Note: Descriptions are shown in the official language in which they were submitted.
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TITLE OF THE I1WENTION
MULTIPARAMETRIC FLUORESCENCE
s IN SITU HYBRIDIZATION
FIELD OF THE INVENTION
The present invention relates to nucleic acid chemisi:y, and more specifically
to reagents and methods for accomplishing multiplex image analysis of
chromosomes
and chromosomal fragments. The invention may be used to diagnose chromosomal
abnormalities, infectious agents, etc. This invention was made in part using
Government funds. The Government has certain rights in this invention.
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation-in-part of U.S. Patent Application Serial
No.
08/640,657 (filed May 1, 1996), which is a continuation in part of U.S. Patent
Application Serial No. 08/580,717 (filed December 29, 1995), which is a
continuation
in part of U.S. Patent Application Serial No. 08/s77,622 (filed December 22,
1995),
all of which applications are herein incorporated by reference in their
entirety.
BACKGROUND OF THE INVENTION
The determination of the presence and condition of chromosomes and
chromosomal fragments in a biological sample is of immense importance in the
diagnosis of disease. Traditionally, such determinations have been done
manually by
inspecting metaphase chromosomal preparations that have been treated with
specialized stains to reveal characteristic banding patterns. Unfortunately,
the
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interpretation of such banding patterns requires substantial skill and is
technically
difficult. Hence, alternate methods of analyzing chromosomal presence and
arrangement have been sought.
One alternative approach to the problem of chromosome identification has
involved the use of labeled chromosome-specific oligonucleotide probes to
label
repetitive sequences of interphase chromosomes (Cremer, T. et al., Hum. Genet.
74:346-352 (1986); Cremer, T. et al., Exper. Cell Res. l7b:119-220 (1988)).
Such
methods have been shown to be useful in the prenatal diagnosis of Down's
Syndrome,
as well as in the detection of chromosomal abnormalities associated with tumor
cell
lO lines. Chromosome-specific probes of repetitive DNA that localize to
discrete sub-
regions of a chromosome are, however, unsuitable for analyses of many types of
chromosomal abnormalities (e.g., translocations or deletions).
Ward, D.C. et al. (PCT Application WO/05789, herein incorporated by
reference) discloses a chromosomal in situ suppression ("CTSS") hybridization
method
for specifically labeling selected mammalian chromosomes in a manner that
permits
the recognition of chromosomal aberrations. In that method, sample DNA i~
denatured and permitted to hybridize with a mixture of fluorescently labeled
chromosome-specific probes having high genetic complexity and unlabeled non-
specific competitor probes. Chromosomal images were obtained as described by
Manuelidis, L. et al. (Chromosoma 96:397-410 ( 1988), herein incorporated by
reference). The method provides a rapid and highly specific assessment of
individual
mammalian chromosomes. The method permits, by judicious selection of
appropriate
probes and/or labels, the visualization of sub-regions of some or all of the
chromosomes in a preparation. For example, by using more than one probe, each
specific for a sub-region of a target chromosome, the method permits the
simultaneous
analysis of several sub-regions on that chromosome. The number of available
fluorophores limits the number of chromosomes or chromosomal sub-regions that
can
be simultaneously visualized.
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As described in PCT Application WO/05789, a "combinatorial" variation of
the CISS method can be employed. In the simplest case, two fluors permit three
different chromosomes or chromosomal sub-regions to be simultaneously
visualized.
In this variation, a hybridization probe mixture is made from a single set of
probe
sequences composed of two halves, each separately labeled with a different
fluorophore. Upon hybridization, the two fluorophores produce a third
fluorescence
signal that is optically distinguishable from the color of the individual
fluorophores.
Extension of this approach to Boolean combinations of n fluorophores permits
the
labeling of 2n-1 chromosomes.
Ried, T. et al. (Proc. Natl. Acad. Sci. (U.S.A.) 89:1388-1392 ( 1992), herein
incorporated by reference) describes the use of an epi-fluorescent microscope
equipped
with a digital imaging camera and computer software to "pseudocolor" the
fluorescence patterns obtained from simultaneous in situ hybridization with
seven
probes using three fluorophores. The use of wavelength-selective filters
allows one to
isolate and collect separate gray scale images of each fluorophore. These
images can
be subsequently merged via appropriate software. The sensitivity and linearity
of
CCD cameras surmounts the technical difficulties inherent in color film-based
photomicroscopy.
Although such efforts have increased the number of chromosomes that can be
simultaneously detected and analyzed using in situ hybridization methods, it
would be
highly desirable to define a set of fluorophores having distinguishable
emission spectra
to permit the simultaneous detection and analysis of large numbers of
different
chromosomes and chromosomal sub-regions. The present invention provides such
reagents as well as methods and apparatus for their use.
SUMMARY OF THE INVENTION
The invention concerns reagents and methods for combinatorial labeling of
nucleic acid probes sufficient to permit the visualization and simultaneous
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identification of all 22 autosomal human chromosomes and the human X and Y
chromosomes, or defined sub-regions thereof. Such specific labeling of entire
chromosomes or defined sub-regions thereof is referred to as "painting." The
invention further concerns reagents and methods for combinatorial labeling of
nucleic
acid probes sufficient to permit the characterization of bacteria, viruses
and/or lower
eukaryotes that may be present in a clinical or non-clinical preparation.
In detail, the invention concerns a set of combinatorially labeled
oligonucleotide probes comprised of a first and a second subset of probes,
wherein:
(A) each member of the first subset of probes comprises a plurality of an
oligonucleotide: (i) being linked or coupled to a predetermined label
distinguishable from the label of any other member of the first or
second subsets of probes, and (ii) being capable of specifically
hybridizing with one predetermined autosomal or sex chromosome of a
human karyotype;
the first subset of probes set having sufficient members to be capable of
specifically hybridizing each autosomal or sex chromosome of the:
human karyotype to at least one member; and
(B) each member of the second subset of probes comprises a plurality of an
oligonucleotide: (i) being linked or coupled to a predetermined label
distinguishable from the label of any other member of the first or
second subset, and (ii) being capable of specifically hybridizing with
one extra-chromosomal polynucleotide copy of a predetermined region
of an autosomal or sex chromosome of the human karyotype.
The invention further concerns a set of combinatorially labeled
oligonucleotide
probes comprised of a first subset of genotypic probes and a second subset of
phenotypic probes, wherein:
(A) each member of the first subset of genotypic probes comprises a
plurality of an oligonucleotide: (i) being linked or coupled to a
predetermined label distinguishable from the label of any other member
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of the first or second subsets of probes, and (ii) being capable of
specifically hybridizing with a region of a nucleic acid of a preselected
bacterium, virus or lower eukaryote;
the first subset of probes set having sufficient members to be capable of
distinguishing the preselected bacterium, virus, or lower eukaryote from
other bacteria, viruses, or lower eukaryotes; and
(B) each member of the second subset of phenotypic probes comprises a
plurality of an oligonucleotide: {i) being linked or coupled to a
predetermined label distinguishable from the label of any other member
of the first or second subset, and (ii) being capable of specifically
hybridizing with a predetermined polynucleotide region of the
chromosome of the preselected bacterium, virus, or lower eukaryote, or
an extra-chromosomal copy thereof so as to permit the determination of
whether the preselected bacterium, virus, or lower eukaryote exhibits a
preselected phenotype.
The invention additionally concerns a method of simultaneously identifyinb
and distinguishing the individual autosomal and sex chromosomes of a human
karyotype which comprises the steps:
(n contacting a preparation of the chromosomes, in single-stranded form,
under conditions sufficient to permit nucleic acid hybridization to occur
with a set of combinatorially labeled oligonucleotide probes comprised
of a first and a second subset of probes, wherein:
(A) each member of the first subset of probes comprises a plurality
of an oligonucleotide: (i) being linked or coupled to a
predetermined label distinguishable from the Iabel of any other
member of the first or second subsets of probes, and (ii) being
capable of specifically hybridizing with one predetermined
autosomal or sex chromosome of a human karyotype;
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the first subset of probes set having sufficient members to be
capable of specifically hybridizing each autosomal or sex
chromosome of the human karyotype to at least one member;
and
(B) each member of the second subset of probes comprises a
plurality of an oligonucleotide: (i) being linked or coupled to a
predetermined label distinguishable from the label of any other
member of the first or second subset, and (ii) being capable of
specifically hybridizing with an a predetermined extra-
chromosomal polynucleotide copy of a region of an autosomal
or sex chromosome of the human karyotype.
(In for each chromosome of the preparation hybridized to a member of the
first subset of probes, detecting and identifying the predetermined Iabel
of that member and correlating the identity of the label of that member
with the identity of the autosomal or sex chromosome of the human
karyotype with which that member specifically hybridizes, to thereby
identify the chromosome hybridized to the member;
(III) repeating step (II) until each autosomal and sex chromosome of the
human karyotype has been identified in the preparation
(N) for each member of the second subset of probes hybridized to a
predetermined extra-chromosomal polynucleotide copy of a region of
an autosomal or sex chromosome detecting and identifying the
predetermined label of that member and correlating the identity of the
label of that member with the identity of the region of the autosomal or
sex chromosome of the human karyotype with which that member
specifically hybridizes, to thereby identify the region of the autosomal
or sex chromosome hybridized to the member;
(V) repeating step (IV) for each member of the second subset of probes.
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The invention additionally concerns a method of simultaneously identifying
and distinguishing a preselected bacterium, virus, or lower eukaryote from
other
bacteria, viruses or lower eukaryotes that may be present in a sample which
comprises
the steps:
(17 contacting a preparation suspected to contain the preselected bacterium,
virus, or lower eukaryote, under conditions sufficient to permit in situ
nucleic acid hybridization to occur, with a set of combinatorially
labeled oligonucleotide probes comprised of a first subset of genotypic
probes and a second subset of phenotypic probes, wherein:
(A) each member of the first subset of genotypic probes comprises a
plurality of an oligonucleotide: (i) being linked or coupled to a
predetermined label distinguishable from the label of any other
member of the first or second subsets of probes, and (ii) being
capable of specifically hybridizing with a region of a nucleic
acid of the preselected bacterium, virus or lower eukaryote;
the first subset of probes set having sufficient members to be
capable of distinguishing the preselected bacterium, virus, or
lower eukaryote from other bacteria, viruses, or lower
eukaryotes present in the preparation; and
(B) each member of the second subset of probes comprises a
plurality of an oligonucleotide: (i) being linked or coupled to a
predetermined label distinguishable from the label of any other
member of the first or second subset, and (ii) being capable of
specifically hybridizing with a predetermined polynucleotide
region of the nucleic acid of the preselected bacterium, virus, or
lower eukaryote, or an extra-chromosomal copy thereof so as to
permit the determination of whether the preselected bacterium,
virus, or lower eukaryote exhibits a preselected phenotype.
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(In for each member of the first subset of probes hybridized to a region of a
chromosome of a preselected bacterium, virus or lower eukaryote,
detecting and identifying the predetermined label of that member and
correlating the identity of the label of that member with the identity of
the bacterium, virus or lower eukaryote with which that member
specifically hybridizes, to thereby identify the bacterium, virus or lower
eukaryote hybridized to the member;
(iTl) repeating step (lJn for each member of the first subset of probes;
(N) for each member of the second subset of probes hybridized to a
predetermined polynucleotide region of the chromosome of the
preselected bacterium, virus, or lower eukaryote, or an extra-
chromosomal copy thereof, detecting and identifying the predetermined
label of that member and correlating the identity of the label of that
member with the identity of the predetermined region, to therei;y
identify the presence of the predetermined region on a chromosome of
the preselected bacteria, virus or lower eukaryote;
(V) repeating step (N) for each member of the second subset of probes.
The invention particularly contemplates the embodiments in which the
members of the above sets of probes are detectably labeled with fluorophores,
and,
wherein at least one member of the set is combinatorially labeled with either
one, two,
three, four or five fluorophores selected from the group consisting of the
fluorophores
FTTC, Cy3, Cy3.5, CyS, Cy5.5 and Cy7, and wherein each member of the set is
labeled
with at least one fluorophore selected from the fluorophore group.
The invention additionally provides a set of combinatorially labeled
oligonucleotide probes, each member thereof: (i) having a predetermined label
distinguishable from the label of any other member of the set, and (ii) being
capable of
specifically hybridizing with a telomeric region of one predetermined
autosomal or sex
chromosome of a human karyotype;
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the set having sufficient members to be capable of specifically hybridizing
each
autosomal or sex chromosome of the human karyotype to at least one member.
The invention additionally provides a method of simultaneously identifying
and distinguishing the individual autosomal and sex chromosomes of a human
karyotype which comprises the steps:
(a) contacting a preparation of the chromosomes, in single-stranded form,
under conditions sufficient to permit nucleic acid hybridization to occur
with a set of combinatorially labeled oligonucleotide probes, each
member thereof: (i) having a predetermined label distinguishable from
IO the label of any other member of the set, and (ii) being capable of
specifically hybridizing with a telomeric region of one predetermined
autosomal or sex chromosome of a human karyotype; the set having
sufficient members to be capable of specifically hybridizing each
autosomal or sex chromosome of the human karyotype to at least one
member; wherein the contacting thereby causes at least one of each
autosomal or sex chromosome of the preparation to become hybridized
to at least one member of the set of probes;
(b) for each chromosome of the preparation hybridized to a member of the
set of probes, detecting and identifying the predetermined label of that
member and correlating the identity of the label of that member with
the identity of the autosomal or sex chromosome of the human
karyotype with which that member specifically hybridizes, to thereby
identify the chromosome hybridized to the member; and
(c) repeating step (b} until each autosomal and sex chromosome of the
human karyotype has been identified in the preparation.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 provides a schematic illustration of a CCD camera and microscope
employed in accordance with the present methods.
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Figure 2 shows the raw data from a karyotypic analysis of chromosomes from a
bone marrow patient (BM2486). Adjacent to each source image is a chromosome
"mask" generated by the software program. In Figure 2, panels A and B are the
DAPI
image and mask; panels C and D are FITC image and mask; panels E and F are Cy3
image and mask; panels G and H are Cy3.5 image and mask; panels I and J are
Cy~
image and mask; and panels K and L are Cy7 image and mask.
Figures 3A and 3B show the identification of individual chromosomes by
spectral signature of patient BM2486. Figure 2 is the same photograph as
Figure 3A,
except that it is gray scale pseudocolored. Figure 3B displays the karyotypic
array of
the chromosomes.
Figure 4 shows the differentiation of bacteria by in situ hybridization.
Panels
A-E represent in situ hybridization assay results on a laboratory derived
mixture of
three bacteria (F. nucleatum, A. actinomycetemcomitans and E. corrodens) using
a
mixture of genomic DNA probes for each organism. Panel A shows all the
bacteria
present in the microscope field detected by (DAPn, a general DNA binding
fluorophore. Panel B shows F. nucleatum using Cascade Blue detection. Panel C
shows A. actinomycetemcomitans using FTTC detection. Panel D shows E.
corrodens
using Rhodamine detection. Panel E is a computer-merged composite of panels B-
D
showing the differentiation of each bacteria in the sample. Panel F is a
similar analysis
of a mixture of C. gingivalis (using rhodamine detection) and P. intennedia
(using
FTTC detection) when hybridized with a combination of genomic DNA probes for
those organisms. Panel G shows an in situ hybridization assay for a
combination of
seven different bacteria hybridized with a mixture of seven genomic DNA
probes. In
panel G, the numbers ( 1 )-(7) identify the bacteria. F. nucleatum ( 1 ), E,
corrodens (2)
and A. actinnmycetemcomitans (3) are shown in blue (Cascade Blue detection).
P.
gingivalis (4) and C. ochracea (5) are shown in red (Rhadamine detection),
whereas P.
intermedia (6) and C. gingivalis (7) are seen in yellow (FTTC detection).
Panel H
represents an in situ hybridization assay for the presence of A.
actinomycetemcomitans
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(using FTTC detection) in a plaque sample obtained from a patient with
localized
juvenile periodontist.
Figure SA shows a normal male metaphase chromosomal spread after
hybridization with a 24-color set of telomere-specific probes, shown as a
pseudocolorized image. Figure SB shows the final karyotype generated on the
basis of
the boolean spectral signature of the telomere-specific probes.
Figure 6A shows the hybridization pattern of the chromosome 8 subtelomeric
YACs (telomere-specific probes) on a normal metaphase chromosomal pread.
Figure
6B shows the hybridization pattern of the same probes (as those used in Figure
6A) on
a metaphase chromosomal spread from a patient with a myeloproliferative
disorder.
Previous cytogenetic analysis of this patient using G-banding revealed a
trisomy 8 as
the only change; the M-FISH telomere-specific probes show a split telomere
signal on
the short arms of two of the three chromosome 8, indicating an additional
change: an
inversion in this region.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
A. Overview of the Invention
Fluorescence in situ hybridization (FISH) is used in a variety of areas of
research and clinical diagnostics (Gray, J.W. et al., Curr Opin Biotech 3:623-
631
( 1992); Xing, Y. et al., In: The Causes and Consequences of Chromosomal
Aberrations. LR. Kirsch Ed. CRC Press, Boca Raton, pages 3-28 (1993)). For the
study of the chromosomal and suprachromosomal organization of the cell nucleus
it is
an indispensable tool (Cremer, T. et al., In: Cold Spring Harbor Symposia on
Quantitative Biology, Volume LVIIl, pp. 777-792, Cold Spring Harbor Laboratory
Press, NY ( 1994)). Most importantly FISH offers the capacity for
multiparameter
discrimination. This allows the simultaneous visualization of several DNA
probes
using either a combinatorial (Nederlof, P.M. et al., Cytometry 10:20-27 (
1989);
Nederlof, P.M. et al., Cytometry II:I26-I31 (1990); Ried, T. et al., Proc Natl
Acad
Sci (U.S.A.) 89:1388-1392 (1992a); Ried, T. et al., Hum Mol Genet 1:307-313
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( 1992b); Lengauer, C. et al., Hum Mol Genet 2:505-512 ( 1993); Popp, S. et
al.,
Human Genetics 92:527-532 ( 1993); Wiegant, J. et al., Cytogenet Cell Genet
63:73-76
(1993)) or a ratio labeling (Dauwerse, J.G. et al., Hum Mol Genet 1:593-
598(1992);
Nederlof, P.M. et al., Cytometn~ !3:839-845 ( 1992); du Manoir, S. et al., Hum
Genet
90:590-610 (1993)) strategy. Up to twelve DNA probes have been visualized
(Dauwerse, J.G. et al., Hum Mol Genet 1:593-598 ( 1992)). Consequently, the
goal of
24 different colors has long been sought (Ledbetter, D.H., Hum Mol Genet 5:297-
299
( 1992)). Twenty-four different colors are an important threshold because they
would
allow the simultaneous visualization of the 22 autosomes and both sex
chromosomes.
Beside improved karyotyging, the possibility of simultaneously hybridizinb 24
different and distinguishable DNA probes would allow the addressing of a large
number of important biological questions. However, the previously published
multicolor systems lacked the versatility for an extension to 24 colors and
only proof
of principle experiments were ever published.
The present invention results, in part, from the realization of
multiparametric
fluorescence in situ hybridization to achieve the simultaneous visualization
of 2~
different genetic targets with a combinatorial labeling strategy. This
strategy permits
discrimination between many more target sequences than there are spectrally
distinguishable labels. The simplest way to implement such labeling is using a
simple
"Boolean" combination, i.e., a fluor is either completely absent (i.e. the
value of "0"
will be assigned) or present in unit amount (value of 1 ). For a single fluor
A, there is
only one useful combination (A= 1) and for two fluors A and B, there are 3
useful
combinations (A=1B=0; A=OB=1; A=1B=1). There are 7 combinations of 3 fluors,
15 combinations of 4 fluors, 31 combinations of 5 fluors, 63 combinations of 6
fluors,
and so on (n fluorophores permitting the labeling of 2n-1 chromosomes). Thus,
to
uniquely identify all 24 chromosome types in the human genome using chromosome
painting probe sets, only 5 distinguishable fluors are needed (31 total
combinations).
If each probe set is labeled with one or more of five spectrally distinct
fluorophores in
a combinatorial fashion, simple Boolean combination can be used to identify
each
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DNA probe by a spectral signature dictated by its fluorophore composition
(Speicher,
M.R. et al, Narure Genet. 12:368-375 ( 1996), which reference is herein
incorporated
by reference).
B. Terminology of the Invention
The invention concerns a set of combinatorially labeled oligonucleotide
probes,
each member thereof: (i) having a predetermined label distinguishable from the
label
of any other member of the set, and (ii) being capable of specifically
hybridizing with
one predetermined autosomal or sex chromosome of a human karyotype. In the
most
preferred embodiment, the set will have a sufficient number of members to be
capable
of specifically and distinguishably hybridizing each autosomal or sex
chromosome of
said human karyotype to at least one member. As used herein, the term
"karyotype"
denotes the compliment of chromosomes found in a normal or aberrant cell. In a
normal cells, the number of chromosomes is 46, comprising 22 pairs of
autosomal
chromosomes and 2 sex chromosomes (either 2 X chromosomes (if female) or an X
and Y chromosome (if male)). The labels are said to be distinguishable in that
the
particular label of any one member of the set (and the identity of that
member) differ
from the particular label and identity of any other member of the set. Since
each
probe member is capable of specifically hybridizing to only one chromosome (or
sub-
chromosomal region) and since the identity of the label and probe are known in
advance, the detection of a particular label associated with an unidentified
chromosomal region means that the probe bearing that label has become
hybridized to
the unidentified chromosomal region. Since the chromosome to which that probe
specifically hybridizes is known, the detection of a distinguishable label
permits the
identification of the chromosomal region.
More specifically, the invention concerns fluors that can be used to label
oligonucleotide probes so that such probes may be used in multiparametric
fluorescence in situ hybridization. As used herein, a "fluor" or "fluorophore"
is a
reagent capable of emitting a detectable fluorescent signal upon excitation.
Most
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preferably, the fluor is coupled directly to the pyrimidine or purine ring of
the
nucleotides of the probe (Ried, T. et al. (Proc. Natl. Acad. Sci. (U.S.A.)
89:1388-1392
( 1992), herein incorporated by reference; U.S. Patent Nos. 4,687,732;
4,711,955:
5,328,824; and 5,449,767, each herein incorporated by reference.
Alternatively, the
fluor may be indirectly coupled to the nucleotide, as for example, by
conjugating the
fluor to a ligand capable of binding to a modified nucleotide residue. The
most
preferred Iigands for this purpose are avidin, streptavidin, biotin-binding
antibodies
and digoxigenin-binding antibodies. Methods for performing such conjugation
are
described by Pinkel, D. et al., Proc. Nat'l. Acad. Sci. (U.S.A.) 83:2934-2938
( 1986),
herein incorporated by reference).
1'he term "multiparametric fluorescence" denotes the combinatorial use of
multiple fluors to simultaneously label the same chromosome or sub-chromosomal
fragment, and their detection and characterization. Chromosomes or sub-
chromosomal fragments are said to be simultaneously labeled if they are
exposed to
more than a single chromosome-specific probe under conditions sufficient to
permit
each chromosome-specific probe to independently hybridize to its target
chromosome.
As used herein, it is thus unnecessary for all such hybridization reactions to
commence
and conclude at the same instant. The simultaneous labeling permitted by the
present
invention is thus in contrast to protocols in which chromosomes are exposed to
only a
single chromosome-specific probe at a time.
The simultaneous detection and characterization permitted by the present
invention denotes an ability to detect multiple (and most preferably all) of
the
autosomal and/or sex chromosomes in a sample, without any need to add further
reagent, or probe after the detection of the first chromosome.
In the simplest embodiment, digital images of the chromosomes are obtained
for each fluorophore employed, thereby providing a series of gray scale
fluorescence
intensities associated with each fluorophore and each chromosome. The final
image is
obtained by pseudocoloring the blended gray scale intensities for each
chromosome.
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The invention thus provides a method of simultaneously identifying and
distinguishing the individual autosomal and sex chromosomes of a human
karyotype
which comprises contacting a preparation of chromosoraes, that has been
previously
treated to render it in single-stranded form, with the above-described set of
combinatorially labeled oligonucleotide probes, under conditions sufficient to
permit
nucleic acid hybridization to occur.
Such treatment causes at least one of each autosomal or sex chromosome of the
preparation to become hybridized to at least one member of said set of probes.
For
each chromosome of the preparation hybridized to a member of the set of probes
one
next detects and identifies the predetermined label of that member and
correlates the
identity of the label of that member with the identity of the autosomal or sex
chromosome of said human karyotype with which that member specifically
hybridizes.
This process identifies the chromosome hybridized to the member. This last
step is
repeated until each or a desired number of autosomal and sex chromosome of the
human karyotype has been identified in the preparation.
The oligonucleotide probes used in accordance with the methods of the present
invention are of either of two general characteristics. In one embodiment,
such probes
are chromosome or sub-chromosome specific (i.e., they hybridize to DNA of a
particular chromosome at lower cotl/2 than with DNA of other chromosomes;
cot~/2
being the time required for one half of an initial concentration (co) of probe
to
hybridize to its complement). Alternatively, such probes are feature (e.g.,
telomere,
centromere, etc.) specific. Such probes, being proximal to the telomere, are
capable of
defining and identifying translocations that may be so close to the
chromosomal
termini as to be otherwise cryptic.
Both types of probes may be used if desired. Sources of such probes are
available from the American Type Culture Collection, and similar depositories.
The oligonucleotide probes used in accordance with the methods of the present
invention are of a size sufficient to permit probe penetration and to optimize
reannealing hybridization. In general, labeled DNA fragments smaller than 500
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nucleotides in length, and more preferably of approximately 150-250
nucleotides in
length, probes are employed. Probes of such length can be made by synthetic or
semi-
synthetic means, or can be obtained from longer polvnucleotides using
restriction
endonucieases or other techniques suitable for fragmenting DNA molecules.
Alternatively, longer probes (such as polynucleotides) may be employed.
Most preferably, the oligonucleotide probes are synthesized so as to contain
biotinylated or otherwise modified nucleotide residues. Methods for
accomplishing
such biotinylation or modification are described in U.S. Patent Nos.
4,687,732;
4,711,955; 5,328,824; and 5.449,767, each herein incorporated by reference.
Biotinylated nucleotides and probes are obtainable from Enzo Biochem,
Boehringer
Mannheim, Amersham and other companies. In brief, such biotinylated or
otherwise
modified nucleotides are produced by reacting a nucleoside or nucleotide with
a
mercuric salt under conditions sufficient to form a mercurated nucleoside or
nucleotide
derivative. The mercurated product is then reacted in the presence of a
palladium
catalyst with a moiety (e.g., a biotin group) having a reactive terminal group
and
comprising three or more carbon atoms. This reaction adds the moiety to the
purine c:r
pyrimidine ring of the nucleoside or nucleotide.
In a highly preferred embodiment, such modified probes are used in
conjunction with competitor DNA in the manner described by Ward et al.
(W090/05789), herein incorporated by reference. Competitor DNA is DNA that
acts
to suppress hybridization signals from ubiquitous repeated sequences present
in human
and other mammalian DNAs. In the case of human DNA, alu or kpn fragments can
be
employed, as described by Ward et al. (W090/05789). Initially, probe DNA
bearing a
detectable label and competitor DNA are combined under conditions sufficient
to
permit hybridization to occur between molecules having complementary
sequences.
As used herein, two sequences are said to be able to hybridize to one another
if they
are complementary and are thus capable of forming a stable anti-parallel
double-
stranded nucleic acid structure. Conditions of nucleic acid hybridization
suitable for
forming such double stranded structures are described by Maniatis, T., et al.
(In:
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Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratories, Cold
Spring Harbor, NY ( 1982)), by Haymes, B.D., et al. (In: Nucleic Acid
H~~bridi~ation.
,4 Practical Approach, IRL Press, Washington, DC (1985), and by Ried, T. et
al.
(Proc. Natl. Acad. Sci. (U:S.A.) 89:1388-1392 (1992)). For the purpose of the
present
invention, the sequences need not exhibit precise complementarily, but need
only be
sufficiently complementary in sequence to be able to form a stable double-
stranded
structure. Thus, departures from complete complementarily are permissible, so
long as
such departures are not sufficient to completely preclude hybridization and
formation
of a double-stranded structure.
The quantity of probe DNA combined with competitor DNA is adjusted to
reflect the relative DNA content of the chromosome target. For example, as
disclosed
by Ward et al. (W090/05789), chromosome 1 contains approximately 5.3 times as
much DNA as is present in chromosome 21. 'Thus, a proportionally higher probe
concentration would be employed when using chromosome 1 specific probes.
The resulting hybridization mixture is then treated (e.g., by heating) to
denature
the DNA present and is incubated at approximately 37 °C for a time
sufficient to
promote partial reannealing. The sample containing chromosomal DNA to be
identified is also heated to render it susceptible to being hybridized to the
probe. The
hybridization mixture and the sample are then combined, under conditions
sufficient to
permit hybridization to occur. Thereafter, the detection and analysis of the
hybridized
product is conducted by detecting the fluorophore label of the probe in any of
the
methods described below.
In an alternative embodiment, a modification of the method of Ried T. et al.
(Proc. Natl. Acad. Sci. (U.S.A.) 89:1388-1392 (1992), herein incorporated by
reference) is employed. Thus, probes are labeled either directly (e.g., with
fluorescein)
or indirectly (e.g., with biotinylated nucleotides or other types of labels),
and permitted
to hybridize to chromosomal DNA. After hybridization, the hybridized complexes
are
incubated in the presence of streptavidin, that had been conjugated to one or
more
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floors. The streptavidin binds to the biotinylated probe of the hybridized
complex
thereby permitting detection of the complex, as described below.
C. The Preferred Fluorophores of the Invention
By labeling with two or more floors in combination, it is possible to
discriminate between many more objects than there are available floors. The
simplest
way to implement such labeling is by Boolean combination, i.e., a floor is
either
completely absent (0) or present in unit amount (1). For a single floor A,
there is only
one useful combination (A=I). For two floors A, B there are 3 useful
combinations
(A=1, B=0; A=0, B=l; A=1, B=I). For three floors A, B, C, there are %
combinations
(A=l, B=0, C=0; A=0, B=1, C=0; A=0, B=0, C=1; A=1, B=1, C=0; A=1, B=0, C=1;
A=0, B=1, C=1; A=1, B=1, C=1). There are 15 combinations of 4 floors, 31
combinations of 5 floors, 63 combinations of 6 floors, and so on.
To uniquely code all 24 chromosome types in the human genome, 5
distinguishable combinatorial floors are needed. With a 5-floor set, 15
chromosomes
can be distinguished using combinations of 4 of the 5 floors. The labeling of
the
remaining 9 chromosomes requires all five floors to be used combinatorially.
Seven of
the available S-floor combinations are not required. Thus, there is a certain
amount of
latitude available to avoid any 5-floor combination that might prove
particularly hard
to resolve. In particular, quaternary or quinternary combinations may be
avoided.
One aspect of the present invention concerns the identification of a set of
seven
floors that are be well resolvable by the excitation-emission contrast (EEC)
method.
As indicated above, mufti-floor combinatorial labeling depends in general on
acquiring and analyzing the spectral signature of each object i.e., obtaining
the relative
weighting coefficients of the component floors. Because full spectroscopic
analysis of
mixed floor spectra (e.g., by interferometry) is not yet sufficiently
developed, the
method chosen was conventional bandwidth-restricted widefieid imaging using
epi-
fluorescence triplets, viz. excitation filter, dichroic reflector and emission
bandpass
filter. The limited spectral bandwidth available for imaging (roughly 380-750
nm),
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and the extensive overlap between the spectra of organic floors, makes
separating
multiple floors spectroscopically during the imaging step a significant
technical
challenge.
To make software segmentation of the source images as straightforward as
practicably possible, a target figure of <10% crosstalk between any given
floor and the
two adjacent channels was set. Computer modeling indicated that for DAPI plus
the
five combinatorial floors FITC, Cy3, Cy3.5, CyS, Cy5.5, this level of contrast
cannot
be attained using either excitation selection or emission selection alone, no
matter how
narrow the filter bandwidths. Thus, both excitation selection and emission
selection
must be invoked simultaneously. This is referred to as excitation-emission
contrast
(EEC).
Contrast ratio plots were first computed for each of the floors vs. its two
neighbors. These plots indicate regions where pairwise contrast is high enough
to be
useful. A constraint on the practically attainable contrast is that regions of
high
contrast generally lie far down the flanks of at least one of the spectra
i.e., where
excitation and/or emission are strongly sub-optimum. Further, to attain the
required
degree of selectivity it is necessary to use filters of bandwidths in the
range S-15 nm
(cf. approx. 50 nm for 'standard' filter sets). Together, these impose a
severe
sensitivity penalty. The goal of 10% maximum crosstalk represents an
acceptable,
practical compromise between sensitivity and selectivity.
A fundamental asymmetry exists between excitation contrast and emission
contrast. For low-noise detectors such as the cooled CCD, restricting the
excitation
bandwidth has little effect on attainable image S/N ratio; the only penalty is
the need
for longer exposure times. Restricting the emission bandwidth is very
undesirable,
however, since every fluorescence photon blocked by the filter represents
irreversible
photochemical bleaching of the floor. For this reason, the highest practicable
contrast
was invoked on the excitation side. A suitable emission filter was then found
to give
the necessary EEC ratios. Filter selection is additionally constrained by the
fact that
each channel must adequately reject both adjacent channels simultaneously:
improving
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one may significantly degrade the other. Good contrast was attainable in
practice for
all fluors except the Cy5.5/Cy5 pair, which is marginal. For this reason, Cy7
was later
substituted for Cy5.5. Other considerations relating to choice of filters
include:
1. Commercial narrow-band interference filters may have a large amount
of wedging i.e., non-parallelism between the top and bottom faces.
This results in large image shifts (up to several microns equivalent).
The shift is a vector characteristic of each filter and its orientation in the
epicube. Thus, automatic compensation for image displacement is a
necessary part of the processing software.
2. Manufacturing variations of a few nm in peak wavelength and FWHM
specifications can have significant effects on the EEC ratios. Filter
errors to long wavelength may be fine-tuned by tilting, but this option is
severely curtailed in the case of emission filters because of increased
image aberrations and worsened pixel shifts. There is no equivalent
way to compensate short-wavelength errors.
3. the need to prevent infra-red light emitted by the arc lamp from
reaching the detector. Silicon CCD's are extremely sensitive in this
region. Filter sets for the blue and midvisible fluors were found not to
need additional IR blocking, but loss of image contrast due to spurious
IR was found to be a serious problem for the red - far red - near IR
fluors. Heat filters routinely used in microscopy (e.g., Schott BG-38
glass) are completely inadequate to alleviate this problem. Thus,
extensive additional blocking was required. However, available
commercial interference filters for infra-red blocking filters also
transmit poorly in the near UV, and thus cannot be inserted in the
excitation path. Instead, it was found necessary to put the IR blocking
filters into the emission path. To minimize loss of image quality by
insertion of these filters in the image path, they are placed inside the
CCD camera, immediately in front of the window. In practice, two
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interchangeable filters were chosen, one for use with CyS, Cy5.5 (Oriel
#58893; 740 nm cutoff) and one for use with Cy7 (Oriel 58895: 790 nm
cutoff).
The first member of the set of floors is the counterstain DAPI, which gives a
weak G-like banding pattern. Five of the remaining six floors may be used
combinatorially to paint the entire human chromosome set. All are available as
avidin
conjugates (for secondary detection of biotinylated probe libraries) or
directly linked to
dtJTP (for direct labeling).
Thus, a set of six floors and corresponding optical filters spaced across the
spectral interval 350-750 nm was identified that achieve a high discrimination
between
all possible floor pairs. These floors comprise the preferred floors of the
present
invention and are: 4'-6-diamidino 2-phenyl indole (DAPI}, fluorescein (FTTC),
and the
new generation cyanine dyes Cy3, Cy3.5, CyS, Cy5.5 and Cy7. Of these Cy3,
Cy3.5,
Cy5 and Cy7 are particularly preferred, The absorption and emission maxima for
the
respective floors are: DAPI (Absorption maximum: 350 nm; Emission maximum: 456
nm), F1TC (Absorption maximum: 490 nm; Emission maximum: 520 nm), Cy3
(Absorption maximum: 554 nm; Emission maximum: 568 nm}, Cy3.5 (Absorption
maximum: 581 nm; Emission maximum: 588 nm), Cy5 (Absorption maximum: 652
nm; Emission maximum: 672 nm), Cy7 (Absorption maximum: 755 nm; Emission
maximum: 778 nm). Complete properties of selected fluorescent labeling
reagents are
provided by Waggoner, A. (Methods in Enzymology 246:362-373 ( 1995) herein
incorporated by reference). In light of the above, it is readily apparent that
other
fluorophores and filter combinations having adequate spectral resolution can
alternatively be employed in accordance with the methods of the present
invention.
D. Methods for the Detection of Fluorescent In situ Hybridization
1. The Theory of Fluorescence Detection
Of the various methods for contrast generation in site-specific labeling,
fluorescence is arguably the most powerful, because of its high absolute
sensitivity and
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multiparameter discrimination capability. Modem electronic cameras used in
combination with high numerical aperture microscope objectives and state of
the art
optical filters are capable of imaging structures labeled with as little as a
10-100 floor
molecules per pixel. Thus, floor-tagged single-copy DNA sequences as small as
a few
hundred bases in size are detectable under favorable conditions. The
availability of
families of spectrally distino ishable floors makes simultaneous imaging of
several
different targets in the same specimen possible, either directly or through
combinatorial or analog multiplex methods. In principle, mufti-floor
discrimination
may be based on differential excitation of the floors, differential emission,
fluorescence lifetime differences, or on more complex but still analyzable
observables
such as fluorescence anisotropy. This discussion assumes an epi-imaging
geometry.
Table I describes the symbols and operators relevant to the theoretical
considerations
of fluorescence.
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Table 1
InstrumentUnits Definition
Parameter
photon s'1 ntt~ spectral distribution of source,
1 assumed to be an
isotro is radiator over 4a steradians
dimensionless collection of efficiency of condenser
optics
photon s-1 m'2 spectral distribution of photons
nm-I in collimated
beam of excitation light impinging
on excitation
filter F1
dimensionless transmittance function of excitation
filter F1
photon s'1 cm'' spectral distribution of photons
nm'I in collimated
b eam of excitation light emerging
from FI an~i
im in in on dichroic beam~~litter
DB 1
dimensionless reflectance function of dichroic
beamsplitter DB I
dimensionless transmittance function of dichroic
beamsplitter
DB 1
1~13(a,) photon s'I cm-2nm--Ispectral distribution of photons
in collimated
beam of fluorescence emerging
from DB 1 and
im in in on emission filter F2
~I ~(~) dimensionless transmittance function of emission
filter F2
', 1~14(a,)photon s'1 cm'2 spectral distribution of photons
nm-I in collimated
beam of excitation light emerging
from F2 and
entering the entrance a il of
the ob'ective lens
dimensionless linear magnification factor of
objective lens
photon s' 1 cm'2spectral distribution of photons
nrrr I in focused beam
of excitation lieht traversin
the s ecimen lane
dimensionless quantum efficiency of detector
pixel.lt'I magnification factor of final
image at detector
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Table
1 (continued)
Floor Units Definition
~
Parameter
M-1 c~ 1 ~ molar decadic extinction coefficient
of floor Fa.
Z s decay lifetime of excited state
of floor Fa.
~FaU) cm 1 photon absorption cross-section
of floor Fa.
Fa dimensionless fluorescence quantum efficiency
of floor Fa.
dimensionless normalized spectral distribution
of fluorescence
from floor Fa.
Whether a given excitation rate at pixel location p will give an acceptable
fluorescence signaUnoise ratio (defined as S/N = (signal mean] / [variance due
to all
noise sources]) in a given integration period depends on the number of floor
molecules
S within p, their quantum yield and photochemical stability, and the quantum
efficiency
and noise performance of the detector.
In the limit of weak excitation (y(~).6F(~)«'C-1 ), the rate of excitation of
the
N molecules of floor F within pixel p in object space is:
R(P)=N~iVJs(~"P).6F(~).d~.s-1
yrs(~) is the spectral distribution of exciting light passing through the
focal plane of
the microscope (photon nm-l.cm-2.s-1). It is approximately given by
~s(~')~~s~f I (~,).R(~,).oc2.~t, where a= diameter of objective lens entry
pupil / diameter
of collimated beam from condenser. The integral is taken over the bandwidth
(i) for
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which the fluor has non-zero absorption cross section 6F(~), which is related
to the
molar decadic extinction coefficient ~F(~,) by the expression:
6p(~) = 3.825 x 10-21 tF(~.) cm2.
In practice, JiyrS(~~P). d~, can be measured with a bolometric detector such
as a
calibrated micro-thermopile placed at or near the focal plane of the objective
lens.
For a perfectly noiseless detector and a non-bleachable fluor, the S/N of each
pixel increases indefinitely as (photons detected)»2. How rapidly SIN
increases
depends on excitation strength, but the relationship between S/N and dose does
not.
The effect of non-zero bleaching constant is to change the tt~ function to an
asymptotic function, the form of which depends on the bleaching mechanism.
However, because the bleaching rate and the signal strength are linearly
related, the
asymptote once again does not depend at all on excitation rate, although the
speed of
approach to the asymptote does. If finite camera noise is added to
photobleaching, it is
found that the S/N climbs to a maximum value, then falls as the fluo~ is
exhausted.
1 S Now both the kinetics and the peak S/N depend of excitation rate, in
general the faster
the excitation the higher the maximum attainable S/N. However, for
contemporary
cooled CCD cameras the dark noise is so low that it can be virtually ignored
on the
timescale of bleaching (typically a few minutes); a more important factor in
determining ultimate SIN is the stray light background (esp. from nonspecific
luminescences and leakage of excitation light).
Note that although the microscope objective compresses the excitation beam,
in a nonconfocal microscope it does not focus it to a point and so has no
bearing on the
fluorescence image resolving power (incoherent emitter).
The commonest excitation source for fluorescence microscopy is the high
pressure short mercury arc, whose spectrum consists of pressure-broadened
lines from
the UV to the middle region of the visible spectrum (principal wavelengths are
334.1
nm, 365.6 nm, 404.7 mm, 435.8 nm, 546.1 nm. 577.9 nm), superimposed on a
weaker
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thermal continuum. Many fluorophores have excitation spectra that overlap one
or
other of the mercury lines to an acceptable extent. Others (of which the best
known is
FTTC) do not, but may be adequately excited by the continuum if a wide enough
excitation bandwidth is employed. Another common source is the high pressure
xenon
short arc, which produces an almost uniform continuum from ca. 300 nm: to
beyond
900 nm. However, the power nm-1, is almost everywhere less than the mercury
continuum for the same arc wattage. If high intensity light with no structure
is
required (e.g., for fluorescence ratio imaging) a high CW power or pulsed
quartz
halogen lamp outperforms the xenon beyond about 450 nm. Certain floors are
well
matched to laser excitation (e.g., Ar+ @488 nm for FITC, He-Ne @ 632.8 nm for
CyS,
semiconductor diode - pumped YAG @ 680 nm for Cy5.5).
In single-floor imaging, use of the available spectral bandwidth is rarely
stringent. The excitation filter FI and dichroic beamsplitter DB1 can usually
be chosen
to give adequate overlap between the source spectrum and the floor excitation
spectrum. If an arc line is available, the Fl bandpass need be no wider than
the line. If
not, and part of the thermal continuum must be used, the wider the F1 bandpass
the
greater will be the available excitation flux. However, with low noise
integrating
detectors the goal of high excitation efficiency is generally secondary to the
need to
exclude excitation light from the emission path. This limits how close the
excitation
and emission bandpasses can be placed to one another, and hence constrains the
excitation bandwidth. For floors with small Stokes' shifts, high quality
filters with
very steep skirts are required. The excitation filter must be rigorously
'blocked' on the
long wavelength side, and have no pinholes, scratches, or light leaks around
the edge.
Dichroic beamsplitters are currently much less 'evolved' than bandpass
interference filters, meaning that the slopes of their transmit <-> reflect
transitions are
far less than the skirt slopes of premium notch filters, and there may be
large spectral
intervals where they oscillate between intermediate states of partial
reflectance and
transmittance. The main purpose of a dichroic beamsplitter is to improve the
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combined efficiency of excitation and emission, rather than to define the
wavelength
response of the instrument.
The resolvability of overlapping fluors in imaging microscopy may depend
critically on the degree of excitation contrast that can be achieved (see C,
below). The
variation with wavelength of the ratio of the extinction coefficient of two
fluors is the
excitation contrast spectrum. It can readily be calculated from the digitized
absorption
spectra. Depending on the overlap of the absorptions, their ratio spectrum may
either
show a distinct peak or may grow indefinitely large. In either case, it is
usually
possible to choose an excitation wavelength that favors one fluor over another
to a
useful extent (from a factor of 3-4 fold up to a hundredfold or more). Some
difficulties in obtaining high contrast mufti-fluor images include:
a. The excitation wavelengths required for high contrast imaging are often
far from the absorbance peaks. Thus, there may be a high degree of
intensity trade-off to obtain high signal contrast vs. other fluors.
b. From the above, standard filter sets cannot be used.
c. Arc source spectral lines that are useful for exciting single fluors may
not give high contrast discrimination against adjacent fluors.
d. When using a broadband source or the continuum spectrum of an arc
source, the need for narrow excitation bandwidth may reduce the
excitation flux to problematically low levels. It is sometimes possible
to relax the constraints on excitation wavelength for the sake of more
efficient excitation.
Collecting the fluorescence of F and imaging it onto the detector with high
efficiency is the principal design goal of the emission optics. Operationally,
it is even
more important to have an efficient emission path than an efficient excitation
path.
The reason is that inefficient excitation causes inefficient photobleaching;
the only
penalty is a long image integration time (assuming a low-noise detector). On
the other
hand, any fluorescence lost on its way to the detector represents
photobleaching
without concomitant increase in the information content of the image.
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The detector pixel p accumulates signal (detected photons) at a rate:
F(P) = G. R(P).~F-~iiT(~)~~()~).~d(~,).d~..s-1
The integral is over the bandwidth (ii) for which the detector quantum
efficiency ~d(~,) > 0. G represents the efficiency with which the optics
gather the
fluorescence and transmit it to the detector; it may be assumed to be
wavelength
independent to first order. The principal factor in G is the numerical
aperture (NA) of
the objective lens, which determines the fraction of the isotropically
radiated
fluorescence collected by the imaging system (~ = l/n.siwl(6) = l/n.siwl(NA/n)
,
where 8 is the half angle subtended by the objective lens from its focal
point. For an
oil immersion lens with NA = 1.3; n = 1.515; ~ = 0.328;). NA additionally
determines
the spatial resolving power of the microscope, because it scales the
dimensions of the
Frauenhofer diffraction pattern produced in the image plane by a point source
in the
specimen plane. Several 'rules' are in use for specifying the resolving power
of a lens,
depending on how much overlap of the Airy discs of two adjacent objects is
deemed to
constitute the threshold of resolution. The commonly used Rayleigh criterion
is r =
1.22aJ2NA (e.g., for the above NA = 1.3 lens working at S00 nm, r = 0.24 p).
For a noise-free detector, image 'noise' at p is determined by the statistical
variance in the number S(p) of fluorescent photons detected in time interval
Ot. The
only detector characteristic that has any bearing on this is its quantum
efficiency ~d(~,).
In the absence of photobleaching (probability of destruction per hit IIb = 0),
S(p) = F(p).Ot, with variance S(P)1/2 , i.e., S/N = S(p)~/2. Image quality
will therefore
increase indefinitely with Ot, though at an ever decreasing rate.
In the presence of photobleaching (IIb ~ 0), S(p) is an integral of the form:
S(P) =F(P) jOtf(t).dt
where f(t) is the photobleaching decay function. SIN rises along a more or
less
complex path to an asymptotic value that corresponds to total exhaustion of
the fluor.
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For the case of a unimolecular photobleaching process this would be an
exponential
function. i.e., N(p.t) = Np(1-exp-[k~kJ.t) where kb = TIb-1 is the
photobleaching
constant and Ek represents all other processes by which the excited state of F
is
deactivated. Note that the normalized asymptote in this first-order system
depends
only on k~k, and is independent of the strength of the ercitation. Thus, the
extent of
bleaching is exponentially related to the accumulated excitation dose, but is
independent of the path. In reality, however, bleaching of fluors in solution
may be
mechanistically and kinetically much more complex. A common mechanism involves
ring opening following peroxidation of the fluor excited state, e.g., by ~ 02
or 022-.
This type of bleaching may be considerably slowed by rigorous deoxygenation or
by
the use of oxygen radical scavengers (i.e., antifade agents) such as tertiary
amines (p-
phenylene diamine or DABCO). Nevertheless, other (as yet poorly characterized}
irreversible processes are not excluded, including reactions with impurities.
A nonideal detector contributes noise of many kinds, detailed analysis of
which
may be intractable. The simplest noise component is fluctuation in the so-
called 'dark
current,' i.e., the flow of thermally excited carriers within the detector. If
this noise is
assumed to be random, it adds to the photon shot noise in RMS fashion. Thus,
if the
mean photogenerated signal is F s-1 and the mean dark count is D s-1, the S/N
after
time Ot is F.( 0t}t~l (F+D)t~; S/N still increases as (~t)t~, but more slowly
than for
a noiseless detector. When photobleaching is present, however, the situation
is
entirely different. In this case, S/N rises initially as (Ot)», but at some
point reaches
a shallow maximum and then begins to fall again. as the fluorescent signal
declines but
the thermal noise power remains constant. In this case, it is in principle
desirable to
continuously monitor the S/N of the image, and terminate the exposure when the
peak
is reached. Most commercial digital imaging systems make no provision for
this.
Fortunately, state of the art cooled CCD cameras have so little dark noise per
pixel
(typically <0.01 electron s-1 in inverted clock mode) that SIN would not begin
to fall
until almost complete exhaustion of the fluor. In practice, autofluorescences
and stray
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light dominate system performance long before the noise threshold of the CCD
is
reached.
The principal design goals for a single-fluor imaging system are:
i . To achieve an adequate rate of excitation of the fluor (F).
2. To collect the fluorescence of F and image it onto the detector with high
efficiency and with the necessary spatial resolution.
3. To prevent reflected and/or scattered excitation light from reaching the
detector.
Design of the emission channel for a single fluor is straightforward. The
dichroic beamsplitter transition wavelength is specified at for example, 20 mn
to the
red of the excitation passband. This ensures a high level of rejection of
exciting light
reflected and/or scattered from the specimen and/or microscope optics. The
emission
filter cut-on is usually considerably steeper than the dichroic edge, and so
can be
placed practically coincident with it. The most efficient emission filter is a
Long-pass
IS element. The preferred filter of this type is Schott glass, which transmits
upwards of
90% of all fluorescence to the red of its cut-on, while rejecting other Iight
(especiall~~
any excitation light that gets through the dichroic beamsplitter) to very high
order -
typically > 105. However, it is usually inadvisable to leave the emission
channel 'wide
open' into the near infrared, especially with silicon detectors which have
high
sensitivity there.
The multiparametric imaging of the present invention not only increases the
throughput of information about the system under observation and makes more
efficient use of the biological material, but also can reveal spatial and
temporal
correlations that might otherwise be difficult to establish reliably. When a
large
number of different objects must be visualized, two or more labels can be used
combinatorially, which permits discrimination between many more object types
than
there are spectrally distinguishable labels. Some examples of multi-fluor
imaging are:
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a. The co-distribution of proteins in structures such as microtubule
networks may readily be visualized using immunolabels linked to
different fluors.
b. Multiple genes may be simultaneously mapped by fluorescence in situ
hybridization (FISH) to a single metaphase chromosome spread. Such
signals cannot usually be discriminated reliably on the basis of intensity
alone, and are usually morphologically identical (diffraction-limited
points). However, they are readily discriminated by discrete or
combinatorial mufti-fluor labeling.
c. Identification of small chromosomal translocations is most readily done
by painting with chromosome specific DNA probe libraries linked to
separable fluors, used either singly or combinatorially.
d. Analysis of mixed populations of morphologically identical bacteria
can also be achieved using species-specific DNA or ribosomal RNA
probes coupled to separable fluors.
The primary design goal of a mufti-fluor imager (in addition to those for of a
single fluor imagery is to spectrally resolve the fluorescence at any pixel
location into
components corresponding to each fluor. Methods for spectrally resolving
complex
signals in fluorescent microscope images are outlined below.
There are several ways to resolve spectrally complex signals, i.e., to
determine
which fluors contribute to the fluorescence at a given pixel location. The
most general
method in principle is to spectrally disperse the 2D image along a third axis,
orthogonal to the x,y axes. This amounts to imaging through an optical system
with a
very large amount of chromatic aberration, such that at each position on the z
axis
there is an image x,y that contains only a small spectral bandwidth ~,+0~,. An
area
detector with very small depth of field (such as a spatially filtered confocal
imagery
could then be moved incrementally along the z axis to obtain a family of
images, each
containing its own small spectral interval. A trace through the images at
given x,y
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would constitute an emission spectrum for that pixel. Unfortunately,
implementing
such a scheme is technically very difficult.
If the spectrum of only a small number of objects within the image is required
(such as individual stars in a telescope image), a solution is to extract the
light
corresponding to each object with a probe (e.g., a fiber optic) and disperse
it with an
imaging spectrograph onto a 1-dimensional array detector. To be useful in
microscopy, such a device would have to be arbitrarily positionable in the
field, and
have an adjustable acceptance area.
The most reasonable method for full spectral analysis in microscopy is to
image through a variable narrow band filter. An image is recorded at each
wavelength; intensity values at a given pixel location through the series
represent a
weighted emission spectrum that can be fit to a linear combination of the
known
spectra of the component floors. The coefficients are products of the relative
molar
amounts of the floors with their extinction coefficients at the exciting
wavelength and
their fluorescence quantum yields. If the last two are known, the first is
obtainable
from the fit. In general, it is necessary to take several such image sets, at
severa:
excitation wavelengths, to get a unique fit. With enough iterations, this
process
generates a 3D surface of intensity values as a function of both excitation
and emission
wavelength. This comprises a complete spectral signature of the pixel, giving
a very
highly constrained solution for the relative amounts of its component floors.
The mole
ratios could be mapped back onto the x,y coordinates of the image, with
appropriate
pseudocolor coding, to give a 'composition map'. This general (and
quantitative)
method has a number of technical difficulties, although none of them is
insurmountable. The first is that a large number of images are required to
evaluate a
single microscope field. Imaging time is long, and extensive differential
photobleaching of the floors would make it impossible to achieve a self
consistent
"fit" to the spectral data. Instrument stability is also an issue,
particularly with arc
sources, the output spectra of which change throughout their Life. Finally,
the amount
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of computation required to generate a composition map would realistically
limit the
analysis to small image regions only.
For most applications, there is no need of a full-blown spectral analysis
capability because the fluors to be analyzed are known ahead of time. Thus, it
is only
necessary to be able to identify which fluor is which, and, for multiplex
imaging, to
ascertain the relative amount of each present. A preferred approach to this
category of
problems involves the use filter sets that achieve a high degree of selective
excitation
and visualization of each component fluor. The ideal system of this type would
perfectly isolate each fluorophore, i.e., "one image - one fluor" and all off
diagonal
elements in the matrix of intensity coefficients would be zero. However, the
facts that
the spectra of typical fluorophores are 10-30% of the total available
bandwidth, and
emission filters must have significant bandwidths to pass usable amounts of
light place
severe limitations on the attainable degree of spectral isolation.
Nevertheless, it is not
difficult to achieve useful levels of contrast between suitably chosen fluors,
such that
residual crosstalk can be removed numerically.
The excitation-emission contrast (EEC) approach is in principle applicable t~
analysis of images involving multiple fluors with fine-grained distributions
of mole
fraction (e.g., fluorescence ratio imaging), subject to image SIN and the
limitations of
differential bleaching rates and source instability. However, it is
particularly simple
for the limiting case of combinatorial labeling, in which fluors are
multiplexed in a
strictly Boolean fashion (present = 1, or absent = 0). In this case, it is
necessary only
to be able to reliably discriminate between a 1 and a 0, for which a 10:1
intensity ratio
between any fluor and its neighbors would be sufficient. However, for many
useful
fluors this is not achievable on the basis of excitation contrast or emission
contrast
alone. Simultaneous selection on the basis of both excitation and emission are
required.
In some circumstances it is required to excite and image several fluors
simultaneously, e.g., for direct viewing and/or color video recording.
Excitation and
emission optics that have no wavelength selectivity cannot be used, because
the
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excitation light scattered into the detector would overwhelm the fluorescence
by
several orders of magnitude. One solution is to use multiple-bandpass filters
designed
for the specific set of fluors to be used. The excitation filter defines
narrow passbands
that overlap the fluor excitation spectra. The emission filter defines similar
passbands
that interdigitate between the excitation bands and overlap the floor emission
spectra
(the reddest floor could use a long-pass filter). The dichroic beamsplitter
alternates
between reflect (overlapping the excitation passbands) and transmit
(overlapping the
emission passbands).
Use of a multi-pass filter set also has an advantage even when multicolor
visualization is not required, viz. the absence of geometric displacement
(pixel shifts)
between signals passing through the several emission passbands. However, when
used
with a greyscale imaging system, it is necessary to make either the excitation
filter or
the emission filter switchable, in order to determine which greyscale signal
corresponds to which floor. The preferred choice is to switch the excitation
filter,
because this causes no image displacement.
A second theoretical solution would be to excite at a wavelength where all the
floors absorb. This is often possible because many floors are excitable to
states higher
than S 1 using photons in the middle UV, but because of internal relaxation
processes
give 'normal' fluorescence. For example, many laser dyes can be excited at the
nitrogen laser wavelength, 337 nm, far to the blue of their visible
absorbances. It
would be straightforward to block such exciting light from the emission path,
using a
long-pass filter (e.g., 380 nm), while allowing all fluorescences to
simultaneously
reach the detector. Drawbacks to the use of UV excitation include increased
rates of
photochemical decomposition of the floor, and the expense of suitable UV
optics.
Thus, the method has not found widespread use.
The mufti-bandpass method has the limitation that construction of multiple
bandpass elements giving adequate contrast between more than 3 floors is
extremely
difficult. A generally more powerful approach is to construct optimized filter
sets for
each floor, and switch them as needed. In the case of a single floor, the
primary goal
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on the excitation side is high excitation flux, to give a bright image. When
imaging
multiple fluors, however, this becomes secondary to the goal of achieving high
contrast between fluors. In the weak overlap limit, fluors may be imaged by
sequentially switching filters that are designed using the same criteria as
single-fluor
sets (except that long-pass emission filters are proscribed for all but the
longest-
wavelength fluor). Crosstalk of a few percent is usually allowable, and can be
compensated numerically if necessary. More generally, though, generation of
adequate contrast between fluors requires both strongly selective excitation
and
emission. The optimum wavelengths can be found by ratioing the spectra of
adjacent
fluors. These wavelengths may be far from the excitation maxima, implying a
significant tradeoff between signal brightness and contrast. Standard filter
sets cannot
be used. The need for precisely placed excitation makes it less likely that a
strong
excitation line will be available, and also reduces the flux available from a
continuum
source. It may be necessary to pulse the broadband source (arc or filament
lamp) to
transiently very high output levels, or to supplement it with laser sources.
The principal technical problem with serial imaging is image displacemect
when filters are changed i.e., the coordinate systems of the individual
members of an
image series are not in precise registration. This problem arises from
nonidealities in
the emission channel optics. Two displacement components can be identified:
i. a reproducible offset that is unique to each filter set. This component is
a fixed
vector, and arises mainly from imperfect parallelism (i.e., wedging) between
the top
and bottom faces of the emission bandpass filter. There is also a small
component due
to wedging in the dichroic beamsplitter, but since this element is very thin
the effect is
minor. Since the wedging vector is a constant for each filter set, it can be
automatically removed in the computer. The size of the offset can also be
reduced to
very small values (<0.1 N) by selecting emission filters for a high degree of
parallelism
e.g., by measurement in a laser autocollimator.
ii. a random component due to vibration and hysteresis in the filter switching
mechanism. The magnitude of the noise depends on the filter switching
mechanism.
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The worst are manual push-pull sliders, particularly when the operator
actuates them
using uncompensated forces. The best are motorized filter cassettes, in which
all
mechanical torques act against the microscope body.
Note that both the constant and random components of the image offset noise
are mininuzed by using an objective lens of the highest possible
magnification, and the
minimum amount of magnification in the camera projection oprics. Image
displacements encountered in optical microscopy are linear vectors only; there
is no
evidence for rotation or significant changes in scale (magnification).
Design considerations for high contrast imaging of multiple overlapping floors
are similar to those already outlined for the excitation channel. By
calculating the ratio
of each floor's emission spectrum to that of its neighbors, it is possible to
identify
spectral regions of high contrast that can be defined with narrowband filters.
High
contrast is often achievable only at the cost of throwing away significant
amounts of
the fluorescence (90% or more). Inefficiency in the fluorescence channel is
much
more damaging to image S/N than excitation Inefficiency. Thus, where possible,
selective excitation is the preferred method of achieving contrast between
multiple
floors. In difficult cases, however, both excitation and emission contrast are
required.
2. Optical Filter
As indicated, in a preferred embodiment of the invention, the detection of
floor
is accomplished using optical filters, in a modification of the method of
Ried, T. et al.
(Proc. Natl. Acad. Sci. (U.S.A.) 89:1388-1392 (1992), herein incorporated by
reference).
Imaging DAPI
4',6-diamidino-2-phenylindole (DAPS is a commonly used DNA counterstain
that intercalates preferentially into AT-rich regions of chromosomes and so
gives rise
to a weak G-type banding pattern. It is a very bright floor (E = 3.3 x 104 M-~
cm-~ at
347 nm, with approx. 20-fold enhancement of fluorescence quantum yield when
intercalated into the minor groove of double-stranded DNA), and is relatively
resistant
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to photobleaching. The following points are relevant to imaging DAPI with high
contrast against its neighbors:
a. DAPI has very broad excitation and emission spectra, and a very large
Stokes
shift. Thus, although the DAPI excitation maximum (347 nm) is to the blue of
Cascade Blue (CB), the fluorescence of DAPI peaks to the red of Cascade
Blue, and actually overlaps quite strongly with FTTC.
b. The usual excitation wavelength for DAPI (Hg 366 nm line) cannot be used in
this application, because it is almost isosbestic for DAPI and CB and thus
gives
no excitation contrast.
c. The peak of the DAP1/CB excitation contrast spectrum is at 320 nm, which is
too far into the UV to pass through the microscope optics. An acceptable
compromise is to excite with the Hg 334.1 nm line, which the microscope
transmits with about 30% efficiency. The Ealing 35-2989 interference filter
has an appropriate bandpass. The excitation contrast ratio for DAPUCB at
334.1 nm has an absolute value of 4Ø
d. To further increase the DAPI/CB selectivity, emission contrast must be used
is
addition to excitation contrast. Note that DAPI emits the bulk of its
fluorescence to the red of CB. The wavelength of maximum emission contrast
for DAPI vs. both CB and FTTC is 490 nm. A suitable imaging-grade filter is
the Omega 485DF22. There are no mercury lines within its bandpass, so good
DAPI images with low flare are expected. The calculated emission contrast
between DAPI and both Cascade Blue and FITC is 6.8. Hence, the overall
contrast achievable for DAPI vs. Cascade Blue is approximately 27-fold. The
overall contrast of DAPI vs. FITC is very much higher than this, because at
the
DAPI excitation wavelength the excitation of FTTC is close to zero.
e. The Omega 450DRLP02 dichroic beamsplitter is very well matched to the
proposed excitation and emission filters.
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Imaging Cascade Blue
Cascade Blue (CB) has a broad, two-peak excitation spectrum that overlaps
DAPI extensively, though not its neighbor on the red side (FTTC). The Stokes
shift for
CB is very small, i.e., there is very extensive overlap between its excitation
and
emission spectra. These factors combine to make imaging CB in the presence of
DAPI problematical. To summarize:
a. The peak of the CB/DAPI excitation contrast spectrum is at 396-404 nm.
However, because of the very small Stokes' shift of CB this is very close to
the
emission contrast peak (408 nm). Since it is more important for image S/N to
collect the emission with high efficiency than to excite with high efficiency,
the
400-410 nm region is reserved for the CB fluorescence. The best compromise
for CB excitation is the Omega 380HT15, which overlaps with the Hg 366 nm
line enough to provide good excitation strength.
b. To achieve any emission contrast at all vs. DAPI, the emission filter must
be
narrow and very carefully placed. A suitable filter is the Omega 405DF10, but
the theoretical maximum contrast is only 2.5. Thus, imaging CB while
excluding DAPI is expected to be marginal.
c. Cascade Blue exhibits high excitation contrast vs. FTTC (contrast ratio
with the
Omega 380HTI5 filter = 6).
d. The emission contrast ratio for CB/FTTC goes to very large values below 490
nm, and is essentially infinite at the position of the 405DF10 filter.
The above considerations indicate that it will be possible to use either DAPI
or
Cascade Blue, but not both together unless the DAPI counterstain is weak.
Cascade
Blue is well separable from FTTC and the other five combinatorial fluors
considered
herein.
Imaging FITC
The following points are relevant to high-contrast imaging of FITC:
a. The excitation spectrum of FTTC has insignificant overlap with that of
Cascade
Blue (contrast parameter Rb for FITC/CB becomes extremely large beyond 420
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nm). This makes it possible to excite FTTC on the high frequency shoulder of
its absorbance, so avoiding appreciable excitation of Cy3.
b. There is no mercury line that overlaps well with the FTTC excitation
spectrum.
Thus, it is necessary to use the continuum to excite this floor, as with
single
s floor imaging. The gap between FITC and CB makes it possible to use a very
wide excitation filter; the Omega 455DF70 is well suited to this function.
c. The Omega 455DF70 bandpass also corresponds fortuitously to the maximum
in the excitation contrast ratio for F1TC/Cy3 (460 nm; absolute extinction
ratio
Ra = 8.8).
d. The emission spectrum of FTTC overlaps that of Cy3 (on the red side) and
both
DAPI and CB (on the blue side) to an appreciable extent. However, because
the conditions defined for FTTC excitation do not excite DAPI or Cascade Blue
at all, they are not relevant. The FITC/Cy3 emission contrast ratio goes to
very
high values below 540 nm. Thus, the Omega 530DF30 filter gives very high
emission contrast, which compounds the high excitation contrast ratio for FITC
vs. Cy3 (8.8) given by the 455DF70. It therefore appears possible to image
FITC very cleanly.
Imaging Cy3
The following points are relevant to high-contrast imaging of Cy3:
a. The absorbance peak of Cy3 is at 551 nm, at which wavelength the excitation
of FTTC is essentially zero (contrast parameter Rb jumps to extremely high
values to the red of 525 nm).
b. The Cy3 extinction peak overlaps strongly with the Hg 546.1 nm line.
c. The excitation contrast ratio for Cy3/Cy3.5 is everywhere small, and varies
weakly with wavelength. At 551 nm, the absolute value of the excitation
contrast for Cy3 vs. Cy3.5 is less than 2, and it only rises significantly far
to
the blue where the Cy3 ahsorbance is very low and FITC absorbance is high.
In fact, the apparent rise in Ra around 460-470 nm may be an artifact of the
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low absolute precision of the spectra in that region. From the above
discussion, it is clear that the excitation contrast available for Cy3 vs.
Cy3.5 is
too low to be useful.
d. The emission contrast ratio for Cy3/Cy3.5 rises abruptly below 570 nm. At
the
567 nm peak of the Cy3 fluorescence, the absolute value of Sa is
approximately 6. This, combined with the factor 2 in the excitation contrast
parameter, should just about meet the goal of a 10-fold discrimination between
Cy3 and Cy3.5. The Ealing 35-3722 narrowband interference f lter is suitable,
although it does overlap the Hg 577/579 line significantly. Any stray light
from this line getting through the 546DF10 filter is, however, expected to be
well blocked by the chosen dichroic, Omega 560DRLP02.
e. The inability to differentially excite Cy3 vs. Cy3.5 means wasteful
bleaching of
Cy3.5 during imaging of Cy3.
Imaging Cy3.5
The following points summarize high contrast imaging with this dye:
a. The excitation contrast ratio between Cy3.~ and Cy3 rises to very high
values
beyond approximately 565 nm. This region includes the peak of the Cy3.5
exc~tauon spectrum.
b. The mercury arc line at 577/579 nm is almost exactly coincident with the
peak
of the Cy3.5 absorbance. At this wavelength the excitation contrast ratio is
approximately 25, i.e., very strong selective excitation of Cy3.5 relative to
Cy3
is possible. An ideal filter for this purpose is the Ealing 35-3763. Note that
the
high contrast is mainly a consequence of the Hg line position, not the filter
bandwidth.
c. At the Hg 577/579 excitation wavelength, the excitation contrast ratio for
Cy3.5 relative to Cy5 is also quite large (absolute value approximately 8.0).
d. The emission contrast parameter for Cy3.5 vs. Cy3 is small at all
wavelengths
where the Cy3.5 emission is usefully strong, i.e., isolation of Cy3.5 from Cy3
must rely mainly on excitation contrast.
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e. The emission contrast for Cy3.5 vs. Cy5 is also large over a considerable
spectral interval (and rises to very high values below 640 nm). This permits a
fairly broadband filter to be used to image Cy3.5; a suitable element is the
Omega 615DF45. Almost no bleadthrough of either Cy3 or Cy5 into the Cy3.5
channel is expected with the above combination of excitation and emission
conditions.
f. The Omega 590DRLP02 is a suitable dichroic for this channel.
Imaging Cy~
The following points are relevant to imaging with this dye:
ZO a. Beyond 600 nm, the excitation contrast ratio for Cy5 vs. Cy3.5 gets very
large,
i.e., very high excitation contrast relative to Cy3.5 is possible.
b. The placement of the Cy5 excitation is determined by the extinction ratio
relative to Cy5.5 rather than Cy3.5. The Cy5/Cy5.5 excitation contrast
parameter peaks at 650 nm with a numerical value of 2.25. Thus, analogous
with the Cy3/Cy3.5 pair, excitation contrast for Cy5/Cy5.5 is poor.
c. There is no Hg line available for exciting CyS. Thus, with an arc source,
the
continuum must be used, analogously with FTTC excitation. The "official"
filter for exciting Cy5 in this way is the Omega 640DF20, which will give an
excitation contrast with Cy5 of about 1.8.
d. A much brighter source for exciting Cy5 is the He-Ne laser (632.8 nm). It
does
not, however, improve excitation contrast vs. Cy5.5.
e. The emission contrast for Cy5 vs. Cy3.5 peaks at 673 nm, just to the red of
the
fluorescence intensity peak. The closest available filter is the Omega 660DF32
where the emission contrast ratio is approximately 3.1. This compounds the
very high excitation contrast for Cy5 vs. Cy3.
f. The emission contrast for Cy5 vs. Cy5.5 goes to very large values at
wavelengths shorter than 675 nm. The Omega 660DF32 filter is ideally set up
to take full advantage of this. Unfortunately, it is uncomfortably close to
the
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640DF20 exciter, so some flare from reflected/scattered excitation light is to
be
expected. Use of the He-Ne laser would remove this problem.
g. The best available dichroic beamsplitter for Cy5 imaging is the Omega
645DRLP02, particularly if a He-Ne is used as the excitation source.
Imaging CyS.~
CY5.5 is the penultimate dye of the set, and is very well separated from Cy7.
Thus, only its contrast relative to Cy5 need be considered in detail:
a. The contrast parameter Rb for the Cy5.5/Cy~ pair rises to large values to
the
red of 670 nm. Thus, it is possible to achieve very high excitation contrast
for
this pair of fluors (analogously to Cy3.5/Cy3).
b. As with CyS, the Hg arc is a poor source for exciting Cy5.5. The best
available
source is a 680 nm diode-pumped frequency doubled YAG microlaser
(Amoco}, which coincides with the peak of the Cy5.5 absorbance. At 680 nm,
the excitation contrast ratio for Cy5.5/Cy5 is 5. 1. A suitable excitation
filter is
the Ealing 35-4068.
c. The emission contrast ratio between Cy5.5 and Cy5 peaks at 705 nm,
approximately 3 nm to the red of the Cy5.5 intensity curve. The numerical
value for Sb at this point is 4. If the Omega 700EFLP longpass emission filter
is used, a contrast ratio of approximately 3 (averaged out to 800 nm) is
expected. This, combined with the high excitation contrast, makes imaging
Cy5.5 very clean.
d. At the expense of slightly lower emission contrast (this would not be
significant) and some loss of intensity, a bandpass filter such as the Ealing
35-
6345 could be substituted for the Omega 700EFLP. The potential advantage
would be reduction of the infra-red background . i.e., overall improved image
contrast.
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Imaging Cy7
Cy7 is the reddest dye of the set. The excitation and emission spectra are
well
separated from Cy5.5, and are well matched to the Omega
740DF25/770DRLP02/780EFLP triplet. The Oriel 58895 is an appropriate IR
blocker
for Cy7.
Filters selected for imaging the DAPI, FITC, Cy3, Cy3.5, CyS, Cy5.5, Cy7
fluor set are summarized in the Table 2 below. None of these filter sets
correspond to
the filter sets supplied by manufacturers of conventional fluorescence
microscopes as
narrow band excitation and fluorescence detection is mandatory to achieve
sufficient
contrast.
Table
2
Epicube Filter
Configuration
Fluor (for 75 W Xe
Arc Source)
Excitation Dichroic Emission IR Blocking
Bandpass BeamsplitterBandpass
Filter Filter
', DAPIZeiss Zeiss Zxiss None
365 nm 395 nm >397 nm
F1TC Omeea Omega Omega BG38
455DF70 SOSDRLP02 530DF30
Cy3 Omega Omega Ealing BG38
546DF10 560DRLP02 35-3722
Cy3.5 Ealing Omega Zeiss BG38
35-3763 590DRLP02 630/30
Cy5 Omega Omega Omega Orie158893
640DF20 645DRLP02 670DF32
Cy5.5 Ealing Omega Omega Orie158893
35-4068 DRLP02 700EFLP
Cy7 Omega Omega Omega Orie158895
740DF25 770DRLP02 780EFLP
Characteristics of the microscope system are described in detail by Ballard
S.G.
et al. (J. Histochem. Cytochem. 41:1755-1759 (1993), herein incorporated by
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reference). A high pressure 75W DC xenon arc (XBO) was used as an excitation
source because of its approximately constant spectral power distribution. A
~eiss
Axioskop microscope workstation equipped with a cooled CCD camera
(Photometrics
CH250) was employed. The objective lens was a 63x 1.25NA Plan Neofluar which
should be "plan" and apochromatic with a high numerical aperture. The filter
sets
selected were able to discriminate between the six fluors with a maximum
contrast
(Table 2). To minimize the crosstalk between fluorophores, the filter sets
were
selected on the basis of maximum spectral discrimination rather than maximum
photon throughput. Image exposure times were varied to adjust for photon flux
differences and flux excitation cross-sections. Narrow band excitation. and
fluorescence detection is mandatory to achieve sufficient contrast.
Appropriate
excitation and emission filter sets were used to optically discriminated these
fluors
(Table 2).
The combinatorial labeling strategy relies on accurate measurements of
IS intensity values for each fluorophore. Critical features are accurate
alignment of the
different images, correction of chromatic aberrations, and specific
quantitation of eac:1
fluorophore. Because simple manual image manipulation could not realize these
demands new software was developed in our lab. This program comprises the
following steps in sequential order: ( 1 ) correction of the geometric image
shift; (2)
calculation of a DAPI segmentation mask; (3) for each combinatorial fluor,
calculation
and subtraction of background intensity values, calculation of a threshold
value and
creation of a segmentation mask; (4) use of this segmentation mask of each
fluor to
establish a "Boolean" signature of each probe; (5) for each chromosome,
display of the
chromosome material next to the DAPI image; (6) create a composite gray value
image, where each labeled object is encoded with a unique gray value; (7)
final
presentation of the results using a look-up-table (LUT) that assigns each gray
value a
particular color
The above-described program was developed on the basis of an image analysis
package (BDS-Image) implemented on a Macintosh Quadra 900. Image shifts caused
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by optical and mechanical imperfections were corrected by the alignment of the
gravity center (center of mass) of a single chromosome in each image according
to a
procedure described by Waggoner, A. et al. (Methods Cell Biol 30:449-478 (
1989))
and modified (du Manoir, S. et al., Cytometry 9:4-9 ( 1995); du Manoir, S. et
al.,
Cytometry 9:21-49 (1995), all herein incorporated by reference). The DAPI
image was
used to define the morphological boundary of each chromosome. Accurate
chromosome segmentation was achieved by pre-filtering the images through a top-
hat
filter (Smith, T.G., et al., J. Neurosci Methods 26:75-81 (1988); modified in
du
Manoir, S. et al., Cytometry 9:4-9 (1995); du Manoir, S. et al., Cytometn~
9:21-49
(1995)). The mode of the gray level histogram of the top-hat filtered DAPI
image was
used as the threshold. For each fluor background was eliminated by subtracting
the
mean interchromosomal fluorescence intensity from the image. The mean of the
chromosomal fluorescence intensities was used to calculate the threshold for
the
individual segmentation mask of each fluor. Individual DNA targets were
assigned
distinct gray values depending on the "Boolean" signature of each probe, i.e.
combination of fluors used to label this DNA probe. In a final step a look-up
table
was used to assign each DNA target a pseudocolor depending on this gray value,
for
display.
Switching the filter sets in excitation and emission path as well as the
dichroic
mirror was done manually, but a computer-controlled electro-mechanical
solution will
allow automation of the procedure.
3. Optical Combs
In an alternative embodiment, selective excitation of multiple fluors and
analysis of fluorescence spectral signatures can be carried out using
dispersion optics
rather than wavelength-selective transmission filters. Such optics may be used
to
create filters of any passband characteristic, including short-pass, long-
pass, single
bandpass and multiple bandpass functions. In this method, a dispersion element
(prism or grating) is used in conjunction with a wavelength-selective spatial
filter to
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create the desired spectral response. The combination is referred to herein as
a "comb
filter." Using a comb filter, the spectral distribution of the exciting light
may be
tailored for optimum simultaneous excitation of multiple fluors. The inverse
comb
filter may also be used to selectively block from the CCD camera only the
wavelengths
used for excitation: the remaining wavelength intervals (corresponding to the
gaps
between teeth of the comb) are available for spectral analysis of the
fluorescence
emitted by the fluors. This analysis constitutes the spectral signature.
4. Interferometers
In lieu cf using the optical filters described above, an interferometer may be
used in conjunction with an epi-fluorescent microscope. A light source for
excitation
of fluorescence that is either coherent {e.g. an Argon laser) or incoherent
(e.g. a
Mercury arc lamp) may be used. A Mercury-Xenon mixed gas arc lamp is preferred
due to its intense Mercury lines and broad Xenon visible and near-infrared
continuum.
Although any of a variety of interferometer designs (such as Michelson
interferometer) may be employed, the use of a Sagnac interferometer is
preferred. The
Sagnac interferometer has a larger acceptance angle, greater entendue, and is
less
sensitive to alignment, vibration, and temperature variations than a similar
Michelson
interferometer.
The Sagnac interferometer is a common path interferometer. An
interferometer consists of two or more interfering beams of light. In a common
path
interferometer there are two beams each traveling the same path but in
opposite
directions. The optical paths are produced by reflecting light through
beamsplitters,
for example.
Multiple beam interferometers operate by dividing the optical energy from a
light source into two substantially equal beams of light. The two beams of
light are
combined after one is permitted to pass through a sample and the interference
pattern
(the changes in intensity of the combined light caused by the interference of
two
beams) is detected.
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In the Sagnac interferometer, the light source is also divided into two
substantially equal harts. Changing the angle of incidence of light on the
beamsplitter,
(by rotation of the interferometer, or rotation of an optic, such as a
galvanometer
driven mirror within the interferometer) causes the optical path length to be
changed
along one optical axis of the interferometer. This produces a fringe pattern
along one
axis of the detector, for example a CCD detector. The other axis of the
detector can
sample gray scale. As the optical path length is scanned, by rotating the
interferometer
or a mirror, the fringe pattern produces an interferogram at each pixel. The
Fourier
Transform of this interferogram yields the spectrum of light falling on that
pixel of the
CCD. Thus, an advantage of the Sagnac interferometer is that it produces an
optical
path difference across an entire field of view, rather than at a single point.
Disturbances, such as a small shift of one of the optical elements, effect
both
beams in the same way, and hence have no effect on the measurement. This
mechanical stability also makes the interferometer relatively insensitive to
temperature
1 ~ changes as well. Thus, another advantage of the common path interferometer
is its
intrinsic stability. Sagnac interferometers and their use are well known (see,
fc.r
example, U.S. Patent Nos. 3,924,952; 4,410,275; 4,529,312; 4,637,722;
4,671,658;
4,687,330; 4,836,676 and 5,108,183).
In one implementation of a Sagnac interferometer (J. Bruce Rafert et al.,
"Monolithic Fourier-Transform imaging spectrometer", Applied Optics, November
1995), the acceptance angle of the interferometer is determined to be:
q = 2 n tan-1(w/8a)
where w/a = tan 30°, w is the aperture width of the interferometer, a
is the length of
each leg, and n is the index of refraction of the interferometer glass. The
input beam
to the interferometer need not be collimated.
The interference pattern or interferogram is most preferably detected with a
CCD camera (such as a Princeton Instruments frame transfer CCD camera) capable
of
512 X 512 pixels or larger. Since the interferogram in a Sagnac interferometer
has an
angular dependence, each pixel of the CCD detector measures a small interval
of the
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interferogram. The fringe spacing of the interferogram is set such that a
pixel on the
CCD detector can adequately sample the interferobaram. The Optical Path
Difference
(OPD) that a pixel can span, in order to properly sample the interferogram is
given by
the relation:
OPDpixel = ~min~4
where din is the shortest wavelength in the spectrum to be measured by the
interferometer. This OPDpixeI determines the theoretical limit of the
resolving power
of the interferometer.
As the OPD is being changed by the rotating mirror, the interferogram is being
moved across the CCD detector, such that the maximum optical path difference
is then
given by the relation,
OPDmax = N(OPDpixel)
where N is the linear dimension of the CCI~ detector in pixels. Each angular
displacement of the light incident on the interferometer beamsplitter may then
correspond to one or several OPDpixel's~ And, in the case of a CCD detector,
one
frame of CCD data is required to sample this angular displacement.
Finally then, each pixel comprises an interferogram which contains within it
information about the spectrum of light falling on the pixel, the intensity of
light
falling on that pixel, and the x and y coordinates of the pixel. The spectrum
of light
may be recovered from the interferogram by the use of a computational Fourier
Transform algorithm.
In practice, because of the limited dynamic range of CCD's, typically about
10,000:1, the light used to excite the fluorescence must be blocked from
entering the
interferometer. This excitation light is often 108 to 102 more intense than
the
fluorescence that is emitted from the sample. Without blocking by using
optical
filtering, this excitation light would saturate the CCD. However, this filter
need only
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be fabricated so as to block the excitation, all other wavelengths may be
allowed to
pass.
In one embodiment, ultra violet (UV) Iight is used to excite the fluorescent
probes. The UV light may be easily blocked with a long pass interference
filter
allowing the visible and near-infrared colors to pass through to the
interferometer.
This embodiment has the advantage that UV will excite many of the fluorescent
dyes
currently in use. This embodiment also has the advantage that it will allow
better than
90% transmittance of the visible fluorescence to the interferometer. The
disadvantage
of UV is that it photobleaches the dyes faster than visible light.
Both the input and the output lens of the interferometer are preferably very
high efficiency camera lenses, and do not significantly effect the efficiency
of imaging.
The focus of the image within the interferometer is most preferably adjusted
so as to
be constant for the variable powers of the zoom eyepiece, and thus a
microscope
having the characteristic of infinite image distance (such as Olympus AX70
microscope) are preferred.
The above-described interferometer possesses certain advantages over optical
filters. One key advantage is that all the light emitted by fluorescence is
theoretically
available for detection, whereas the transmittance of an interference f Iter
is limited.
Another advantage is that since the filters do not have to be changed, there
is no image
shift due to the non-parallelism of filters.
E. Uses of the Present Invention
The capacity of the FISH (Fluorescent In situ Hybridization) methods and
reagents of the present invention to detect and analyze chromosomal
abnormalities,
such as translocations, inversions, duplications, etc. can be used for a large
number of
applications.
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1. Applications Relating to the Cytogenetic Diagnosis of
Genetic Disease
Among the primary applications of the present invention is the cvtogenetic
diagnosis of genetic disease, such as the pre- or past-natal diagnosis of
disease,
complex tumor karyotyping, the analysis of cryptic transiocations (especailly
through
the use of telomere-specific probes). It provides a novel method for automated
chromosome identification and analysis. A large number of diseases (prenatal
disease,
cancers (especially BRCA1 or BRCA2 associated breast cancer), leukemias,
Down's
Syndrome, etc.) are characterized by rearrangements and other chromosomal
abnormalities that can be discerned using the methods of the invention.
Chromosome karyotyping by conventional cytogenetic banding methods is
both time consuming, expensive and not easily automated. The detection of
recurring
genetic changes in solid tumor tissues by karyotyping are particularly
problematic:
because of the difficulty in routinely preparing metaphase spreads of
sufficient quality
and quantity and the complex nature of many of the chromosomal changes, which
make marker chromosome identification based solely on banding patterns
extremely
difficult. Indeed, attempts to automate karyotype analysis over the past
twenty years
(e.g., pattern matching, eigen analysis) have failed because robust computer
algorithms
could not be developed to reliably decipher complex banding patterns,
particularly
those of extensively rearranged chromosomes.
It has been proposed that the next generation of cytogenetic techniques would
be far superior by using bands that are defined molecularly by hybridization
of probes
or probe sets each labeled with a different color (Nederlof, P.M. et al.,
Cytometry
11:126-131 ( 1990); Nederlof, P.M. et al., Cytometry 13:839-845 ( 1992);
lxngauer, C.
et al., Hum Mol Genet 2:505-512 ( 1993)). This would provide a high
versatility and
would constitute a quantum leap well comparable to the introduction of
chromosome
banding and high resolution analysis of chromosomes in prometaphase.
Advantages
of the FISH karyotype are the instant identification of the chromosomal origin
of
marker chromosomes, double-minutes and homology staining regions ("HSRs")
.,
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Even "poor quality" chromosome spreads can be evaluated. If desired, one could
design a probe set for particular applications or for particular clinical
applications. e.g.
hematologic diseases, pre- or postnatal diagnosis. The development of specif c
probe
sets that stain particular regions of chromosomes (e.g. telomeric regions) for
the
S identification of cryptic translocations would overcome limitations of the
whole
chromosome painting probes. Similarly, such probes could be used to generate
multicolor "barcodes" on individual chromosomes thereby facilitating the
automated
analysis of karyotype. Probes can also be designed that would be specific for
a
particular arm of a chromosome, thereby permitting a molecular
characterization of
translocation breakpoints, hot spots of recombination, etc. Other applications
would
include rapid evolutionary studies, provided that the protocols for multicolor
FISH on
human chromosomes can be adjusted, as expected, for applications on other
species.
2. Applications Relating to the Detection of Infectious Agents
The methods of the invention may also be used to assess the presence or
absence of infectious agents (treponema pallidum, rickettsia, borrelia,
hepatitis virus,
HN, influenza virus, herpes, Group B streptococcus, diarrhea-causing agents,
pathogens causing acute meningitis, etc.) in tissue, or in blood or blood
products. This
can be accomplished by employing labeled probes specific for such agents.
Moreover,
by employing serotype-specific probes, the methods of the present invention
permit the
rapid serotyping of such agents, or the determination of whether any such
agents carry
drug resistance determinants. The methods of the present invention may be used
to
assess chromosomal abnormalities caused by exposure to radiation (such as
personnel
exposed to the radioactivity of nuclear power plants).
The methods of the present invention may be used to quantitate
microorganisms that are difficult to propagate (such as anaerobic
microorganisms
involved in periodontal disease). The methods of the present invention provide
a
means for the rapid diagnosis of acute bacterial meningitis. Just as one can
employ
serotype-specific probes to perform serological analysis, one can employ
probes that
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are specific to particular drug resistance determinants, and thereby rapidly
determine
not only the presence and identity of an infectious agent, but also its
susceptibility or
resistance to particular antibiotics.
3. Additional Applications
The methods of the present invention further permit simultaneous mapping of a
large number of different DNA probes. With this technique the analysis of
chromosomal number and architecture in individual intact cells becomes
accessible.
Interphase cytogenetics is already possible with small region specific probes,
e.g.
YAC-clones. The accuracy of such analysis could be increased by a th.:ee
dimensional
analysis using a laser scanning microscope, or more preferably, a CCD camera
system
with a Z-axis stepping motor coupled with 3-dimensional (3-D) image
deconvolution
software. Suitable 3-D image deconvoiution software is obtainable from Imstar
Corp.
(Paris, France) or Scanalytics, Inc. (Boston). In addition, the use of such
scanning
microscope systems would ultimately allow one to visualize all whole-
chromosome
painting probes, and telomere- or centromere-specific probes sets, in
interphase nuclei
and questions relating to intranuclear chromosomal organization as a function
of
developmental status, cell cycle or disease state could be addressed.
Different models
about the chromosomal organization in interphase nuclei could finally be
explored.
Although conventional laser scanning microscopes currently do not allow the
excitation of some of the fluorophores used, other, more appropriate fluors or
devices
may be employed including 3 dimensional deconvoluting imaging systems..
Extended to non-mitotic cells, the methods of the present invention enable one
to examine chromosome architecture or quantitate the chromosome contents of
nuclei
in single hybridization experiments. Questions relating to intranuclear
chromosomal
organization as a function of developmental status, cell cycle or disease
state can
accordingly be addressed. In addition, the ability to quantitatively assess
the levels of
multiple mRNAs or proteins in a single cell or to determine if they exhibit
different
intracellular distributions could prove extremely useful in addressing a
myriad of
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interesting biological questions. The multiparametric imaging of the present
invention
does not merely increases the throughput of information, it also makes more
efficient
use of the biological material. Thus, it can reveal spatial and temporal
correlations as
well as mosaicisms that might otherwise be difficult to establish reliably.
Since the
intracellular distribution of mRNAs and protein is not known a priori to be
spatially
distinct, as in the case for the infra-nuclear chromosome domains, it will not
be
possible to use the combinatorial labeling strategies in these experiments.
However,
many mRNA and protein antigens can be spectrally resolved and detected using a
multiplex format with n fluors. Thus, for the first time the intracellular
distribution of
oncoproteins or tumor suppressor proteins can be determined within the same
cell
simultaneously.
F. Automated Karyotypic Analyses
One aspect of the present invention relates to automated, preferably computer-
facilitated, karyotypic analyses. As described above, in one embodiment of the
invention, the chromosomes of a particular karyotype are pseudo-colored to
thereby
facilitate the assignment of the chromosomes, or the recognition of
translocations,
deletions, etc. In one sub-embodiment thereof, the digitized images of the
chromosomes may be stored in a computer-readable storage device (such as a
magnetic or optical disk) to facilitate their comparison with other
chromosomal images
or their transmission and study. In this regard, probes may be employed that
are
translocation specific or specific to sub-chromosomal elements or regions,
such that
the pseudocolorized chromosomes or chromosomal elements are displayed to the
investigator as chromosomal images depicting a cytogenetic banding pattern
(for
example, the cytogenetic banding pattern of the metaphase chromosomes of the
patient). The position and sizes of individual bands is preferably digitized
and stored
so that an image of the chromosome may be stored on a computer. Similarly, the
precise position of any translocation or other karyotypic abnormality can be
discerned
and stored.
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The methods of the present invention thus permit karyotypic analyses to be
conducted more widely and more accurately than was previously feasible. The
present
invention may thus be used to systematically correlate karyotypic
abnormalities with
disease or conditions. For example, karyotypes of asymptomatic individuals can
be
obtained and evaluated in light of any subsequent illness (e.g., cancer,
Alzheimer's
disease, etc.) or condition (e.g., hypertension, atherosclerosis, etc.) in
order to permit a
correlation to be made between a patient's karyotype and his or her
predisposition to
different diseases and conditions. Similarly, karyotypes of individuals having
diagnosed diseases or conditions can be obtained and evaluated in light of the
extent of
any subsequent progression or remission of the disease or condition so as to
permit a
correlation to be made between a particular karyotype and the future course of
a
disease or condition.
In a further sub-embodiment, a computer or other digital signal analyzer may
be employed to orient and arrange the chromosomal images as well as assigning
and
identifying the chromosomes of the karyotype. Thus, a computer or other data
processor will, upon assigning a particular chromosome to a particular
designation (for
example, upon assigning that a particular chromosomal image is the image of
the
chromosome 7 of the karyotype being evaluated), group the assigned chromosome
with its homologue (e.g., the second chromosome 7 of the patient's karyotype)
and
generate, via a printer, monitor, or other output means, an ordered array of
chromosomal images (such as one in which each autosomal chromosome is paired
with its homologue, and in which the sex chromosomes X and Y are paired
together).
In one sub-embodiment, the chromosomal images of such arrays will be the
pseudocolorized images discussed above. Alternatively, such psudocoloring may
be
internal to the process of assigning chromosomal identity, and not displayed
in the
output of the computer or digital signal analyzer. Rather, in this sub-
embodiment, the
output generated will be the light-microscope visible cytogenetic banding
pattern of
the metaphase chromosomes of the patient whose karyotype is being evaluated.
In a
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further sub-embodiment, a scale (in Morgans or other suitable units) will be
superimposed upon the chromosomal images.
Having now generally described the invention, the same will be more readily
understood through reference to the following examples which are prow ided by
way of
illustration, and are not intended to be limiting of the present invention,
unless
specified.
Example 1
Combinatorial Labeling of Chromosomes
1~ In order to test the feasibility to produce 24 colors chromosome painting
probes
representing the 22 autosomes and the two sex chromosomes were used. The DNA
probes used were generated by microdissection.
Microdissected probes (National Center for Human Genome Research,
Bethesda, MD) give a very uniform labeling of the target region. The detailed
protocols for microdissection and PCR amplification are described by Telenius
et al.
(Telenius, H. et al., Genes, Chromosomes & Cancer 4:257-263 ( 1992); Telenius,
H. er
al., Genornics 1.x:718-725 ( 1992); Meltzer, P.S. et al., Nature Genetics 1:24-
28
( 1992); Guan, X.Y. et al., Hum Mol Genet 2:1117-1121 ( 1993); Guan, X.Y. et
al.,
Genomics 22:101-107 ( 1994); Guan, X.Y. et al., Hum Genetics 95:637-640 (
1995), all
herein incorporated by reference). For some chromosomes different DNA-probes
for
the p- and the q-arms were available, namely 2, 4, 5, 10, 11, 16, 18, and Y.
For all
other chromosomes microdissected probes painting the entire chromosome were
used.
The first member of the set of preferred fluors, DAPI, was used as a general
DNA counterstain. The remaining fluors: fluorescein, Cy3, Cy3.5 (emission and
excitation spectra are between Cy3 and Cy5), Cy5 (Mujumdar, R.B. et al.,
Cytometry
10: 11-19 ( 1989), and Cy7 (emitting to the red of Cy5 (Ernst, L.A., et al.,
Cytometry
10:3-10 ( 1989)), were used to combinatorially label different probes (Table
3).
Distinctive features of these dyes are high extinction coefficients, quantum
yields, and
photostabilities. Fluorescein is a xanthene dye with an extinction coefficient
around
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70,000 Lmol cm and quantum yields in optimal buffers around 0.7. The
respective
values for cyanines are 1-200,000 and 0.3 (Waggoner, A., Methods in Enzvmology
24b:362-373 ( 1995)).
After microdisection, the probes were subjected to a PCR amplification and
labeled by nick translation. Fluorescein (Wiegant, J. et al., Nuc Acids Res
19:3237
3241 ( 1991 )), Cy3, and Cy5 were directly Linked to DIJTP for direct
labeling. Cy3.5
and Cy7 were available as avidin or anti-digoxin conjugates for secondary
detection of
biotinylated or digoxinigated probes. They were synthesized using conventional
N
succinamide ester coupling chemistry. For each probe one to three separate
nick
translation reactions were necessary, each with a single labeled fluor-labeled
triphosphate or biotin or digoxigenin (Table 3).
Table
3
Fluor Chromosome
1
2
3
4
5
6
7
8
9
10
11
12
F1TC X X X X
C 3 X X X X X
C 3.5 X X X X X
C 5 X X X X
C 7 X X X X
Fluor Chromosome
14
13
15
16
17
18
192021
22
X
Y
FTTC _ _ X X X X X
X X
C 3 X X X X X
C 3.5 X X X X X X
C 5 X X X X X X
C 7 X X X X X X
As expected probes labeled with equal amounts of different fluors did not give
equivalent signal intensities for each fluor reflecting the fact that the
filter sets were
selected to maximize spectral discrimination rather than photon throughput. In
order
to diminish signal intensity -differentials, probe concentrations for the
hybridization
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mix had to be established carefully in a large number of control experiments.
Hybridization conditions were optimized for these multiplex probes. Thus,
probes
were denatured and hybridized for two to three nights at 37 °C to
metaphase
chromosome spreads in a conventional 50% formamide hybridization cocktail. The
slides were washed at 45 °C in 50% formamide/2 x SSC three times
followed by three
washes at 60 °C in 0. 1 x SSC to remove excess probe. After a blocking
step in 4 x
SSC/3% bovine serum albumin for 30 min at 37 °C the biotinylated
probes were
detected with avidin Cy3.5 and the dig-labeled probes with anti-dig-Cy7.
Fluorescein-
dUTP, Cy3-dUT'P, and Cy5-dUTP did not require any immunological detection
step.
After final washes at 45 °C with 4 x SSC/0.1% Tween 20 three times,
moernting
medium and a coverslip were applied and the hybridization signals from each
floor
imaged using the filters sets listed in Table 3.
Figure 1 provides a schematic illustration of the CCD camera and microscope
employed in accordance with the present methods.
Figure 2 shows the raw data from a karyotypic analysis of chromosomes from a
bone marrow patient (BM2486). Adjacent to each source image is a chromosome
"mask" generated by the software program. In Figure 2, panels A and B are the
DAPI
image and mask; panels C and D are FTTC image and mask; panels E and F are Cy3
image and mask; panels G and H are Cy3.5 image and mask; panels I and J are
Cy5
image and mask; and panels K and L are Cy7 image and mask.
Figures 3A and 3B show the identification of individual chromosomes by
spectral signature. Figure 3A is the same photograph as Figure 2, except that
it is gray
scale pseudocolored. Figure 3B displays the karyotypic array of the
chromosomes.
The exceptional power of the methods of the present invention are illustrated
by the
ease with which the translocation of chromosomes 5 and 8 are identified in
Figures 3A
and 3B, relative to conventional non-chromosome specific karyotype analysis.
The above experiment demonstrates that five floors can be spectrally
discriminated to produce at least twenty four different colors. The
combinatorial
labeling schemes need not be as complex as previously thought, because using S
floors
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for probe labeling, only 9 painting probes need to be labeled with as many as
three
fluors. A sixth fluor for probe labeling would allow up to 63 possible fluor
combinations. Such a high number of different targets will not be required for
most
applications, but would allow the selection of combinations with the best
spectral
signature. These above-described protocols allow highly reliable and
reproducible
multicolor FISH.
Example 2
Rapid Analysis of Multiple Oral Bacteria
in Mixed Cell Populations Using Multiparametric Fluorescence In situ
Hybridization (M-FISH)
In situ hybridization has been recognized as having potential application as a
means of analyzing individual bacterial cells in the absence of culture.
DeLong, E.F.
et al. (Science 243:1360-1363 (1989)), for example, used oiigonucleotide
probes
complementary to the 16s ribosomal RNA (rRNA) sequences to differentiate an
archaebacterium (Metharcosarcina acetivorans) from the eubacteria Bacillus
megaterium and Proteus vulgaris. Van Den Berg, F.M. et al. (J. Clin. Pathol.
42:995-
1000 ( 1989)) used total genomic DNA to detect Campylobacter pylori in stomacn
tissue. Amann, R. et al. (Nature 351:161-164 (I991)) detected Holospora
obtccsa in
the macronucleus of the protozoan Paramecium caudatum.
Prior to the present invention, however, the use of in situ hybridization to
differentiate microorganisms in clinical specimens with mixed populations,
especially
in a quantitative fashion, was difficult and time consuming. This is
particularly true of
bacteria with fastidious growth requirements, in which culture methods may
result in
the selective enrichment of a subset of the organisms present in the specimen.
A. Analysis of Non-Cross Hybridizing Microbial Species
To further illustrate the present invention, the M-FISH methods of the present
invention are used used to speciate bacteria involved in the microbial
etiology of
periodontal disease.
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Several methods have previously been used to establish the microbial etiology
of periodontal disease. Well over 200 bacterial species can be identified in
one
sublingual plaque sample using labor-intensive cultural methods (Dzink, J.L.
et al., J.
Clin. Periodont. 12:648-659 (1985); Moore, W.E.C.: J. Periodont. Res. 22:335-
341
(1987); Movre, W.E.C., Infect. Immunity 38:651-667 (1987)). This diversity has
hampered the association of specific microbes in the etiology of the various
forms and
stages of inflammatory periodontal diseases. Direct assessment, both
qualitatively and
quantitatively, of plaque bacteria would obviate the need for culturing and
its attendant
difficulties. DNA probes (French, C.K. et al., Oral Microbiol. Imma~nol. 2:58-
62
( 1986)) have been widely used in a blot hybridization format as a direct
method for
bacterial assessment. However, this method does not allow for the direct
visualization
of the bacterial or provide information on the relative abundance of a
particular
bacterial within a complex mixture.
In order to more simply illustrate the capacity of the invention to speciate
microbial strains, a test for in situ hybridization specificity is conducted
using a
mixture of bacteria that are morphologically distinct and non-cross
hybridizing. Using
such a mixture, one can immediately differentiate positive hybridization from
that of
non-specific binding by correlating the correct morphology with the correct
probe.
Thus, Fusobacteriunt nucleatum (25586), Porphyromonas gingivalis (33277),
Eikenella corrodens (23834), Prevotella intermedia (25611 ), and
Actinobacillus
actinomycetemcomitants (29522) were obtained from the American Type Culture
Collection. Bethesda, MD, and Capnocytophaga species, C. ochracea (C25) and C.
gingivalis (DR2001 ) were obtained from The National Institute of Dental
Research,
Bethesda, MD.
Total genomic DNAs were isolated from F. nucleatum, P. ~ingivalis, E.
corrodens, P. intermedia, A. actinomycetemcomitans, C. gingivalis, and C.
ochracea.
using the procedure of Chassy, B.M. et al., Appl. Environ Microbiol. 39:153-
158
(1980)) as modified by Donkersloot, J.A. et al., J. Bacteriol. 162:1075-1078
(I985)).
Each DNA was labeled with 32P and tested for crosshybridization with each of
the
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other DNAs by dot blot hybridization. In all cases, the total genomic DNA
probes
show specificity for the bacteria from which it was derived; no hybridization
with any
of the other bacterial DNAs was detected. Since the genomic DNA probes were
highly specific in the dot blot assays, they were used directly for the in
situ
identification of these bacteria in reconstructed mixtures.
Total genomic bacterial DNAs are labeled by nick translation using biotin-I1-
dUTP (BIO), digoxigenin-11-dUTP (DIG) or dinitrophenol-I1-dUTP (DNP) as
described by Lichter, P. et al., Hum. Genet. 80:224-234 ( 1988), herein
incorporated by
reference). Unincorporated nucleotides are removed using a Sephadex G-50 spin
column equilibrated with lOmM Tris-HCI/1 mIVI EDTA/0.1% SDS, pH 8Ø Labeled
DNAs (2-6 pg) are ethanol precipitated and redissolved in 100% deionized
formamide. F. nucleatum DNA is labeled with BIO-11-dUTP (BIO) and detected
with
Cascade Blue avidin; A. actinomycetemcomitans DNA is labeled with DNP-11-LT1'P
(DNP) and detected indirectly with an FTTC conjugated antibody; and E.
corrodens
DNA is labeled with DIG-11-dUTP (DIG) and detected with a Rhodamine labeled
anti-DIG antibody.
To prepare slides for in situ hybridization, small aliquots (1-2 ml) of
cultured
bacteria are centrifuged at 1,200 xg in an HBI microcentrifuge. The cell
pellet is
suspended in phosphate buffered saline (PBS) and adjusted to approximately 10;
bacterial per pl. Aliquots of both pure cultures and synthetic mixtures are
covalently
bound to activated glass slides prepared as described by Maples, J.A. (Amer.
J. Clin.
Pathol. 83:356-363 ( 1985), herein incorporated by reference). Alternatively,
bacterial
are spotted directly onto positively (+) charged slides and air dried.
Suitable slides are
commercially available from Fisher Scientific, Pittsburgh, PA (cat. #12-550-
15), and
retain all the bacterial strains listed above throughout the in situ
hybridization
procedure with high efficiency. All slides are then fixed in Carnoy's B
Fixative
(Ethanol: Chloroform: Acetic Acid, 6:3:1 ) for 5 minutes.
The bacteria are hybridized simultaneously with the three differentially
labeled
genomic DNAs. Each sample is hybridized using 15 pl of hybridization solution
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which contains 50 ng of labeled probe and DNAse-treated salmon sperm DNA (15
Ng)
in 50% (vol/voi) deionized formamide/2xSSC (0.3 M sodium chloride/0.03 M
sodium
citrate, pH 7.0)/5% dextran sulfate. The solution is applied to the sample,
covered
with a coverslip and sealed with rubber cement. Both bacterial DNA and labeled
DNA probe are denatured by heating at 80°C for 5-8 minutes in a oven.
These steps
are sufficient to perrneabilize the bacteria far probe assessability.
The DNAs were allowed to reassociate by incubating the slides at 37°C
for 18
hrs in a moist chamber. Posthybridization washings, blocking and detection
were as
described by Lichter et al. (7). Briefly, the slides were washed 3X in 50%
formamide/2XSSC at 42°C for 5 minutes, and then washed 3X in O.1XSSC at
60°C
for 5 minutes. The slides were then incubated in a solution of 3% bovine serum
albumin/4XSSC (blocking solution) for 30 minutes at 37°C.
Biotinylated probes were detected using fluorescein isothiocyanate-(FTTC)-
avidin DCS (Vector Laboratories, Burlingame, CA, 5 ~tg/ml) Cascade Blue-avidin
(Molecular Probes Inc., Eugene, OR, 10 p.g/ml)) or Rhodamine-avidin
(Boehringer
Mannheim, Indianapolis, IN, 10 pg/ml).). Digoxigenin-labeled probes were
detected
using FITC or Rhodamine conjugated sheep anti-digoxigenin Fab fragments
(Boehringer Mannheim, 2 ~tg/m!). Dinitrophenol labeled probe was detected by
incubating with rat anti-DNP antibodies (Novagen, Madison, WI, 1:500
dilution)) and
then with goat anti-rat FITC-conjugated antibodies (Sigma, St. Louis, MO, 1
p.g/ml).
In some experiments, bacterial DNA was counterstained with 4,6-diamidino-2-
phenylindole (DAPn at a concentration of 200 ng/ml.
For detection, the fluorochrome-conjugated antibodies or avidin are diluted
into a solution of 4xSSC/1% BSA and 0.1% tween-20 (200 p.l/slide) and
incubated
with the sample at 37°C for 30 minutes in the dark. The slides are then
washed 3
times in 4xSSC/0. I % tween-20 at 42°C prior to viewing via
epifluorescence
microscopy.
Epifluorescence microscopy is conducted using a Zeiss Axioskop-20 wide-
field microscope with a 63x NA 1.25 Plan Neofluar oil immersion objective and
a
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50W high pressure mercury arc lamp. Images are projected with a Zeiss SFL-10
photo-eyepiece onto a cooled charged-coupled device (CCD) camera (Photometrics
CH220; 512x512pixel array). Effective magnification is set by changing the
microscope-camera distance, using a bellows. The 8-bit greyscale images are
recorded
sequentially using DAPI, FTTC and Rhodamine filter sets (manufactured by C.
Zeiss,
Inc., Germany) to minimize image offsets. Camera control, greyscale image
acquisition and image pre-processing are done using an Apple Macintosh 11 x
computer preferably running custom software (Dr. Marshall Long; Yale
University).
Images are pseudocolored, aligned and merged using a Macintoch 11 ci computer
equipped with an accelerated 24-bit graphics system (SuperMac Spectrum 24
PDQ).
The merging software, "Gene Join Max Pix" (Yale University) is preferably
employed.
This software assigns a user definable pseudocoIor and greyscale value to each
of the
source images, and then generates an output ("merged") image based on the most
intense source image at each pixel location. Thus, objects with higher scaled
brightness (e.g., probe signals) override the DAPI counterstain signals in the
corresponding image location. Objects displayed in merged image retain the
color
assigned to them and therefore appear distinct and easily identifiable. Merged
images
were photographed directly from the computer monitor.
A mixed sample of F. nucleatum, E. corrodens and A. actinomycetemcomitans,
in a 1:1:1 ratio was used as a test sample. As can be seen in Figure 4, the
three
bacteria are distinguishable on the basis of morphology when stained with
DAPI.
Figure 4 shows the differentiation of bacteria by in situ hybridization.
Panels
A-E represent in situ hybridization assay results on a laboratory derived
mixture of
three bacteria (F. nucleatum, A. actinomycetemcomitans and E. corrodens) using
a
mixture of genomic DNA probes for each organism. Panel A shows all the
bacteria
present in the microscope field detected by (DAPI), a general DNA binding
fluorophore. F. nucleatum is seen using Cascade $lue detection (panel B), A.
actinomycetemcomitans with FTTC detection (panel C) and E. corrodens using
Rhodamine detection (panel D). Panel E is a computer-merged composite of
panels
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B-D showing the differentiation of each bacteria in the sample. Note that a
single A.
actinomvetemcomitans cell is detected in a clump of E. corrodens (panel E)
that could
not be identified by DAPI staining (panel A). Panel F is a similar analysis of
a mixture
of C. gingivalis (Rhodamine) and P. intermedia (FTTC) when hybridized with a
combination of genomic DNA probes for those organisms. Panel G shows an in
situ
hybridization assay for a combination of seven different bacteria hybridized
with a
mixture of seven genomic DNA probes. F. nucleatum ( 1 ), E. corrodens (2) and
A.
actinomycetemcomitans (3) are shown in blue (Cascade Blue detection). C.
gingivalis
(4) and C. ochracea (5) are shown in red (Rhodamine detection), whereas P.
intermedia (6) and C. gingivalis (7) are seen in yellow (FITC detection).
Phase
contrast microscopy was used to assist in determining morphological
differences
between E. corrodens and A. actinomycetemcomitans which were not readily
evident
through the hybridization generated signals. Panel H represents an in situ
hybridization assay for the presence of A. actinomycetemcomitans (FTTC
detection) in
a plaque sample obtained from a patient with localized juvenile periodontist.
When assaying for Cascade Blue, only the F. nucleatum bacteria (Figure 4,
panel B) were seen, when assaying for FTTC only A. actinomycetemcomitans
bacteria
(Figure 4, panel C) were visible, and when assaying for Rhodanune, only the E.
corrodens bacteria (Figure 4, panel D) were positive. The three separate
fluorophore
images were then merged to generate the composite image that is shown in
Figure 4,
panel E. Each of the morphologically distinct bacteria exhibit the correct
fluorescence
specificity.
The four additional bacterial species are included in subsequent experiments.
These bacteria are combined pairwise such that each mixture again would differ
morphologically. Figure 4, panel F shows a combined analysis of P. intermedia
(BIO
label and FTTC detection) and C. gingivalis (DIG label and Rhodamine
detection). An
analysis of C. ochracea and P. gingivalis, whose morphology is similar to the
P.
intermedia and C. ochracea and P. gingivalis, whose morphology is similar to
the P.
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intermedia and C. gingivalis pair, is also performed. In both cases, absolute
specificity
of the probe for its cognate bacteria is demonstrated.
Since the above experiments indicate that none of the bacteria cross-
hybridized
and that each species of bacteria, as grouped, could be distinguished by
morphology as
well as by hybridization via the binding of a specific fluorochrome, it was
reasoned
that all seven bacteria could be detected in a single mixed sample. The
experimental
design was to label the DNA from the bacteria within each of the three
morphologically distinct groups with the same "reporter" (BIO, DIG or DNP).
The
three groups would be initially differentiated by hybridization and the
specificity of the
fluorescence signal. The bacteria within a group could then be
different,'_ated on the
basis of morphology.
To demonstrate this capacity of the present invention, P. intermedia and C.
gingivalis DNAs were labeled with DIG, F. nucleatum, A. actinomycetemcomitans,
and E. corrodens DNAs were labeled with BIO whereas P. gingivalis and C.
ochracea
DNAs were labeled with DNP. All seven probes were then combined and used for
in
situ hybridization on mixed bacterial samples containing all organisms (Figure
~,
panel G). In Figure 4, panel G, the F. nucleatum, E. corodens, A.
actinomycetemcomitans group is shown using Cascade Blue (BIO detection) and
its
members are numbered l, 2 and 3 respectively; the P. gingivalis and C.
ochracea
group is seen with Rhodamine (DIG detection) and these bacteria are numbered 4
and
5 respectively; the G gingivalis, P. intermedia group is identified with FITC
(DNP
detection) and they are numbered 6 and 7 respectively. All seven bacteria are
clearly
differentiated using the combination of hybridization fluorescence and
morphology.
While morphological identification could be made simply on the basis of size,
shape
and color of the hybridization signal, more definitive morphological
characterization is
made by examination of the sample using phase-contrast optics.
To determine how few hybridization-positive bacteria could be detected in a
sample, 4.0 ~1 of E. corrodens containing 105 bacteria/~tl is mixed with 1.0
~tl of
serial dilutions of A. actinomycetemcomitans and the 5 ~tl mixture applied to
slides.
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The sample is then tested for the presence of A. actinomycetemcomitans using a
BIO-
labeled probe for that bacterium. Following the hybridization, the total
bacteria on the
slide are visualized using Propidium Iodide and the hybridization-positive
bacteria are
detected using Avidin-FTTC. The results of this experiment show that single A.
actinomycetemcomitans cells could be detected in the presence of an excess of
at least
I0~ E. corrodens cells. Thus, it is apparent that hybridization positive
bacteria can be
identified readily even when they constitute only a minor fraction of a mixed
cell
population.
Clinical samples are not usually as free of contaminating debris as laboratory
derived mixtures of bacteria. To test the potential utility of M-FISH for the
analysis of
clinical specimens, a dental plaque sample from a patient with localized
juvenile
periodontitis, in which prior studies (Slots, J., Dent. Res. 84:1 (1976);
Slots,1. et al., J.
Clin Periodontal. 13:570-577 ( 1986); Zambon, 3. et al., J. Periodontal.
54:707 ( 1983)
had suggested the involvement of A. actinomycetemcomitans as the microbial
pathogen is employed. Figure 4, panel H shows the results of an in situ
hybridization
experiment with this patient's sample using BIO-labeled A.
actinomycetemcomitans
DNA and detecting with FTTC-conjugated avidin. As can be seen, a large number
of
A. actinomycetemcomitans or other closely related organisms were detected.
Quantitative studies indicate that more than 50% of the individually
identifiable
bacteria seen in the specimen hybridized with the A. actinomycetemcomitans DNA
probe. Cultural studies on this sample showed the presence of A.
actinomycetemcomitans.
All of the above experiments employ a standard overnight hybridization time
(- 18 hours) to differentiate the bacteria of interest. Even though such
analyses can
often be done faster than the traditional methods of culturing and biochemical
testing,
a number of experimental parameters could be adjusted to give a more rapid
assay. It
is well known that hybridization time is directly related to probe
concentration. Thus,
by increasing probe concentration, hybridization time can be proportionately
reduced.
A mixture of F. nucleatum and A. actinomycetemcomitans is therefore analyzed
using
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a 10-fold higher concentration of probe than previously used (from 50 ng to
0.5 pg per
slide). At the higher probe concentration no specific or non-specific
hybridization is
observed with undenatured probe and bacterial DNA. However, when the probe and
bacterial DNA are denatured simultaneously, and the washing cycles begun
immediately, some of the DNAs hybridized during the time necessary for
subsequent
sample processing.
In this experiment, in which 0.5 ~,g of A. actinomycetemcomitans probe is
introduced to a sample containing both .A. actinomycetemcomitans and F.
nucleatum
bacteria using a 2.5 minute incubation at 37°C prior to initiating the
washing cycles,
the hybridization signal is as strong and as specific as that observable in
experiments
utilizing 50 ng of probe and hybridizing for 18 hours. Furthermore, by
reducing the
blocking and antibody detection times to 20 minutes each, a total assay time
of 1.5
hours is achieved. Further reduction in assay times clearly can be obtained
using
probe DNA directly labeled with the fluorophore detector.
The above experiments provide a relatively rapid and simple method for
identifying individual bacteria in mixed microbial populations using M-FISH.
The
method provides a quantitative assessment of specific microorganisms without
the
necessity of culturing. The technique can be applied to samples containing
only a
limited number of organisms and a single hybridization-positive bacteria can
be
detected quite easily in a 104- fold excess of hybridization-negative
bacteria. In
addition, as exemplified by the overlapping and "hidden" A.
actinomycetemcomitans
cell among the group of E. corrodens cells (Figure 4, panel E), bacterial
specified that
are not clearly identified by a non-specific analysis (i.e., DAPI staining of
DNA,
Figure 4, panel A) can be detected by the hybridization-specific assay. Since
both the
hybridization specificity and the morphological characteristics of bacteria
can be
assessed simultaneously by this procedure, the number of different bacterial
species
can be increased stilt further by judicious selection of probe labeling and
fluorophore
detector combinations.
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However, morphological characteristics are not always a reliable indicator of
bacterial identity since many different bacterial species appear
morphologically similar
and some are pleomorphic depending on the culturing methods. For example, A.
actinomycetemcomitans are generally coccobacillary in clinical specimens but
can
assume rod and even filamentous forms upon in vitro passage and in clinical
specimens. Thus, additional fluorescence colors would preferably be employed
to
expand the number of different bacteria that can be analyzed simultaneously
and
identified unequivocally.
The intensity of the hybridization signal is extremely strong using either
whole
genomic DNA or subtracted (suppressed) probes. Although data is collected by
digital
imaging, hybridization signals can be detected readily by eye and recorded by
conventional photographic methods. However, the sensitivity and photon
counting
capabilities of CCD-camera based imaging system offer considerable advantages
fnr
further refining microbial analysis by in situ hybridization. Detection of
single copy
sequences for specific toxin or drug resistance genes in individual bacteria
should be
feasible; individual human and murine genes have been visualized, both in
metaphase
chromosomes and interphase nuclei by FISH (Lichter, P. et al., Hum. Genet.
80:224-
234 (1988)). The level of cross-homology between bacterial strains also can be
rapidly
assessed by quantitating the photon output of the hybridization signal from
individual
bacteria in a specimen. Finally, and most importantly, digital imaging is
essential to
fully exploit the potential of combinatorial fluorescence.
As exemplified here, total genomic DNA can often provide adequately specific
probes, thus eliminating the usual cloning and screening efforts necessary for
probe
production. Although not required in these studies because of the absence of
cross
hybridization between the bacterial genomes, suppression hybridization
techniques
(Lichter, P. et al., Hum. Genet. 80:224-234 ( 1988)) can be used to squelch
cross-
hybridization between organisms that share partial sequence homology. For
example,
C. orchracea and C. sputigena exhibit 22-23% sequence homology (Zambon, J. et
al.,
J. Periodontal. 54:707 ( 1983)) yet specific hybridization to C. orchracea can
be
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obtained with genomic DNA by preannealing the C. ochracea probe with an excess
of
C. sputigena DNA just prior to the in situ hybridization reaction.
In sum, total genomic DNAs from seven anaerobic and facultative oral bacteria
recognized as pathogens in periodontal disease {Fusobacterium nucleatum,
Porphyromonas (Bacteroides) gingivalis, Prevotella intermedia, Eikenella
corrodens,
Actinobacillus actinomycetemcomitans, Capnocytophaga gingivalis and
Capnocytophaga ochracea) are labeled non-isotopically and hybridized in situ
to
reconstructed bacterial mixtures. Each probe is hybridized uniquely to the
bacterium
from which it was derived. Three bacterial strains are differentiated
simultaneously by
labeling DNA with different reporter groups and visualizing the hybridization
signal
with reporter-specific detector proteins labeled with different fluorophores.
By
assessing bacterial morphology in conjunction with the hybridization signal
each
bacteria in a mixture of all seven organisms can be identified. The total
assay time is
as short as 1.5 hours. A dental plaque sample from a patient with localized
juvenile
periodontitis, an oral mixed microbial infection, reveals numerous
hybridization
positive bacteria when probed with DNA from A. actinomycetemcomitans, the
putative
microbial pathogen for this disorder. This example demonstrates the usefulness
of the
in situ hybridization methods of the present invention for the identification
of
individual non-cross hybridizing bacteria in mixed cell populations.
The seven bacterial species analyzed above are facultative or obligate
anaerobes that are often found in association with inflammatory periodontal
disease.
The role of these microorganisms is uncertain due to the lack of rapid and
reliable
methods to identify specifies. Direct analysis of oral microbial specimens
circumvents
biases imposed by culture methods and may allow a better understanding of the
role of
these bacteria in the pathogenisis of periodontal disease. Data on the
detection of A.
actinomycetemcomitans in a specimen from a patient with localized juvenile
periodontitis indicates that the above-described in situ hybridization
procedure
facilitates such studies. It also complements traditional culture methods for
the
identification of a broad spectrum of microorganisms, especially notoriously
slow
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growing bacterial such as anaerobes or mycobacterium. For example, swabs taken
from early culture plates may be streaked on a slide and assayed by in situ
hybridization using probes for suspected pathogens of interest.
The present invention permits one to increase further the number of
simultaneously detectable bacteria without increasing the number of available
fluorophores by using combinatorial fluorescence imaging (Nederlof, P.M. et
al.,
Cytometry 11:126-131 ( 1990)). By labeling a single probe with two (or more)
different reporter molecules, the resultant hybridized probe will be detected
by more
than one of the fluorescent detectors and the signal will appear in more than
one of the
separate flurorescence images. When these separate pseudocolored images are
merged
into a composite image (as in Figure 4 panels E, F and G), fluorescence
signals
appearing at the same site on two images will be "blended" and generate a new
pseudocolar that is distinguishable from either of the originals. In this
fashion, three
fluorophores have been used combinationally to visualize seven probes
simultaneously, each appearing as a distinct color (Ried, T. et al., Proc Natl
Acad Sci
(USA) 89:1388-1392 (1992)). By applying this combinatorial fluorescence
imaging
strategy to bacterial identification, one can obtain a distinct pseudocolor
for each of the
seven bacterial species analyzed above.
The above example demonstrates the feasibility of differentiating multiple
bacterial species (such as those found in the periodontal microflora) using
multiparametric fluorescence in situ hybridization (M-FISH) and digital
imaging
microscopy. By monitoring both hybridization signal specificity and bacterial
morphology, seven different bacteria are readily and simultaneously
distinguished in a
mixed population using only three distinct fluorophores. Such methods are
readily
applicable to the detection of bacteria (as well as viruses, and/or lower
eukaryotes) that
may be present in other clinical samples (in eluding those that contain
complex
mixtures of microflora (e.g., stool, saliva, throat swabs, sputum, vaginal
secretions,
etc.) and those that are normally (e.g., central spinal fluid (meningitis),
blood (sepsis),
etc.) or non-clinical samples (food products, water reservoirs, ecosytems,
etc.).
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Furthermore, by expanding the number of spectrally resolvable fluors used in
combinatorial fashion, the number of different types of bacteria, viruses
and/or lower
eukaryotes that can be analyzed in a mixed population can be markedly
increased.
B. Analysis of Cross Hybridizing Microbial Species
As indicated above, using differentially labeled total genomic DNA probes, the
in situ hybridization and digital imaging fluorescence microscopy methods of
the
present invention can simultaneously differentiate seven microorganisms in a
laboratory derived mixed sample. This technique provides both single cell
detection
sensitivity and the ability to correlate hybridization signal specificity with
microbial
morphology. The relative abundance of a specific organism in a complex mixture
or
bacteria, can be readily assessed and a single hybridization positive bacteria
can be
detected in an excess of 104 other bacteria. Moreover, the assays can be
completed in
less than 1.5 hours. However, none of the genomic DNAs from the seven bacteria
used in the above study exhibited cross-hybridization.
To illustrate the capacity of the present invention to differentiate among
related
(i.e., cross-hybridizing) microflora, a study is performed using
Capnocytophaga DF 1
strains: C. gingivalis (DR2001 ), C. sputigena (D4), and C. ochracea (C25)
Such
strains exhibit cross-hybridization (Rubin, S.1., Eur. J. Clin. Microbiol.
3:253-257
( 1984)). The strains are obtained from the N1H Institute of Dental Research,
Bethesda,
MD. Bacterial DNA is isolated as described above. Probes are made as follows:
total
genomic bacterial DNAs is labeled by nick translation using biotin-11-dUTP
(BIO),
digoxigenin-11-dUTP (DIG) or dinitrophenol-11-dUTP (DNP) as described by
Lichter, P. et al. (Hum. Genet. 80:224-234 ( 1988)). Unincorporated
nucleotides are
removed using a Sephadex G-50 spin column equilibrated with 10 mM Tris-HCl/1
mM EDTA/0.1% SDS, pH 8Ø Labeled DNAs (2-6 Irg) are ethanol precipitated and
redesolved in 100% deionized formamide.
The genus Capnocytophaga encompasses a group of fusiform, gram-negative,
capnophilic, fermatative, gliding bacteria (Ledbetter, E.R. et al., Arch.
Microbiol.
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122:17-27 ( 1979)) that are common inhabitants of the oropharyngeal flora
(Holdeman,
L.V. et al., J. Periodon. Res. 20:475-483 (1985)). C. gingivalis. C ochracea
and C.
sputigena have been implicated in gingivitis in children (Moore, W.E.C. et
al., Infect.
Immun. 46:1-6 (1984)) and periodontitis in juvenile diabetics and adults
(Dzink, J.L et
al., J. Clin. Periodont. 12:648-659 (1985); Mashimo, P.A. et al., J.
Periodont. 54:420-
430 ( 1983); Slots, J. et al., J. Dent. Res. 63:414-421 ( 1984)). These three
bacteria are w
members of the first of three groups of "Dysgonic Fermenters" (DF-1 ) defined
by the
Center for Disease Control (Speck, H. et al., Zbl. Bakt. Hyg. A. 266:390-402 (
1987)).
The DF-2 group contain species designated as C. canimorsus and C. cynodeami,
although these bacteria appear both phenotypically and genetically distinct
from the
Capnocytophaga species of the DF-1 group (Brenner, D.J. et al., J. Clin.
Microbiol.
27:231-235 ( 1989)). Few of the microbes comprising the DF-3 group have been
characterized, however it is thought that they are closely related to the
Capr~.ocytophaga DF-1 species (Gill, V.J. et al., J. Clin. Microbiol. 29:1589-
1592
( 1991 )). Members from all three DF groups have been identified as the
etiologic
agents found in some cases of septicemia, joint infections and endocarditis
(Arlet, G.
et al., Ann. Biol. Clin. 44: 373-379. ( 1986); Juhl, G. et al., J. Infect.
Dis. 149:654
(1984); Mallow, A. et al., J. Infect. Dis. 152:223-234 (1985); Mosher, C.B. et
al., J.
Clin. Microb. 24:161-162 ( 1986); Parenti, D.M. et al., J. Infect. Dis.
151:140-147
( 1985); Ratner. H., Clin Microbiol 6:10 ( 1984); Rummens, J.L. et al., J.
Clin.
Microbiol. 75:376 (i985); von Graevenitz, A., Eur. J. Clin. Microbiol. 3:223-
224
( 1984)). The increased spectrum of oral and infra-oral infections caused by
Capnocytophaga DF-1 species shows the pathogenic potential of this genus and
may
reflect differences in clinical significance of the respective species in
medically
important infections and periodontal disease (von Graevenitz, A., Eur. J.
Clin.
Microbiol. 3:223-224 ( 1984)}. Conventional methods of identifying bacteria
from oral
sources requires first, isolation of single bacterial colonies from the oral
microbial
milieu, and second, the assignment of isolates to genus and species based upon
cellular
morphology, gram stain, fermentation of carbohydrates and hydrolysis of
various
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substrates. These procedures require considerable time and expertise, taking
upwards
of 2 weeks to identify single species from clinical samples. Such conventional
methods often fail to isolate Capnocytophaga from oral samples because these
bacteria
are slow growing, fastidious as to growth conditions and are therefore
overgrown by
other oral flora (Mashimo, P.A. et al., J. Periodont. 54:420-430 (1983);
Rummens,
J.L. et al., J. Clin. Microbiol. i 5:3 76 ( 1985)). Moreover, such methods may
fail to
discriminate C ochracea and C. sputigena unambiguously due to phenotypic
similarities (Gill, V.J. et al., J. Clin. Microbiol. 29:1589-1592 (1991)).
Recent methods of microbial identification using enzymatic tests (Heltsberg,
O. et al., Eur. J. Clin. Microbiol. 3:236-240 ( 1984); Kristiansen, J.E. et
al., F,ur. J.
Clin. Microbiol.3:236-240 ( 1984)), specific immunologic or molecular probes
(Murray, P.A. et al., Oral Microbiol. Immunol. 6:34-40 ( 1991 ); Smith, G.L.F.
et al.,
Oral Microbiol. Immunol. 4:41-46 (1989)) have been used with some success for
identifying bacteria in clinical oral samples. With the exception of enzymatic
techniques, these methods have not been applied to the identification of
Capnocytophaga spp. Enzymatic tests still require the isolation of pure
colonies cf
bacteria, whereas immunologic and molecular methods can be performed directly
on
an oral sample, thereby overcoming the difficulties of isolating pure colonies
of
fastidious microorganisms. Generally, molecular methods rely on the use of DNA
probes, formatted as a dot-blot hybridization assay, for the identification of
a particular
microorganism in a clinical sample (Parenti, D.M. et al., J. Infect. Dis.
151:140-147
( 1985)). A major drawback of this method is that DNA from 10'~ to 106
microorganisms of interest are required for such assays, therefore bacteria in
low
abundance in a mixed population are likely not to be detected. In addition,
cross-
hybridization, which is common in closely related species, can impede
identification to
the species level.
It had been previously reported (Williams, B.L. et al., Arch. Microbiol.
122:35-
39 ( 1979)) that C. gingivalis, C. ochracea, and C., sputigena DNAs exhibit up
to 22%
sequence homology, based on reassociation kinetics. Using conventional in situ
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hybridization methods, such extensive homology would appear to preclude the
use of
total genome DNA probes for the speciation of Capnocytophaga strains and
necessitate the production and characterization of species-specific subgenomic
clones.
However, one of the attributes of the computerized digital imaging camera
system of
the present invention is the ability to collect hybridization data in the form
of
quantitative fluorescence images and to process such image data with
appropriate
computer software.
The intensity of the hybridization signal (i.e., fluorescence photon output)
from
a bacteria ("bacteria A") A will be greatest with a genomic DNA derived from
that
bacteria. Related strains which share some DNA sequences in common with a DNA
probe of genomic DNA for bacteria A (genomic DNA probe A) will give less
intense
hybridization signals, which directly reflect the extent of sequence homology.
Unrelated bacteria that share little or no sequence homology to bacteria .A
will yield
virtually no fluorescence signal at all. Once the relative extent of DNA
sequence
homology between stains has been established, cross-hybridization noise can be
electronically suppressed (preferably via appropriate computer software and
image
thresholding techniques) thus directly leading to bacterial identification.
Computer thresholding of fluorescence images is used to establish the extent
of
cross-hybridization (i.e., sequence homology) between the three Cagnocytophaga
species. In this experiment, pure samples of each bacteria are separately
hybridized
with biotinylated DNA probes from all three species. For example, three slides
are
prepared that contain only C. ocl:racea cells. The first slide is hybridized
in situ using
BIO-labeled C. ochracea genomic DNA, the second slide is hybridized in situ
using
BIO-labeled C. gingivalis genomic DNA and the third slide is hybridized in
situ using
BIO-labeled C. sputigena genomic DNA. The slides are then washed and
hybridization positive bacteria are identified using F1TC-avidin.
C. ochracea and C. gingivalis did not crosshybridize with one another,
however C. sputigena cross-hybridized with both C. gingivalis and C. ochracea.
A
computer analysis of the intensity of the fluorescent hybridization signals
indicates that
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C. sputigena had significant sequence homology with C. ochracea (- 22%) but
much
less (- 5%) with C. gingivalis.
To demonstrate how image thresholding can be used for bacterial speciation, a
slide with a mixture of C. sputigena and C. orchracea is hybridized with Bio-
labeled
genomic DNA from C. sputigena. To prepare such slides for in situ
hybridization,
small aliquots (1-2 ml) of cultured bacteria are centrifuged at 1,200 xg in a
HBI
microcentrifuge. The cell pellet is suspended in phosphate buffered saline
(PBS) and
adjusted to approximately 103 bacteria per lrl. Aliquots of both pure cultures
and
synthetic mixtures are spotted directly onto commercially available (Fisher
Scientific,
cat #12-550-15) positively (+) charged slides and air dried. All slides are
then fixed in
(,arnoy's B Fixative (Ethanol: Chloroform: Acetic Acid, 6:3:1 ) for 5 minutes.
Hybridization is accomplished using 15 111 of hybridization solution which
contained 50 ng of labeled probe and DNAse treated salmon sperm DNA (15 pl;)
in
50% (vol/vol) deionized formamide/2xSSC (0.3 M sodium chloride/0.03 M sodium
citrate, pH 7.0)/ 5% dextran sulfate. The solution is applied to the sample,
covered
with a coverslip and sealed with rubber cement. Both bacterial DNA and labeled
DNA
probe are denatured by heating at 80°C for 5-8 minutes in an oven.
These steps are
sufficient to permeabilize the bacteria for probe assessability.
The DNAs are allowed to reassociate by incubating the slides at 37°C
for 2.5
min. in a moist chamber. Posthybridization washings, blocking and detection
are
conducted as described by Lichter, P. et al. (Hum. Genet. 80:224-234 ( 1988)).
Briefly,
the slides are washed 3 times in 50% formamide/ 2xSSC at 42°C for 5
minutes and
then washed 3 times in O.IxSSC at 60°C for 5 minutes. The slides are
then incubated
in a solution of 3% bovine serum albumin/4xSSC (blocking solution) for 30
minutes
at 37°C.
Biotinylated probes are detected using fluorescein isothiocyanate-(FITC)-
avidin DCS (Vector Laboratories, 5 ~tg/ml), Cascade Blue-avidin (Molecular
Probes
Inc., 10 pg/ml) or Rhodamine-avidin (Boehringer Mannheim, 10 pg/ml).
Digoxigenin-labeled probes are detected using FTTC or Rhodamine conjugated
sheep
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anti-digoxigenin Fab fragments (Boehringer Mannheim, 2 ~tg/ml). Dinitrophenol
labeled probe is detected by incubating with rat anti-DNP antibodies (Novagen,
1:500
dilution)) and then with goat anti-rat F1TC-conjugated antibody (Sigma, 1
ug/ml). In
some experiments, bacterial DNA was counterstained with 4,6-diamidino-2-
phenylindole (DAPn at a concentration of 200 ng/ml. For detection,
fluorochrome-
conjugated antibodies or avidin are diluted into a solution of 4xSSC/1% BSA
and
0.1% tween-20 (200 Nl/slide) and incubated with the sample at 37°C for
30 minutes in
the dark. The slides are then washed 3 times in 4xSSC/0. I % tween-20 at
42°C prior to
viewing.
Hybridization is visualized via epifluorescence microscopy using a Zeiss
A;:ioskop-20 wide-field microscope with a 63x NA 1.25 Plan Neofluar oil
immersion
objective and a SOW high pressure mercury arc lamp. Images are projected with
a
Zeiss SFL-10 photo-eyepiece onto a cooled charged-coupled device (CCD) camera
(Photometries CH220; 512x512 pixel array). Effective magnification is set by
changing the microscope-camera distance, using a bellows. The 8-bit greyscale
images
are recorded sequentially using DAPI, FTTC and Rhodamine filter sets,
manufactured
by C. Zeiss, Inc., Genmany to minimize image offsets. Camera control,
greyscale
image acquisition and image pre-processing are done as described above.
All bacteria are found to be hybridization positive in this experiment,
although
some appear to fluorescence more intensely than others. After the fluorescence
image
had been thresholded (using the Gene Join Max Pix software; Yale University)
to
remove components of the image that exhibited less than 25% of the maximum
fluorescence intensity, only a subset of the original bacteria appear
hybridization
positive. These bacteria are all C. sputigena because only they exhibit 100%
homology to the probe. This general strategy can be used to speciate multiple
related
microorganisms, provided the level of their sequence relatedness is known so
that
appropriate thresholding parameters can be applied. Bacteria exhibiting up to
40%
sequence homology can be discriminated with genomic DNA probes using this
technique.
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The Gene Join Max Pix software can be used in a second way to achieve
bacterial speciation with genomic DNA probes. This embodiment exploits the
fact
that when separate fluorescence images (e.g., fluorescein and rhodamine) are
merged
to form a composite image; the source image with the greatest fluorescent
intensity
(i.e., photon output) at each pixel location will be color-dominant in the
merged
image. This attribute permits the use of multiple differentially labeled
genomic DNA
probes simultaneously to probe a single or mixed bacterial population. Since
each
bacteria in a mixture will exhibit the greatest fluorescence signal with the
genomic
probe that is 100% homologous, the image merging process results in effective
speciation.
This is demonstrated by the following experiment. A mixture of the three
Capnocytophaga strains is hybridized using a 1:I:1 of all three genomic DNA
probes,
each labeled with a different reporter. C. ochrac:a DNA was labeled with DNP-
11-
dUTP for detection with FTTC (yellow green), C. gingivalis Di~tA was labeled
with
biotin-11-dUTP for detection with Cascade Blue (blue) and C. sputigena DNA was
labeled with DIG-11-dUTP for detection with Rhodamine (red). The image
generated
by Cascade Blue shows intense signals for some bacteria present in the sample,
presumably C. gingivalis cells and weak signals for other bacteria. The FTTC
fluorescence image produced by the C. ochracea probe and the Rhodamine
fluorescence imge produced by the C. sputigena probe also show both intense
signals
for some bacteria and weak signals for others, reflecting interspecies
hybridization.
However, each bacterium shows an intense signal in one of the three images and
a
weaker (or absent) signal in the other images (reflecting interspecies
hybridization).
Accordingly, through the use of thresholding (i.e., using the signal generated
to
determine which probe gave the more dominant signal with which bacteria), the
method enables one to determine the species of each bacterium.
Thus, in contrast to traditional methods of culturing and testing biochemical
products for the differentiation and/or speciation of a bacteria, that are
often difficult
and time consuming, the in sitcc hybridization methods of the present
invention provide
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a rapid and simple method for the speciation of cross-hybridizing
microorganisms,
such as the oral Capnocytophaga. The basis of this method relies on the
finding that
all microorganisms within different genera and species have unique DNA
sequences
within their genome. Closely related organisms will have some DNA sequences in
common, and the amount of such cross-hybridizing sequences will directly
affect their
ability to be differentiated by in situ hybridization.
In this analysis, the fluorescence images obtained from in situ hybridization
with each detector (e.g., Rhodamine, FTTC or Cascade Blue), which reflect the
amount
of hybridization of a bacterium to each of the genomic DNA probes, are
digitalized via
computer. If a bacteria has no cross-hybridizing sequence with any of the
other
bacteria it will hybridize only to its homologous DNA and thereby be detected
by only
a single detector and its presence will be found in only the image
corresponding to that
detector. However, if a bacteria does have cross-hybridizing sequences with
one of the
other bacteria, then it will be detected by the DNA probes from each bacteria
and its
presence will be seen in the corresponding images for each probe (detectors).
However, the intensity of the signal generated in each image will be directly
proportional to the amount of hybridization of a bacterium with a DNA probe.
The
corresponding images from each detector are pseudocolored and overlaid
(merged) to
form a composite image. Since any given pixel on the monitor can only be a
single
color, only the most intense signal at each pixel is shown, and displayed as
the
corresponding pseudocolor.
Whereas two of the Capnocytophaga species (C gingivalis and C ochracea) of
the above experiment show no cross-hybridization, the third species (C
sputigena)
cross-hybridized with both of the preceding organisms. The amount of cross-
hybridization as estimated by a computer analysis of the intensity of the
hybridization
signal following in situ hybridization is in good agreement with that found in
the
literature ( 22-23% for C. sputigena and C. ochracea and 9-11% for C.
sputigena and
C. gingivalis (Heltberg, O. et al., Eur. J. Clin. Microbiol. 3:236-240 (
1984)). The 22-
23% crosshybridization of C. sputigena and G ochracea is evident in the
composite
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image within some of the C. sputigena cells, but not evident in the C.
ochracea cells.
This may reflect differences in how the cross-hybridizing sequences are
arranged
within those genomes.
This example further demonstrates that for an in situ hybridization analysis,
the
DNA probe need not be totally specific. Cross-hybridizing sequences must only
be
reduced below the level necessary for showing specificity. The level of unique
sequences necessary for specificity would desirably be ascertained for each
bacteria of
interest, since it would probably be affected by not only the total number of
cross-
hybridizing sequences, but also how those sequences are arranged within a
genome.
However, some microorganisms, such as Neisseria meningitis and N. gonorrhoea
show greater than 80% sequence homology (Kingsbury, D.T., J. Bacteriol. 94:870-
874
(1967)). Such large amounts of cross-hybridizing sequences may result in an
ambiguous analysis using in situ hybridization with total genomic DNA probes
that
can be addressed by the methods of the present invention.
Thus, in sum, multiparametric fluorescence in situ hybridization (M-FISH) and
computer assisted digital imaging microscopy have been used to develop a
rapi,i
method for differentiating C. gingivalis, C ochracea and C. Sputigena with
total
genomic DNA probes. Although these bacteria exhibit up to 22% sequence
homology,
cross-hybridization signals can be completely suppressed. Speciation of these
three
Capnocytophaga species can be achieved in as little as 30-90 minutes, markedly
faster
than the 2-3 weeks required using conventional microbiological methods. The
general
discrimination strategy reported here is applicable to the broad spectrum of
microbial
pathogens for which an extent of sequence homology is known.
EXAMPLE 3
Synergistic Use of Nucleic Acid Amplification Technology in Concert
with M-FISH Analysis
Nucleic acid amplification technology has greatly increased the ability to
investigate detailed questions about the genotype or the transcriptional
phenotype in
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small biological samples, and has provided the impetus for many significant
advances
in biology, especially in the field of genetics.
One aspect of the present invention concerns the use of the above-described
multiparametric fluorescence in situ hybridization (M-FISH) methods in concert
with
one or more methods of nucleic acid amplification to facilitate the detection
and
characterization of mutations, chromosomal elements, chromosomal
rearrangements,
and infectious agents (e.g., viruses, proviruses, etc.) that may be present in
small
amount or proportion in a sample undergoing analysis. For example, through the
use
of fluorophore combinations designed for multiparametric color coding, a 6-
fluor
tagging approach permits the simultaneous identification of 63 different types
of
signals by virtue of their unique spectral signatures.
As described above, the M-FISH method of the present invention can
distinguish and identify all of the chromosomes of a human karyotype. However,
by
ampl_fying a specific gene, allele or chromosomal element, and employing one
or
more labeled probes that are capable of specifically hybridizing to the
amplified
sequences, the invention permits the determination of whether a particular
karyotyFe
contains the gene, allele or chromosomal element in question. By way of
illustration,
the use of a probe specific for a deletion, rearrangement, insertion or other
mutation
characteristic of a genetic disease (e.g., hemophilia, cystic fibrosis, breast
or other
cancer, etc.) it is possible to diagnose whether a patient exhibits the
genetic disease
(e.g., to diagnose cystic fibrosis, etc.). Similarly, the invention permits
one to
determine whether a patient's chromosomes carry a recessive allele associated
with
genetic disease. Likewise, the invention permits one to determine whether a
patient is
predisposed to a disease by virtue of the presence of a genetic lesion (e.g.,
a mutation
in the apoE, p53, rb, or brcall2 genes) associated with a future disease state
(such as
Alzheimer's Disease, heart disease, cancer, etc.).
Similarly, as applied to the investigation of microbes (e.g., bacteria,
viruses,
and lower eukaryotes), the M-FISH method of the present invention can
distinguish
and identify the species of microorganisms present in a clinical or non-
clinical sample.
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However, by amplifying a specific gene, allele or chromosomal element, and
employing one or more labeled probes that are capable of specifically
hybridizing to
the amplified sequences, the invention permits the determination of whether a
particular microorganism contains the specific gene, allele or chromosomal
element.
Thus, for example, by using a probe specific for an antibiotic resistance
determinant, a
cellular antigen, a toxin, etc., the combined use of M-FISH and nucleic acid
amplification permits a determination of whether a particular microbial or
viral strain
is resistant to an antibiotic, or is pathogenic, or expresses a toxin. Such
combination
of methodologies thus permits the serotyping and subspeciation of pathogens
without
any requirement of culturing andlor purification.
The M-FISH methods of the present invention particularly concern probe sets
and methods that are sufficient to characterize both the genotypic and
phenotypic
charateristics of micobes present in a preparation. A genotypic probe is one
capable of
specifically hybridizing to phylogenetically related species of microbes;
hybridization
of such a probe to nucleic acid of a preparation thus reveals whether a
preselected
microbe, or its cross-hybridizing {i.e., phylogenetically) related microbes
are present in
such a preparation. A phenotypic probe is one capable of specifically
hybridizing to
genes, or genetic elements associated with a particular phenotype (e.g.,
antibiotic
resistance, toxin production, antigen presentation, etc.).
The invention particularly contemplates the multiparametric use of multiple
fluors, and particularly concerns the embodiments in which 2, 3, 4, 5, 6 or
more
fluorophores are employed so as to permit the detection and/or
characterization of 3, 7,
15, 31, 63 or more combinations of genotypic or phenotypic elements. Thus, for
example, if five fluors are employed, 31 genotypic/phenotypic elements can be
probed.
Any combination of such elements can be employed. For example, 25 of such
elements can be genotypic elements (thus permitting the identification of 25
different
species of microbes), whereas 6 of such elements can be genotypic (for example
permitting the determination of whether any of the 25 identified species are
resistant to
any of 4 antibiotics or present any of 2 toxins). Similarly, 10 of such
elements can be
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genotypic elements (thus permitting the identification of 10 different species
of
microbes), whereas 21 of such elements can be genotypic (for example
permitting the
determination of whether any of the 10 identified species are resistant to any
of 9
antibiotics, present any of 3 toxins, present any of 4 surface antigens, and
express any
of 5 genes).
Any of a wide group of bacteria (e.g., E. coli strains and strains of other
enterics (e.g., Salmonella), Clostridria, Vibrio, Corynebacteria, Listeria,
Bacilli
(especially B. anthracis), Staphylococcus Streptococci (especially beta-
hemolytic
Streptococci and S. pneumoniae), Borrelia, Mycobacterium (especially M.
tuberculosi); Neisseria (especially N. gonorrhoeae), Trepanoma. bacteria
implicated
in periodontal disease (e.g., Fusobacteria, Porphyromonas, Eikenella,
Prevotella,
Actinobacillus, and Capnocytophaga species), etc.), viruses (e.g.,
parvoviruses,
papoviruses, herpesviruses, togaviruses, retroviruses (especially HIV),
rhabdoviruses,
influenza viruses, etc.), and lower eukaryotes (fungi (e.g., Dermatophytes;
Pneumocystis, Trypanosome; etc.), yeast, helminths, nematodes, etc.) can be
detected
and characterized using such a method.
Significantly, the combined use of a nucleic acid amplification method and the
multiparametric fluorescence in situ hybridization methods of the present
invention
can be used to explore the quiescence or expression state of cells. Thus, by
employing
probes specific to RNA produced by, for example, growing cells, cells
expressing
tumor antigens or hormones, etc., the methods of the present invention can
determine
not merely the presence of tumor cells, but the extent of their malignancy.
Such
mRNA profiling may be conducted even in circumstances in which standard cDNA
hybridization approaches may not be sensitive enough to detect changes in the
concentration of low abundancy gene products.
While the combined use of a nucleic acid amplification method and the
multiparametric fluorescence in situ hybridization methods of the present
invention
can employ any suitable amplification method or methods (e.g., Polymerase
Chain
Reaction (Mullis, K. et al., Cold Spring Harbor Symp. Quant. Biol. 51:263-273
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( 1986); Erlich H. et al., EP 50,424; EP 84,796, EP 258,017, EP 237,362;
Mullis, K.,
EP 201,184; Mullis K. et al., US 4,683,202; Erlich, H., US 4,582.788; Saiki,
R. et al.,
US 4,683,194 and Higuchi, R. "PCR Technology," Ehrlich, H. (ed.), Stockton
Press,
NY, 1989, pp 61-68), Strand Displacement Amplification (Walker, G.T. et al.,
Proc.
Natl. Acad. Sci. (U.S.A.) 89:392-396 ( 1992); Walker et al., US 5,270,184;
Walker,
US 5,455,166); Ligase Chain Reaction (Segev, W090/01069; Birkenmeyer,
W093/00447), etc.), the preferred method of amplification is the Rolling
Circle
Amplification (RCA) method (Caplan, M. et al.. PCT Patent Application
Publication
No. WO 97/19193; herein incorporated by reference in its entirety).
Rolling Circle Amplification (RCA) is an amplification strategy in which
nucleic acid amplification is driven by a DNA polymerase that can replicate
circularized oligonucleotide probes with either linear or geometric kinetics,
under
isothermal conditions. In detail, RCA involves incubating at least one one
rolling
circle replication primer (RCRP) with at least one amplification target circle
(ATC).
The ATC comprises a single stranded circular DNA molecule that contains a
region
complementary to the RCRP, such that the RCRP can hybridize to the ATC and
mediate amplification in the presence of a DNA polymerase.
In the presence of two primers, one hybridizing to the "+" strand, the other
to
the "=' strand of DNA, a complex pattern of DNA strand displacement ensues
that
generates 109 or more copies of each circle in 90 minutes, enabling detection
of point
mutations in human genomic DNA. This expanding cascade of strand displacement
and fragment-generation events is termed "DNA Hyperbranching," and the special
rolling circle amplification driven by two primers is termed "Hyperbranched-
RCA" or
"HRCA." Using a single primer, RCA generates hundreds of tandemly linked
copies
of a covalently closed circle in a few minutes.
It is preferred to conduct HRCA using exo(-) Vent DNA polymerase, or the
large fragment of Bst DNA polymerase (Aliotta, J.M. et al., Genet. Anal.
12:185-195
( 1996); Thomas, D.C. et al., Clin. Cheri~. 43:?219 Abs 38 ( 1997); both
herein
incorporated by reference), and by the Sequenase 2.0 variant of T7 DNA
polymerase.
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It had been shown that Sequenase supports rolling circle amplification of
circular
oligonucleotides, albeit relatively slowly (Fire. A. et al., Proc. Natl. Acad.
Sci. IUSA)
92:4641-4645 ( 1995); Liu, D. et al., J. Amer. Chem. Soc. 118:1587-1594 (
1996)). The
efficiency of RCA and HRCA reactions is increased by the addition of proteins
that
bind single-stranded DNA. The addition of E. coli single-strand binding
protein (SSB)
stimulates Sequenase-catalyzed HRCA, while phage T4 gene-32 protein stimulates
Vent exo(-) catalyzed HRCA. In contrast, the Bst polymerase does not require
such
ssingIe-strand binding proteins for maximal activity.
If matrix-associated, the DNA product remains bound at the site of synthesis,
where it may be tagged, condensed, and imaged as a point light source. Linear
oIigonucieotide probes bound covalently on glass surfaces can generate RCA
signals,
whose color indicates the allele status of the target, depending on the
outcome of
specific, target-directed ligation events. Single molecule counting by RCA
provides a
powerful mutation detection method for studies using oligonucieotide arrays,
and is
particularly amenable for the analysis of rare somatic mutations.
In a further embodiment, the combined M-FISH / nucleic acid amplification
methods of the invention (especially RCA/HRCA) may be used to detect
circularizable
oligonucleotides, called "padlock probes" (Landegren, U. et al., Methods 9:84-
90
( 1996); Landegren, U. et al., Ann Med. 29:585-590 ( 1997); (Niisson, M. et
al., Science
265:2085-2088 ( 1994); Nilsson, M. et al., Nat. Genet. 16: 252-255 ( 1997),
all herein
incorporated by reference) bound to single copy genes in cytological
preparations.
Multiple applications exist for the above-described gap-fill reaction, in
which
target-complementary sequences are incorporated into circles by copying and
covalent
closure. In principle, any DNA sequence thus captured into a circular DNA may
be
amplified by RCA or HRCA. The reaction may thus be advantageously used in
situations where it is desirable to interrogate the sequence incorporated into
a padlock
probe at some point after RCA. Longer sequences, such as microsatelIite
repeats,
should also be capable of being copied into circularizable probes for
amplification and
analysis. Subsequent to such copying, since there is little likelihood that
rolling circle
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amplification will modify the number of repeats incorporated into a
circularized probe
(Fire, A. et al., Proc. Natl. Acad. Sci. (USA) 92:4641-4645 (1995)), direct
measurement of repeat size would be feasible.
In particular, any of three alternative HRCA/RCA strategies for allele
discrimination utilizing Iigation of circularizable DNA probes and rolling
circle
replication --"gap-probe RCA," "polymerase-mediated gap-fill RCA," and "ligase
mediated extension of immobilized probe RCA" --may be employed in concert with
the M-FISH methods of the present invention. The polymerase mediated "gap-
fill"
reaction is preferred over the "gap probe" ligation reaction for solution
studies of
complex genomes. This is because the gap oligonucleotides, which cannot be
washed
away in a solution assay, are preferably used at relatively high
concentrations for the
ligation step, and may therefore interfere with the HRCA reaction, inducing
the
formation of amplicon artefacts. In contrast, the gag Iigatior reaction is
considered
preferable for allele analysis of DNA in cytological specimens. Here, the
spe:;ificity of
ligation should be enhanced relative to probes without a gap because three
different
sequence recognition events and two independent ligation events must occur
before
"padlock" closure. The third assay method, ligase-mediated extension of an
oligonucleotide linked to a solid surface, provides a totally novel approach
to quantify
individual hybridization/ligation events and to score rare somatic mutations.
In a highly preferred embodiment of HRCA/RCA, the DNA generated by RCA
is labeled with combinatorially labeled fluorescent DNP-oligonucleotide tags
that
hybridize at multiple sites in the tandem DNA sequence. The "decorated" DNA,
labeled by specific encoding tags, is then condensed into a small object by
cross-
linking with a multivalent antibody (e.g., anti-DNP IgM). The wild-type
specific
primer generates RCA products which can hybridize to fluorescein-labeled DNP-
oligonucleotide tags, while the mutant RCA products hybridize to Cy3-labeled
DNP-
oligonucleotides. The process of Condensation of Amplified Circles after
Hybridization of Encoding Tags is termed "CACHET." The use of CACHET, in
concert with the M-FISH methods of the present invention is particularly
preferred.
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EXAMPLE 4
Use of M-FISH Analysis to Evaluate Telomere Integrity
Conventional analyses of chromosomes using, for example, Giemsa banding of
metaphase chromosomes have several salient deficiencies. In particular,
karyotypic
changes involving chromosome fragments that have very similar banding patterns
can
be impossible to resolve using this method (Saccone, E. et al., Proc. Natl.
Acad. Sci.
(USA) 89:4913-4917 (1992)). Such a Ending is particularly true of the
telomeric
bands which are virtually all Giemsa negative and which are also the most gene-
rich
bands within the genome. The correct identification of telomeric regions is
very
important because telomeres may be involved in breakage and healing events
that can
result in terminal deletion, gene amplification or cryptic translocations.
Indeed, there is an increasing number of clinical cases that have been found
to
be the result of cryptic cranslocations such as hemoglobin H (Lamb, J et al.,
Lancet
2:819-824 ( 1989)), Cri-du-Chat (Overhauser, J. et al., Amer. J. Hum. Genet.
45:296-
303 (1989)), Wolf Hirschhorn (Altherr, M.R. et al, Amer. J. Hum. Genet.
49:1235-
12342 ( 199I )), and Miller-Dieker lissencephaly syndrome (Kuwano, A. et al.,
Amer. J.
Hum. Genet. 49:707-714 ( 1991 )}. In addition, cryptic translocations occur in
up to 6%
of the population with mild to moderate mental retardation who have no
detectable
chromosomal changes upon karyotyping. Many of these genetic changes go
undetected using standard cytogenetic analysis.
As indicated above, the present invention provides an ability to
simultaneously
identify the twenty-four different human chromosomes in a metaphase spread by
hybridizing a complete set of chromosome-specific DNA probes, each labeled
with a
different combination of dyes. One aspect of the present invention is the
recognition
that, through the use of telomere-specific probes, the methods of the present
invention
may be used to identify translocations that would be non-identifiable (i.e.,
cryptic)
through the use of conventional methods. As such, the present invention
provides, for
the first time, a simple screening test to assess the integrity of telomeric
regions using
a single hybridization reaction. Such multiplex hybridization assays
significantly
CA 02329253 2000-11-30
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- -86-
improve the ability to detect terminal deletions and cryptic chromosomal
rearrangements, and thereby facilitate the identification of structural
abnormalities that
elude detection with conventional cytogenetic banding or multicolor whole-
chromosome painting methods. Thus, this aspect of the present invention
extends
prior efforts in both karyotyping technology (see, e.g., Speicher, M.R. et
al., Nature
Genet 12:368-375 ( 1996); Speicher, M.R. et al., Bioimaging -x:52-64 ( 1996);
Schrock,
E. et al., Science 273:494-497 (1996)) and telomere integrity assay technology
(see,
e.g., Ledbetter, D.H., Amer. J. Hum. Gener. 51:451-456 (1992)); all herein
incorporated by reference).
To illustrate this capacity of the invention, a set of YAC clones containing
human DNA sequences specific for the sub-telomeric region of each chromosome
arm
is assembled. This probe set employed a microdissected probe for the short arm
of the
Y chromosome (Guar, X.Y., Nature Genet. 12:10-11 (1996), herein incorporated
by
reference) because no suitable sub-telomeric YAC for the short arm could be
identified. Similarly, because of extensive cross-hybridization, the probe set
did not
include probes for the p arms of the acrocentric chromosomes 13, 14, 15, 21
and 22.
The probes are combinatorially labeled such that the telomeric regions of
these
chromosomes are visualized in different pseudocolors based upon their unique
fluorophore composition.
The p arm and q arm telomere proximal probes for each chromosome are
labeled with the same "color code," for example both chromosome 1 telomeres
are
labeled in color "a," while the chromosome 2 telomeres are labeled in color
"b," ete.
Thus, any cytogenetically cryptic chromosome translocation occurring on a non-
acrocentric chromosome are visualized in the metaphase spreads as a chromosome
harboring 2 colors rather than a single color per chromosome. Although the 24
color
telomere integrity assays utilize a complex probe mixture, such probe
cocktails can be
hybridized with high reproducibility. Such hybridization is visualized, as
described
above, with, for example, an epifluorescence microscope equipped with an
appropriate
filter set and a cooled CCD-camera, etc. The fluors and high contrast filters
are the
CA 02329253 2000-11-30
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-87-
same as those described above. In brief, fluorescein (FLU), Cy3, and Cy5 are
linked to
dUTP for direct labelling; Cy3.5 and Cy7 are available as avidin or
antidigoxigenin
conjugates for secondary detection of biotin- or digoxigenin-labelled probes.
For each
probe, 1-3 separate nick translations are performed.
The telomeric probe set may be composed of a mixture of YACs, Half Yacs
(Vocero-Akbani, A. et al., Genomics 36:492-506 (1996); Macini, R.A. et al.,
Genomic
Res. 5:225-232 ( 1995)), and CEPH-YACs (Chumakov, LM. et al., Nature 377:175-
297 ( 1995)), chosen to contain the most teloineric genetic markers. Half YACs
are
generated from specialized YAC libraries enriched for telomeric sequences. The
term
"half YACs" refers to the fact that one of the vector telomere sequences of
the
molecule is provided by human DNA rather than yeast DNA. Half YACs contain sub-
telomere repeats that are known to reside on different chromosomes thus, in
some
ins!ances, they hybridize to multiple telomeres. However, specific M-FISH
signals
can be obtained from half YACs, for example through the use of DNA
amplification
(using, for example, PCR with Alu primers to reduce the relative
representation of
repeat sequences); addition of Cot-1 DNA to the hybridization cocktail will
suppress
hybridization of any residual repetitive sequences. Suitable YACS, half YACs
and/or
CePH-YACs are described by Vocero-Akhani, A. et al. (Genomics 36:492-506
( 1996)), by the National Intitutes of Health and Institute of Molecular
Medicine
Collaboration (Nature Genet 14:86-89 (1996)) and by Bray-Ward, P. et al.
(Genomics
36:1-14 (1996)); all of which references are herein incorporated by reference.
The
fluors used to label telomeric regions are shown in Table 4. In Table 4, the
references
are ( 1 ) Vocero-Akbani, A. et al., Genomics 36:492-506 ( 1996); (2) National
Intitutes
of Health and Institute of Molecular Medicine Collaboration (Nature Genet
14:86-89
(1996)); (3) Bray-Ward, P. et al. (Genomics 36:1-14 (1996)); (4) Bray-Ward, P.
et al.
(Genomics 36:104-111 ( 1996)); (5)Guan, X.Y., Nature Genet. 12:10-11 ( 1996)),
all
herein incorporated by reference.
CA 02329253 2000-11-30
WO 99/62926 PCT/US99/12107
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CA 02329253 2000-11-30
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CA 02329253 2000-11-30
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CA 02329253 2000-11-30
WO 99/62926 PCT/US99/12107
-92-
In order to test the reproducibility of specific signals with half YAC probes,
each is carefully tested in single color experiments on normal metaphase
spreads
from at least five different donors. These studies permit the assessment of
polymorphisms as well as the frequency or cross-hybridizations to other sub-
s telomeric repeats. Suitable half-YACs perform very reproducibly; in a few
cases
where occasional cross-hybridization occur, the nonspecific signals usually
had very
low fluorescence intensities and thus can be easily discriminated from a real
signal.
CEPH-YACs were selected to contain the most telomeric genetic markers.
Because the distance from the true telomeric end of the chromosome is unknown,
these probes were tested by FTSH fur their distal band location. For six
telomeric
regions (2p, 2q, Sq, 7q, 8p, 8q) the hybridization of a single sub-telomeric
YAC
yielded low fluorescence intensities. Therefore two (Sq, 7q, 8p, 8q) or three
(2p, 2q)
subtelomeric YACs were pooled in order to increase the signal. Alu-
fingerprinting
analysis was used to confirm that the YACs overlap.
A mn~iified version of the previously published M-FISH software for whole
chromosome painting probes was used for probe discrimination (Speicher, M.R.
et
al., Bioimaging 4:52-64 ( 1996); Schrock, E. et al., Science 273:494-497 (
1996));
Yale University). In brief, to correct image shifts caused by optical and
mechanical
imperfections, whole chromosome painting probes, one for each fluor used in
the
experiment, are hybridized simultaneously with the pool of sub-telomeric YACs.
Pixel shift correction of the chromosome painting probes is done as described
above,
however the chromosome paints are not shown on the final images to facilitate
the
color discrimination of the sub-telomeric probes. Instead of calculating a
specific
threshold for each fluor, based on the whole chromosome paint signals, the
terminal
regions of the chromosomes are specifically analyzed. Chromosome ends are
identified and segmentation masks with different thresholds are calculated and
checked for regions with increased fluorescence intensities. This is necessary
because the simultaneous hybridization of multiple small region-specific
probes
results in signals that are often in different focal planes. In the absence of
Z-axis
CA 02329253 2000-11-30
WO 99/62926 PCT/US99/1210~
-93-
optical sectioning and image merging, this results in some YAC probes giving
significantly reduced fluorescence intensity values. If a specific telomeric
region is
not labeled with a particular fluor, no increased fluorescence intensity peaks
is
observed in at least one of the segmentation masks. The calculated fluor
segmentation masks were not overlayed with the DAPI segmentation mask that
delineates the chromosomal boundaries (since some of the telomeric FISH
signals
can lie outside the DAPI segment mask). Individual YAC-clones first are
assigned
distinct gray values depending on the Boolean signature of each probe or the
combination of fluors used to label it (Speicher, M.R. et al., Bioimaging 4:52-
64
( 1996); Schrock, E. et al., Science 273:494-497 ( 1996)). A look-up-table
then is
used to assign each DNA target a psuedocolor depending on this gray value.
Finally,
this pseudocolored image was overlayed onto the DAPI-stained chromosome image
that was assigned a light blue color.
In order to optimize the experimental parameters for hybridizing such a large
number of combinatorially labeled YAC clones simultaneously, the multiplex
telomere probe set is hybridized to normal metaphase spreads from peripheral
blooti
lymphocytes. Similar to the results described above obtained with whole
chromosome painting probes, YAC probes that are labeled with equal amounts of
different fluors do not always give equal signal intensities for each fluor.
To
diminish signal intensity differentials, probe concentrations for the
hybridization mix
and a reliable combinatorial labeling scheme are therefore established by
control
experiments. YACs yielding large fluorescence signals are preferentially
labeled
with three different fluors while YACs yielding rather weak signals were
labeled
with one fluor only.
Using such probes, a 24 color telomere integrity assay is conducted on
karyotypes of 10 normal male and female donors. No indication of polymorphisms
are detected thereby suggesting that cryptic translocations are extremely
infrequent in
normal populations. A typical metaphase spread is observed (Figure SA), and a
CA 02329253 2000-11-30
WO 99/62926 PCT/US99/12107
-94-
normal karyotype based on the boolean signature of our subtelomere probes is
attained as expected (Figure SB).
In the process of testing the 24 color telomere integrity assay, the probe set
is
hybridized on metaphase spreads from a patient with a myeloproliferative
disorder.
Karyotyping using G-bands reveals trisomy 8 as the only detectable cytogenetic
change. This is verified by M-FISH using chromosome specific painting probes.
The telomere integrity assay, however, yields an intriguing hybridization
pattern on
the chromosomes 8. The trisomy 8 is verified in all 10 metaphase spreads
analyzed.
However, on 8 of these 10 metaphase spreads a split telomere signal is
observed on
two chromosomes 8, indicating an inversion in this region (Figure 6). This
analysis
demonstrates the power of this new technique to detect structural
abnormalities that
are undetectable by standard cytogenetic methods or 24-color techniques using
chromosome specific painting probes.
Cases where the application of the telomere-integrity assay may be very
rewarding include evaluating karyotvpes of patients with mental retardation, a
combination of mental retardation and dysmorphic features, and cancer cells
that are
known to have a high genomic instability. The latter cells should be highly
predisposed to sub-telomeric translocation events. Screening for cryptic
translocations can not be done efficiently with chromosome specific painting
probes
because they were not designed to detect subtle deletion or rearrangment
events.
In a further embodiment of the above methods, the telomere-specific probe
sets can be improved using PCR-assisted chromatography (Craig, J. et al., Hum.
Genet 100:472-476 ( 1997), herein incorporated by reference). This tool
removes
repetitive DNA, including Ehe polymorphic repetitive sequences from half-YACs
or
other M-FISH probes, and thereby facilitates the generation of probes that
will
hybridize specifically in the absence of Cot-1 suppressor DNA.
While the invention has been described in connection with specific
embodiments thereof, it will be understood that it is capable of further
modifications
and this application is intended to cover any variations, uses, or adaptations
of the
CA 02329253 2000-11-30
WO 99/62926 PCT/US99/1Z107
-95-
invention following, in general, the principles of the invention and including
such
departures from the present disclosure as come within known or customary
practice
within the art to which the invention pertains and as may be applied to the
essential
features hereinbefore set forth and as follows in the scope of the appended
claims.
