
Example 2. Labeling of Genomic DNA
Genomic DNA for test and reference samples substantially is prepared
substantially
as above, and is substantially free of RNA and protein. The DNA is pretreated
to obtain small
more uniform pieces, is differentially labeled, and is hybridized to a slide
having an array
spotted as described herein, washed, and scanned and analyzed for detection of
chromosomal
abnormalities.
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Pretreatment includes digestion with a DNase, preferably a four base pair
cutter such
as EcoRI, to reduce the size of the genomic DNA. About one microgram of sample
DNA is
incubated with EcoRI (2 microliters or 20 units) for about 16h at 37°
C. Extent of completion
of the reaction is analyzed by electrophoresis in 0.8% Agarose, and is
deternlined to be
complete if a relatively homogeneous smear from 600 by to > 20 kb is observed.
If the
digestion is complete, the reaction is terminated by incubating the tube at
72° C for 10 min.
DNA is re-purified by phenol/chloroform extraction and ethanol precipitation
as described
herein, or by equivalent means (Zymo Research kit, DNA Clean and Concentrator
TM-5, Cat.
No. D4005, Hornby, Ontario, Canada).
An aliquot of each of a test sample and a reference sample DNA is labeled with
each
of Cy3TM-dCTP and CySTM-dCTP, to facilitate co-hybridization. About 500
micrograms of
re-purified digested DNA in 25 microliters of water is added to 20 microliters
of 2.SX
random primer reaction mix (Gibco/BRL BioPrime labeling kit, Gibco/BRL,
Bethesda MD).
Samples are mixed, and incubated at 100°C for 5 min, then incubated on
ice for 5 min. To
each tube of DNA, 2.5 microliters of Spectral Labeling Buffer (Spectral
Genomics, Inc.,
Houston, TX), and 1.5 microliters of Gy3TM-dCTP (1 mm stock) or CySTM-dCTP (1
mm
stock) is added. Then 1 microliter of Klenow fragment (from the Gibco/BRL
BioPrime
Labeling kit) is added, the solution is mixed and re-collected by brief
centrifugation, and the
sample is incubated at 37°C for about 1.5 to 2 hours. Samples are
placed on ice, and
analyzed by electrophoresis (using 0.8% Agarose) to determne probe size, which
should have
a range of about 100 by to about 500 bp. The reaction is ternlinated by
addition of 5
microliters of O.SM EDTA, pH 8.0, and incubated at 72°C for 10 min,
followed by placing
the samples on ice. Tube contents are ready for hybridization, or can be
stored at -20°C until
required.
For hybridization, two mixtures of labeled test sample and labeled reference
sample
are prepared. Tube contents of Cy3TM-labeled test sample DNA is mixed with
tube contents
of CySTM-labeled reference DNA, and tube contents of Cy3TM-labeled reference
DNA is
mised with tube contents of CySTM-labeled test DNA. Spectral Hybridization
Buffer (45
microliters; Spectral Genomics, Inc., Houston, TX) is added to each of the
tubes, and the
contents are precipitated by adding 11.3 microliters of SM NaCl and 110
microliters of room
temperature isoproponol. Samples are mixed and incubated in the dark at room
temperature,'
for about 10-15 min, centrifuged at maximum speed for 10 min preferably in the
dark, and
supernatants are aspirated. Pellets are rinsed with 500 microliters of 70%
ethanol and air-
dried briefly in the dark, 10 microliters of sterile water is added, and tubes
are incubated at
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room temperature for 5 min and thoroughly mixed. When pellets are dissolved,
30 microliters
of Spectral Hybridization Buffer II (Spectral Genomics Inc., Houston, TX) are
added, and
tube contents are mixed by repeated pipetting. Samples are denatured by
incubation at 72° for
min.
Example 3. Optimization of Printing of Nucleic Acid Spots as a Function of
Ionic Strength
Spots are generally printed robotically on a glass slide in a pattern, in
duplicate
blocks, each block containing hundreds of spots. Following drying, each spot
can be
examined microscopically to assess quality of deposition of the biological
materials. High
10 quality is indicated by a smooth, uniform appearance of a high proportion
of the printed
spots, and is reflected in a uniform pattern of hybridization. Low quality is
indicated by spots
that appear as "mountains" such that sample is deposited non-uniformly in the
center, or as
several "hills" or in an X-shaped configuration across diameters of the spot,
and is reflected
in a similarly distributed hybridization.
To determine the effect of ionic strength of printing buffer on the quality of
the spots
in arrays on glass slides, six samples of BAC DNA were prepared as shown in
Table 1.
Table 1. Buffer component concentrations of experimental printing buffers, in
millimolar*
Component:Tube #: 2 3 4 5 6
1
NaOH 25 51.25 77.5 104 130 156.25
TRIS-HCl 50 102.5 155 208 260 312.5
EDTA 5 10.3 15.5 20.8 26 31.2
* All buffers were prepared from standard stock solutions of 400 mm NaOH and
1M TRIS-
HCI, O.1M EDTA, pH 4.3.
An aliquot of DNA was added to each of tubes 1 through 6, and spots were
printed on
glass slides and were dried by standard procedures.
Examination of printed slides showed best quality, i.e., uniform distribution
of DNA
deposited on the greatest proportion of printed spots, of those printed at
conditions found in
tube #3; slides with spots printed in buffers with tubes #1, #2, #4 and #5
were of poorer
quality appearance and gave poorer quality of hybridization than did those
printed with buffer
of tube #3. These intermediate ionic strengths in tubes #2-5 have a higher
than ionic strengths
than the previously used buffer (tube #1). Optimum appearance of the printed
spot was
reflected in greatest reliability and reproducibility of hybridization of
reference samples, and
was seen with spots printed in the buffer of tube #3.
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Production of buffer for printing of spots was, as a result of data shown
above,
changed for further printing by mixing equal volumes of two stock solutions:
solution I is 150
mM NaOH, and solution II is 300 mM TRIS-HCI, 30 mM EDTA, pH of 4.3. Final
concentrations of components of this printing buffer are: 75 mM NaOH, 150 mM
TRIS-HCI,
and 15 mM EDTA.
Example 4. Preparation of S~mtenic Arrays
DNA is prepared as above from characterized BAC clones having DNA inserts that
are syntenic for each mouse chromosome and for each human chromosome of
interest. The
DNA is deposited (printed) in an array of addressible locations (spots) on a
glass slide. The
arrayed DNA can be from a normal chromosome of a human and a normal chromosome
of
any other species such as a mouse, or from a chromosome of a human having a
known
disease, or a mouse having a mouse disease that is a model of a human disease,
such as lung
cancer, mesothelioma, adenocarcinoma, and prostate cancer. The presence both
of human and
another species, i.e., mouse syntenic sequences that are homologous to the
human sequences,
constitutes a syntenic array.
Syntenic arrays are printed also for other combinations of species DNA, such
as
human-rat; human-chimpanzee; human-dog, and the like.
Test and reference samples (positive and negative controls) are prepared from
genomic DNA of diseased and normal subjects, not necessarily limited to the
subjects of
those species identifted by having the array of immobilized DNA. Each is
labeled with a first
and a second fluorescent dye, and two mixtures of test and reference samples
are made. Each
of the two mixtures is separately hybridized to an array of the multiple
arrays on the surface.
Example 5. Detection of Chromosomal Abnormalities using Ratio Plot Anal. skis
Fig. 1 a is a ratio plot of a sample of patient DNA hybridized to immobilized
BAC
clones of chromosome 18, using a linear display of ratio of DNA from the 25
BAC clones, in
order from left to right of p to q arms. Two iterations of the hybridization
were performed,
one in which the hybridization was performed with a mixture of test DNA (from
the patient)
labeled with Cy3TM and reference normal DNA is labeled with CySTM; and a
second iteration
in which the with a mixture of test DNA (from the patient) labeled with CySTM
and reference
normal DNA is labeled with Cy3TM. After data are analyzed, the ratio of
Cy5:Cy3 is
determined and is plotted as shown in the Figs.
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At the q terminus of chromosome 18, the patient data indicates a deletion, as
both
normalized functions are increasing in value for the same three q-terminus BAC
hybridizations (both show a simultaneous deviation from a modal value of 1.0,
regardless of
whether the test sample or reference sample is the numerator of the ratio). A
similar ratio plot
shown in Fig. lb indicates that DNA at the q-terminus of chromosome 4 the same
patient
carnes an insertion, as both functions deviate in opposite directions for two
BAC clones, with
the blue (reference sample in numerator) DNA being greater than the red (test
sample DNA
in numerator) hybridization.
Fig. 2 shows a ratio plot of the X chromosome of a different male patient,
which by
the computations described herein indicates that there is an insertion
(amplification) of
sequences found at the p-ter of this chromosome. The amplification is shown to
extend over
at least three BAC clones. The deviations shown at the q-ter of this
chromosome are
considered random and not significant, as only one of the functions deviates
to any extent
from a modal value of 1Ø
Example 6. Testing of Cancer Tissue for Chromosomal Abnormalities Using
Syntenic Array
Cancer cells of strains of mice having particular cancer conditions, such as
lung
cancer, rnesothelioma, adenocarcinoma, and prostate cancer, are used to make
DNA test
samples. Test samples are prepared from tissue obtained from individual mice
having
different stages of each of the diseases. The DNA is fragmented, labeled, and
hybridized as
described herein. Abnormalities associated with each of these conditions are
determined from
ratio plots of hybridization data, using a syntenic array having probe
elements for each of
human and mouse syntenic sequences from cloned BAC chromosomes that are highly
homologous in humans and mice.
Presence, extent, and location of these abnormalities are then determined
using test
sample DNA obtained from human patients having various stages of the disease,
such that a
diagnostic instrument capable of diagnosing and prognosing the stage of the
cancer is
established. The data show not only the location of the cancer-induced changes
on the mouse
chromosome set, but also show the location of such changes that would occur in
humans, and
at loci for homologous sequences on the human chromosome set.
Example 7. Toxicological Testing Wwntenic Array
Groups of test animals (or test cells), for example, rodents such as rates,
mice, or
guinea pigs, are exposed to a chemical. In one experiment, each test animal
member of a
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group is injected with a concentration of one or more chemicals to be tested
for genotoxic or
mutational activities, with different groups receiving each of different
doses, and one group
receiving only carrier or solvent (negative control), so that a range of
concentrations of the
composition is tested to establish a dose response curve. A genotoxic activity
is detected as
causing one or more of deletion/insertion mutations, or translocation of DNA
from one locus
and chromosome to another locus on the same or a different chromosome, or
amplification of
a region of a chromosome. In another experiment, cells in culture are exposed
to a range of
each of the chemicals, individually, or in combinations such as in groups of
10 chemicals in a
single tube, i.e., related chemicals are tested in sibling groups which can be
further analyzed
individually or in smaller groups to obtain a correlation with genotoxicity.
Following exposing or contacting the cells or organisms with the one or more
chemical compositions for varying extents of time and/or concentrations, DNA
is prepared
from a cell sample, e.g., from a somatic tissue from an autopsy or biopsy,
e.g., from ovarian
or testicular tissue such as a blood sample, or from the cells in culture that
were exposed to
the chemical. The DNA is fragmented and labeled as described above with each
of two
fluorescent dyes or equivalent detectible labels, for test sample to be mixed
with oppositely
labeled reference sample, and the mixtures are each hybridized to an iteration
of a syntenic
array having both the rodent, e.g., mouse and human genomes, or cloned nucleic
acids from
particular chromosomes, arrayed as probe elements. The data show extent and
location of
DNA damage in the test sample from test animals or cells, both in the rodent
chromosome
set, and for homologous sequences on the human chromosome set.
A number of embodiments of the invention have been described. Nevertheless, it
will
be understood that various modifications may be made without departing from
the spirit and
scope of the invention. Accordingly, other embodiments are within the scope of
the following
claims.
Example 8. Syntenic Arrays for Analyzing Chromosomal Abnormalities in a Trans
enic
Animal
A large variety of transgenic animals are available for research and testing
purposes.
An animal lacking a gene function, in this example, a mouse strain having a
disruption in a
gene encoding nitric oxide synthase (NOS), can be used for screening purposes,
to identify
compositions capable remediating a phenotype (LT.S. patent number 6,310,270
issued Oct. 30,
2001).
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A DNA sample is prepared from a blood sample of each animal in treated and
control
groups, and is labeled with each of two fluorescent dyes as described in
examples above, and
mixtures of labeled samples and differently labeled reference DNA are prepared
as above.
Treated and control animal DNA mixtures are hybridized to the syntenic chips
having each of
human and mouse BAC cloned DNA in each array block.
Results indicate which of those compositions that are active in remediating
the
disrupted phenotype, NOS, that have not caused chromosomal abnormalities in
the test
animals. It is envisioned that the majority of compositions do not cause
chromosomal
abnormalities, so an additional positive control group of animals administered
an agent
known to cause chromosomal abnormalities, such as benzene, is included. In
this example,
compositions are screened both for a pharmacological activity, remediation of
lack of NOS,
and for induction of chromosomal abnormality, i.e., teratogenicity and
mutagenicity, in the
same groups of test and control animals. The syntenic chip readout contains
mouse and
human elements, and as it is envisioned that any chromosomal abnormality
observed in the
mouse in comparison to mouse reference DNA has occurred de novo, the
chromosomal
abnormality is simultaneously analyzed both with respect to the mouse
chromosome and
genome, and to homologous elements of the human genome on the single surface
of the
syntenic chip.
Example 9. Syntenic Arrays for Anal.~g Chromosomal Abnormalities in an Animal
Model
of Human Disease
Groups of non-obese diabetic (NOD) mice are administered a each of variety of
compositions, to determine whether these are capable of remediating or
preventing type I or
insulin-deficient diabetes. Agents are administered intravenously.
At the end of the treatment protocol, a DNA sample is prepared from a blood
sample
of each animal in treated and control groups, and is labeled with each of two
fluorescent dyes
as described in examples above, and mixtures of labeled samples and
differently labeled
reference DNA are prepared as above. Treated and control animal DNA mixtures
are
hybridized to syntenic chips having each of human and mouse BAC cloned DNA in
each
array block.
Results indicate which of those compositions that are active in remediating
the model
disease, diabetes, that have not caused chromosomal abnormalities in the test
animals. It is
envisioned that the majority of compositions will not cause chromosomal
abnormalities, so a
positive control group of animals administered an agent known to cause
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abnormalities can be included in the example. In this manner compositions have
been
screened both for a pharmacological activity, remediation of diabetes, and for
mutagenicity,
in the same groups of test and control animals. The syntenic chip readout
contains mouse
and human elements, and as it is envisioned that any chromosomal abnormality
observed in
the mouse in comparison to mouse reference DNA has occurred de novo, the
chromosomal
abnormality is simultaneously analyzed both with respect to the mouse
chromosome and
genome, and to homologous elements of the human genome on the single surface
of the
syntenic chip.
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