Note: Descriptions are shown in the official language in which they were submitted.
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METHODS AND COMPOSITIONS FOR
CHROMOSOME-SPECIFIC STAINING
FIELD OF THE INVENTION
The invention relates generally to the field of cytogenetics, and more
particularly, to the field of molecular cytogenetics. The invention concerns
methods for identifying and classifying chromosomes. Still more
particularly, this invention concerns nucleic acid probes which can be
designed by the processes described herein to produce staining distributions
that can extend along one or more whole chromosomes, and/or along a
region or regions on one or more chromosomes, including staining patterns
that extend over the whole genome. Staining patterns can be tailored for any
desired cytogenetic application, including prenatal, tumor and disease related
cytogenetic applications, among others. The invention provides for
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compositions of nucleic acid probes and for methods of staining
chromosomes therewith to identify normal chromosomes and chromosomal
abnormalities in metaphase spreads and in interphase nuclei. The probe-
produced staining patterns of this invention facilitate the microscopic and/or
flow cytometric identification of normal and abnormal chromosomes and the
characterization of the genetic nature of particular abnormalities.
Although most of the examples herein concern human
chromosomes and much of the language herein is directed to human
concerns, the concept of using nucleic acid probes for staining or painting
chromosomes is applicable to chromosomes from any source indudin~; both
plants and animals.
BACKGROUND OF THE INVENTION
Chromosome abnormalities are associated with genetic disorders,
degenerative diseases, and exposure to agents known to cause degenerative
diseases, particularly cancer, German, "Studying Human Chromosomes
Today," American Scientist, Vol. 58, pp. I82-202 (1970); Yunis, "The
Chromosomal Basis of Human Neoplasia," Science, Vol. 221, pp. 227-236
(1983); and German, "Clinical Implication of Chromosome Breakage," in
Genetic Damage in Man Caused by Environmental Agents, Berg, Ed., pp. 65-86
(Academic Press, New York,1979). Chromosomal abnormalities can be of
several types, including: extra or missing individual chromosomes, extra or
missing portions of a chromosome (segmental duplications or deletions),
breaks, rings and chromosomal rearrangements, among others.
Chromosomal or genetic rearrangements include translocations (transfer of a
piece from one chromosome onto another chromosome), dicentrics
,~.~5~.~~~T4",',~arv.~~.~..~~.»~.~.,.e.n...nraawm~~;~.~,sa~z5 ~.-- ._. ...-
»,~~, ._ " ~,_", y....__..... ..... w,~",~,..._..... .......
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(chromosomes with two centromeres), inversions (reversal in polarity of a
chromosomal segment), insertions, amplifications, and deletions.
Detectable chromosomal abnormalities occur with a frequency of one
in every 250 human births. Abnormalities that involve deletions or
additions of chromosomal material alter the gene balance of an organism and
generally lead to fetal death or to serious mental and physical defects. Down
syndrome can be caused by having three copies of chromosome 21 instead of
the normal 2. This syndrome is an example of a condition caused by
abnormal chromosome number, or aneuploidy. Down syndrome can also be
caused by a segmental duplication of a subregion on chromosome 21 (such as,
21 q22), which can be present on chromosome 21 or on another chromosome.
Edward syndrome (18+), Patau syndrome (13+), Turner syndrome (X0) and
Kleinfelter syndrome (XXY) are among the most common numerical
aberrations. (Epstein, The Consequences of Chromosome imbalance:
Principles, Mechanisms and Models (Cambridge Univ. Press 1986); Jacobs,
Am. T. Epidemiol,105:180 (1977); and Lubs et al., Science,169:495 (1970).]
Retinoblastoma (del 13q14), Prader-Willi syndrome (del 15q11- qI3),
Wilm's tumor (del 11pI3) and Cri-du-chat syndrome (del 5p) are examples of
important disease linked structural aberrations. [Nora and Fraser,
Medical Genetics: Principles and Practice, (Lea and Febiger 1989).]
Measures of the frequency of structurally aberrant chromosomes, for
example, dicentric chromosomes, caused by clastogenic agents, such as,
ionizing radiation or chemical mutagens, are widely used as quantitative
indicators of genetic damage caused by such agents, Biochemical Indicators of
Radiation Injury in Man (International Atomic Energy Agency, Vienna,
1971); and Berg, Ed. Genetic Damage in Man Caused by Environmental
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A-gents (Academic Press, New York, 1979). A host of potentially carcinogenic
and teratogeruc chemicals are widely distributed in the environment because
of industrial and agricultural activity. These chemicals include pesticides,
and a range of industrial wastes and by-products, such as halogenated
hydrocarbons, vinyl chloride, benzene, arsenic, and the like, Kraybill et al.,
Eds., Environmental Cancer (Hemisphere Publishing Corporation, New York,
1977). Sensitive measures of chromosomal breaks and other abnormalities
could form the basis of improved dosimetxic and risk assessment
methodologies for evaluating the consequences of exposure to such
occupational and environmental agents.
Current procedures for genetic screening and biological dosimetry
involve the analysis of karyotypes. A karyotype is the particular chromosome
complement of an individual. or of a related group of individuals, as defined
both by the number and morphology of the chromosomes usually in mitotic
metaphase. It includes such things as total chromosome number, copy
number of individual chromosome types (e.g., the number of copies of
chromosome X), and chromosomal morphology, e.g., as measured by length,
centromeric index, connectedness, or the like. Chromosomal abnormalities
can be detected by examination of karyotypes. Karyotypes are conventionally
determined by staining an organism's metaphase, or otherwise condensed
(for example, by premature chromosome condensation) chromosomes.
Condensed chromosomes are used because, until recently, it has not been
possible to visualize interphase chromosomes due to their dispersed
condition and the lack of visible boundaries between them in the cell
nucleus.
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A number of cytological techniques based upon chemical stains have
been developed which produce longitudinal patterns on condensed
chromosomes, generally referred to as bands. The banding pattern of each
chromosome within an organism usually permits unambiguous
identification of each chromosome type, Latt, "Optical Studies of Metaphase
Chromosome Organization;' Annual Review of Bio~h,3isics and
Bioengineering, Vol. 5, pp. I-37 (1976). Accurate detection of some important
chromosomal abnormalities, such as translocations and inversions, has
required such banding analysis.
Unfortunately, such conventional banding analysis requires cell
culturing and preparation of high quality metaphase spreads, which is time
consuming and labor intensive, and frequently difficult or impossible. For
example, cells from many tumor types are difficult to culture, and it is not
clear that the cultured cells are representative of the original tumor cell
population. Fetal cells capable of being cultured need to be obtained by
invasive means and need to be cultured for several weeks to obtain enough
metaphase cells for analysis. In many cases, the banding patterns on the
abnormal chromosomes do not permit unambiguous identification of the
portions of the normal chromosomes that make them up. Such
identification may be important to indicate the location of important genes
involved in the abnormality. Further, the sensitivity and resolving power of
current methods of karyotyping are limited by the fact that multiple
chromosomes or chromosomal regions have highly similar staining
characteristics, and that abnormalities (such as deletions) which involve only
a fraction of a band are not detectable. Therefore, such methods are
substantially limited for the diagnosis and detailed analysis of contiguous
.___. __
av.~ .~,. _ ______ _ .__ _,"" -.._ _ _ _ ._.
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gene syndromes, such as partial trisomy, Prader-Willi syndrome [Emanuel,
Am. T. Hum. Genet., 43:575 (1988); Schmickel, I. Pediatr.,109:231 (1986)] and
retinoblastoma [Sparkes, Biochem. Biopl~s. Acta., 780:95 (1985)].
Thus, conventional banding analysis has several important
limitations, which include the following. 1) It is labor intensive, time
consuming, and requires a highly trained analyst. 2) It can be applied only to
condensed chromosomes. 3) It does not allow for the detection of structural
aberrations involving less than 3-15 megabases (Mb), depending upon the
nature of the aberration and the resolution of the banding technique
[Landegren et al., Science, 242:229 (1988)]. This invention provides for probe
compositions and methods to overcome such limitations of conventional
banding analysis.
The chemical staining procedures of the prior art provide patterns
over a genome for reasons not well understood and which cannot be
modified as required for use in different applications. Such chemical staining
patterns were used to map the binding site of probes. However, only
occasionally, and with great effort, was in situ hybridization used to obtain
some information about the position of a lesion, for example, a breakpoint
relative to a particular DNA sequence. The present invention overcomes the
inflexibility of chemical staining in that it stains a genome in a pattern
based
upon nucleic acid sequence; therefore the pattern can be altered as required
by
changing the nucleic acid sequence of the probe. The probe-produced staining
patterns of this invention provide reliable fundamental landmarks which are
useful in cytogenetic analysis.
Automated detection of structural abnormalities of chromosomes
with image analysis of chemically stained bands would require the
~____..~_ .
.~ _.. __ _.._.__ ~ __ _ . ._
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development of a system that can detect and interpret the banding patterns
produced on metaphase chromosomes by conventional techniques. It has
proven to be very difficult to identify reliably by automated means normal
chromosomes that have been chemically stained; it is much more difficult to
differentiate abnormal chromosomes having structural abnormalities, such
as, translocations. Effective automated detection of translocations in
conventionally banded chromosomes has not been accomplished after over a
decade of intensive work. 'The probe-produced banding patterns of this
invention are suitable for such automated detection arid analysis.
In recent years rapid advances have taken place in the study of
chromosome structure and its relation to genetic content and DNA
composition. In part, the progress has come in the form of improved
methods of gene mapping based on the availability of large quantities of pure
DNA and RNA fragments f~r probes produced by genetic engineering
techniques, e.g., Kao, "Somatic Cell Genetics and Gene Mapping,"
International Review of C, toy, Vol. 85, pp. 109-I46 (1983), and
D'Eustachio et al., "Somatic Cell Genetics in Gene Families," Science,
Vol. 220, pp. 9, I9-924 (1983). The probes for gene mapping comprise labeled
fragments of single-stranded ox double-stranded DNA or RNA which are
hybridized to complementary sites on chromosomal DNA. With such probes
it has been crucially important to produce pure, or homogeneous, probes to
minimize hybridizations at locations other than at the site of interest,
Henderson, "Cytological Hybridization to Mammalian Chromosomes,"
International Review of C,~olo~.y, Vol. 76, pp. 1-46 (1982).
The hybridization process involves unravelling, or melting, the
double-stranded nucleic acids of the probe and target by heating, or other
.u. ~.~_~. _ -,~ _ '_.._ _ _~"._ _ -
CA 02449414 2003-12-11
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means (unless the probe and target are single-stranded nucleic acids). This
step is sometimes referred to as denaturing the nucleic acid. When the
mixture of probe and target nucleic acids cool, strands having complementary
bases recombine, or anneal. When a probe anneals with a target nucleic acid,
the probe's location on the target can be detected by a label carried by the
probe
or by some intrinsic characteristics of the probe or probe-target duplex. When
the target nucleic acid remains in its natural biological setting, e.g., DNA
in
chromosomes, mRNA in cytoplasm, portions of chromosomes or cell nuclei
(albeit fixed or altered by preparative techniques), the hybridization process
is
referred to as in situ hybridization.
In situ hybridization probes were initially limited to identifying the
location of genes or other well defined nucleic acid sequences on
chromosomes or in cells. Comparisons of the mapping of single-copy probes
to normal and abnormal chromosomes were used to examine chromosomal
abnormalities. Cannizzaro et al., C,yto~;enetics and Cell Genetics, 39:173-178
(1985). Distribution of the multiple binding sites of repetitive probes could
also be determined.
Hybridization with probes which have one target site in a haploid
genome, single-copy or unique sequence probes, has been used to map the
locations of particular genes in the genome [Harper and Saunders,
"Localization of the Human Insulin Gene to the Distal End of the Short Arm
of Chromosome 11," Proc. Natl. Acad. Sci., Vol. 78, pp. 4458-4460 (1981); Kao
et
al., "Assignment of the Structural Gene Coding for Albumin to Chromosome
4," Human Genetics, Vol. 62, pp. 337-341 (1982)]; but such hybridizations are
not reliable when the size of the target site is small. As the amount of
target
sequence for low complexity single-copy probes is small, only a portion of the
..._...__ .,~~~~~,M,,.s.....~~...~.~n.~,...~.,_.~,_~,~ac.~.,_. _._.._..._
~~_~...___..
CA 02449414 2003-12-11
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potential target sites in a population of cells form hybrids with the probe.
Therefore, mapping the location of the specific binding site of the probe has
been complicated by background signals produced by non-specific binding of
the probe and also by noise in the detection system (for example,
autoradiography or immunochemistry). The unreliability of signals for such
prior art single-copy probes has required statistical analysis of the
positions of
apparent hybridization signals in multiple cells to map the specific binding
site of the probe.
Different repetitive sequences may have different distributions on
chromosomes. They may be spread over all chromosomes as in the just cited
reference, or they may be concentrated in compact regions of the genome,
such as, on the cenfromeres of the chromosomes, or they may have other
distributions. In some cases, such a repetitive sequence is predominantly
located on a single chromosome, and therefore is a chromosome-specific
repetitive sequence. [Willard et al., "Isolation and Characterization of a
Major
Tandem Repeat Family from the Human X Chromosome," Nucleic Acids
Research. Vol. 1I, pp. 2017 2033 (1983).]
A probe for repetitive sequences shared by all chromosomes can be
used to discriminate between chromosomes of different species if the
sequence is specific to one of the species. Total genomic DNA from one
species which is rich in such repetitive sequences can be used in this manner.
[Pinkel et al. (III), PNAS USA, 83:2934 (1986); Manuelidis, Hum. Genet.,
71:288
(1985) and Durnam et al., Somatic Cell Molec. Genet.,11:571 (1985.]
Recently, there has been an increased availability of probes for
repeated sequences (repetitive probes) that hybridize intensely and
specifically
to selected chromosomes. [Trask et al., Hum. Genet., X8:251 (1988) and
__ ._ _ _ .w,,. :,,~~ a~.~,~~",,~. . =- __ _ . _ _._._~T _..__-___.___
CA 02449414 2003-12-11
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references cited therein.] Such probes are now available for over half of the
human chromosomes. In general, they bind to repeated sequences on
compact regions of the target chromosome near the centromere. However,
one probe has been reported that hybridizes to human chromosome 1p36, and
there are several probes that hybridize to human chromosome Yq.
Hybridization with such probes permits rapid identification of chromosomes
in metaphase spreads, determination of the number of copies of selected
chromosomes in interphase nuclei [Pinkel et al. (I), PNAS USA, 83:2934
(I986); Pinkel et al. (II), Cold Spring Harbor Sip. Quant. Biol., 51:151
(1986)
and Cremer et al., Hum. Genet, 74:346 (1986)] and determination of the
relative positions of chromosomes in interphase nuclei [Trask et al., su ra~
Pinkel et al. (I), supra; Pinkel et al. (II), supra; Manuelidis; PNAS USA,
81:3123
(1984); Rappold et al., Hum. Genet., 67:317 (1984); Schardin et al., Hum.
Genet.,
71:282 (I985); and Manuelidis, Hum. Genet., 71:288 (1985)].
However, many applications are still limited by the lack of
appropriate probes. For example, until the methods described herein were
invented, probes with sufficient specificity for prenatal diagnosis were not
available for chromosome 13 or 21. Further, repetitive probes are not very
useful for detection of structural aberrations since the probability is low
that
the aberrations will involve the region to which the probe hybridizes.
This invention overcomes the prior art limitations on the use of
probes and dramatically enhances the application of in situ hybridization for
cytogeneHc analysis. As indicated above, prior art probes have not been
useful for in-depth cytogenetic analysis. Low complexity single-copy probes
do not at this stage of hybridization technology generate reliable signals.
Although repetitive probes do provide reliable signals, such signals cannot be
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tailored for different applications because of the fixed distribution of
repetitive sequences in a genome. The probes of this invention combine the
hybridization reliability of repetitive probes with the flexibility of being
able to
tailor the binding pattern of the probe to any desired application..
The enhanced capabilities of the probes of this invention come from
their increased complexity. Increasing the complexity of a probe increases the
probability, and therefore the intensity, of hybridization to the target
region,
but also increases the probability of non-specific hybridizations resulting in
background signals. However, within the concept of this invention, it was
considered that such background signals would be distributed approximately
randomly over the genome. Therefore, the net result is that the target region
could be visualized with increased contrast against such background signals.
Exemplified herein are probes in an approximate complexity range of
from about 50,000 bases (50 kb) to hundreds of millions of bases. Such
representative probes are for compact loci and whole human chromosomes.
Prior to this invention, probes employed for in situ hybridization techniques
had complexities below 40 kb, and more typically on the order of a few kb.
Staining chromosomal material with the probes of this invention is
significantly different from the chemical staining of the prior art. The
specificity of the probe produced staining of this invention arises from an
entirely new source -- the nucleic acid sequences in a genome. Thus, staining
patterns of this invention can be designed to highlight fundamental genetic
information important to particular applications.
The procedures of this invention to construct probes of any desired
specificity provide significant advances in a broad spectrum of cytogenetic
studies. The analysis can be carried out on metaphase chromosomes and
.. "~"",~.~, ~. __ ~ _ _
CA 02449414 2003-12-11
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interphase nuclei. The techniques of this invention can be especially
advantageous for applications where high-quality banding by conventional
methods is difficult or suspected of yielding biased information, e.g., in
tumor
cytogenetics. Reagents targeted to sites of lesions known to be diagnostically
or prognostieally important, such as tumor type-specific translocations and
deletions, among other tumor specific genetic arrangements, permit rapid
recognition of such abnormalities. Where speed of analysis is the
predominant concern, e.g., detection of low-frequency chromosomal
aberrations induced by toxic environmental agents, the compositions of this
invention permit a dramatic increase in detection efficiency in comparison to
previous techniques based on conventional chromosome banding.
Further, prenatal screening for disease-linked chromosome
aberrations (e.g., trisomy 21) is enhanced by the rapid detection of such
aberrations by the methods and compositions of this invention. Interphase
aneuploidy analysis according to this invention is particularly significant
for
prenatal diagnosis in that it yields more rapid results than are available by
cell
culture methods. Further, fetal cells separated from maternal blood, which
cannot be cultured by routine procedures and therefore cannot be analysed by
conventional karyotyping techniques; can be examined by the methods and
compositions of this invention. In addition, the intensity, contrast and Color
combinations of the staining patterns, coupled with the ability to tailor the
patterns for particular applications, enhance the opportunities for automated
cytogenetic analysis, for example, by flow cytometry or computerized
microscopy and image analysis.
This application specifically claims chromosome specific reagents for
the detection of genetic rearrangements and methods of using such reagents
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to detect such rearrangements. Representative genetic rearrangements so
detected are those that produce a fusion gene - BCR-ABL - that is diagnostic
for chrome myelogenous leukemia (CML).
Chronic rnyelogenous leukemia (CML) is a neoplastic proliferation of
bone marrow cells genetically characterized by the fusion of the BCR and ABL
genes on chromosomes 9 and 22. That fusion usually involves a reciprocal
translocation t(9;22)(q34;q11), which produces the cytogenetically distinctive
Philadelphia chromosome (Ph1). However, more complex rearrangements
may cause BCR-ABL fusion. At the molecular level, fusion can be detected by
Southern analysis or by in vitro amplification of the mRNA from the fusion
gene using the polymerase chain reaction (PCR). Those techniques are
sensitive but cannot be applied to single cells.
Clearly, a sensitive method for detecting chromosomal abnormalities
and, more specifically, genetic rearrangements, such as, for example, the
tumor specific arrangements associated with CML, would be a highly useful
tool for genetic screening. This invention provides such tools.
The following references are indicated in the ensuing text by numbers
as indicated:
I. A. de Klein et al., Nature 300, 765 (1982).
2. J. Groffen et al., Cell 36,93 (1984).
3. N. Heisterkamp et al., Nature 306,239 (1983).
4. E. Shtivelman et al., Blood 69,971 (1987).
5. j. B. Konopka, S. M. Watanabe, O.N. Witte, Cell 37,1035 (1984).
6. Y. Ben-Neriah et al., Science 233,212 (1986).
7. P. C. Nowell and D. A. Hungerford, Science 132,1497 (1960).
~,.w_ _ _ ._ _____
CA 02449414 2003-12-11
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8. J. D. Rowley, Nature 243,290 (June 1973).
9. G.Grosveld et al., Mol Cell Biol 6,607 (1986).
I0. E. Canaani et al., Lancet 1, 593 (1984).
I1. R. P. Gale and E. Canaani, Proc Nat1 Acad Sci USA 81,5648 (1984).
12. Konopka J. B. et al., Proc Natl Acad Sci USA 82:1810 (1985).
13. P. Benn et al., Cancer Genet Cytogenet 29,1 (1987).
14. S. Abe et al., Cancer Genet Cytogenet 38,61 (1989)
I5. M. Shtalrid et al., Blood '72, 485 (1988).
16. I. Dube et al., Genes Chromosomes and Cancer I,I06 (I989).
17. A. J. Fishleder, B. Shadrach and C. Tuttle, Leukemia 3:10,746 (1989)
18. C. R. Bartram et al.; J Exp Med 164 (5):1389 (1986).
19. S. Hiroswa et al., Am L Hematol 28,133 (I988).
20. M. S. Lee et al., Blood 73 (8):2I65 (19$9).
22. E.S. Kawasaki et al., Proc Natl Acad Sci USA 85,5698 (I988).
22. M. S. Roth et al., Blood 74, 882 (1989).
23. A. L. Hooberman et al., Blood 74, 1101 (1989).
24. C. A. Westbrook et al., Blood 71 (3):697-702 (1988).
25. B. Trask, D. Pinkel, and G. van den Engh, Genomics 5,710 (1989).
26. S. J. Collins and M. T. Groudine, Proc Natl Acad Sci USA 80, 4813 (I983).
27. D. Pinkel et al., Proc Natl Acad Sci USA 83,2934 (1986).
28. D. Pinkel, T. Straume and J. W. Gray., Proc Natl Acad Sci USA 85, 9138
(1988).
29. B. Trask and J. Hamlin, Genes and Development, 3:1913 (I989).
. __ .
CA 02449414 2003-12-11
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30. J. B. Lawrence, C.A. Villnave and RH. Singer, Cell 42,51 (1988).
31. G. D. Johnson and J. G. Nogueria j. Immunol. Methods 43, 349 (I981).
32. Hegewisch-Becker et al., J. Cell. Biochem. (Suppl.) 13E, 289 (1989).
34. Heisterkamp et al., Nature, 315:758 (I985).
35. Heisterkamp et' al, J. Molec. Appl. Genet., 2:57 (1983).
Fusion of the proto-oncogene c-ABL from the long arm of
chromosome 9 with the BCR gene of chromosome 22 is a consistent finding
in CML (1-3). That genetic change leads to formation of a BCR ABL transcript
that is translated to form a 210 kd protein present in virtually all cases of
CML
(4-6). In 90% of the cases, the fusion gene results from a reciprocal
translocation involving chromosomes 9 and 22 producing a cytogenetically
distinct small acrocentric chromosome called the Philadelphia (Phl)
chromosome (7-12), Fig. 8. However, standard cytogenetics does not have the
resolution to distinguish closely spaced breakpoints, such as those
characteristic of CML and acute lymphocytic leukemia (ALL), and misses
fusions produced by more complex rearrangements. Mapping and cloning of
the breakpoint regions in both genes has lead to molecular techniques capable
of demonstrating BCR ABL fusion in CML cases where the Phl chromosome
could not be detected cytogenetically (13-16). Southern analysis for BCR
rearrangements has become the standard for diagnosis of CML. More
recently; fusion has been detected by in vitro amplification of a cDNA
transcript copied from CML mRNA using reverse transcriptase (17-23). That
. . _ n.~~~. .s. ~ ~-..o~r$~......~..~..x~,n ~~-....,....~~.~
___~.~afx~rm,~.m~..~____ ._
CA 02449414 2003-12-11
_ ~ ~, -'I6-
technique permits detection of BCR-ABL transcript from CML cells present at
low frequencies. Both of those techniques utilize nueleic acid obtained from
cell populations so that correlation between genotype and phenotype for
individual cells is not possible.
Described herein are chromosome-specific reagents and methods to
detect genetic rearrangements, such as those exemplified herein for the
BCR-ABL fusion, that supply information unavailable by existing techniques.
SUMMARY OF THE 1NVENTTON
This invention concerns methods of staining chromosomal material
based upon nucleic acid sequence that employ one or more nucleic acid
probes. Said methods produce staining patterns that can be tailored for
specific cytogenetic analyses. It is further an object of this invention to
produce nucleic and probes that are useful for cytogenetic analysis, that
stain
chromosomal material with reliable signals. Such probes are appropriate for
in situ hybridization. Preferred nucleic. acid probes for certain applications
of
this invention are those of sufficient complexity to stain reliably each of
two
or more target sites.
The invention provides methods and compositions for staining
chromosomal material. The probe compositions of this invention at the
current state of hybridization techniques are typically of high complexity,
usually greater than about 50 kb of complexity, the complexity depending
upon the application for which the probe is designed. In particular,
chromosome specific staining reagents are provided which comprise
heterogeneous mixtures of nucleic acid fragments, each fragment having a
substantial fraction of its sequences substantially complementary to a portion
CA 02449414 2003-12-11
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of the nucleic acid for which specific staining is desired -- the target
nucleic
acid, preferably the target chromosomal material. Tn general, the nucleic acid
fragments are labeled by means as exemplified herein and indicated infra.
However, the nucleic and fragments need not be directly labeled in order for
the binding of probe fragments to the target to be detected; for example, such
nucleic and binding can be detected by anti-RNA/DNA duplex antibodies and
antibodies to thymidine dimers. The nucleic acid fragments of the
heterogenous mixtures include double-stranded ~or single-stranded RNA or
DNA.
This invention concerns chromosome specific reagents and methods
of staining targeted chromosomal material that is in the vicinity of a
suspected genetic rearrangement. Such genetic rearrangement include but are
not limited to translocations, inversions, deletions, amplifications and
insertions. When such a genetic rearrangement is associated with a disease,
such chromosome specific reagents are referred to as disease specific reagents
or probes. When such a genetic rearrangement is associated with cancer, such
reagents are referred to as tumor specific reagents or probes.
This invention provides for nucleic acid probes that reliably stain
targeted chromosomal materials in the vicinity of one or more suspected
genetic rearrangements. Such nucleic aeid probes useful for the detection of
genetic rearrangements are typically of high complexity. Sueh nucleic acid
probes preferably comprise nucleic acid sequences that are substantially
homologous to nucleic acid sequences in chromosomal regions that flank
and/or extend partially or fully across breakpoints associated with genetic
rearrangements.
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This invention further provides for methods and reagents to
distinguish between cytogenetically similar but genetically different
chromosomal rearrangements.
Specifically herein exemplified are chromosome specific regents and
methods to detect genetic rearrangements, e.g., translocations, deletions,
amplifications and insertions, that produce the BCR-ABL fusion which is
diagnostic for chronic myelogenous leukemia (CML). Such chromosome
specific reagents for the diagnosis of CML contain nucleic acid sequences
which are substantially homologous to chromosomal sequences in the
vicinity of the translocation breakpoint regions of chromosomal regions 9q34
and 22qI1 associated with CML.
Those reagents produce a staining pattern which is distinctively
altered when the BCR-ABL fusion characteristic of CML occurs. Figure 11
graphically demonstrates a variety of staining patterns which, along with
other potential staining patterns, are altered in the presence of a genetic
rearrangement, such as, the BCR-ABL fusion.
The presence of a genetic rearrangement can be determined by
applying the reagents of this invention according to methods herein described
and observing the proximity of and/or other characteristics of the signals of
the staining patterns produced.
Preferably, the chromosome specific reagents used to detect CML of
this invention comprise nucleic acid sequences having a complexity of from
about SO kilobases (kb) to about 1 megabase (Mb), more preferably from about
50 kb to about 750 kb, and still more preferably from about 200 kb to about
400 kb.
_ _ _ ~.~,~~ . ._....~,P.,. _ _.
CA 02449414 2003-12-11
_ qy _
This invention further provides for methods of distinguishing
between suspected genetic rearrangements that occur in relatively close
proximity in a genome wherein the chromosome specific reagents comprise
nucleic acid sequences substantially homologous to nucleic acid sequences in
the vicinity of said suspected genetic rearrangements. An example of such a
differentiation between two potential genetic rearrangements is the
differential diagnosis of CML from acute lymphocytic leukemia (ALL).
This invention still further provides methods and reagents for
producing staining patterns in a patient who is afflicted with a disease
associated genetic rearrangement, such as those associated with the BCR-ABL
fusion in CML, wherein said staining patterns are predictive and/or
indicative of the response of a patient to various therapeutic regimens, such
as chemotherapy, radiation, surgery, and transplantation, such as bone
marrow transplantation. Such staining patterns can be azseful in monitoring
the status of such a patient, preferably on a cell by cell basis, and can be
predictive of a disease recurrence for a patient that is in remission.
Computer
assisted microscopic analysis can assist in the interpretation of staining
patterns of this invention, and the invention provides for methods wherein
computer assisted microscopic analysis is used in testing patient cells on a
call
by cell basis,. for e.g., to search for residual disease in a patient.
Still further, this invention provides for methods and reagents to
determine the molecular basis of genetic disease, and to detect specific
genetically based diseases.
Still further, this invention provides for methods and reagents for
detecting contiguous gene syndromes comprising the in situ hybridization of
nucleic acid probes which comprise sequences which are substantially
_._ mT.w._ ~..,~.~.~....__.. ._.._,.~~,~, w_.. T _.____ _
CA 02449414 2003-12-11
Q_
homologous to nucleic acid sequences characteristic of one or more
components of a contiguous gene syndrome. Representative of such a
contiguous gene syndrome is Down syndrome.
Also provided are methods of simultaneously detecting genetic
rearrangements of multiple loci in a genome comprising in situ hybridization
of high complexity nucleic acid probes comprising nucleic acid sequences that
are substantially homologous to nucleic acid sequences in multiple loci in a
genome.
Still further provided are methods of searching for genetic
rearrangements in a genome. For example, conventional banding analysis
may indicate an abnormality in a chromosomal region of a genome under
examination. Methods of this invention may include the application
of nucleic acid probes, produced from the vicinity of that chromosomal
region of a normal genome, by in situ hybridization to cells containing the
abnormality to detail the exact location and kind of genetic rearrangement of
said abnormality by observation of the staining patterns so produced.
The invention still further provides for high complexity nucleic acid
probes which have been optirruzed for rapid, efficient and automated
detection of genetic rearrangements.
One way to produce a probe of high complexity is to pool several or
many clones, for example, phage, plasmid, cosmid, and/or YAC elones,
among others, wherein each clone contains an insert that is capable of
hybridizing to some part of the target in a genome. Another way to produce
such a probe is to use the polymerase chain reaction (PCR).
Heterogeneous in reference to the mixture of labeled nucleic acid
fragments means that the staining reagents comprise many copies each of
CA 02449414 2003-12-11
-21-
fragments having different sequences and/or sizes (e.g., from the different
DNA clones pooled to make the probe). In preparation for use, these
fragments may be cut, randomly or specifically, to adjust the size
distribution
of the pieces of nucleic acid participating in the hybridization reaction.
As discussed more fully below, preferably the heterogeneous probe
mixtures are substantially free from nucleic acid sequences with hybridization
capacity to non-target nucleic acid. Most of such sequences bind to repetitive
sequences which are shared by the target and non-target nucleic acids, that
is,
shared repetitive sequences.
Methods to remove undesirable nucleic acid sequences and/or to
disable the hybridization capacity of such sequences are discussed more fully
below. [See Section II]. Such methods include but are not limited to the
selective removal or screening of shared repetitive sequences from the probe;
careful selection of nucleic acid sequences for inclusion in the probe;
blocking
shared repetitive sequences by the addition of unlabeled genomic DNA, or,
more carefully selecting nucleic acid sequences for inclusion in the blocking
mixture; incubating the probe mixture for sufficient time for reassociation of
high copy repetitive sequences, or the like.
Preferably, the staining reagents of the invention are applied to
interphase or metaphase chromosomal DNA by in situ hybridization, and the
chromosomes are identified or classified, i.e., karyotyped, by detecting the
presence of the Iabel, such as biotin or 3H, on the nucleic acid fragments
comprising the staining reagent.
The invention includes chromosome staining reagents for the total
genomic complement of chromosomes, staining reagents specific to single
chromosomes, staining reagents specific to subsets of chromosomes, and
CA 02449414 2003-12-11
- 22 -
staining reagents specific to subregions within single or multiple
chromosomes. The term "chromosome-specific," is understood to
encompass all of these embodiments of the staining reagents of the
invention. The term is also understood to encompass staining reagents made
from and directed against both normal and abnormal chromosome types.
A preferred method of making the chromosome-specific staining
reagents of the invention includes: I) isolating chromosomal DNA from a
particular chromosome type or target region or regions in the genome,
2) amplifying the isolated DNA to form a heterogeneous mixture of nucleic
acid fragments, 3) disabling the hybridization capacity of or removing shared
repeated sequences in the nucleic acid fragments, and 4) labeling the nucleic
acid fragments to form a heterogeneous mixture of labeled nucleic and
fragments. As described more fully below, the ordering of the steps for
particular embodiments varies according to the particular means adopted for
carrying out the steps.
The present invention addresses problems associated with
karyotyping chromosomes, especially for diagnostic and dosimetric
applications. In particular, the invention overcomes problems which arise
because of the lack of stains that are sufficiently chromosome-specific by
providing reagents comprising heterogeneous mixtures of nucleic acid
fragments that can be hybridized to the target DNA and/or RNA, e.g., the
target chromosomes, target subsets of chromosomes, or target regions of
specific chromosomes. The staining technique of the invention opens up the
possibility of rapid and highly sensitive detection of chromosomal
abnormalities, particularly genetic rearrangements, in both metaphase and
interphase cells using standard clinical and laboratory equipment and
CA 02449414 2003-12-11 _ -.__.
-23-
improved analysis using automated techniques. It has direct application in
genetic screening, cancer diagnosis, and biological dosimetry.
This invention further specifically provides for methods and nucleic
acid probes for staining fetal chromosomal material, whether condensed, as
in metaphase, or dispersed as in interphase. Still further, the invention
provides for a non-embryo-invasive method of karyotyping the
chromosomal material of fetal cells, wherein the fetal cells have been
separated from maternal blood. Such fetal cells are preferably leukocytes
and/or cytotrophoblasts. Exemplary nucleic acid probes are high complexity
probes chromosome-specific for chromosome types I3, IS and/or 21.
Representative probes comprise chromosome-specific Bluescribe plasmid
libraries from which a sufficient number of shared repetitive sequences have
been removed or the hybridization capacity thereof has been disabled prior to
and/or during hybridization with the target fetal chromosomes.
This invention still further provides for test kits comprising
appropriate nucleic acid probes for use in tumor cytogenetics, in the
defection
of disease related loa, in the analysis of structural abnormalities, for
example
translocatians, among other genetic rearrangements, and for biological
dosimetry.
This invention further provides for prenatal screening kits
comprising appropriate nucleic acid probes of this invention. 'This invention
also provides for test kits comprising high complexity probes for the
detection
of genetic rearrangements, and specifically for those producing the BCR-ABL
fusion characteristic of CML.
The methods and compositions of this invention permit staining of
chromosomal material with patterns appropriate for a desired application.
*'rrademark
.-.,~,,.~n_v._
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CA 02449414 2003-12-11
The pattern may extend over some regions of one or more chromosomes, or
over some or all the chromosomes of a genome and may comprise multiple
distinguishable sections, distinguishable, for example, by multiple colors.
Alternatively, the pattern may be focused on a particular portion or portions
of a genome, such as a portion or portions potentially containing a deletion
or
breakpoint that is diagnostically or prognosHcally important for one or more
tumors, or on those portions of chromosomes having significance for
prenatal diagnosis.
The staining patterns may be adjusted fox the analysis method
employed, for example, either a human observer or automated equipment,
such as, flow cytometers or computer assisted microscopy. The patterns may
be chosen to be appropriate for analysis of condensed chromosomes or
dispersed chromosomal material.
The invention further provides for automated means of detecting
and analyzing chromosomal abnormalities, particularly genetic
rearrangements, as indicated by the staining patterns produced according to
this invention.
Another object of the present invention is to provide an alternative
method to currently available techniques for preparing and applying non-
selfcomplementary single stranded DNA hybridization probes.
Another object of the invention is to improve the signal-to-noise
ratios attainable in in situ hybridization by reducing nonspecific and
mismatched binding of probe.
Another object of the invention is to provide a method of denaturing
double stranded target DNA for application of hybridization probe which
CA 02449414 2003-12-11
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minimizes single stranded regions available for hybridization that are
noncomplementary to probe sequences.
DNA fragments from which probes can be constructed by treating
with a restriction endonuclease which generates a collection of restriction
fragments having "sticky" ends, or staggered cuts, characteristic of the
endonuclease used. That is, the two fragment ends introduced by a cut each
consist of a protruding strand and a recessed strand. The restriction
fragments
are inserted into vectors which have been engineered to accept that type of
restriction fragment; and the vectors are transfected into host organisms
which are grown to increase the number of restriction fragments. Next the
vectors are separated from the host organisms, and the restriction fragments
are excised and separated from the vectors. On each end of the restriction
fragments the recessed strands are digested by an appropriate exonuclease.
Digestion is not allowed to go to completion. The exonuclease treated
restriction fragments are then used as template/primers for DNA polymerase
which replaces the digested strand in the presence of a labeled precursor.
Examples of enzymes suitable for this process are exonuclease III followed by
treatment with the large fragment of DNA polymerase I; or T4 DNA
polymerise, which can perform both functions by changes in reaction
conditions. After synthesis is completed, the restriction fragments. are
broken
into smaller fragments such that the labeled portions of the original
restriction fragment remain substantially intact. The smaller fragments are
denatured, and the labeled strands are separated from the unlabeled strands to
form the hybridization probes.
Under this method of using single-stranded probes, before application
of the hybridization probe to the target DNA, the target DNA is first treated
CA 02449414 2003-12-11
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with the same restriction endonuclease used to excise the probe DNA from
the cloning vector. This treatment breaks the target DNA into a collection of
restriction fragments having tails at each end characteristic of the
restriction
endonuclease. Next the target DNA is treated with an exonuclease which
removes the recessed strand, thereby exposing single stranded DNA in the
vicinity of the cut introduced by the restriction endonuclease. Finally, the
hybridization probe is applied to the target DNA, e.g., using standard in situ
hybridization protocols, as described more fully below.
An important feature of the single stranded probe method is treating
the cloned probe DNA and the target DNA with the same restriction
endonuclease. This ensures that the single stranded DNA of the target is
complementary to the labeled strand of the probe. Of course many segments
of the target in addition to the correct binding sites will be made single
stranded because there are many restriction cuts, but there will be much less
total single stranded target than would be made by indiscriminant
denaturation. In addition, target :DNA rendered single stranded in this
manner cannot reanneal with itself and thus block access to the probe.
Another important (but not critical) feature of such a method is the
selection of a label which permits iabeied strands to be separated from
unlabeled strands. Preferably precursors are labeled by biotinylation, and the
labeled strands are separated from unlabeled strands by affinity
chromatography.
....~.~ ..x~..n_.~. ~_.-..~.....~~~"",~~,~~,~,~~...~..~.. __ ~..W
~":~.~....._.__._ . _ ________. _ .__
CA 02449414 2003-12-11
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BRIEF DESCRIPTION OF THE DRAWINGS
Figures 1A, B and C and Figures 2A and 2B illustrate the
hybridization of a chromosome-specific 21 library to human metaphase
spread wherein the inserts were cloned in Lambda phage Charon 21A. The
hybridization capacity of the high copy repetitive sequences in the library
was
reduced by the addition of unlabeled genomic DNA to the hybridization
mixture. The probe was labeled with biotin, which was detected with green
FITC-avidin (fluorescein isothiocyanate avidin). All of the DNA in the
chromosomes was stained with the blue fluorescent dye DAPI (4,6-diamidino-
2-phenylindole).
Figure 1A is a binary image of the DAPI stain in the human
metaphase spread obtained by using a TV camera attached to a fluorescence
microscope. Filters appropriate for DAPI visualization were used. Computer
processing of the image shows all portions above a chosen threshold intensity
as white, and the rest as black.
Figure IB is a binary image of the FITC staining of the same human
metaphase spread as in Figure 1A. The image was processed as in Figure IA
but the filter was changed in the microscope such that the FITC attached to
the probe is visible rather than the DAPI.
Figure 1C is a binary image of the chromosome 21s alone,
nonspecifically stained objects (which are smaller) having been removed by
standard image processing techniques on the binary image of Figure IB.
Figure 2A is a color photograph of the DAPI stain in a human
metaphase spread which was prepared and hybridized contemporaneously
with the spread shown in the computer generated binary images of
Figures 1A, B and C.
CA 02449414 2003-12-11
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Figure 2B is a photograph of the fluorescein attached to the
DNA probe in the same human metaphase spread as shown in Figure 2A. It
was obtained by changing the filters in the fluorescence microscope to excite
fluorescein rather than DAPI. The photograph is comparable to the binary
image of Figure I B.
Figure 3 is a photograph of a human metaphase spread prepared and
hybridized contemporaneously with the spreads shown in Figures 1A, B and
C and ZA and B. The procedures used were the same except that PI
(propidium iodide) instead of DAPI, was used to stain all the chromosomes.
Both PI and fluorescein stains can be viewed with the same microscope filters.
Color film was used such that the propidium iodide counterstain appears red
and the fluorescein of the probe appears yellow on the calor film.
Figure 4A shows the hybridization of the chromosome 4-specific
library in Bluescribe plasmids (the library pBS-4) to a human metaphase
spread wherein no unlabeled human genomic DNA was used, and wherein
the hybridization mixture was applied immediately after denaturation. Both
copies of chromosome 4 are seen as slightly brighter than the other
chromosomes. The small arrows indicate regions that are unstained with the
probe. As in Figure 3 and as in the rest of the Figures below, PI is the
counterstain and fluorescein is used to label the probe.
Figure 4B shows the hybridization of pBS-4 to a human metaphase
spread wherein unlabeled human genomic DNA was used during the
hybridization (Q = 2 of genomic DNA; the meaning of Q is explained infra .
Quantitative image analysis shows that the intensity per unit length of the
chomosome 4s is about 20X that of the other chromosomes. The
chromosome 4s are yellow; the other chromosomes are red due to the
CA 02449414 2003-12-11
- 29 -
propidium iodide counterstain. Two layers of avidin-fluorescein
isothiocyanate have been used to make the target chromosomes sufficiently
bright to be measured accurately. However, the number 4 chromosomes can
be recognized easily after a single layer is applied.
Figure 4C shows the same spread as in Figure 4B but through a filter
that passes only the fluorescein isothiocyanate fluorescence.
Figure 4D shows the detection of a radiation-induced translocation
(arrows) involving chromosome 4s in a human metaphase spread wherein
pBS-4 specific libraries are used. The contrast ratio is about 5X.
Figure 4E shows that normal and two derivative chromosomes
resulting from a translocation between chromosome 4 and 11 (in cell line
RS4;11) can be detected by the compositions and methods of this invention in
interphase nuclei. They appear as three distinct domains.
Figure 4F shows the hybridization of the chromosome 2I-specific
library in Bluescribe plasmids (the library pBS-21) to a metaphase spread of a
trisomy 2I cell line. A small amount of hybridization is visible near the
centromeres of the other acrocentric chromosomes.
Figure 4G shows the same hybridization as in Figure 4F but with
interphase nuclei. Clearly shown are the three chromosome 21 domains.
Figure 4H shows the hybridization with a.pool of 120 single copy
probes from chromosome 4 to a human metaphase spread. The number 4
chromosomes are indicated by arrows.
Figure 5 shows the hybridization of a yeast artificial chromosome
(YAC) clone containing a 580 kb insert of human DNA to a human
metaphase spread. A yellow fluorescein band on each of the chromosome I2s
(at 12q21.I) is visible against the propidium iodide counterstain.
CA 02449414 2003-12-11
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Figure 6 shows the hybridization of DNA from a human/hamster
hybrid cell containing one copy of human chromosome 19 to a human
metaphase spread. A little to the right of the photograph's center are the two
chromosome 19s which are brighter than the other chromosomes in the
spread.
Figure 7 illustrates a representative method of using the polymerase
chain reaction (PCR) to produce probes of this invention which are reduced
in repetitive sequences.
Figure 8 illustrates the locations of probes to the CML breakpoint and
corresponding pattern of staining in both normal and CML metaphase and
interphase nuclei.
The left side shows schematic representations of the BCR gene on
chromosome 22, the ABL gene of chromosome 9, and the BCR-ABL fusion
gene on the Philadelphia chromosome. Also shown are the locations of CML
breakpoints and their relation to the probes (32). The right shows
hybridization patterns expected for the c-hu-ABL, and PEM12 probes to
normal and CML metaphase spreads and interphase nuclei.
Figure 9 shows fluorescence in-situ hybridization (FISH) in
metaphase spreads and interphase nuclei. Panels A and B show ABL and
BCR hybridization to normal metaphase spreads. The ABL signal (A) is
localized to the telomeric portion of 9q and the BCR signal (B) is localized
near the centromere of 22q. Panel C shows that abl staining is localized to
the
telomeric region of Philadelphia chromosome in a case of CML with 46XY, t
(9:22) (q34;q11). Panel D shows that abI staining is interstitial on the
derivative 22 chromosome arising from an insertional event in a case of CML
with 46XY ins (22:9)(q11;q34). Panel E illustrates that the K562 Bell line
CA 02449414 2003-12-11
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presents multiple signals localized to a region of the interphase nucleus.
Identical staining pattern was seen with BCR probe indicating BCR -ABL
fusion gene amplification. Panel F presents a metaphase spread from the
K562 cell line showing fusion gene amplification localized to a single
chromosome.
Figure 10 illustrates fluorescence in-situ hybridization in CML
interphase nuclei with ABL , and BCR probes visualized
simultaneously through a double band pass filter. Cells from a CML patient
show the signals resulting from the hybridization to the
BCR-ABL fusion gene and ~ hybridization signals to the
normal BCR and ABL genes on chromosomes 22 and 9.
Figure 11 illustrates some exemplary probe strategies for detection of
structural aberrations. The design of the binding pattern, colors etc., of the
probe can be optimized for detection of genetic abnormalities in metaphase
and/or interphase cells. Different patterns may have advantages for
particular applications. The drawings in Figure 1I illustrate some of the
patterns useful for detection of some abnormalities. 'The examples are
representative and not meant to be exhaustive; different patterns can be
combined to allow for the detection of multiple abnormalities in the same
cell.
In the drawings of Figure 11, the metaphase chromosomes are shown
with probe bound to both chromatids. The interphase nuclei are pictured to
be in a stage of the cell cycle prior to replication of the portion of the
chromosome to which the probe binds; thus there is only one chromatid for
each interphase chromosome. When the probe binding is restricted to only a
portion of a chromosome, the signal is indicated as either a black or white
~,:~:~,....__~. ._. _____. ___~.
CA 02449414 2003-12-11
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circle. Sueh a representation is employed to indicate different colors or
otherwise distinguishable characteristics of the staining. Patterns containing
more than two distinguishable characteristics (three colors, different ratios
of
colors etc.) permit more complex staining patterns than those illustrated.
Chromosomal locations of the breakpoints in the DIeTA are indicated with
horizontal lines next to the abnormal chromosomes.
a. Section a) represents the use of a probe which stains a whole
chromosome. Such a probe can be used to detect a translocation that occurs
anywhere along the chromosome. The color photograph of Figure I2 shows
use of such a stain for chromosome 22 to detect a translocation, in this case
that which occurs with CML. Such an approach to staining is not very useful
in interphase nuclei since the region of the nucleus that is stained is
relatively large; overlaps in the stained regions can make interpretation
difficult in many nuclei.
b. Section b) represents the reduction of the stained region of the
chromosome shown in a) to that in the vicinity of a breakpoint, providing
information focused on events in that region. The staining pattern can be
continuous or discontinuous across the breakpoint, just so that some binding
is on both sides of the breakpoint. Such a staining pattern requires only one
"color", but gives no information about which other genomic region may be
involved in the exchange.
c. Section c) represents the use of a probe which binds to sequences
which come together as a result of the rearrangement and allows for the
detection in metaphase and interphase cells. In this case the different
sequences are stained with different "colors". Such a staining pattern is that
used in the examples of Section VIII of the this application.
______ ____._. .~, . ~~ ~,:~~,~ ~m,~~-w_.______ . m~-..,~-u,.7,..nw~ ~__. _. .
__~__~___
CA 02449414 2003-12-11
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d. Section d) represents an extension of c) by including staining of
both sides of both breakpoints involved in the rearrangement. Different
"colors" are used as indicated. The additional information supplied by the
more complex staining pattern may assist with interpretation of the nuclei. It
might also permit recognition of an apparent insertional event as discussed
herein.
e. Section e) represents the detection of an inversion in one
homologue of a chromosome.
f. Section f) represents a staining pattern useful in the detection of a
deletion. A deletion could also be detected with a probe that stains only the
deleted region; however, lack of probe binding may be due to reasons other
than deletion of the target sequence. The flanking regions stained a different
"color" serve as controls for hybridization.
Figure 12 illustrates a staining pattern to detect a rearrangement by
staining a whole chromosome, in this case a rearrangement of chromosome
22 associated with CML. The metaphase spread of this figure is from a CIvIL
cell that has been stained with a probe which binds all alang chromosome 22.
Probe-stained regions appear yellow. The rest of the DNA has been stained
with the red-fluorescing chemical stain propidium iodide. The entirely
yellow chromosome is a normal copy of chromosome 22. Just below said
normal chromosome 22 is the Philadelphia chromosome, a small part yellow
and part red chromosome. Below and to the right of the Philadelphia
chromosome is the abnormal chromosome 9 (red) with the distal part of
chromosome 22 (yellow) attached. The photograph of this figure illustrates
the staining pattern represented in part a) of the previous figure.
CA 02449414 2003-12-11
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DETAILED DESCRIPTION OF THE INVENTION
This invention concerns the use of nucleic acid probes to stain
targeted chromosomal material in patterns which can extend along one or
more whole chromosomes, and/or along one or more regions on one or
more chromosomes, including patterns which extend over an entire genome.
The staining reagents of this invention facilitate the microscopic and/or flow
cytometric identification of normal and aberrant chromosomes and provide
for the characterization of the genetic nature of particular abnormalities,
such
as, genetic rearrangements. The term "chromosome-specific" is herein
defined to encompass the terms "target specific" and "region specific", that
is,
when the staining composition is directed to one chromosome, it is
chromosome-specific, but it is also chromosome-specific when it is directed,
for example, to multiple regions on multiple chromosomes, or to a region of
only one chromosome, or to regions across the entire genome. The term
chromosome-specific originated from the use of recombinant DNA libraries
made by cloning DNA from a single normal chromosome type as the source
material for the initial probes of this invention. Libraries made from DNA
from regions of one or more chromosomes are sources of DNA for probes for
that region or those regions of the genome. The probes produced from such
source material are region-specific probes but are also encompassed within
the broader phrase "chromosome-specific" probes. The term "target specific"
is interchangeably used herein with the term "chromosome-specific".
The word "specific°' as commonly used in the art has two somewhat
different meanings. The practice is followed herein. "Specific" may refer to
the origin of a nucleic acid sequence or to the pattern with which it will
hybridize to a genome as part of a staining reagent. For example, isolation
CA 02449414 2003-12-11 ""-~-...._
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and cloning of DNA from a specified chromosome results in a "chromosome-
specific library". [Eg., Van Dilla et ai., "Human Chromosome-Specific DNA
Libraries: Construction and Availability," Biotechnolo~~y, 4:537 (1986).)
However, such a library contains sequences that are shared with other
chromosomes. Such shared sequences are not chromosome-specific to the
chromosome from which they were derived in their hybridization properties
since they will bind to more than the chromosome of origin. A sequence is
"chromosome-specific" if it binds only to the desired portion of a genome.
Such sequences include single-copy sequences contained in the target or
repetitive sequences, in which the copies are contained predominantly in the
target.
"Chromosome-specific" in modifying °'staining reagent" refers to the
overall hybridization pattern of the nucleic acid sequences that comprise the
reagent. A staining reagent is chromosome-specific if useful contrast between
the target and non-target chromosomal material is achieved (that is, that the
target can be adequately visualized).
A probe is herein defined to be a collection of nucleic acid fragments
whose hybridization to the target can be detected. The probe is labeled as
described below so that its binding to the target can be visualized. The probe
is produced from some source of nucleic and sequences, for example, a
collection of clones or a collection of poiymerase chain reaction (PCR)
products. The source nucleic acid may then be processed in some way, for
example, by removal of repetitive sequences or blocking them with unlabeled
nucleic acid with complementary sequence, so that hybridization with the
resulting probe produces staining of sufficient contrast on the target. Thus,
the word probe may be used herein to refer not only to the detectable nucleic
_n .....~.A~_ _.__....~w..~~."~a:. ~ ~_._____.______.r ~..._ri.__ ___
CA 02449414 2003-12=11 _.~..._._~_T_.__. _.___,
-36-
acid, but also to the detectable nucleic acid in the form in which it is
applied to
the target, for example, with the blocking nucleic acid, etc. The blocking
nucleic acid may also be mentioned separately. What "probe" refers to
specifically should be clear from the context in which the word is used.
When two or more nucleic acid probes of this invention are mixed
together, they produce a new probe which when hybridized to a target
according to the methods of this invention, produces a staining pattern that
is
a combination of the staining patterns individually produced by the
component probes thereof. Thus, the terms "probe" and "probes" (that is, the
singular and plural forms) can be used interchangeably within the context of a
staining pattern produced. For example, if one probe of this invention
produces a dot on chromosome 9, and another probe produces a band on
chromosome II, together the two probes form a probe which produces a
dot/band staining pattern.
The term "labeled" is herein used to indicate that there is some
method to visualize the bound probe, whether or not the probe directly
carries some modified constituent. Section III infra describes various means
of directly labeling the probe and other labeling means by which the bound
probe can be detected.
The terms "staining" or "painting°' are herein defined to mean
hybridizing a probe of this invention to a genome or segment thereof, such
that the probe reliably binds to the targeted chromosomal material therein
and the bound probe is capable of being visualized. The terms "staining" or
"painting" are used interchangeably. The patterns resulting from "staining"
or "painting" are useful for cytogenetic analysis, mare particularly,
molecular
cytogenetic analysis. The staining patterns facilitate the microscopic and/or
_._.. ....______... . _._.__._ .~...~~~.,~~~"~~,."~~~__.._._ __ ..~
,,,°d._.-.._._..__. __ ___._. ___..__.
CA 02449414 2003-12-11
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flow cytometric identification of normal and abnormal chromosomes and the
characterization of the genetic nature of particular abnormalities. Section
III
infra describes methods of rendering the probe visible. Since multiple
compatible methods of probe visualization are available, the binding patterns
of different components of the probe can be distinguished-for example, by
color. Thus, this invention as capable of producing any desired staining
pattern on the chromosomes visualized with one or more colors (a multi-
color staining pattern) and/'or other indicator methods. The term "staining"
as defined herein does not include the concept of staining chromosomes with
chemicals as in conventional karotyping methods although such
conventional stains may be used in conjunction with the probes of this
invention to allow visualization of those parts of the genome where the
probe does not bind. The use of DAPI and propidium iodide for such a
purpose is illustrated in the figures.
The phrase "high complexity" is defined herein to mean that the
probe, thereby modified contains on the order of 50;000 (50 kb) or greater, up
to many millions or several billions, of bases of nucleic acid sequences which
are not repeated in the probe. For example, representative high complexity
nucleic acid probes of this invention can have a complexity greater than 50
kb,
greater than 100,000 bases (100 kb), greater than 200,000 (200 kb), greater
than
500,000 bases (500 kb), greater than one million bases (1 Mb), greater than
2 Mb, greater than 10 Mb, greater than 100 Mb, greater than 500 Mb, greater
than 1 billion bases and still further greater than several billion bases.
The term "complexity" is defined herein according to the standard for
nucleic acid complexity as established by Britten et al., Methods of Enzymol.,
29:363 (1974). See also Cantor and Schimmel, Biophysical Chemistry: Part III:
CA 02449414 2003-12-11
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The Behavior of Biological Macromolecules, at 1228-1230 (Freeman and Co.
1980) for further explanation and exemplification of nucleic acid complexity.
The complexity preferred for a probe composition of this invention is
dependent upon the application for which it is designed. In general, the
larger the target area, the more complex is the probe. It is anticipated that
the
complexity of a probe needed to produce a desired pattern of landmarks on a
chromosome will decrease as hybridization sensitivity increases, as progress
is
made in hybridization technology. As the sensitivity increases, the
reliability
of the signal from smaller target sites will increase. Therefore, whereas from
about a 40 kb to about a 100 kb target sequence may be presently necessary to
provide a reliable, easily detectable signal; smaller target sequences should
provide reliable signals in the future. Therefore, as hybridization
sensitivity
increases, a probe of a certain complexity, for example, I00 kb, should enable
the user to detect considerably more loci in a genome than are presently
reliably detected; thus, more information will be obtained with a probe of the
same rnmplexity. The term "complexity" therefore refers to the complexity of
the total probe no matter how many visually distinct loci are to be detected,
that is, regardless of the distribution of the target sites over the genome.
As indicated above, with current hybridization techniques it is
possible to obtain a reliable, easily detectable signal with a probe of about
40 kb
to about 100 kb (eg. the probe insert capacity of one or a few cosmids)
targeted
to a compact point in the genome. Thus, for example, a complexity in the
range of approximately 100 kb now permits hybridization to both sides of a
tumor-specific translocation. The portion of the probe targeted to one side of
the breakpoint can be labeled differently from that targeted to the other side
of
the breakpoint so that the two sides can be differentiated with different
colors,
CA 02449414 2003-12-11
_~g..
for example. Proportionately increasing the complexity of the probe permits
analysis of multiple compact regions of the genome simultaneously. The
conventional banding patterns produced by chemical stains may be replaced
accorcling to this invention with a series of probe-based, color coded (for
example), reference points along each chromosome or significant regions
thereof.
Uniform staining of an extended contiguous region of a genome, for
example, a whole chromosome, requires a probe complexity proportional to
but substantially less than, the complexity of the target region. The
complexity required is only that necessary to provide a reliable,
substantially
uniform signal on the target. Section V.B, infra, demonstrates that
fluorescent staining of human chromosome 2I, which contains about
50 megabases (Mb) of DNA, is sufficient with a probe complexity of about I
Mb. Figure 4H illustrates hybridization of about 400 kb of probe to human
chromosome 4, which contains about 200 Mb of DNA. In that case, gaps
between the hybridization of individual elements of the probe are visible.
Figures 4B and 4F demonstrate the results achieved with probes made up of
entire libraries for chromosomes 4 and 21, respectively. The chromosomes
are stained much more densely as shown in Figures 4B and 4F than with the
lower complexity probe comprising single-copy nucleic acid sequences used to
produce the pattern of Figure 4H.
Increasing the complexity beyond the minimum required for
adequate staining is not detrimental as long as the total nucleic acid
concentration in the probe remains below the point where hybridization is
impaired. The decrease in concentration of a portion of a sequence in the
probe is compensated for by the increase in the number of target sites. In
fact,
CA 02449414 2003-12-11
-40-
when using double-stranded probes, it is preferred to maintain a relatively
low concentration of each portion of sequence to inhibit reassociation before
said portion of sequence can find a binding site in the target.
The staining patterns of this invention comprise one or more
"bands". The term '°band" is herein defined as a reference point in a
genome
which comprises a target nucleic acid sequence bound to a probe component,
which duplex is detectable by some indicator means, and which at its
narrowest dimension provides for a reliable signal under the conditions and
protocols of the hybridization and the instrumentation, among other
variables, used. A band can extend from the narrow dimension, of a sequence
providing a reliable signal to a whole chromosome to multiple regions on a
number of chromosomes.
The probe-produced bands 'of this invention are to be distinguished
from bands produced by chemical staining as indicated above in the
Back r~ ound. The probe-produced bands of this invention are based upon
nucleic acid sequence whereas the bands produced by chemical staining
depend on natural characteristics of the chromosomes, but not the actual
nucleic acid sequence. Further, the banding patterns produced by chemical
staining are only interpretable in terms of metaphase chromosomes whereas
the probe-produced bands of this invention are useful both for metaphase
and interphase chromosorxtes.
One method of forming the probes of the present invention is to pool
many different low complexity probes. Such a probe would then comprise a
"heterogeneous mixture'° of individual cloned sequences. The number of
clones required depends on the extent of the target area and the capacity of
the
CA 02449414 2003-12-11
-41 -
cloning vector. If the target is made up of several discrete, compact loci,
that
is, single spots at the limit of microscopic resolution, then about
40 kb, more preferably 100 kb, for each spot gives a reliable signal given
current techniques. The portion of the probe for each spot may be made up
from, for example, a single insert from a yeast artificial chromosome (YAC),
from several cosmids each containing 35-40 kb or probe sequence, or from
about 25 plasmids each with 4 kb of sequence.
Representative heterogeneous mixtures of clones exemplified herein
include phage (Figures l, 2 and 3), and plasmids (Figure 4). Yeast artificial
chromosomes (YACS) (Figure 5), and a single human chromosome in an
inter-species hybrid cell (Figure 6) are examples of high complexity probes
for
single loci and an entire chromosome that can be propagated as a single clone.
A base sequence at any point in the genome can be classified as either
"single-copy" or "repetitive". For practical purposes the sequence needs to be
long enough so that a complementary probe sequence can form a stable
hybrid with the target sequence under the hybridization conditions being
used. Such a length is typically in the range of several tens to hundreds of
nucleotides.
A "single-copy sequence" is that wherein only one copy of the target
nucleic acid sequence is present in the haploid genoxne. "Single-copy
sequences" are also known in the art as "unique sequences". A "repetitive
sequence" is that wherein there are more than one copy of the same target
nucleic acid sequence in the genome. Each copy of a repetitive sequence need
not be identical to all the others. The important feature is that the sequence
be sufficiently similar to the other members of the family of repetitive
sequences such that under the hybridization conditions being used, the same
CA 02449414 2003-12-11
-42-
fragment of probe nucleic acid is capable of forming stable hybrids with each
copy. A "shared repetitive sequence" is a sequence with some copies in the
target region of the genome, and some elsewhere.
When the adjectives "single-copy", "repetiti.ve", "shared repetitive",
among other such modifiers, are used to describe sequences in the probe, they
refer to the type of sequence in the target to which the probe sequence will
bind. Thus, "a repetitive probe" is one that binds to a repetitive sequence in
the target; and "a single-copy probe" binds to a single-copy target sequence.
Repetitive sequences occur in multiple copies in the haploid genome.
The number of copies can range from two to hundreds of thousands, wherein
the Alu family of repetitive DNA are exemplary of the latter numerous
variety. The copies of a repeat may be clustered or interspersed throughout
the genome. Repeats may be clustered in one or more locations in the
genome, for example, repetitive sequences occurring near the centromeres of
each chromosome, and variable number tandem repeats (VNTRs)
[Nakamura et al, Scienee, 235:1616 (1987)]; or the repeats may be distributed
over a single chromosome [for example, repeats found only on the X
chromosome as described by Bardoni et al., C~genet. Cell Genet., 46:575
(I987)]; or the repeats may be distributed over all the chromosomes, for
example, the Alu family of repetitive sequences.
Herein, the terms repetitive sequences, repeated sequences and
repeats are used interchangeably.
Shared repetitive sequences can be clustered or interspersed.
Clustered repetitive sequences include tandem repeats which are so named
because they are contiguous on the DNA molecule which forms the backbone
of a chromosome. Clustered repeats are associated with well-defined regions
CA 02449414 2003-12-11
-43-
of one or more chromosomes, e.g., the centromeric region. If one or more
clustered repeats form a sizable fraction of a chromosome, and are shared
with one or more non-target regions of the genome and are consequently
removed from the heterogeneous mixture of fragments employed in the
invention or the hybridization capacity thereof is disabled, perfect
uniformity
of staining of the target region may not be possible. That situation is
comprehended by the use of the term "substantially uniform" in reference to
the binding of the heterogeneous mixture of labeled nucleic acid fragments to
the target.
Chromosome-specific staining of the current invention is
accomplished by using nucleic acid fragments that hybridize to sequences
specific to the target. These sequences may be either single-copy or
repetitive,
wherein the copies of the repeat occur predominantly in the target area.
Figure 4H and the results of the work detailed in section V infra indicate
that
probes can be made of single-copy sequences. However, in probes such as that
of Figure 4B, low-copy chromosome-specific repeats [Nakamura et al., and
Bardoni et al., supra] may contribute to the hybridization as well.
If nucleic acid fragments complementary to non-target regions of the
genome are included in the probe, for example, shared repetitive sequences or
non-specific sequences, their hybridization capacity needs to be sufficiently
disabled or their prevalence sufficiently reduced, so that adequate staining
contrast can be obtained. Section V and Figure 4H show examples of
hybridization with probes that contain pools of clones in which each clone
has been individually selected so that it hybridizes to single-copy sequences
or
very low copy repetitive sequences. The remaining figures illustrate use of
probes that contain fragments that could have hybridized to high-copy
CA 02449414 2003-12-11
repetitive sequences, but which have had the hybridization capacity of such
sequences disabled.
The nucleic acid probes of this invention need not be absolutely
specific for the targeted portion of the genome. They are intended to produce
"staining contrast". "Contrast" is quantified by the ratio of the stain
intensity
of the target region of the genome to that of the other portions of the
genome.
For example, a DNA library produced by cloning a particular chromosome,
such as those listed in Table I, can be used as a probe capable of staining
the
entire chromosome. The library contains sequences found only on that
chromosome, and sequences shared with other chromosomes. In a
simplified (approximately true to life) model of the human genome, about
half of the chromosomal DNA falls into each class. If hybridization with the
whole library were capable of saturating all of the binding sites, the target
chromosome would be twice as bright (contrast ratio of 2) as the others since
it
would contain signal from the specific and shared sequences in the probe,
whereas the other chromosome would only have signal from the shared
sequences. Thus, only a modest decrease in hybridization of the shared
sequences in the probe would substantially enhance the contrast.
Contaminating sequences which only hybridize to non-targeted sequences, for
example, impurities in a library, can be tolerated in the probe to the extent
that said sequences do not reduce the staining contrast below useful levels.
In reality all of the target sites may not be saturated during the
hybridization, and many other mechanisms contribute to producing staining
contrast, but this model illustrates one general consideration in using probes
targeted at a large portion of a genome.
~ 02449414 2003-12-11 J~' .s~,j~,Q.~~ ~.,....._~.,.._ _ -.... .-...-"...v--
.__.. _ _. _.
-45-
The required contrast depends on the application for which the probe
is designed. When visualizing chromosomes and nuclei, etc.,
microscopically, a contrast ratio of two or greater is often sufficient for
identifying whole chromosomes. In Figures 4D-F, the contrast ratio is 3-5.
The smaller the individual segments of the target region, the greater the
contrast needs to be to permit reliable recognition of the target relative to
the
fluctuations in staining of the non-targeted regions. When quantifying the
amount of target region present in a cell nucleus by fluorescence intensity
measurements using flow cytometry or quantitative microscopy, the required
contrast ratio is on the order of 1/T or greater on average for the genome,
where T is the fraction of the genome contained in the targeted region.
When the contrast ratio is equal to 1 /T, half of the total fluorescence
intensity
comes from the target region and half from the rest of the genome. For
example, when using a high complexity probe for chromosome I, which
comprises about 10% of the genome, the required contrast ratio is on the
order of 10, that is, for the chromosome 1 fluorescence intensity to equal
that
of the rest of the genome.
Background staining by the probe, that is, to the non-target region of
the genome, may not be uniform. Figure 4F shows that a chromosome ~I
spetific probe contains probe fragments that hybridize weakly to compact
regions near the centromeres of other acrocentric human chromosomes.
This degree of non-specificity does not inhibit its use in the illustrated
applications. For other applications, removal of or further disabling the
hybridization capacity of the probe fragments that bind to these sequences
may be necessary.
CA 02449414 2003-12-11
U 3
-4b-
For other applications, repetitive sequences that bind to centromeres,
for example, alpha-satellite sequences, and/or telomeres can be part of the
chromosome-specific staining reagents wherein the target includes some or
all of the centromeres and/or telomeres in a genome along with perhaps
other chromosomal regions. Exemplary of such an application would be that
wherein the staining reagent is designed to detect random structural
aberrations caused by clastogenic agents that result in dicentric chromosomes
and other structural abnormalities, such as translocations. Addition of
sequences which bind to all centromeres in a genome, for example to the
probe used to create the staining pattern of Figure 4D, would allow more
reliable distinguishing between dicentrics and translocations:
Application of staining reagents of this invention to a genome results
in a substantially uniform distribution of probe hybridized to the targeted
regions of a genome. The distribution of bound probe is deemed
"substantially uniform" if the targeted regions of the genome can be
visualized with useful contrast. For example, a target is substantially
uniformly stained in the case wherein it is a series of visually separated
loci if
mast of the loci are visible in most of the cells.
"Substantial proportions" in reference #o the base sequences of
nucleic acid fragments that are comple- mentary to chromosomal DNA
means that the complementarity is extensive enough so that the fragments
form stable hybrids with the chromosomal DNA under the hybridization
conditions used. In particular, the term comprehends the situation where the
nucleic acid fragments of the heterogeneous mixture possess some regions of
sequence that are not perfectly complementary to target chromosomal
CA 02449414'2003-12-11 . ..___. __. _ _~ _~ ___ _,-
-47-
material. The stringency can be adjusted to control the precision of the
complementarity required for hybridization.
The phrase "metaphase chromosomes" is herein defined to mean not
only chromosomes condensed in the metaphase stage of mitosis but includes
any condensed chromosomes, for example, those condensed by premature
chromosome condensation.
To disable the hybridization capacity of a nucleic acid sequence is
herein sometimes abbreviated as "disabling the nucleic acid sequence".
The methods and reagents of this invention find a particularly
appropriate application in the field of diagnostic cytogenetics, particularly
in
the field of diagnostic interphase cytogenetics. Detecting genetic
rearrangements that are associated with a disease, such as cancer, are a
specific
application of the chromosome specific reagents and staining methods of this
invention.
Contiguous gene syndromes are an example of the genetic
rearrangements that the probes and methods of this invention can identify.
Contiguous gene syndromes are characterized by the presence of several
closely spaced genes which are in multiple and/or reduced copy number.
Down syndrome is an example of a contiguous gene syndrome wherein an
extra copy of a chromosomal region containing several genes is present.
Particularly described herein is the application of chromosome
specific reagents and methods for detecting genetic rearrangements that
produce the BCR-ABL fusion associated with CML. Such reagents are
exemplary of disease specific, in this case tumor specific, probes which can
be
labeled, directly and/or indirectly, such that they are visualizable when
bound
to the targeted chromosomal material, which in the case of CML, is the
_ . ._.. ..M__.____ . .._~.~., ~~. ~.~_~. ~.m",.~~m _~...~..~~___ ..
CA 02449414 2003-12-11
- 48 _
vicinity of the translocation breakpoint regions of chromosomal regions 9q34
and 22qI1 known to be associated with CML. In the examples provided in
t
Section VIII of this application, the probes are labeled such that a dual
color
fluorescence is produced in the staining pattern of said probes upon in situ
hybridization [fluorescent in situ hybridisation (FISH)]; however, staining
patterns can be produced in many colors as well as other types of signals, and
any visualization means to signal the probe bound to ifs target can be used in
the methods of this invention.
Section VIII herein describes representative methods and reagents of
this invention to detect genetic rearrangements. The examples of Section VIII
concern genetic rearrangements that produce the BCR-ABL fusion that is
characteristic of CML. The approach in such examples is based on FISH with
probes from chromosomes 9 and 22 that flank the fused BCR and ABL
sequences in essentially all cases of CML (Figure 8). The probes when
hybridized to the chromosomal material of both normal and abnormal Bells
produce staining patterns that are different as illustrated in Figures 8-12.
The
staining patterns produced by such exemplary probes are different in normal
and abnormal cells; the staining pattern present when the genetic
rearrangement occurs is distinctively altered from That of the staining
pattern
shown by hybridizing the probes to chromosomal anaterial that does not
contain the genetic rearrangement. Further, staining patterns are
distinctively different for one type of genetic rearrangement versus another.
For example, the staining patterns produced upon hybridization of nucleic
acid probes of this invention to chromosomal material containing a genetic
rearrangement associated with ALL is distinctively different from that
produced upon hybridization of such probes to chromosomal material
__._____..~-.~. a ....~.._ _.. .,-,~"..~.W. _.._..__ ._
CA 02449414 2003-12-11
-49-
containing the BCR-ABL fusion characteristic of CML. Thus, the methods
and reagents of this invention provide for differential diagnosis of related
diseases.
The examples of Section VIII provide for the diagnosis of CML based
upon the proximity of the fluorescent signals in the staining patterns, and
rely upon a I micron cutoff point for determination of the presence of a
fusion. The proximity distance of signals is only one characteristic, among
many others, of signals that can be used to detect the presence of a genetic
rearrangement. Further, the proximity distance is dependent on the
particular cell preparation techniques employed and the size of the nuclei
therein, and for a particular cell preparation is relative depending on the
distance between signals in normal and abnormal cells.
The staining patterns exemplified in the examples of Section VIII are
representative of one type of probe strategy. Many other probe strategies can
be employed. Figure 11 illustrates some other exemplary probe strategies for
detecting genetic rearrangements, the patterns of which can be modified and
optimized and otherwise varied to detect particular genetic rearrangements.
Use of other disease specific reagents of this invention would be
analogous to the methods detailed in Section VIII for CML. For example, the
diagnosis and study of acute lymphocytic leukemia (ALL) may be ,
accomplished by replacing the BCR probe (PEM12) of Section VIII with a probe
from the 5' end of the BCR gene. ALL is of particular interest because the Ph'
chromosome is the most common cytogenetic abnormality in that disease,
and the presence of such a chromosome is indicative of a very aggressive
neoplasm.
CA 02449414 2003-12-11
-50-
The methods and reagents herein exemplified, particularly in Section
VIII, provide for the means to distinguish between cytogenetically similar but
genetically different diseases. "Cytogenetically" in that particular context
refers to a similarity determined by conventional banding analysis. CML and
ALL are in that context cytogenetically similar in that conventional banding
analysis can not distinguish them because the breakpoints associated with
each are so close together in the human genome.
Further, this invention provides methods and reagents that can be
used in a cytogenetic research mode for the study of the molecular bases of
genetic disease. For example, if an abnormality in a person's karyotype is
noted by conventional banding analysis, the probes and reagents of this
invention can be used to detect any genetic rearrangements in the vicinity of
said abnormality. The underlying molecular basis of the abnormality can be
determined by the methods and reagents of this invention, and the resulting
differences at the genetic level may be indicative of different treatment
plans
and prognostically important. The underlying genetic rearrangements may
be found to be consistently associated with a set of phenotypic
characteristics
in a population.
The following sections provide examples of making and using the
staining compositions of this invention and are for purposes of illustration
only and not meant to limit the invention in any way. The following
abbreviations are used.
CA 02449414 2003-12-11
-51-
Abbreviations
B N - bicarbonate buffer with NP-40
DAPI - 4,6-diamidino-2-phenylindole
DCS - as in fluorescein-avidin DCS (a commercially
available cell
sorter grade
of fluorescein
Avidin
D)
AAF - N-acetoxy-N-2-acetyl-aminofluorene
EDTA - ethylenediaminetetraacetate
FACS - fluorescence-activated cell sorting
FITC - fluorescein isothiocyanate
IB - isolation buffer
NP-40 - non-ionic detergent commercially available from
Sigma as Nonidet P-40 (St. Louis, MO)
PBS - phosphate-buffered saline
PI - propidium iodide
PMSF - phenylmethylsulfonyl fluoride
PN - mixture of 0.1 M NaH2PC74 and 0.1 M
buffer Na2HP04, pH 8; 0.1 % NP-40
PNM - Pn buffer plus 5% nonfat dry milk (centrifuged);
buffer 0.02% Na azide
SDS - sodium dodecyl sulfate
SSC - 0.15 M NaCI /0.015 M Na citrate, pH 7
VNTR - variable number tandem repeat
CA 02449414 2003-12-11
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I. Methods of Preparing Chromosome-Specific Staining Reagents
LA. Isolation of Chromosome-Specific DNA and
Formation of DNA Fragment Libraries.
The first step in a preferred method of making the compositions of
the invention is isolating chromosome-specific DNA (which term includes
target-specific and/or region-specific DNA, as indicated above, wherein
specific refers to the origin of the DNA). This step includes first isolating
a
sufficient quantity of the particular chromosome type or chromosomal
subregion to which the staining composition is directed, then extracting the
DNA from the isolated chromosomes) or chromosomal subregion(s). Here
"sufficient quantity" means sufficient for earrying out subsequent steps of
the
method. Preferably, the extracted DNA is used to create a library of DNA
inserts by cloning using standard genetie engineering techniques.
Preferred cloning vectors include, but are not limited to, yeast
artificial chromosomes (YACS), plasmids, bacteriophages and cosmids.
Preferred plasmids are Bluescribe plasmids; preferred bacteriophages are
lambda insertion vectors, more preferably Charon 4A, Charon 21A,
Charon 35, Charon 40 and CEM11; and preferred cosmids include Lawrist 4,
Lawrist 5 and sCosl.
As indicated above, the DNA can be isolated from any source.
Chromosome-specific staining reagents can be made from both plant and
animal DNA according to the methods of this invention. Important sources
of animal DNA are mammals, particularly primates or rodents wherein
primate sources are more particularly human and monkey, and rodent
sources are more particularly rats or mice, and more particularly mice.
____ __w~~_ _ ___..__~.~-~~,..:.,~_-._. ~~. .,", _ __. ,e__~_______
CA 02449414 2003-12-11
- 53 -
1. Isolating DNA from an Entire Chromosome. A preferred means for
isolating particular whole chromosomes (specific chromosome types) is by
direct
flow sorting [fluorescence-activated cell sorting (FACS)] of metaphase
chromosomes with or without the use of interspecific hybrid cell systems. For
some species, every chromosome can be isolated by currently available sorting
techniques. Most, but not all, human chromosomes are currently isolatable by
flow
sorting from human cells, Carrano et al., "Measurement and Purification of
Human
Chromosomes by Flow Cytometry and Sorting," Proc. Natl. Acad. Sci., Vol. 76,
pp.
1382-1384 (1979). Thus, for isolation of some human chromosomes, use of the
IO humanlrodent hybrid cell system may be necessary, see Kao, "Somatic Cell
Genetics and Gene Mapping," International Review of Cy_tolo~c.v., Vol. 85, pp.
109-146 (1983), for a review; and Gusella et al., "isolation and Localization
of
DNA Segments from Specific Human Chromosomes," Proc. Natl.. Acad. Sci., Vol.
77, pp. 2829-2833 (1980). Chromosome sorting can be done by commercially
IS available fluorescence-activated sorting machines, e.g., Becton Dickinson
FACS-II, Coulter Epics V sorter, or special purpose sorters optimized for
chromosome sorting or like instrument.
DNA is extracted from the isolated chromosomes by standard techniques,
e.g., Marmur, "A Procedure for the Isolation of Deoxyribonucleic Acid from
20 Micro-Organisms,'° L Mol. Biol., Vol. 3, pp. 208-218 (1961 ); or
Maniatis et al.,
Molecular Gloningr A Laboratonr Manual (Cold Spring Harbor Laborat~ry, 1982)
pp. 280-281.
Generation of insert libraries from the isolated chromosome-specific DNA is
carried out using standard genetic engineering techniques, e.g.,
CA 02449414 2003-12-11
-54-
Davies et al., "Cloning of a Representative Genomic Library of the Human X
Chromosome After Sorting by Flow Cytometry," Na. furs, Vol. 293, pp. 374-376
(1981 ); Krumlauf et al., "construction and Characterization of Genomic
Libraries
from Specific Human Chromosomes," Proc. Natl. Acad. Sci., Vol. 79, pp.
2971-2975 (1982); Lawn et al., "The Isolation and Characterization of Linked
Delta-and-Beta-Globin Genes from a Cloned Library of Human DNA." ~ej_I, Vol.
15, pp. 1157-1174 (1978); and Maniatis et al., "Molecular Cloning: A
Laboratory
Manual," (Cold Springs Harbor Laboratory, 1982), pp. 256-308, Van Dilla et
al.,
:id, Fuscoe, en , 52:291 (1987); and Fuscoe et al., C~toaenet. Cell Genet,
43:79
(1986).
Recombinant DNA libraries for each of the human chromosomes have
been constructed by the National Laboratory Gene Library Project and are
available from the American Type Culture Collection. [Van Dilla et al.,
Biotechnology, 4:537 (1986). Small insert-containing libraries were
constructed by
complete digestion of flow sorted human chromosome genomic DNA with Hin 111
or EcoRl and cloning into the Lambda insertion vector Charon 21A. The vector
is
capable of accepting human inserts of up to 9.1 kb in size. Thus, Hindlll (or
EcoRl)
restriction fragments greater than 9.1 kb will not be recovered from these
libraries.
The observed average insert size in these. libraries is approximately 4 kb. A
representative list of the Hindlll chromosome-specific libraries with their
ATCC
accession numbers are shown in Table 1.
_.___.~, ~~ .-~~,.,.~"~"-.~,e~.~._....n._.a.GT.~..,:;~.,_._~~,~..a ~
~.~.Y..__~.~.__ ___
CA 02449414 2003-12-11
-55-
TABLE 1
HUMAN CHROMt?SOME - SPECIFIC GENOMIC LIBRARIES
IN' CHARON 22A VECTOR
CHROMOSOME ATCC # LIBRARY
1 57753 LLO1NS01
1 57754 LL01NS02
2 57744 LL02NS01
3 57751 LL03NS01
4 57700 LL04NS01
4 57745 LL04NS02
57746 LL05NSOI
6 57701 LL06NS01
7 57755 LL07NSOI
8 57702 LL08NS02
9 57703 LL09NSOI
57736 LLIONS01
I1 57704 LL1INS01
12 57756 LL12NS01
13 57705 LLI3NSOI
13 57757 LLI3NS02
I4 57706 LL14NSOI
14/I5 57707 LL99NSOI
57737 LL15NSOI
16 57758 LLI6NS03
I7 57759 LLI7NS02
18 57710 LLI8NS01
19 57711 LLI9NSOI
57712 LL20NS01
21 57713 LL21NS02
22 57714 LL22NSOI
X 57747 LLOXNSOI
Y 57715 LLOYNS01
CA 02449414 2003-12-11
-56-
Alternatively, the extracted DNA from a sorted chromosome type can be
amplified by the polymerase chain reaction (PCR) rather than cloning the
extracted DNA in a vector or propagating it in a cell line. Appropriate tails
are
added to the extracted DNA in preparation for PCR, F~eferences for such PCR
procedures are set out in Section I.B infra.
Other possible methods of isolating the desired sequences from hybrid cells
include those of Schmeckpeper et al., "Partial Purification and
Characterization of
DNA from Human X Chromosome," Proc. Natl. Acad. Sci.. Vol. 76, pp. 6525-6528
(1979); or Olsen et al., a r (in Backgroundl.
I0 2. Isolating DNA from a PQ ion Qf~hromosome Among the methods that
can be used for isolating region-specific chromosomal DNA include the
selection
of an appropriate chromosomal region from DNA that has previously been
mapped, for example, from a library of mapped cosmids; the sorting of
derivative
chromosomes, for example, by FACS; the microdissection of selected
chromosomal material, subtractive hybridization; identification of an
appropriate
hybrid cell containing a desired chromosomal fragment, extracting and
amplifying
the DNA, and selecting the desired amplified DNA; and the selection of
appropriate chromosomal material from radiation hybrids. The standard genetic
engineering techniques outlined above in subsection I.A.1 are used in such
procedures well-known to those in the art. Amplification of the region-
specific DNA
can be perfomled by cloning in an appropriate vector, propagating in an
appoopriate cell line,, andlor by the use of PCR (see I.B infra).
A preferred method of isolating chromosomal region-specific DNA is to ase
mapped short DNA sequences to probe a library of longer DNA
_ __._.~ m..."~~p ~~..~.~.......__.~_ ____.~.~~~,.*~,~,,~M~.~~*..~._~_._._.
~.~._.~_.. __. __ ._____ _ .__..
CA 02449414 2003-12-11
sequences, wherein the latter library has usually been cloned in a different
vector. For example, a probe cloned in a plasmid can be used to probe a
cosmid or yeast artificial chromosome (YAC) library. By using an initial seed
probe, overlapping clones in the larger insert library can be found (a process
called "walking"), and a higher complexity probe can be produced for reliable
staining of the chromosomal region surrounding the seed probe. Ultimately,
when an entire genome for a species has been mapped (for example, by the
Human Genome Project for the human species), ordered clones for the entire
genome of the species will be available. One can then easily select the
appropriate clones to form a probe of the desired specificity.
Another method of isolating DNA from a chromosomal region or
regions (or also a whole chromosome) is to propagate such a chromosomal
region or regions in an appropriate cell line (for example, a hybrid cell line
such as a human/hamster hybrid cell), extract the DNA from the cell line and
clone it in an appropriate vector and select clones containing human DNA to
form a library. When a hybrid cell is used, the chromosomes in the hybrid
cell containing the human chromosomal material may be separated by flow
sorting (FACS) prior to cloning to increase the frequency of human clones in
the library. Still further, total DNA from the hybrid cell can be isolated and
labeled without further cloning and used as a probe, as exemplified in
Figure 6.
3. Single-Stranded Probes. In some cases, it is preferable that the
nucleic acid fragments of the heterogeneous mixture consist of single-
stranded RNA or DNA. Under some conditions, the binding efficiency of
single-stranded nucleic acid probes has been found to be higher during in situ
hybridization, e.g., Cox et al., "Detection of mRNAs in Sea Urchin Embryos by
CA 02449414 2003-12-11
_~8_
In Situ Hybridization Using Asymmetric RNA Probes," Developmental
Biolo Vol. 101, pp. 485-502 (1984):
Standard methods are used to generate RNA fragments from isolated
DNA fragments. For example, a method developed by Green et al., described
in Cell, Vol. 32, pp. 68I-694 (1983), is commercialy available from Promega
Biotec (Madison, WI) under the tradename "Riboprobe." Other transcription
kits suitable for use with the present invention are available from United
States Biochemical Corporation (Cleveland, OH) under the tradename
"Genescribe." Single-stranded DNA probes can be produced with the single-
stranded bacteriophage MI3, also available in kit form, e.g. Bethesda Research
Labora- tories (Gaithersburg, MD). The hybridization5 illustrated in Figure 4
were performed with the libraries of Table 1 subcloned into the Bluescribe
plasmid vector (Stratagene, La Jolla, CA). The Bluescribe plasmid contains
RNA promoters which permit production of single-stranded probes.
Section IX, infra provides methods for preparing and applying non-
self-complementary single-stranded nucleic acid probes that improve signal-
to-noise ratios attainable in in situ hybridization by reducing non-specific
and
mismatched binding of the probe. That section further provides for methods
of denaturing double-stranded target nucleic acid which minimizes single-
stranded regions available for hybridization that are non-complementary to
probe sequences. Briefly, probe is constructed by treating DNA with a
restriction enzyme and an exonuclease to form template/primers for a DNA
polymerase. The digested strand is resynthesized in the presence of labeled
nucleoside triphosphate precursor, and the labeled single-stranded fragments
are separated from the resynthesized fragments to form the probe. The target
CA 02449414 2003-12-11
-59-
nucleic acid is treated with the same restriction enzyme used to construct the
probe, and is treated with an exonuclease before application of the probe.
LB. PCR
Another method of producing probes of this invention includes the
use of the polymerase chain reaction [PCR]. [For an explanation of the
mechanics of PCR, see Saiki et al., Science, 230:1350 (1985) and U.S. Patent
Nos. 4,683,195, 4,683,202 (both issued July 28, 198 and 4,800,159 (issued
January 24, 1989).] Target-specific nucleic acid sequences, isolated as
indicated
above, can be amplified by PCR to produce target-specific sequences which are
reduced in or free of repetitive sequences. The PCR primers used for such a
procedure are for the ends of the repetitive sequences, resulting in
amplification of sequences flanked by the repeats.
Figure 7 illustrates such a method of using PCR wherein the
representative repetitive sequence is Alu. If only short segments are
amplified, it is probable that such sequences are free of other repeats, thus
providing DNA reduced in repetitive sequences.
One can further suppress production of repetitive sequences in such a
PCR procedure by first hybridizing complementary sequences to said
repetitive sequence wherein said complementary sequences have extended
non-complementary flanking ends or are terminated in nucleotides which do
not permit extension by the polymerase. The non-complementary ends of
the blocking sequences prevent the blocking sequences from acting as a PCR
primer during the PCR process.
_ _ _~ ~_ ___.
CA 02449414 2003-12-11
- 60 -
II. Removal of Repetitive Sequences andfor Disabling
the Hybridization Ca~aci , of Repetitive Sequences
Typically a probe of the current invention is produced in a number of
steps including: obtaining saurce nucleic acid sequences that are
complementary to the target region of the genome, labeling and otherwise
processing them so that they will hybridize efficiently to the target and can
be
detected after they bind, and treating them to either disable the
hybridization
capacity or remove a sufficient proportion of shared repetitive sequences, or
both disable and remove such sequences. The order of these steps depends on
the specific procedures employed.
The following methods can be used to remove shared repetitive
sequences and/or disable the hybridization capacity of such shared repetitive
sequences. Such methods are representative and are expressed schematically
in terms of procedures well known to those of ordinary skill the art, and
which can be modified and extended according to parameters and procedures
well known to those in the art.
1. Single-copy probes. A single-copy probe consists of nucleic acid
fragments that are complementary to single-copy sequences contained in the
target region of the genome. One method of constructing such a probe is to
start with a DNA library produced by cloning the target region. Some of the
clones in the library will contain DNA whose entire sequence is single-copy;
others will contain repetitive sequences; and still others will have portions
of
single-copy and repetitive sequences. Selection, on a clone by clone basis,
and
pooling of those clones containing only single-copy sequences will result in a
probe that will hybridize specifically to the target region. The single-copy
nature of a clone can ultimately be established by Southern hybridization
__. ~____ _ .___
..__ ._" ; .~...~_.__... _
CA 02449414 2003-12-11
-61-
using standard techniques. figure 4H shows hybridization with I20 clones
selected in this way from a chromosome 4 library.
Southern analysis is very time consuming and labor intensive.
Therefore, less perfect but more efficient screening methods for obtaining
candidate single-copy clones are useful In Section ~,l.B, examples of
improved methods are provided for screening individual phage and pfasmid
clones for the presence of repetitive DNA using hybridization with genomic
DNA. The screening of plasmid clones is more efficient, and approximately
80% of selected clones contain only single-copy sequences; the remainder
contain low-copy repeats. However, probes produced in this way can produce
adequate staining contrast, indicating that the low-copy repetitive sequences
can be tolerated in the probe (see subsection 3 of this section).
A disadvantage of clone by clone procedures is that a clone is
discarded even if only a portion of the sequence it contains is repetitive.
The
larger the length of the cloned nucleic acid, the greater the chance that it
will
contain a repetitive sequence. Therefore, when nucleic acid is propagated in a
vector that contains Iarge inserts such as a cosmid, YAC, or in a cell line,
such
as hybrid cells, it may be advantageous to subclone it in smaller pieces
before
the single-copy selection is performed. The selection procedures just outlined
above do not discriminate between shared and specific repetitive sequences;
clones with detectable repetitive sequences of either type are not. used in
the
probe.
2. Individual tesfing of h3rbridization properties. The hybridization
specificity of a piece of nucleic acid, for example, a clone, can be-tested by
in
situ hybridization. If under appropriate hybridization conditions it binds to
single-copy or repetitive sequences specific for the desired target region, it
can
CA 02449414 2003-12-11 ""'' :- - ".-_.._.. _
- 62 -
be included in the probe. Many sequences with specific hybridization
characteristics are already known, such as chromosome-specific repetitive
sequences CTrask et al., supra, (1988) and references therein], VNTRs,
numerous mapped single copy sequences. More are continuously being
mapped. Such sequences can be included in a probe of this invention.
3. Bulk Procedures. In many genomes, such as the human genome, a
major portion of shared repetitive DNA is contained in a few families of
highly repeated sequences such as Alu. A probe that is substantially free of
such high-copy repetitive sequences will produce useful staining contrast in
many applications. Such a probe can be produced from some source of
nucleic acid sequences, for example, the libraries of Table I, with relatively
simple bulk procedures. Therefore, such bulk procedures are the preferred
methods for such applications.
These methods primarily exploit the fact that the hybridization rate of
complementary nucleic acid strands increases as their concentration increases.
Thus, if a heterogeneous mixture of nucleic acid fragments is denatured and
incubated under conditions that permit hybridization, the sequences present
at high concentration will become double-stranded more rapidly than the
others. The double-stranded nucleic acid can then be removed and the
remainder used as a probe. Alternatively, the partially hybridized mixture
can be used as the probe, the double-stranded sequences being unable to bind
to the target. The following are methods representative of bulk procedures
that are useful for producing the target-specific staining of this invention.
3a. Self-reassociation of the probe. Double-stranded probe nucleic
acid in the hybridization mixture is denatured and then incubated under
hybridization conditions for a time sufficient for the high-copy sequences in
___ ____.__.___~____~..~, __ ~ ,.
CA 02449414 2003-12-11 __. ' I ...._.~ _.._.._... .
the probe to become substantially double-stranded. The hybridization
Bnixture is then applied to the sample. The remaining labeled single-stranded
copies of the highly repeated sequences bind throughout the sample
producing a weak; widely distributed signal. The binding of the multiplicity
o~ low-copy sequences specific for the target region of the genome produce an
easily distinguishable specific signal. .
Such a method is exemplified in Section VLB (infra) with
chromosome-specific libraries for chromosomes 4 and 2I (pBS4 and pBS21) as
probes for those chromosomes. [Pinkel et al., PNAs ~U.SA), 85:9138-9142
(December 2988)]. The hybridization mix, containing a probe concentration in
the range of 1-IO ng/ul was heated to denature the probe and incubated at
37°C for 24 hours prior to application to the sample.
3b. Use of blocking nucleic acid. Unlabeled nucleic acid sequences
which are complementary to those sequences in the probe whose
hybridization capacity it is desired to inhibit are added to the hybridization
mixture. The probe and blocking nucleic acid are denatured, if necessary, and
incubated under appropriate hybridization conditions. The sequences to be
blocked become double-stranded more rapidly than the others, and therefore
are unable to bind to the target when the hybridization mixture is applied to
the target. In some cases, the blocking reaction occurs so quickly that the
incubation period can be very short, and adequate results can be obtained if
the hybridization mix is applied to the target immediately after denaturation.
A blocking method is generally described by Sealy et al., "Removal of Repeat
Sequences form Hybridization Probes", Nucleic Acid Research, 13:1905 (1985)
Examples of blocking nucleic
CA 02449414 2003-12-11 .__.__ ....._ ' -- ..,.~.
acids include genomic DNA, a high-copy fraction of genomic DNA and
particular sequences as outlined below (i-iii).
3b.i. Genomic DNA. Genomic DNA contains all of the nucleic acid
sequences of the organism in proportion to their copy-number in the
genome. Thus, adding genomic DNA to the hybridization mixture increases
the concentration of the high-copy repeat sequences more than low-mpy
sequences, and therefore is more effective at blocking the former. However,
the genomic DNA does contain copies of the sequences that are specific to the
target and so will also reduce the desired chromosome-specific binding if too
much is added. Guidelines to determine how much genomic DNA to add
(see 3.e. Concept of Q, infra) and examples of using genomic blocking DNA
are provided below. The blocking effectiveness of genomic DNA can be
enhanced under some conditions by adjusting the timing of its addition to
the hybridization mix; examples of such timing adjustments are provided
with Protocol I and Protocol II hybridizations illustrated in Figures 4B
through E (Protocol I) and Figure 4F (Protocol II) and detailed in Section VI,
infra.
3b.ii. H. igh-copy fraction of genomic DNA. The difficulty with use of
genomic DNA, is that it also blocks the hybridization of the low-copy
sequences, which are predominantly the sequences that give the desired target
staining. Thus, fractionating the genomic DNA to obtain only the high-copy
sequences and using them for blocking overcomes this difficulty. Such
fractionation can be done, for example, with hydroxyapatite as described
below (3c.i).
3b.iii. Specified sequences. The blocking of a particular sequence in
the probe can be accomplished by adding many unlabeled copies of that
CA 02449414 2003-12-11
-65-
sequence. For example, Alu sequences in the probe can be blocked by adding
cloned Alu DNA. Blocking DNA made from a mixture of a few clones
containing the highest copy sequences in the human genome can be used
effectively with chromosome-specific libraries for example, those of Table I.
Alternatively, unlabeled nucleic acid sequences from one or more
chromosome-specific libraries could be used to block a probe containing
labeled sequences from one or more other chromosome-specific libraries.
The shared sequences would be blocked whereas sequences occurring only on
the target chromosome would be unaffected. Figure 4F shows that genomic
DNA was not effective in completely blocking the hybridization of a sequence
or sequences shared by human chromosome 21 and the centromeric regions
of the other human acrocentric chromosomes. When a clone or clones
containing such a sequence or sequences is or are eventually isolated,
unlabeled DNA produced therefrom could be added to the genomic blocking
DNA to improve the specificity of the staining.
3c. Removal of Sequences.
3c.i. Hydrox~rapatite. Single- and double-stranded nucleic acids
have different binding characteristics to hydroxyapatite. Such characteristics
provide a basis commonly used for fractionating nucleic acids.
Hydroxyapatite is commerically available (eg. Bio-Rad Laboratories,
Richmond, CA). The fraction of genomic DNA containing sequences with a
particular degree of repetition, from the highest copy-number to single-copy,
can be obtained by denaturing genomic DNA, allowing it to reassociate under
appropriate conditions to a particular value of Cot, followed by separation
using hydroxyapatite. The single- and double-stranded nucleic acid can also
be discriminated by use of SI nuclease. Such techniques and the concept of
,. ;
CA 02449414 2003-12-11
-66-
Cot are explained in Britten et al., "Analysis of Repeating DNA Sequences by
Reassociation", in Method ~s 'n En~Xmology, Vol. 29, pp. 363-418 (1974).
The single-stranded nucleic acid fraction produced in 3a. or 3b. above can
be separated by hydroxyapatite and used as a probe. Thus, the sequences that
have been blocked {that become double-stranded) are physically removed. The
probe can then be stored until needed. The probe can then be used without
additional blocking nucleic acid; or its staining contrast can perhaps be
improved
by additonal blocking.
3c.ii. Reaction with irhmobilized nucleic acid. Removal of particular
sequences can
IO also be, accomplished by attaching single-stranded "absorbing" nucleic acid
sequences to a solid support. Single-stranded source nucleic acid is
hybridized to
the immobilized nucleic acid. After the hybridization, the unbound sequences
are
collected and used as the probe. For example, human genomic DNA can be used
to absorb repetitive sequences from human probes. One such mefihod is
described by Brison et al., "General Method for Cloning Amplified DNA by
Differential Screening with Genomic Probes," I~I~.I~~uiar and Cellular
BioI~Vol,
2, pp. 578-587 (1982). Briefly, minimally sheared human genomic DNA is bound
to
diazonium cellulose or a like support. The source DNA, appropriately cut into
fragments, is hybridized against the immobilized DNA to Cot values in the
range of
about 1 to 100. The preferred stringency of the hybridization conditions may
vary
depending on the base composition of the DNA. Such a procedure could remove
repetitive sequences from chromosome-specific libraries, for example, those of
Table I, to produce a probe capable of staining a whole human chromosome.
m. .... ._... ", ~,." .«..,, ,~:~,, ~ , . ,~,~ ,~ ,....___~.a.".~......~
.,."..n "...~.~__ .. .... ._ ..... . _ ._. _
~~~.-....
_.
CA 02449414 2003-12-11
-67-
3d. Blocking non-targeted sequences in the targeted genome.
Blocking of non-targeted binding sites in the targeted genome by
hybridization with unlabeled complementary sequences will prevent binding
of labeled sequences in the probe that have the potential to bind to those
sites.
For example, hybridization with unlabeled genomic DNA will render the
high-copy repetitive sequences in the target genome double-stranded. Labeled
copies of such sequences in the probe will not be able to bind when the probe
is subsequently applied.
In practice, several mechanisms combine to produce the staining
contrast. For example, when blocking DNA is added to the probe as in 3b
above, that which remains single-stranded when the probe is applied to the
target can bind to and block the target sequences. Tf the incubation of the
probe with the blocking DlrTA is minimal, then the genomic DNA
simultaneously blocks the probe and competes with the probe for binding
sites in the target.
3e. Concept of As mentioned in section 3b.i above, it is necessary
to add the correct amount of genomic DNA to achieve the best compromise
between inhibiting the hybridization capacity of high-copy repeats in the
probe and reduczng the desired signal intensity by inhibition of the binding
of
the target-specific sequences. The following discussion pertains to .use of
genomic blocking DNA with probes produced by cloning or otherwise
replicating stretches of DNA from the target region of the genome. Thus, the
probe contains a representative sampling of the single-copy, chromosome-
specific repetitive sequences, and shared repetitive sequences found in the
target. Such a probe might range in complexity from 100 kb of sequence
derived from a small region of the genome, for example several closely
CA 02449414 2003-12-11 i._._ __
-68-
spaced cosmid clones; to many millions of bases, for example a combination
of multiple libraries from Table I. The discussion below is illustrative and
can be extended to other situations where different blocking nucleic acids are
used. The following discussion of Q is designed only to give general
guidelines as to how to proceed.
The addition of unlabeled genomic DNA to a hybridization mix
containing labeled probe sequences increases the concentration of all of the
sequences, but increases the concentration of the shared sequences by a larger
factor than the concentration of the target-specific sequences because the
shared sequences are found elsewhere in the genome, whereas the target-
specific sequences are not. Thus, the reassociation of the shared sequences is
preferentially enhanced so that the hybridization of the labeled copies of the
shared sequences to the target is preferentially inhibited.
To quantity this concept, first consider one of the sequences, repeat or
single-copy, that hybridize specifically to the ith chromosome in a
hybridization mixture containing a mass mp of probe DNA from the ith
chromosome library of Table 1 (for example) and mb of unlabeled genomic
DNA. The number of labeled copies of the sequence is proportional to xnp.
However, the number of unlabeled copies is proportional to fimb, where fi is
the fraction of genomic DNA contained on the ith chromosome. Thus, the
ratio of unlabeled to labeled copies of each of the sequences specific for the
target chromosome, is fimb/mp, which is defined herein as Q. For normal
human chromosomes, O.Q16<_fi<0.08 [Mendelsohn et al., Science, 179:1126
(1973)). For representative examples described in Section VLB (infra ,
f4 = 0.066 and f21 = 0.016. For a probe targeted at a region comprised of L
base
_ _..,_ ~.~~~"".~.,w,.: ~~ ~_.___~ _ . .n.,.~~.a..,._ -..__... _ _..____. _._
_ __ ._
CA 02449414 2003-12-11
_g9_
pairs, fi=L/G where G is the number of base pairs in a genome (approximately
3 x 109 bases for humans and other mammals). Thus, Q = (L/G) (mb/mp).
Now consider a shared sequence that is distributed more-or-less
uniformly over the genome, for example, Alu. The number of labeled copies
is proportional to mp, whereas the number of unlabeled copies is
proportional to mb. Thus, the ratio of unlabeled to labeled copies is
mb/mp = Q/fi. This is true for all uniformly distributed sequences, regardless
of copy number. Thus adding genomic DNA increases the concentration of
each specific sequence by the factor I + Q, whereas each uniformly distributed
sequence is increased by the larger factor 1 + Q/fi. Thus, the reassociation
rates of the shared sequences are increased by a larger factor than those of
the
specific sequences by the addition of genomic DNA.
It can be shown that roughly half of the beneficial effect of genomic
DNA on relative reassociation rates is achieved when Q =1, and, by Q = 5,
there is essentially no more benefit to be gained by further increases. Thus,
the protocol I hybridizations of Section VLB infra keep Q<_5.
To illustrate the use of genomic blacking DNA, it is convenient to
consider a model of a genome wherein 50% of the DNA is comprised of
specific sequences (both repetitive and single-copy) and the other 50% of the
DNA is comprised of shared repetitive sequences that are distributed
uniformly over the genome. Thus, according to the model, if the target is L
bases (that is, the probe contains fragments representing L bases of the
target
area or areas of the genorne), sequences containing L/2 bases will be specific
to
the target, and L/2 will be shared with the entire genome.
_.. ____. _._.~...~......~~.,~wu___.~_.,.~a.., .~.~.~. a",
CA 02449414 2003-12-11
~i
_7d_
III. Labeling_,the Nucleic Acid Fragments of the Heterogeneous Mixture.
Several techniques are available for labeling single- and double-
stranded nucleic acid fragments of the heterogeneous mixture. They include
incorporation of radioactive labels, e.g. Harper et al. Chromosoma, Vol 83,
pp.
431-439 (1984); direct attachment of fluorachromes or enzymes, e.g. Smith et
al:, Nucleic Acids Research, Vol. 13, pp. 2399-2412 (1985), and Connolly et
al.,
Nucleic Acids Research, Vol. 13, pp. 4485-4502 (I985); and various chemical
modifications of the nucleic acid fragments that render them detectable
immunochemically or by other affinity reactions, e.g. Tchen et al.,
"Chemically Modified Nucleic Acids as Immunodetectable Probes in
Hybridization Experiments," Proc. Natl. Acad. Sci., Vol 81, pp. 3466-3470
(1984); Richardson et al., "Biotin and Fluorescent Labeling of RNA Using T4
RNA Ligase," Nucleic Acids Research, Vol. 11, pp. 6167-6184 (1983);
Langer et al., "Enzymatic Synthesis of Biotin-Labeled Polynudeotides: Novel
Nucleic Add Affinity Probes," Proc. Natl. Acad. Sci., Vol. 78, pp. 6633-6637
(I98I); Brigati et al., "Detection of Viral Genomes in Cultured Cells and
Paraffin-Embedded Tissue Sections Using Biotin-Labeled Hybridization
Probes," ViroloQV, Vol. 126, pp. 32-50 (1983); Broker et al., "Electron
Microscopic Visualization of tRNA Genes with Ferritin-Avidin: Biotin
Labels," Nucleic Adds Research, Vol. 5, pp. 363-384 (I978); Bayer et al., "The
Use of the Avidin Biotin Complex as a Tool in Molecular Biology," Methods
of Biochemical Analysis, Vol. 26, pp. I-45 (I980) Kuhlmann, Immunoe_nz~me
Techniques in C~tochemistry (Weinheim, Basel, 1984). Langer-Safer et al.,
PNAS USA, 79: 4381 (1982): Landegent et al., Exp. Cell Res.,153; 61 (1984);
and
Hopman et al., Exp. Cell Res.,169: 357 (1987).
CA 02449414 2003-12-11 - .__ __..~",~ ,___._ ., . _. ___.__,__._____
-m -
Exemplary labeling means include those wherein the probe fragments are
blotinylated, modified with N-acetoxy-N-2-acetylaminofluorene, modified with
filuorescein isothiocyanate, modified with mercurylTNP ligand, sulfonated,
digoxigenenated or contain T-T dimers.
The key feature of "probe labeling" is that the probe bound to the target be
detectable. In some eases, an intrinsic feature of the probe nucleic acid,
rather
than an added feature, can be exploited for this purpose. For example,
antibodies
that specifically recognize RNAIDNA duplexes have been demonstrated to have
the ability to recognize probes made from RNA that are bound to DNA targets
IO [Rudkin and Stollar, Nature 2fi5:472-473 (1977)]. The RNA used for such
probes
is unmodified. Probe nucleic acid fragments can be' extended by adding "tails"
of
modified nucleotides or particular normal nucleotides. When a normal
nucleotide
tall is used, a second hybridization with nucleic acid complementary to the
tail and
containing fluorochromes, enzymes, radioactivity, modified bases, among other
labeling means, allows detection of the bound probe. Such a system is
commercially available from Enzo Biochem (BiobridgeT"" Labeling System; Enzo
Biochem. Inc., New York, ~N.Y.).
Another example of a means to visualize the bound probe wherein the nucleic
acid sequences in the pr~ibe do not directly carry some modified constituent
is the
use of antibodies to thymidine dimers. Nakane et al., ~0 (2):229 (1987),
111ustrate
such a method wherein thymine=thymine dimerized DNA (T-T DNA) was used as a
marker for in situ hybridization. The hybridized T-T DNA was detected
immunohistochemically using rabbit anti-T-T DNA antibody.
._ _.~_ ~ 02449414'2003-12-11 --,~.__ ~ ; ..._._.
I;;
- 72 -
All of the labeling techniques disclosed in the above references may
be preferred under particular circurn- stances.
Further, any labeling techniques
known to those in the art would be useful to Label the staining compositions
of this invention. Several factors govern the choice of labeling means,
including the effect of the label on the rate of hybridization and binding of
the
nucleic acid fragments to the chromosomal DNA, the accessibility of the
bound probe to labeling moieties applied after initial hybridization, the
mutual compatibility of the labeling moieties, the nature and intensity of the
signal generated by the label, the expense and ease in which the label is
applied, and the like.
Several different high complexity probes, each labeled by a different
method, can be used simultaneously. The binding of different probes can
thereby be distinguished, for example, by different colors.
IV: In Situ Hybridization.
Application of the heterogeneous mixture of the invention to
chromosomes is accomplished by standard in situ hybridization techniques..
Several excellent guides to the technique are available, e.g., Gall and
Pardue,
"Nucleic Acid Hybridization in Cytological Preparations;' Methods in
Enz~molo~, Vol. 2I, pp. 4?0-48Q (I98I); Henderson, "Cytological
Hybridization to Mammalian Chromo- somes," International Review of
Cytology, Vol. 76, pp. I-46 (I982); and Angerer, et al., "In Situ
Hybridization to
Cellular RNAs;' in Genetic Engineering: Principles and Methods, Setlow and
Hollaender, Eds., Vol: 7, pp. 43-65 (Plenum Press, New York, 1985).
...m... _.... . ~...~. ~ ~~._ --~r.-s~~.~~,.:",~,..~-.- ~.._~_~__._ ._.
CA 02449414 2003-12-11
-73-
Three factors influence the staining sensitivity of the hybridization
probes: (1) effiaency of hybridization (fraction of target DNA that can be
hybridized by probe), (2) detection efficiency (i.e., the amount of visible
signal
that can be obtained from a given amount of hybridization probe), and (3)
level of noise produced by nonspecific binding of probe or components of the
detection system.
Generally in situ hybridization comprises the following major steps:
(1) fixation of tissue or biological structure to be examined, (2) prehybridi-
zation treatment of the biological structure to increase accessibility of
target
DNA, and to reduce nonspecific binding, (3) hybridization of the
heterogeneous mixture of probe to the DNA in the biological structure or
tissue; (4) posthybridization washes to remove probe not bound in specific
hybrids, and (5) detection of the hybridized probes of the heterogeneous
mixture. The reagents used in each of these steps and their conditions of use
vary depending on the particular situation.
The following comments are meant to serve as a guide for applying
the general steps listed above. Some experimentation may be required to
establish optimal staining conditions for particular applications.
In preparation for the hybridization, the probe, regardless of the
method of its production, may be broken into fragments of the size
appropriate to obtain the best intensity and specificity of hybridization. As
a
general guideline concerning the size of the fragments, one needs to
recognize that if the fragments are too long they are not able to penetrate
into
the target for binding and instead form aggregates that contribute background
noise to the hybridization; however, if the fragments are too short, the
signal
intensity is reduced.
CA 02449414 2003-12-11
- '74 -
Under the conditions of hybridization exemplified in Section VI.B wherein
human genomic DNA is used as an agent to block the hybridization capacity of
the
high copy shared repetitive sequences, the preferred size range of the probe
fragments is from about 200 bases to about 2000 bases, more preferably in the
vicinity of 1 kb. When the size of the probe fragments is in about the 800 to
about
1000 base range, the preferred hybridization temperature is about 30°C
to about
45°C, more preferably about 35°C to about 40°C, and still
more preferably about
37°C; preferred washing temperature range is from about 40°C to
about 50°C,
more preferably about 45°C. .
The size of the probe fragments is checked before hybridization to the
target; preferably the size of the fragments is monitored by electrophoresis,
more
preferably by denaturing agarose gel electrophoresis.
Fixatives include acid alcohol solutions, acid acetone solutions,
Petrunkewitsch's reagent, anal various aldehydes such as formaldehyde,
paraformaldehyde, glutaraldehyde, or the like. Preferably, ethanol-acetic acid
or
methanol-acetic acid solutions in about 3:1 proportions are used to fix the
chromosomes in metaphase spreads. For cells or chromosomes in suspension, a
fixation procedure disclosed by Trask, et al., in ~,c~ence. Vol. 230, pp. 1401-
1402
(1985), is useful: Briefly, K2C03 and dimethyisuberimidate (DMS) are added
(from
a 5x concentrated stock solution, mixed immediately before, use) to a
suspension
containing about 5 x 10fi nuclei/ml. Final K2C03 and DMS concentrations are 20
mM and 3 mM, respectively. After 15 minutes at 25°C, the pH is adjusted
from
10.0 to 8.0 by the addition of 50 microliters of 100 mM citric acid per
milliliter of
suspension. Nuclei are washed once by _
_.-...~..~.~.....
CA 02449414 2003-12-11 ij
_75_
centrifugation (300g, 10 minutes, 4°C in 50 mM KCI, 5 mM Hepes buffer,
at pH
9.0, and 10 MM MgS04).
A preferred fixation procedure for cells or nuclei in suspension is disclosed
by Trask et al., F~lum. genet. 78:251-259 (1988). Briefly, nuclei are fixed
for about
10 minutes at room temperature in 1 % paraformaldehyde .in PBS, 50 mM MgS04,
pH 7.6 and washed twice. Nuclei are .resuspended in isolation buffer (1B) (50
mM
KCI, 5 mM HEPES, 10 mM MgS04, 3 mM dithioerythritol, 0.15 mglml RNase, pH
8.0)10.05% Triton X-100TM at 108/m!.
Frequently before in situ hybridization chromosomes are treated with
agents to remove proteins. Such agents include enzymes or mild acids. Pronase,
pepsin or proteinase K are frequently used enzymes. A representative acid
treatment Is 0.02-0.2 N HCI, followed by high temperature (e.g., 70°C)
washes.
Optimization of deproteinization requires a combination of protease
concentration
and digestion time that maximizes hybridization, but does not cause
unacceptable
loss of morphological detail. Optimum conditions vary according to tissue
types
and method of fixation. Additional fixation after protease treatment may be
useful.
Thus, for particular applications, some experimentation may be required to
optimize protease treatment:
In. some cases pretreatment with RNase may be desirable to remove
residual RNA from the target. Such removal can be accomplished by incubation
of
the fixed chromosomes in 50-100 microgram /milliliter RNase in ZX SSC (where
SSC is a solution of 0.15M NaCI and 0..015M sodium citrate) for a period of 1-
2
hours at room temperature.
..~.w~... __. ..__w.~m..~~.a...~~~.~, ~n~ ~. ~~..~,..._~_-..~...
~....~_....______.__ __
CA 02449414 2003-12-11
-76-
The step of hybridizing the probes of the heterogeneous probe
mixture to the chromosomal DNA involves (1) denaturing the target DNA so
that probes can gain access to complementary single-stranded regions, and
(2) applying the heterogeneous mixture under conditions which allow the
probes to anneal to rnmplementary sites in the target. Methods for
denaturation include incubation in the presence of high pH, low pH, high
temperature, or organic solvents such as formamide, tetraalkylammonium
halides, or the like, at various combinations of concentration and
temperature. Single-stranded DNA in the target can also be produced with
enzymes, such as, Exonudease III [van Dekken et al., Chromosoma (Berl)
97:1-5 (1988)]. The preferred denaturing procedure is incubation for between
about I-10 minutes in formamide at a concentration between about 35-95
percent in 2X SSC and at a temperature between about 25-70°C.
Determination of the optimal incubation time, concentration, and
temperature within these ranges depends on several variables, including the
method of fixation and type of probe nucleic acid (for example, DNA or
RNA).
After. the chromosomal DNA is denatured, the denaturing agents are
typically removed before application of the heterogeneous probe mixture.
Where formamide and heat are the primary denaturing agents, removal is
conveniently accomplished by several washes with a solvent, which solvent
is frequently chilled, such as a 70%, 85%, I00% cold ethanol series.
Alternatively the composition of the denaturant can be adjusted as
appropriate for the in situ hybridization by addition of other consitutents or
washes in appropriate solutions. The probe and target nucleic acid may be
CA 02449414 2003-12-11
denatured simultaneously by applying the hybridization mixture and then
heating to the appropriate temperature.
The ambient physiochemical conditions of the chromosomal DNA
and probe during the time the heterogeneous mixture is applied is referred to
herein as the hybridization conditions, or annealing conditions. Optimal
hybridization conditions for particular applications can be adjusted by
controlling several factors, including concentration of the constituents,
incubation time of chromosomes in the heterogeneous mixture, and the
concentrations, complexities, and lengths of the nucleic acid fragments
making up the heterogeneous mixture. Roughly, the hybridization
conditions must be sufficiently close to the melting temperature to minimize
nonspecific binding. On the other hand, the conditions cannot be so stringent
as to reduce correct hybridizations of complementary sequences below
detectable levels or to require excessively Long incubation times.
The concentrations of nucleic acid in the hybridization mixture is an
important variable. The concentrations must be high enough so that
sufficient hybridization of respective chromosomal binding sites occurs in a
reasonable time (e.g., within hours to several days). hIigher rnncentrations
than that necessary to achieve adequate signals should be avoided so that
nonspecific binding is minimized. An important practical constraint on the
concentration of nucleic acid in the probe in the heterogeneous mixture is
solubility. Upper bounds exist with respect to the fragment concentration,
i.e.,
unit length of nucleic acid per unit volume, that can be maintained in
solution and hybridize effectively.
In the representational examples described in Section VLB (infra , the
total DNA concentration in the hybridization mixture had an upper limit on
.. "_~ _~ _ . _ _ ~..~_~,~. b ,... ~. ~ ,...__. ___ .~~~~."", . ~ .__ .
CA 024494~14 2003-12-11
_7s_
the order of I ug/ul. Probe concentrations in the range of 1-20 ng/ul were
used for such whole chromosome staining. The amount of genomic blocking
DNA was adjusted such that Q was less than 5. At the low end of probe
concentration, adequate signals were obtained with a one hour incubation,
that is, a time period wherein the probe and blocking DNA are maintained
together before application to the targeted material, to block the high-copy
sequences and a 16 hour hybridization. Signals were visible after two hours
of hybridization. The best results (bright signals with highest contrast)
occurred after a 100 hour hybridization, which gage the low-copy target-
specific sequences more opportunity to find binding sites. At the high end of
the probe concentration, bright signals are obtained after hybridizations of
16
hours or less; the contrast was reduced since more labeled repetitive
sequences were included in the probe.
The fixed target object can be treated in several ways either during or
after the hybridization step to reduce nonspecific binding of probe DNA.
Such treatments include adding nonprobe, or "carrier", DNA to the
heterogeneous mixture, using coating solutions, such as Denhardt's solution
(Biochem. Bioph~rs. Res. Commun., Vol. 23, pp. 641-645 (1966), with the
heterogeneous mixture, incubating for several minutes, e.g., 5-20, in
denaturing solvents at a temperature 5-10°C above the hybridization
temperature, and in the case of RNA probes, mild treatment with single
strand RNase (e.g., 5-IO micrograms per millileter RNase) in 2X SSC at room
temperature for I hour).
._"~.~.._...~. ___..__
CA 02449414 2003-12-11
-79-
V. ~,r_~n~s_o~e-Saecific Staining Reagents Com r~ising~
Selected Swig-Copy Seguences
V.A. ll~laking end Usina a Stainina Reaaent Saecific to
Human Chromosome 21
V.A.1, isolation of Chromosome 21 and
Construction of a Chromosome 21-Specific Library
DNA fragments from human chromosome-specific Libraries are available
from the National Laboratory Gene Library Project through the American Type
Culture Collection (ATCC), Rockvitle, MD. DNA fragments from chromosome 21
were generated by the procedure described by Fuscoe et al., in
°'Construction of
Fifteen Human Chromosome-Specific DNA Libraries from Flow-Purified.
Chromosomes," Cytog~enet. C~tl genet.. Vol. 43, pp. 79-86 (1986). Briefly, a
human diploid fibroblast culture was established from newborn foreskin tissue.
Chromosomes of the cells were isolated by the MgSU4 method of van den Engh et
al., ~vfometerv Vol. 5, pp. 108-123 (1984), and stained with the fluorescent
dyes-Hoechst 33258 and Chromomycin A3. Chromosome 21 was purified on the
Lawrence Livermore National Laboratory high speed sorter, described by Peters
et al., ~vtar meter Vol. 6, pp. 290-301 (1985).
After sorting, chromosome concentrations were approximately 4 x 1051m1.
Therefore, prior to DNA extraction, the chromosomes (0.2 - 1.0 x 10g) were
concentrated by centrifugation at 40,000 x g for 30 minutes at 4°C. The
pellet was
then resuspended in 100 microliters of DNA isolation buffer (15 mM NaCI, 10'
mM
EDTA, 10 mM Tris HCI pH 8.0) containing 0.5% SDS and 100 micrograms/mt
proteinase K. After overnight incubation at 37°C, the proteins were.
extracted twice
with phenol:chloroform: ' isoamyl alcohol (25:24:1 ) and once with
chloroform:isoamyl alcohol (24:1 ). Because of
CA 02449414 2003-12-11
_gp_
the small amounfis of DNA, each organic phase was reextracted with a small
amount of 10 mM Tris pH 8.0, 1 mM EDTA (TE). Aqueous layers were combined
and transferred to a Schleich-er and Schuell mini-collodion membrane
(#UH020/25) and dialyzed at room temperature against TE for 6-8 hours. The
purified DNA solution was then digested with 50 units of Hindlll (Bethesda
Research Laboratories, Inc.) in 50 mM NaCI, 10 mM Tris HCI pH 7.5, 10 mM
MgCl2, 1 mM dithiothreitol. After 4 hours at 37°C, the reaction was
stopped by
extractions with phenol and chloroform as described above. The aqueous phase
was dialyzed against water overnight at 4°C in a mini-collodion bag and
then 2
micrograms of Charon 21AT"" arms cleaved with Hindlll and treated with calf
alkaline phosphatase (Boehringer Mannheim) were added. This solution was
concentrated under vacuum to a volume of 50-100 rnicroliters and transferred
to a
0.5 ml.microfuge tube where the DNA was precipitated with one-tenth volume 3M
sodium acetate pH 5.0 and 2 volumes ethanol. The precipitate was collected by
centrifugation, washed with cold 70% ethanol, and dissolved in 10 microliters
of
TE.
After allowing severe! hours for the DNA. to dissolve, 1 microliter of 1 OX
ligase buffer (0.5M Tris HCI pH 7.4, 0.1 M MgCl2, 0.1 M dithiothreitol, 10 mM
ATP,
1 mg/ml bovine serum albumin) and 1 unit of T4 ligase (Bethesda Research
Laboratory, Inc.) were added. The ligation reaction was incubated at
10°C for
16-20 hours and 3 microliter aliquots were packaged into phage particles using
i~
'vitro extracts prepared from _E_. coli strains BHB 2688 and BHB 2690,
.described by
Hohn in Methods in En~rmolcagv Vol. 68, pp. 299-309 (1979) Molecular Cloning:
A Late rator~ Manual. (Cold Spring Harbor Laboratory, New York, 1982).
Briefly,
both extracts were prepared by sonication and combined at the time of in vivQ
packaging. These extracts
_.m.",.~.y' 02449414 2003-12-11 ~.-_.~~
-81-
packaged wild-type lambda DNA at an efficiency of 1-5 x 108 plaque forming
units (pfu) per microgram. The resultant phage were amplified on E. coli
LE392 at a density of approximately 10'1 pfu/ 150 mm dish for 8 hours to
prevent plaques from growing together and to minimize differences in
growth rates of different recombinants. The phage were eluted from the agar
in 10 ml SM buffer (50 mM Tris HCl pH 7.5, 10 mM MgS04, 100 mM NaCI,
0.01 % gelatin) per plate by gentle shaking at 4°C for 12 hours. The
plates were
then rinsed with an additional 4 ml of SM. After pelleting cellular debris,
the
phage suspension was stored over chloroform at 4°C.
V.A.2. Construction and Use of Chromosome 2I-
Specific Stain for Staining Chromosome 21 of Human Lymphocytes
Clones having unique sequence inserts are isolated by the method of
Benton and Davis, Science, Vol. 196, pp. 180-182 (1977). Briefly, about 1000
recombinant phage are isolated at random from the chromosome 21-specific
library. These are transferred to nitrocellulose and probed with nick
translated total genomic human DNA.
Of the clones which do not show strong hybridization, approximately
300 are picked which contain apparent unique sequence DNA. After the
selected clones are amplified, the chromosome 21 insert in each clone is 32P
labeled and hybridized to Southern blots of human genomic DNA digested
with the same enzyme used to construct the chromosome 21 library, i.e.,
Hind III. Unique sequence containing clones are recognized as those that
produce a single band during Southern analysis. Roughly, 100 such clones are
selected for the heterogeneous mixture. The unique sequence clones are
amplified, the inserts are removed by Hind III digestions, and the inserts are
_ ~.M~.. "~~..,-,~ _,-_.~._~.....~..~.~ ~,.~..-___ .__._._..,,F"~,~",n
A~_.m___ ___.....~ w..~~.,-_.___ _ .. _
CA 02449414 2003-12-11 'W
-82-
separated from the phage arms by ge( electrophoresis. The probe DNA fragments
(i.e., the unique sequence inserts) are removed from the gel and biotinylated
by
nick translation (e.g., by a kit available from Bethesda Research
Laboratories).
Labeled DNA fragments are separated from the nick translation reaction using
small spin columns made in 0.5 ml Eppendorph tubes filled with SephadexT"" G-
50
(medium) swollen . in 50 mM Tris, 1 mm EDTA, 0.1 % SDS, at pH 7.5. Human
lymphocyte chromosomes are prepared following Harper et al, Proc. N to i-Acid.
Sei.. Vol. 78, pp. 4458-4460 (1981 ). Metaphase and interphase cells were
washed
3 times in phosphate buffered saline, fixed in methanol-acetic acid (3:1 ) and
dropped onto cleaned microscope slides. Slides are stored in .a nitrogen
atmosphere at -20°C.
Slides carrying interphase cells and/or metaphase spreads are removed
from the ~ nitrogen, heated to 65°C for 4 hours in air, treated with
RNase (100
microgramslml for 1 hour at 37°C), and dehydrated in an ethanol series.
They are
then treated with proteinase K (60 ng/ml at 37°C for 7.5 minutes) and
dehydrated.
The proteinase K concentration is adjusted depending on the cell type and
enzyme lot so that almost no phase microscopic image of the chromosomes
remains on the dry slide. The hybridization mix consists of (final
concentrations)
50 percent formamide, 2X SSC, 10 percent dextran sulfate; 500 micrograms/ml
carrier DNA (sonicated herring sperm D.NA), and 2.0 microgram/ml biotin-
labeled
chromosome 21-specific DNA, This mixture is applied to the slides at a density
of
3 microliters/cm2 under a glass coverslip and sealed with rubber cement. After
overnight Incubation at 37°C, the slides are washed at 45°C (50%
formamide-
2XSSC pH 7, 3 times 3 minutes; followed by 2XSSC pH 7, 5 times 2 minutes)
-_n_r.~....~__..___.~ -....~ ..~_ _ _ ..
--.~.~-. _ _. ~,
CA 02449414 2003-12-11 li~i
-83-
and immersed in BN buffer (0.1 M Na bicarbonate, 0.05 percent NP-40, pH 8).
The slides are never allowed to dry after this point.
The slides are removed from the BN buffer and blocked for 5 minutes
at room temperature with BN buffer containing 5% non-fat dry milk
(Carnation) and 0.02% Na Azide (5 microliter/cm2 under plastic coverslips).
The coverslips are removed, and excess liquid briefly drained and fluorescein-
avidin DCS (3 microgram/ml in BN buffer with 5% milk and 0.02% NaAzide)
is applied (5 microliter/cm2). The same coverslips are replaced and the slides
incubated 20 minutes at 37°C. The slides are then washed 3 times for 2
minutes each in BN buffer at 45°C. The intensity of biotin-linked
fluorescence is amplified by adding a layer of biotinylated goat anti-avidin
antibody (5 microgram/ml in BN buffer with 5% goat serum and 0.02% Na
Azide), followed, after washing as above, by another layer of fluorescein-
avidin DCS. Fluorescein-avidin DCS, goat antiavidin and goat serum are all
available commercially, e.g., Vector Laboratories (Burlingame, CA). After
washing in BN, a fluorescence antifade solution, p-phenylenediamine (1.5
microliter/cm2 of coverslip) is added before observation. It is important to
keep this layer thin for optimum microscopic imaging. This antifade
significantly reduced fluorescein fading and allows continuous microscopic
observation for up to 5 minutes. The DNA counterstains (DAPI or
propidium iodide) are included in the antifade at 0.25-0.5 microgram/ml.
The red-fluorescing DNA-specific dye propidium iodide (PI) is used to
allow simultaneous observation of hybridized probe and total DNA. The
fluorescein and PI are excited at 450-490 nm (Zeiss filter combination
487709).
Increasing the excitation wavelength to 546 nm (Zeiss filter combination
487715) allows observation of the PI only. DAPI, a blue fluorescent DNA-
CA 02449414 2003-12-11
_grl_
specific stain excited in the ultraviolet (Zeiss filter combination 4$770I),
is
used as the counterstain when biotin-labeled and total DNA are observed
separately. Metaphase chromosome 21s are detected by randomly located
spots of yellow distributed over the body of the chromosome.
V.B. Improved Method for Efficiently Selecting
Chromosome 21 Sin le-Copy Sequences
Fuscoe et al., Genomics, 5_:100-109 (I989) provides more efficient
procedures than the method described immediately above (V.A.2) for
selecting large numbers of single-copy sequence or very low copy number
repeat sequence clones from recombinant phage libraries and demonstrates
their use to stain chromosome 21.
Briefly, Bones were selected from the Charon 21A library
LL21NS02 (made from DNA from human chromosome 21) using two basic
procedures. In the first, the phage library was screened in two stages using
methods designed to be more sensitive to the presence of repetitive sequences
in the clones than the method of Section V:A.2. The selected clones were
then subdoned into plasmids. The 450 inserts thus selected form the library
pBS-U2I. The second was in a multistep process in which: 1) Inserts from
LL21NS02 were subcloned into Bluescribe plasmids, 2) plasmids were grown
at high density in bacterial colonies on nitrocellulose filters and 3)
radioactive
human genomic DNA was hybridized to the plasmid DNA on nitrocellulose
filters at low stringency in two steps and 4) plasmids having inserts that
failed
to hybridize were selected as potentially carrying single-copy sequences.
Fifteen hundred and thirty colonies were picked in this manner to form the
library pBS-U21 / 1530.
-., _ _ ,-, .~ ~ ,..,".~~.~"~.3~ ,~~"".~.~ ~~T....._....___._.__~._
__.._.._... -__
CA 02449414 2003-12-11
-85-
Southern analysis indicated that the second procedure was more
effective at recognizing repetitive sequence than the first. Fluorescence in
situ hybridization with DNA from pBS-U21 / 1530 allowed specific, intense
staining of the number 21 chromosomes in metaphase spreads made from
human lymphocytes. Hybridization with pBS-U21 gives less specific staining
of chromosome 21. Details concerning the Fuscoe et al. method of selecting
single-copy sequence or very low repeat sequence probes from recombinant
libraries can be found in found in Fuscoe et al., id.
V.C. Hybridization with a Collection of Chromosome
4 Single-Copv Seauences
Pinkel et al., PNAS (USA), 85:9138-9142 (December 1988) describe the
procedures for preparing chromosome 4 single-copy sequences and then a
protocol (modification of the procedure described in T-'inkel et al; PNAS
(USA), 83:2934-2938 (1986)] for hybridizing said single copy probes to a human
metaphase spread. Figure 4H shows the hybridization with a pool of 120
single-copy probes from chromosome 4 to a human metaphase spread.
VI. Incapacitating Shared Repetitive Sequences
VLA. Chromosome 2I-Specific Stainin Using Blocking DNA
High concentrations of unlabeled human genomic DNA and lambda
phage DNA were used to inhibit the binding of repetitive and vector DNA
sequences to the target chromosomes. Heavy proteinase digestion and
subsequent fixation of the target improved access of probes to target DNA.
CA 02449414 2003-12-11
-86-
Human metaphase spreads were prepared on microscope slides with
standard techniques and stored immediately in a nitrogen atmosphere at -
20°C.
Slides were removed from the freezer and allowed to warm to room
temperature in a nitrogen atmosphere before beginning the staining procedure.
The warmed slides were first treated with 0.6 microgramlml proteinase K in P
buffer (20 mM Tris, 2 mM CaCl2 at pH 7.5) for 7.5 minutes, and washed once in
P
buffer. The amount of proteinase K used needs to be adjusted for different
batches of slides. After denaturing the slides were stored in 2XSSC. A
hybridization mix was prepared which consisted o~f 50% formamide, 10% dextran
sulfate, 1 % Tween 20T"', 2XSSC, 0.5 mglml human genomic DNA, 0.03 mg/ml
lambda DNA, and 3 microgram/ml biotin labeled probe DNA. The probe DNA
consisted of the highest density fraction of phage from the chromosome 21 Hind
III fragment library (ATCC accession number 57713); as determined by a cesium
chloride gradient. (Both insert and phage DNA of the probe were labeled by
nick
translation.) The average ' insert size (amount of chromosome 21 DNA), as
determined by gel electrophoresis was about 5 kilobases. No attempt was made
to
remove repetitive sequences from the inserts or to isolate the inserts from
the
lambda phage vector. The hybridization mix was denatured by heating to
70°C for
5 minutes followed by Incubation at 37°C for 1 hour. The incubation
allows the
human genomic DNA and unlabeled lambda DNA In the hybridization mix to block
the human repetitive sequences and vector sequences in the probe.
The slide containing the human metaphase spread was removed from the 2XSSC
and blotted dry with lens paper. The hybridization mix was immediately applied
to
the slide, a glass cover slip was placed on the slide
CA 02449414 2003-12-11
_87_
with rubber cement, and the slide was incubated overnight at 37°C.
Afterwards preparation of the slides proceeded as described in Section V.B.
(wherein chromosome 21 DNA was stained with fluorescein and total
chromosomal DNA counterstained with DAPI). Figures lA-C illustrate the
results. Figure 1A is a DAPI image of the human metaphase spread obtained
with a computerized image analysis system. It is a binary image showing
everything above threshold as white, and the rest as black. The primary data
was recorded as a gray level image with 256 intensity levels. (Small arrows
indicate the locations of the chromosome 21 s.) Figure 1 B is a fluorescein
image of the same spread as in Figure 1A, again in binary form. (Again, small
arrows indicate the locations of the chromosome 2Is.) Figure 1C illustrates
the positions of the chromosome 21s after other less densely stained objects
were removed by standard image processing techniques.
VLB. Detection of Trisomy 21 and Translocations of
Chromosome 4 Using Bluescribe Plasmid Libraries
As illustrated in Section VLA., a human chromosome-specific library,
including its shared repetitive sequences, can be used to stain that
chromosome if the hybridization capacity of the shared repetitive sequences is
reduced by incubation with unlabeled human genomic DNA. In Section
VLA., the nucleic acid sequences of the heterogeneous mixture were cloned
in the phage vector Charon 21A, in which the ratio of insert of vector DNA is
about 0.1 (4 kb average insert to 40 kb of vector). In this section, we
demonstrate that transferring the same inserts to a smaller cloning vector,
the
about 3 kb Bluescribe plasmid, which increases the ratio of insert to vector
DNA to 0.5, improved the specificity and intensity of the staining.
CA 02449414 2003-12-11
_g8-
As previously discussed, incubation of the probe can be carried out
with the probe alone, with the probe mixed with unlabeled genomic DNA,
and with the probe mixed with unlabeled DNA enriched in all or some
shared repetitive sequences. If unlabeled genomic DNA is added, then it is
important to add enough to incapacitate sufficiently the shared repetitive
sequences in the probe. However, the genomic DNA also contains unlabeled
copies of the sequences, the hybridization of which is desired. As explained
above, Q is herein defined as the ratio of unlabeled to labeled copies of the
chromosome- specific sequences in the hybridization mixture.
Pinkel et al., PNAS (LTSA), 85:9138-9142 (December 1988) describes the
use of unlabeled genomic DNA to competitively inhibit hybridization of
those sequences in a chromosome-specific library that are shared with other
chromosomes. That paper describes materials and methods for fluorescence
in situ hybridization with human chromosome-specific libraries
[chromosome 4 library LL04NS02 subcloned into Bluescribe plasmids (pBS-4);
chromosome 2I library LL21NS02 subcloned into Bluescribe plasmids
(pBS-21)]. The results of those hybridizations are shown in Figure 4 A-C and
4F and G.
VLC. Hybridization of Yeast Artificial Chromosomes ~YACS) to
Human Metaphase Spread
YACS. Seven yeast clones HYI, HY19, HY29, HYAI.A2, HYA3.A2,
HYA3.A9, and HYA9.E6 were obtained from D. Burke (Washington
University, St. Louis, MO). The lengths of the human DNA in the clones
ranged from about 100 kb to about 600 kb. Gel electrophoresis was performed
to verify the size of these inserts. Each of these clones was grown up and
total
_...._ __ . . . _._ . . .~~ w. x.a9~ ~.~r~ ~..~_,~.m. ~ ~_ «.,~ ..~ .>~ ~~, .~
__ _ _____ _ _ __ ____
CA 02449414 2003-12-11
-89-
DNA was isolated. The isolated DNA was biotinylated by nick translation so
that IO-30% of the thymidine was replaced by biotin-lI-dUTP. The
concentration of the total labeled DNA after nick translations is in the range
of IO-20 ng/ul.
Blocking DNA. Human placental DNA (Sigma) was treated with
proteinase K and extracted with phenol and sonicated to a size range of
200-600 bp. Total DNA isolated from yeast not containing an artificial
chromosome was sonicated to a similar size range. Both of these DNA's were
maintained at a concentration of I-10 ug/ul.
Fluorescence in situ hybridization (FISH). Hybridization followed the
procedures of Pinkel et al. (1988), supra with slight modifications. Metaphase
spreads were prepared from methotrexate synchronized cultures according to
the procedures of Harper et al. PNAS (USA) 78: 4458-4460, (198I). Cells were
fixed in methanol/acetic acid, fixed (3:3), dropped onto slides, air dried,
and
stored at -20°C under nitrogen gas until used. The slides were then
immersed
two minutes in 70% formamide/2xSSC to denature the target DNA
sequences, dehydrated in a 70-85-I00% ethanol series, and air dried. (SSC is
O.I5 M NaCI/0.015 M'Na Citrate, pH 7). Ten - I00 ng of biotinylated yeast
DNA, and approximately 1 ug each of unlabeled yeast and human genomic
DNA were then added to the hybridization mix (final volume 10 u1, final
composition 50% formamide/2xSSC/10% dextran sulfate), heated to 70°C
for
min., and then incubated at 37°C for 1 hr to allow the complementary
strands of the more highly repeated sequences to reassociate.
The hybridization mixture was then applied to the slide
(approximately 4 cm2 area) and sealed with rubber cement under a glass cover
slip. After overnight incubation at 37°C the coverslip was removed and
the
CA 02449414 2003-12-11
-90-
slide washed 3 times 3 min each in 50% formamide/2xSSC at 42-45°C, and
once
in PN buffer [mixture of 0.1 M NaH2P04 and 0.1 M Na2HP04 to give pH 8, 0.1
NonidetT"' P-40 (Sigma)]. The bound probe was then detected with alternating
20
min incubations (room temperature in avidin-FITC and goat-anti-avidin
antibody,
both at 5 uglml in PNM buffer (PN buffer plus 5% nonfat dry milk, centrifuged
to
remove solids; 0.02% Na azide). Avidin and anti-avidin incubation were
separated
by 3 washes of 3 min each in PN buffer. Two or three layers of avidin were
applied
(Avidin, DCS, grade, and biotinyfated goat-anti-avidin are obtained from
Vector
Laboratories Inc., Burlingame, CA).
Figure 5 shows the hybridization of HYA3.A2 (580 kb of human DNA) to 12q21.1.
The location of the hybridization was established by using a conventional
fluorescent banding technique employing the DAPII actinomycin D procedure:
Schweizer, "Reverse fluorescent chromosome banding with chromomycin and
DAP1," Chromosoma. 58:307-324 (1976). The hybridization signal forms a band
across the width of each of the chromosome 12s, indicating the morphology of
the
packing of DNA in that region of the chromosome.
~.x,.,... ..,.,. _..._,. >.r....-...,. .x>-.r~,~..c,-. ~ e...."r.F..
.,...".,.>..,~.x....n. -n,.~"oe»,~-t~Px°, .v~m~~M~~~.- -
..._._.~___...v, cv0.'.-~s~~qcsF~xw~xan.~..-.,..e.".~...~...,.~"p,~..-
....,.~__.~-. _.._
CA 02449414 2003-12-11
-9I-
The YAC clone positions are attributed as shown in Table 2 below.
Table 2
YAC Competition Hybridization
YAC Clone Insert Size Localization
HY1 120 Xq23
HY19 450 8q23.3
21 q21.1
HY29 500 14q12
HYA1.A2 250 6q16
HYA3.A2 580 12q21.1
HYA3.A9 600 I4q2I
HYA9.E6 280 1p36:2
3q~
CA 02449414 2003-12-11
_92_
VLD. Hxbridization With Human/Hamster Hybrid Cell
Essentially the same hybridization and staining conditions were used
in this example as for those detailed in the procedure of Pinkel et al.
(1988),
supra and exemplified in Sections V.C. and VLB., supra. In this example, 400
ng of biotin labeled DNA from a hamster-human hybrid cell that contains
one copy of human chromosome 19 was mixed with 1.9 ug of unlabeled
human genomic DNA in 10 u1 of hybridization mix. Hybridization was for
approximately 60 hours at 37°C. Fluorescent staining of the bound probe
and
counterstaining of the chromosomes was as in the other examples above.
Figure 6 shows the results of the hybridization.
VII. Specific Applications.
The present invention allows microscopic and in some cases flow
cytometric detection of genetic abnormalities on a cell by cell basis. The
microscopy can be performed entirely by human observers, or include
various degrees of addititional instrumentation and computational
assistance, up to full automation. The use of instrumentation and
automation for such analyses offers many advantages. Among them are the
use of fluorescent dyes that are invisible to human observers (for example,
infared dyes), and the opportunity to interpret results obtained with multiple
labeling methods which might not be simultaneously visible (for example,
combinations of fluorescent and absorbing stains, autoradiography, etc.)
Quantitative measurements can be used to detect
CA 02449414 2003-12-11
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differences in staining that are not detectable by human observers. As is
described below, automated analysis can also increase the speed with which
cells and chromosomes can be analysed.
The types of cytogenetic abnormalities that can be detected with the
probes of this invention include: Duplication of all or part of a chromosome
type can be detected as an increase in the number or size of distinct
hybridization domains in metaphase spreads or interphase nuclei following
hybridization with a probe for that chromosome type or region, or by an
increase in the amount of bound probe. If the probe is detected by
fluorescence, the amount of bound probe can be determined either flow
cytometrically or by quantitative fluorescence microscopy. Deletion of a
whole chromosome or chromosome region can be detected as a decrease in
the number or size of distinct hybridization domains in metaphase spreads or
interphase nuclei following hybridization with a probe for that chromosome
type or region, or by a decrease in the amount of bound probe. If the probe is
detected by fluorescence, the amount bound can be determined either flow
cytometrically or by quantitative fluorescence microscopy. Translocations,
dicentrics and inversions can be detected in metaphase spreads and
interphase nuclei by the abnormal juxtaposition of hybridization domains
that are normally separate following hybridization with probes that flank or
span the regions) of the chromosomes) that are at the points) of
rearrangement. Translocations involve at least two different chromosome
types and result in derivative chromosomes possessing only one centromere
each. Dicentrics involve at least two different chromosome types and result
in at least one chromosome fragment lacking a centromere and one having
CA 02449414 2003-12-11
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two cenfiromeres. Tnversions involve a reversal of polarity of a portion of a
chromosome.
VILA Banding Analysis
Substantial effort has been devoted during the past thirty years to
development of automated systems (especially computer controlled
microscopes) for automatic chromosome classification and aberration
detection by analysis of metaphase spreads. In recent years, effort has been
directed at automatic classification of chromosomes which have been
chemically stained to produce distinct banding patterns on the various
chromosome types. These efforts have only partly succeeded because of the
subtle differences in banding pattern between chromosome types of
approximately the same size, and because differential contraction of
chromosomes in different metaphase spreads causes a change in the number
and width of the bands visible on chromosomes of each type. The present
invention overcomes these problems by allowing construction of reagents
which produce a staining pattern whose spacing, widths and labeling
differences (for example different colors) are optimized to facilitate
automated
chromosome classification and aberration detection. This is possible because
hybridization probes can be selected as desired along the lengths of the
chromosomes. The size of a band produced by such a reagent may range from
a single small dot to a substantially uniform coverage of one or more whole
chromosomes. Thus the present invention allows construction of a
hybridization probe and use of labeling means, preferably fluorescence, such
that adjacent hybridization domains can be distinguished, for example by
color, so that bands too closely spaced to be resolved spatially can be
detected
"_..._._._._,__""_.~..~ ~ 02449414 2003-12-11 _ .__ .,.~_._.~_.___--_ ..
-95-
spectrally (i.e. if red and green fluorescing bands coalesce, the presence of
the
two bands can be detected by the resulting yellow fluorescence).
The present invention also allows construction of banding patterns
tailored to particular applications. Thus they can be significantly different
in
spacing and color mixture, for example, on chromosomes that are similar in
general shape and size and which have similar banding patterns when
conventional techniques are used. The size, shape and labeling (e.g. color) of
the hybridization bands produced by the probes of the present invention can
be optimized to eliminate errors in machine scoring s o that accurate
automated aberration detection becomes possible. This optimized banding
pattern will also greatly improve visual chromosome classification and
aberration detection.
The ease of recognition of specific translocation breakpoints can be
improved by using a reagent closely targeted to the region of the break. For
example, a high complexity probe of this invention comprising sequences
that hybridize to both sides of the break on a chromosome can be used. The
portion of the probe that binds to one side of the break can be detected
differently than that which binds to the other, for example with different
colors. In such a pattern, a normal chromosome would have the different
colored hybridization regions next to each other, and such bands would
appear close together. A break would separate the probes to different
chromosomes or result in chromosomal fragments, and could be visualized
as much further apart on an average.
....">". ,.~,~"",v,.~,... , . x;...w, ~,...yrtgygo~A.yt.,.~x~'h~"Ty., . ~,~tw.
x.~.--m_..,~ . __.....-.,- ~",.~, _ _.....
CA 02449414 2003-12-11
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VILB Biological Dosimetr~
One approach to biological dosimetry is to measure frequencies of
structurally aberrant chromosomes as an indication of the genetic damage
suffered by individuals exposed to potentially toxic agents. Numerous studies
have indicated the increase in structural aberration frequencies with
increasing exposure to ionizing radiation and other agents, which are called
clastogens. Dicentric chromosomes are most commonly scored because their
distinctive nature allows them to be scored rapidly without banding analysis.
Rapid analysis is important because of the low frequency of such aberrations
in individuals exposed at levels found in workplaces (~2 x IO-3/cell).
Unfortunately, dicentrics are not stably retained so the measured dicentric
frequency decreases with time after exposure. Thus law level exposure over
long periods of time does not result in an elevated dicentric frequency
because
of the continued clearance of these aberrations. Translocations are better
aberrations to score for such dosimetric studies because they are retained
more or less indefinitely. Thus, assessment of genetic damage can be made at
times long after exposure. Translocations are not routinely scored for
biological dosimetry because the difficulty of recognizing them makes scoring
sufficient cells for dosimetry logistically impossible.
The present invention eliminates this difficulty. Specifically,
hybridization with a probe which substantially uniformly stains several
chromosomes (e.g. chromosomes 1, 2, 3 and 4} allows immediate microscopic
identification in metaphase spreads of structural aberrations involving these
chromosomes. Normal chromosomes appear completely stained or
unstained by the probe. Derivative chromosomes resulting from
translocations between targeted and non-targeted chromosomes are
_ _ v _.~..~_-__.
CA 02449414 2003-12-11 '
~1
- 97 -
recognized as being only partly stained, Fig. 4D. Such partially hybridized
chromosomes can be immediately recognized either visually in the
microscope or in an automated manner using computer assisted microscopy.
Discrimination between translocations and dicentrics is facilitated by adding
to the probe, sequences found at all of the chromosome centromeres.
Detection of the centrorneric components of the probe with a labeling means,
for example color, different from that used to detect the rest of the probe
elements allows ready identification of the chromosome centromeres, which
in turn facilitates discrimination between dicentrics and translocations. This
technology dramatically reduces the scoring effort required with previous
techniques so that it becomes feasible to examine tens of thousands of
metaphase spreads as required for low level biological dosimetry.
VILC. Prenatal Dia _ nosis
The most common aberrations found prenatally are trisomies
involving chromosomes 21 (Down syndrome), I8 (Edward syndrome) and 13
(Patau syndrome) and XO (Turner syndrome), XXY (Kleinfelter syndrome)
and XYY disease. Structural aberrations also occur. i lfowever, they are rare
and their clinical significance is often uncertain. Thus, the importance of
detecting these aberrations is questionable. Current techniques for obtaining
fetal cells for conventional karyotyping, such as, amniocentesis and chorionic
villus biopsy yield hundreds to thousands of cells for analysis. These are
usually grown in culture for 2 to 5 weeks to produce sufficient mitotic cells
for
cytogenetic analysis. Once metaphase spreads are prepared, they are analyzed
by rnnventional banding analysis. Such a process can only be carried out by
highly skilled analysts and is time consuming so that the number of analyses
_. _ ----~"~ ---
CA 02449414 2003-12-11
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that can be reliably carried out by even the largest cytogenetics laboratories
is
only a few thousand per year. As a result, prenatal cytogenetic analysis is
usually limited to women whose children are at high risk for genetic disease
(e.g. to women over the age of 35).
The present invention overcomes these difficulties by allowing
simple, rapid identification of common numerical chromosome aberrations
in interphase cells with no or minimal cell culture. Specifically, abnormal
numbers of chromosomes 21, I8, I3, X and Y can be detected in interphase
nuclei by counting numbers of hybridization domains following
hybridization with probes specific for these chromosomes (or for important
regions thereof such as 21q22 for Down syndrome). A hybridization domain
is a compact, distinct region over which the intensity of hybridization is
high.
An increased frequency of cells showing three domains (specifically to greater
than 10%) for chromosomes 21, 18 and I3 indicates the occurrence of Down,
Edward and Patau syndromes, respectively. An increase in the number of
cells showing a single X-specific domain and no Y-specific domain following
hybridization with X-specific and Y-specific probes indicates the occurrence
of
Turner syndrome. An increase in the frequency showing two X-specific
domains and one Y-specific domain indicates Kleinfelter syndrome, and
increase in the frequency of cells showing one X-specific domain and two
Y-specific domains indicates an XYY fetus. Domain counting in interphase
nuclei can be supplemented (or in some cases replaced) by measurement of
the intensity of hybridization using, for example, quantitative fluorescence
microscopy or flow cytometry, since the intensity of hybridization is
approximately proportional to the number of target chromosomes for which
the probe is specific. Numerical aberrations involving several chromosomes
CA 02449414 2003-12-11
_g9_
can be scored simultaneously by detecting the hybridization of the different
chromosomes with different labeling means, for example, different colors.
These aberration detection procedures overcome the need for extensive cell
culture required by procedures since all cells in the population can be
scored.
They eliminate the need fox highly skilled analysts because of the simple,
distinct nature of the hybridization signatures of numerical aberrations.
Further, they are well suited to automated aberration analysis.
The fact that numerical aberrations can be detected in interphase
nuclei also allows cytogenetic analysis of cells that normally cannot be
stimulated into mitosis. Specifically, they allow analysis of fetal cells
found in
maternal peripheral blood. Such a feature is advantageous because it
eliminates the need for invasive fetal cell sampling such as amniocentesis or
chorionic villus biopsy.
As indicated in the Background, the reason such embryo-invasive
methods are necessary is that conventional karyotyping and banding analysis
requires metaphase chromosomes. At this time, there are no accepted
procedures for culturing fetal cells separated from maternal blood to provide
a population of cells having metaphase chromosomes. In that the staining,
reagents of this invention can be employed with interphase nuclei, a non-
embryo-invasive method of karyotyping fetal chromosomes is provided by
this invention.
The first step in such a method is to separate fetal cells that have
passed through the placenta or that have been shed by the placenta into the
maternal blood. The incidence of fetal cells in the maternal bloodstream is
very low, on the order of IO'4 to 106 cells/ml arid quite variable depending
on the time of gestation; however, appropriately marked fetal cells may be
CA 02449414 2003-12-11
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distinguished from maternal cells and concentrated, for example, with high
speed cell sorting.
The presence of cells of a male fetus may be identified by a label, for
example a fluorescent tag, on a chromosome-specific staining reagent for the
Y chromosome. Cells that were apparently either lymphocytes or erythrocyte
precursors that were separated from maternal blood were shown to be
Y-chromatin-positive. [Zillacus et al., Scan. J. Haematol,15: 333 (1975);
Parks
and Herzenberg, Methods in Cell Biology, Vol. 10, pp. 277-295 (Academic
Press, N.Y.,1982); and Siebers et al., Humangenetik, 28: 273 (1975)].
A preferred method of separating fetal cells from maternal blood is
the use of monoclonal antibodies which preferentially have affinity for some
component not present upon the maternal blood cells. Fetal cells may. be
detected by paternal HLA (human leukocyte antigen) markers or by an
antigen on the surface of fetal cells. Preferred immunochemical procedures
to distinguish between fetal and maternal leukocytes on the basis of differing
HLA type use differences at the HLA-A2, -A3; and -B? loci, and further
preferred at the -A2 locus. Further, first and second trimester fetal
trophoblasts may be marked with antibody against the internal cellular
constituent cytokeratin which is not present in maternal leukocytes.
Exemplary monoclanal antibodies are described in the following references.
Herzenberg et al., PNAS, 76: 1453 (1979), reports the isolation of fetal
cells, apparently of lymphoid origin, from maternal blood by fluorescence
activated cell sorting (FACS) wherein the separation was based on the
detection of labeled antibody probes which bind HLA-AZ negative cells in
maternal blood. Male fetal cells separated in that manner were further
identified by quinacrine staining of Y-chromatin.
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Govone et al., Lancet, Oct. 13, 1984: 841, reported the recovery of fetal
trophoblasts from maternal blood by flow cytometry using a monoclonal antibody
termed H315. Said monoclonal reportedly identifiies a glycoprotein expressed
on
the surface of the human syncytiotrophoblast as well as other trophoblast cell
populations, and that is absent from peripheral blood cells.
Kawata et al., J. Exp. Med., 160:653 (1984), discloses a method for
isolating placental cell populations from suspensions of human placenta. The
method uses coordinate two-color and light-scatter FRCS analysis and sorting.
Five different cell populations were isolated on the basis of size and
quantitative
differences in the coordinate expression of cell surface antigens detected by
monoclonal antibodies against an HLA-A, B, C monomorphic determinant
(MB40.5) and against human trophoblasts (anti-Trop-1 and anti-Trop-2).
Loke and Butterworth, J.. Cell Sci:, 76: 189 (1985), describe two monoclonal
antibodies, 18B/A5 and 18A/C4, which are reactive with first trimester
cytotrophoblasts and other fetal epithelial tissues including
syncytiotrophobfasts.
A preferred monoclonal antibody to separate fetal cells from maternal blood
for staining according to this invention is the anti-cytokeratin antibody Cain
5.2,
which is commercially available from Becton-Dickinson (Fran.klin Lakes, N.J.,
USA).
Other preferred monoclonal antibodies for separating fetal cells from
maternal blood are those disclosed in Fisher et al., J. Cell. B-iol. 109 (2):
891-902
(1989). The monoclonal antibodies disclosed therein react
---1-
CA 02449414 2003-12-11
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specifically with antigen on first trimester human cytotrophoblast cells,
which
fetal cells have the highest probability of reaching the maternal circulation.
Briefly, the disclosed monoclonal antibodies were raised by injection of test
animals with cytotrophoblast cells obtained from sections of the placental
bed,
that had been isolated by uterine aspiration. Antibodies raised were subjected
to several cytological screens to select for those antibodies which react with
the cytotrophoblast stem cell layer of first trimester chorionic villi.
Preferred monoclonal antibodies against such first trimester
cytotrophoblast cells disclosed by Fisher et al. include monoclonal antibodies
produced from the following hybridomas deposited at the American Tyupe
Culture Collection (ATCC; Rockville, MD, USA) under the Budapest Treaty:
Hybridoma ATCC Accession #
J1 D8 HBI0096
P1B5 HBI0097
Both hybridoma cultures were received by the ATCC on April 4, 1989 and
reported viable thereby on April 14,1989.
Fisher et al. state that fetal cells isolated from ,maternal blood by use of
said monoclonal antibodies are capable of replication in vitro. Therefore,
fetal cells isolated by the rriethod of Fisher et al., that is, first
trimester fetal
cytotrophoblasts, may provide fetal chromosomal material that is both in
metaphase and in interphase.
The fetal cells, preferably leukocytes and cytotrophoblasts, more
preferably cytotrophoblasts, once marked with an appropriate antibody are
then separated from the maternal cells either directly or by preferably
separating and concentrating said fetal cells by cell sorting or panning. For
CA 02449414 2003-12-11
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example, FACS may be used to separate fluorescently labeled fetal cells, or
flow cytometry may be used.
The fetal cells once separated from the maternal blood can then be
stained according to the methods of this invention with appropriate
chromosome-specific staining reagents of this invention, preferably those of
particular importance for prenatal diagnosis. Preferred staining reagents are
those designed to detect aneuploidy, for example, trisomy of any of several
chromosomes, including chromosome types 21, 18, 13, X and Y and
subregions on such chromosomes, such as, subregion 21q22 on
chromosome 21.
Preferably, a fetal sample for staining analysis according to this
invention comprises at least 10 cells or nuclei, and more preferably about 100
cells or nuclei.
VILD Tumor Cvtogenetics
Numerous studies in recent years have revealed the existence of
structural and numerical chromosome aberrations that are diagnostic for
particular disease phenotypes and that provide clues to the genetic nature of
the disease itself. Prominent examples include the close association between
chronic myelogeneous leukemia and a translocation involving chromosome
9 and 22, the association of a deletion of a portion of 13q14 with
retinoblastoma and the association of a translocation involving
chromosomes 8 and 14 with Burkitts lymphoma. Current progress in
elucidating new tumor specific abnormalities is limited by the difficulty of
produang representative, high quality banded metaphase spreads for
CA 02449414 2003-12-11
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cytogenetic analysis. These problems stem from the fact that many human
tumors are difficult or impossible to grow in culture. Thus, obtaining mitotic
cells is usually difficult. Even if the cells can be grown in culture, there
is the
significant risk that the cells that do grow may not be representative of the
tumorigenic population. That difficulty also impedes the application of
existing genetic knowledge to clinical diagnosis and prognosis.
The present invention overcomes these limitations by allowing
detection of specific structural and numerical aberrations in interphase
nuclei. These aberrations are detected as described supra. Hybridization with
whole chromosome probes will facilitate identification of previously
unknown aberrations thereby allowing rapid development of new
associations between aberrations and disease phenotypes. As the genetic
nature of specific malignancies becomes increasingly well known, the
interphase assays can be made increasingly specific by selecting hybridization
probes targeted to the genetic lesion. Translocations at specific sites on
selected chromosomes can be detected by using hybridization probes.
that closely flank the breakpoints. Use of these probes allows diagnosis of
these specific disease phenotypes. Translocations may be detected in
interphase because they bring together hybridization domains that are
normally separated, or because they separate a hybridization domain into two,
well separated domains. In addition, they may be used to follow the
reduction and reemergence of the malignant cells during the course of
therapy. Interphase analysis is particularly important in such a application
because of the small number of cells that may be present and because they
may be difficult or impossible to stimulate into mitosis..
mFro..~ ..~.,.~. s .. ~.,~.'__ _
CA 02449414 2003-12-11
-I05-
Duplications and deletions, processes involved in gene amplification
and loss of heterozygosity, can also be detected in metaphase spreads and
interphase nuclei using the techniques of this invention. Such processes are
implicated in an increasing number of different tumors.
VIII. Detection of BCR-ABL Fusion in Chronic M eY'lo eg nous
Leukemia (CMU
Probes. This section details a CML assay based upon FISH with probes
from chromosomes 9 and 22 that flank the fused BCR and ABL sequences in
essentially all cases of CML (Figure 8). The BCR and ABL probes used in the
examples of this section were kindly provided by Carol A. Westbrook of the
Department of Medicine, Section of Hematology/Oncology at the University
of Chicago Medical Center in Chicago, Illinois (USA).
The ABL probe on chromosome 9, c-hu-ABL, is a 35-kb cosmid
(pCV105) clone selected to be telomeric to the 200-kb region of ABL between
exons IB and TI in which the breaks occur (24). The BCR probe on
chromosome 22, PEM12, is an 18-kb phage clone (in FMBL3) that contains part
of, and extends centromeric to, the 5.8-kb breakpoint cluster region of the
BCR
gene in which almost all CML breakpoints occur. FISH was carried out using
a biotin labeled ABL probe, detected with the fluorochrome Texas red, and a
digoxigenin labeled BCR probe, detected with the green fluorochrome FITC.
Hybridization of both probes could be observed simultaneously using a
fluorescence microscope equipped with a double band pass filter set (Omega
Optical).
Figure 8 is a schematic representation of the BCR gene on
chromosome 22, the ABL gene of chromosome 9, and the BCR-ABL fusion
1 ~ 02449414 2003-12-11 -- -_.
-IO6-
gene on the Philadelphia chromosome, showing the location of CML
breakpoints and their relation to the probes. Exons of the BCR gene are
depicted as solid boxes. The Roman numeral I refers to the first axon of the
BCR gene; the arabic numerals 1-5 refer to the axons within the breakpoint
cluster region, here indicated by the dashed line. The approximate location of
the I8 kb phage PEMI2 probe (the BCR probe) is indicated by the open
horizontal bar. Since the majority of breakpoints in CML occur between
axons 2 and 4, 15 kb or more of target for PEM12 will remain on the
Philadelphia chromosome. In the classic reciprocal translocation a few kb of
target for PEMI2 (undetectable fluorescent signal) will be found on the
derivative chromosome. The map and exan numbering (not to scale) is
adapted from Heisterkamp et al. (ref. 34, supra).
Exons of the ABL gene are depicted as open vertical bars (not to scale).
The Roman numerals Ia and Tb refer to the alternative first axons, and II to
the second axon. Exon II is approximately 25kb upstream of the end of the 2$
kb cosmid c-hu-abl (the ABL probe). AlI CML breakpoints occur upstream of
axon II, usually between axons Tb and Ia, within a region that is
approximately
200kb in length. Thus, c-hu-abl will always be 25 to 200 kb away from the
fusion junction. The map (not to scale) is adapted from Heisterkamp et al.
(ref. 35, supra). The BCR ABL fusion gene is depicted. In CML, PEM12 will
always lie at the junction, and ~hu-abl will be separated from PEMI2 by 25 to
225kb.
Sample Preparation: CML-4: Peripheral blood was centrifuged for 5
min. Ten drops of interface was diluted with PBS, spun down, fixed in
methanol/acetic acid (3:I), and dropped on slides. CML-2, 3, 7: Five to 10
drops of marrow diluted with PBS to prevent clotting were fixed in
CA 02449414 2003-12-11
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methanol/acetic acid and dropped on slides. CML-I, 4, 5, 6: Peripheral blood
and/or bone marrow was cultured in RPMI 1640 supplemented with 10%
fetal calf serum, an antibiotic mixture (gentamycin 500 mg/ml), and 1%
L-glutamine for 24h. Cultures were synchronized according to J.J. Yunis and
M.E. Chandler 1'ro~in Ciin. Path., 7:267 (1977), and chromosome preparations
followed Gibis and Jackson, Karyo~ram,11:91 (1985).
Hybridization and Detection Protocol. Hybridization followed
procedures described by D. Pinkel et aI. (27), Trask et al. (25), and J. B.
Lawrence et al (30), with modifications. The BCR probe was nick-translated
(Bethesda Research Laboratories Nick-Translation System) with digoxigenin-
11-dUTP (Boehringer Mannheim Biochemicals) with an average
incorporation of 25%Q. The ABL probe was similarly nick-translated with
biotin-11-dUTP (Enzo Diagnostics).
1. Hybridization. Denature target interphase cells and/or metaphase
spreads on glass slides at 72°C in 70% formamide/2xSSC at pH 7 for 2
min.
Dehydrate in an ethanol series (70%, 85%,,and 100% each for 2 min.). Air dry
and place at 37°C (2xSSC is 0.3M NaCI/30 mM sodium citrate). Heat IO ml
of
hybridization mixture containing 2 ng/ml of each probe, 50%
formamide/2xSSC, 10% dextran sulphate, and 1 mg/ml human genomic
DNA (sonicated to 200-600 bp) to 70°C for 5 min. to denature the DNA.
Incubate for 30 min. at 37°C. Place on the warmed slides, cover with a
20 mm
x 20 mm coverslip, seal with rubber cement, and incubate overnight in a
moist chamber at 37°C. Remove coverslips and wash three times for 20
minutes each in 50% formamide/2xSSC pH 7 at 42°C, twice for 20 minutes
each in 2xSSC at 42°C, and finally rinse at room temperature in 4xSSC.
.... . , .~ ... ...., .~~ ~: a.~~. .~.~. . _____ _ _
CA 02449414 2003-12-11
-I08-
2. Detection of Bound Probes: All incubation steps are performed
with approximately 100 ml of solution at room temperature under coverslips.
The biotinylated ABL probe was detected first, then the digoxigenin-labeled
BCR probe.
a. Biotinylated ABL Probe: Preblock with 4xSSC/ 1 % bovine serum
albumin (BSA) for 5 min. Apply Texas Red-avidin (V'ector Laboratories Inc., 2
mg/ml in 4xSSC/1% BSA) for 45 min. Wash in 4xSSC once, 4xSSC/1%
Triton-X 100 (Sigma) and then again in 4xSSC, 5 ~ min. each. Preblock for 5
min, in PNM (PN containing 5%a non-fat dry milk and 0.02% sodium azide
and centrifuged to remove solids. PN is 0.1 M NaH2P~4/O.IM Na2HP04,
0.05% NP40, pH 8). Apply biotinylated goat anti-avidin (Vector Laboratories
Inc., 5 mg/ml in PNM) for 45 min. Wash twice in PN for 5 min. Apply a
second layer of Texas Red-avidin (2 mg/ml in PNM) for 45 min. Wash twice
in PN for 5 min. each.
b. Digoxigenin-Labeled BCR Probe: Preblock with PNM for 5 min.
Apply sheep anti-digoxigenin antibody (obtained from D. Pepper, Boehringer
Mannheim Biochemicals, Indianapolis, IN; 15.4 mg/ml in PNM) for 45 min.
Wash twice in PN for 5 min. each. Preblock with PNM for 5 min. Apply
rabbit-anti-sheep antibody conjugated with FITC (Organon Teknika- Cappel,
1:50 in PNM) for 45 min. Wash twice for 5 min. each in PN. If necessary, the
signal is amplified by preblocking for 5 min, with PNM and applying sheep
anti-rabbit IgG antibody conjugated to FITC (Organon Teknika-Cappel, 1:50 in
PNM) for 45 min. Rinse in PN.
3. Visualization: The slides are mounted fluorescence antifade
solution [G. D. Johnson and J. G. Nogueria, . Immunol. Methods" 43:349
(1981)) (ref. 31, su ra ] containing 1 mg/ml 4',6-amidino-2-phenylindole
n __._ __ .._.._ ... ~~ u~~"~ ,.,* . .*.,~.~...,~~: ~s .~- _ ~,~~".~":.,
.,.*~,..,.,....~.~_ _ _ _..
CA 024n49414 2003-12-11
- 109 -
(DAPI as a counterstain, and examined using a FITCITexas red double-band pass
fitter set (Omega Optical) on a Zeiss AxioskopT"".
The method used for BCR-ABL PCR tested herein was fiat described in
Hegewisch-Becker et al. for CML-3, 4 and 7 (ref. 32, supra), and Kohler et
al:, for
CML-5 and 6 (ref. 33, .supra).
,~~ ABL and BCR hybridization sites were visible on both chromatids
of chromosomes in most metaphase spreads. The ABL probe 'bound to
metaphase spreads from normal individuals (Figure 9 A) near the telomere on
9.q
while the BCR probe bound at 22q11 (Figure 9B). Hybridization with the ABL. or
BCR probe to normal interphase nuclei typically resulted in two tiny
fluorescent
dots corresponding to the target sequence on both chromosome homologues. The
spots were apparently randomly distributed in the two dimensional nuclear
images
and were usually well separated: A few cells showed two doublet hybridization
signals probably a result of hybridization to ,both sister chrornatids of both
homologues in cells which had replicated this region of DNA (i.e., those in
the S
or 'G2- phase of cell cycle}. Duai color FISH of the ABL (red) and BCR (green)
probes to normal -G1 nuclei yielded two red (ABL) and two green (BCR)
hybridization signals.distributed randomly around the nucleus.
The genetic rearrangement of CML brings the DNA sequences homologous.
to the probes together on an abnormal chromosome, usually the Ph', and
together
in the interphase nucleus, as illustrated in Figure 8. The genomic distance
between the probe binding sites in the fusion gene varies among CML cases,
ranging from 25 to 225 kb, but remains the same in all the. cells of a single
leukemic clone. Dual c~lor hybridization with ABL and BCR probes to interphase
CML cells resulted in one red and one green
._.. _._ _.. . . _.....__~ -,~~-,..r~ ~. ..~.,~.,~...,._~....ro.__.
.~~..~,.~.~.-...~__.~~_..~~.~.~_~. _.. __ _..~_._.
CA 02449414 2003-12-11 'i
-110-
hybridization signal located at random in the nucleus, and one red-green
doublet signal in which the separation between the two colors was less than I
micron (or one yellow hybridization signal for hybridization in very close
proximity, see Figure 10). The randomly located red and green signals are
ascribed to hybridization to the ABL and BCR genes on the normal
chromosomes, and the red-green doublet signal to hybridization to the BCR
ABL fusion gene. Interphase mapping studies suggest that DNA sequences
separated by less than 250kb should be separated in ihterphase nuclei by less
than 1 micron (25). As a result, cells showing red and green hybridization
signals separated by greater than 1 micron were scored as normal since this is
consistent with the hybridization sites being on different chromosomes.
However, due to statistical considerations, some normal cells will have red
and green dots close enough together to be scored as abnormal. In these two
dimensional nuclear analyses, 9 out of 750 normal nuclei had red and green
hybridization signals Less than 1 micron of each other. Thus, approximately
1% of normal cells were classified as abnormal.
Table 3 shows the hybridization results for 7 samples from 6 CML
cases along with conventional karyotypes, and other diagnostic results (PCR
and Southern blot data ). All six cases, including 3 that were found to be Phl
negative by banding analysis (CML-5,-6 and -7), showed red-green
hybridization signals separated by less than I micron in greater than 50% of
nuclei examined. In most, the fusion event was visible in almost every cell.
One case (CML-7) showed fusion signals in almost every cell even though
PCR analysis failed to detect the presence of a fusion gene and banding
analysis did not reveal a Philadelphia chromosome.
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CA 02449414 2003-12-11
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~ 02449414 2003-12-11 -~,. .-..__.. _..__
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Hybridization to metaphase spreads was performed in three cases
(CML-I, 5 and -6). All of these showed red and green hybridization signals in
close proximity on a single acrocentric chromosome. In two cases, scored as
t(9:22)(q34;q1I) by banding, the red-green pair was in close proximity to the
telomere of the long arm of a small acrocentric chromosome as expected for
the PhI (Figure 9C). One case (CML-6) was suspected by classical cytogenetics
to have an insertion of chromosomal material at 22q11. Dual color
hybridization to metaphase spreads from this case showed the red-green pair
to be centrally located in a small chromosome (Figure ~D). That result is
consistent with formation of the BCR-ABL fusion gene by an insertion. In
one case (CML-1), two pairs of red-green doublet signals were seen in 3 out
150
(2%) interphase nuclei. That may indicate a double Phl (or double fusion
gene) in those cells. Such an event was not detected by standard cytogenetics,
which was limited to analysis of 25 metaphase spreads. The acquisition of an
additional PhI is the most frequent cytogenetic event accompanying blast
transformation, and its cytogenetic detection may herald disease acceleration.
Simultaneous hybridization with ABL and BCR probes to metaphase
spreads of the CML derived cell line K-562 showed multiple red-green
hybridization sites along both arms of a single acrocentric chromosome.
Hybridization to interphase nuclei showed that the red and green signals
were confined to the same region of the nucleus. That is consistent with their
being localized on a single chromosome. Twelve to fifteen hybridization
pairs were seen in each nucleus indicating corresponding amplification of the
BCR ABL fusion gene (see Figures 9E and 9F). These findings are consistent
with previous Southern blot data showing amplification of the fusion gene in
this cell Line (26).
CA 02449414 2003-12-11
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In summary, analysis of interphase cells for seven CIVIL, and four
normal cell samples using dual color FISH with ABL and BCR probes suggests
the utility of this approach for routine diagnosis of CIvIL and clinical
monitoring of the disease. Among its very important advantages are the
ability to obtain genetic information from individual interphase or
metaphase cells in less than 24 hours. Thus, it can be applied to all cells of
a
population, not just to those that fortuitously or through culture, happen to
be in metaphase. Further, the genotypic analysis can be associated with cell
phenotype, as judged by morphology or other markers, thereby permitting the
study of lineage specificity of cells carrying the CML genotype as well as
assessment of the frequency of cells carrying the abnormality.
Random juxtaposition of red and green signals in two dimensional
images of normal cells, which occurs in about 0.01 of normal cells, sets the
low frequency detection limit. That detection limit may be lowered by more
complete quantitative measurement of the separation and intensity of the
hybridization signals in each nucleus using computerized image analysis.
Such analysis will be particularly important in studying patient populations
in which the cells carrying the BCR-ABL fusion at low frequency (e.g., during
remission, after bone marrow transplantation, during; relapse or in model
systems).
This assay also should be advantageous for detection of CML cells
during therapy when the number of cells available for analysis is low since
only a few cells are required. Finally, simple counting of hybridization spots
allows for the detection and quantitative analysis of amplification of the
BCR-ABL fusion gene as illustrated for the K562 cell line (Figure 9E).
M ,~.,. ~x.~..~, ~,w,~~,e~n~.~~...,",~,_~.~..M..__..~..,~.,~,~,,~",
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CA 02449414 2003-12-11 A"
-1I4 -
Quantitative measurement of fluorescence intensity may assist with such an
analysis.
IX.
Generally the method for preparing and applying single stranded
DNA hybridization probes to double stranded target DNA involves treating
both target DNA and probe DNA with the same restriction endonuclease
followed by digestion of single strands adjacent to the restriction cuts.
Probes
axe constructed by resynthesizing the digested single strands with labeled
nucleotides. The labeled strands are substantially cornplementary to the
undigested single strands of the target DNAs. The double stranded DNA
fragments containing the labeled single strands axe bxoken into smaller pieces
and denatured. The hybridization probes are obtained by separating the
labeled single stranded fragments from the unlabeled fragments.
DNA to be used in the probes is treated with a restriction
endonuclease to form restriction fragments having "sticky" ends. That is, it
is
important that the restriction endonuclease make a staggered cut through the
double stranded DNA. Suitable restriction endonuclease include, but axe not
limited to, Hind III, Bam H1, Eco R1, or the like, all of which are
commercially available, e.g., Promega Biotec (Madison, WI), or Boehringer
Mannheim (Indianapolis, IN). In selecting a restriction endonuclease it is
preferable that the resulting restriction fragments be within a size range
which allows them to be directly inserted into available cloning vectors.
Suitable cloning vectors included plasmids, such as pBR322, and phages, such
as lambda phage, various derivatives of both of these being commercially
CA 02449414 2003-12-11
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available, e.g., Promega Biotec (Madison, VIII), and Boehringer Mannheim
(lndianapolis, lN). Amplified copies of the restriction fragments are
isolated' using
standard techniques, e..g., Maniatis et al., o(~ular Gloning: A Laboratortr
Manual
(Cold Spring Harbor Laboratory, 1982). Alternatively, for some applications
restriction fragments can be obtained from existing libraries. For example,
the
American Type . Culture Collection, Rockville, MD, holds collections of human
chromosome-specific libraries of restriction fragments which are available to
the
.public.
Standard procedures are followed in treating the restriction fragments with
exonuclease, and in enzymatically re-synthesizing the digested strands iw the
presence of labeled .precursors. .In particular the technique disclosed by
James
and Leffak, nal.~iochem., Vol. 141, , pp. 33-37 (1984), is followed. Briefly,
to the
restriction fragments about 3 units of exonuciease Iil are added per microgram
of
DNA in a solution consisting of 100 mM Na.Ci, 50 mM Tris-HCl (pH 8.0), 7 0 mM
IS MgCl2 and 1 mM dithiothreitol, at 37°C. Digestion is terminated by
heating the
sample to 60°C for 5-10 minutes. James and. Laffak report that these
conditions
result in the digestion of about '80-200 nucleotides 'per minute. The actual
digestion rate will vary depending on the source and batch of exonuclease III
as
well as the source of the DNA substrate, e.g., Guo et al., Nucl. Acid~Res..
Vol..10
.pg. 2065. Some experimentation may be necessary to ~abtain labeled stands of
the
desired length. Exonuclease 111 is available commercially, e.g., Boehringer
lVlannheim~ (Indianapolis; IN), or Promega Biotec (Madison, WI). Also, T4
polymerase (BURL, Bethesda, MD) can be used for both the exonuclease and
resynthesis steps.
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The exonuclease treated restriction fragments serve as
primer/templates for a DNA polymerise which re-synthesizes the digested
strands in the presence of labeled precursors. The preferred labeled precursor
is biotinylated uracil, as a substitute for thymidine. Re-synthesis is
accomplished using DNA polymerise I or T4 DNA polymerise following the
procedure of Linger et al., Proc. Nat'1. Acid. Sci., Vol. T8, pp. 6633-6637
(1981)
which, in turn, is an adaption of the basic nick translation technique
disclosed
by Rigby et al., j. Mol. Biol., Vol. 1I3, pg. 237 (1977), e.g., 1 unit E. Coli
polymerise I per microgram of DNA is incubated at 3,7°C in a solution
consisting of 100 mM Nacl, 50 mM Tris-HCl (pH 8.0), 10 mM MgCl2, I mM
dithiothreitol, and 50 mM KCI. Also included in the solution are appropriate
amounts of the triphosphate precursors (one or more of which are labeled),
e.g., 50-100 micromolar of each for 20-50 micrograms per milliliter of
restriction fragments. Under these conditions resynthesis is completed in
about 4t?-60 minutes
The labeled restriction fragments are broken into smaller fragments
to ensure that the labeled regions on either end of the labeled restriction
fragments are separated. (Otherwise, the labeled fragment on one end would
be a part of a larger piece of single stranded DNA which contained
complementary regions to the labeled fragment on the other end). Such
breaking into smaller fragments is accomplished by any number of standard
techniques, e.g. sonication, enzymatic treatment, or the like, Maniatis, et
al.
Molecular Cloning: A Laborator~r Manual (Cold Spring Harbor Laboratory,
1982).
After the labeled restriction fragments are appropriately broken into
smaller pieces, they are denatured and single stranded labeled fragments are
~., ~ 02449414 2003-12-11 v - . __._. ,~..___.~,.m--._.___
-117
separated from unlabeled fragments. The separation can be accomplished in
several ways. Whenever the preferred label, biotin, is used the preferred
separation means is by way of a standard avidin affinity column, e.g. Bayer
and Wilchek, "The Use of the Avidin-Biotin Complex as a Tool in Molecular
Biology;' Methods of Biochemical Analysis, Vol 26, pp.- I-45 (1980); and
Manning et al., Biochemistry, Vol. I6, pp. 1364-1370 (1977). Avidin can be
covalently coupled to a number of different substrates, such as glass,
Sepharose, agarose, and the like, with standard techniques as described in the
above references. Accordingly; Manning et al. and Bayer and Wilchek, pp. 9-
16, are incorporated by reference. Avidin affinity columns are also available
commercially, e.g. Zymed Laboratories, Inc.. (South San Francisco, CA). The
biotinylated probes are removed from the avidin column following the
procedure of Chollet and Kawashima, Nucleic Acids Resources, Vol. 5,
pp.1529-1541 (1985).
Alternatives to the above labeling procedure are available. For
example, after the DNA to be used in the probes is treated with a restriction
endonuclease, the resulting restriction fragments are separated into two
portions. The first portion undergoes treatment as described above. That is,
it
is treated with exonuclease to form template/primers for resynthesizing a
labeled strand of D1VA. The resulting resynthesized restriction fragments are
then broken into smaller pieces, as described above. The label in this case,
need not be biotin. For example, a radioactive label can be used. The second
portion is also treated with an exonuclease, preferably exonuclease ILi.
However, the reaction is allowed to proceed to completion so that each
restriction fragment is converted into two noncomplementary single
stranded pieces approximately half the length of the parent strand. These
*Trademark
CA 02449414 2003-12-11 , . __.
- 118 -
resulting single strands are then covalently linked to DBM paper using
standard
techniques, e.g. Maniatis et al; Molecular Cloningv. A Laboratory Manual (Cold
Spring Harbor Laboratory, 1982) pp. 335-339; and Alwine et al., Methods in
~nzvmoloav. Vol. 68, pp. 220-242 (Academic Press, New York, 1979). The
fragments of the first portion are denatured and combined with the DBM paper
containing the covalently finked fragments of the second portion. Conditions
are
adjusted to permit hybridisation of the labeled. strands' to complementary
strands
covalently linked to the DBM paper. The unlabeled strands from the first
portion
are washed from the paper (there being no complementary strands for them to
hybridize to). After the washing the labeled strands are removed by heating,
for
example, and are ready for use.
Before application of the probe to the target DNA, the target DNA is treated
with the same restriction endonuclease as was used in construction of the
probe.
After restriction endonuclease treatment the target DNA is treated with an
1S exonuclease, preferably exonuclease III or T4 polymerase. Preferably, the
conditions of exonuclease treatment are adjusted so that the lengths of single
stranded regions created are substantially the same as the lengths of the
probe
DNA,
Hybridization of probe to target DNA is carried out using standard
procedures, e.g. Gall and Pardue, Mett~~s,~n E,~z~rmolocyy, Vol. 21, pp. 270-
4.80
(1981); Henderson, Intern~tiQnal F~view of Cy.,tolog~r, Vol. 76, pp. 1-46
(1982);
and Angerer et al., in Genefiic Enaj~eerina ~ Pi rinciples and Methods, Setlow
and
Hollaender, Eds., Vol. 7, pp. 43-65 (Plenum Press, New York, 1985). These
references are useful as guides for the
~.v» .~.._..~ .....~,x..«~,.~.~,".~" .~~,: .~.~.-..-....~.~._
__.._.___..___.__
CA 02449414 2003-12-11
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use of the invention in in situ hybridization. Briefly, probe prepared in
accordance with the invention is combined with several other agents for
reducing nonspecific binding, for maintaining the integrity of the biological
structure being probed, and the like. The resulting mixture is referred to
herein as the hybridization mix. Below; the method is applied. in the
chromosome-specific staining of human chromosome 21.
Hind III restriction fragments of human chromosome 21 are available
from the National Laboratory Gene Library Project through the American
Type Culture Collection, Rockville, MD, Van Dilla et al., "Human
Chromosome-Specific DNA Libraries: Construction and Availability,"
Biotechnology, Vol. 4, pp. 537-552 (1986). Alternately, such fragments can be
produced following the disclosures in Van Dilla et al., cited above, or Fuscoe
et al., "Contraction of Fifteen Human Chromosome-Specific DNA Libraries
from Flow-Purified Chromosomes, " Cr~ogenet Cell Genet., 43:79-86 (1986).
Clones from the library having unique sequence inserts are isolated
by the method of Benton and Davis; Science, VoI. 196, pp. I80-182 (I977).
Briefly, about 1000 recombinant phage are isolated at random from the
chromosome 21-specific library. These are transferred to nitrocellulose and
probed with nick translated total genomic human DNA.
Of the clones which do not show strong hybridization, approximately
300 are picked which contain apparent unique sequence DNA. After the
selected clones are amplified, the chromosome 21 insert in each clone is 32p
labeled and hybridized to Southern blots of human genomic DNA digested
with the same enzyme used to construct the chromosome 21 library, i.e., Hind
III. Unique sequence containing clones are recognized as those that produce a
single band during Southern analysis. Roughly, 100 such clones are selected
CA 02449414 2003-12-11
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for the heterogeneous mixture of probe DNA. The unique sequence clones
are amplified, the inserts are removed by Hind IB digestions, and the inserts
are separated from the phage arms by gel electrophoresis. The probe DNA
fragments (i.e., the unique sequence inserts) are removed from the gel and
treated with exonuclease III as described above, followed by resynthesis in
the
presence of biotinylated UTZ' precursor. The resulting double stranded
fragments are sonicated so that on an average each fragment receives about
1.5-2.0 breaks. The resulting pieces are denatured and the bioHnylated
fragments are isolated by avidin affinity chromotography as described above.
Human lymphocyte chromosomes are prepared following Harper
et al., Proc. Nat'1 Acad. Sci., VoL 78, pp. 4458-4460 (I98I). Metaphase and
interphase cells are washed 3 times in phosphate buffered saline, fixed in
methanol-acetic acid (3:1) and dropped onto cleaned microscope slides. Slides
are stored in a nitrogen atmosphere at -20°C.
Slides carrying interphase cells and/or metaphase spreads are
removed from the nitrogen treated with RNase (I00 micrograms/ml for 1
hour at 37°C), treated for about 1-16 hours with Hind III at
37°C (I0 units M IO
mM Tris-HCI, 50 mM NaCl, IO mM MgCI2, and I4 mM dithioenythritol at
pH 7.6), treated with exonuclease III as described above, and dehydrated in an
ethanol series. They are then treated with proteinase K (60 ng/ml at
37°C for
7.5 minutes) and dehydrated. The proteinase K concentration is adjusted
depending on the cell type and enzyme lot so that almost no phase
microscopic image of the chromosomes remains on the dry slide. The
hybridization mix consists of (final concentrations) 2X SSC (0.15 M NaCI and
0.015 M sodium nitrate) 10 percent dextran sulfate, 500 micrograms/ml carrier
DNA (sonicated herring sperm DNA), and 2.0 microgram/ml biotin-labeled
~~,~~~ _. _ _.. _
CA 02449414 2003-12-11
-I2I-
probe DNA. This mixture is applied to the slides at a density of 3
microliters/cm2 under a glass coverslip and seated with rubber cement. After
overnight incubation at 37°C, the slides are washed at 45°C (50%
formamide-
2XSS pH 7, 3 times 3 minutes; followed by ZXSSC pH 7, 5 times 2 minutes)
and immersed in BN buffer (0.I M Na bicarbonate, 0.05 percent NP-40, pH 8).
The slides are never allowed to dry after this point.
The slides are removed from the BN buffer and blocked for 5 minutes
at room temperature with BN buffer containing 5% non-fat dry milk
(Carnation) and 0.92% Na Azide (5 microliter/em2 under plastic coverslips).
The coverslips are removed, and excess liquid briefly drained and fluorescein-
avidin DCS (3 microgram/ml in BN buffer with 5% milk and 0.02% Na
Azide) is applied (5 microliter/ cm2: The same coverslips are replaced and
the slides incubated 20 minutes at 37°C. The slides are then washed 3
times
for 2 minutes each in BN buffer at 45°C. The intensity of biotin-linked
fluorescence is amplified by adding a layer of biotinylated goat anti-avidin
antibody (5 microgram/ml in BN buffer with 5% goat serum and 0.02%
NaAzide) followed, after washing as above, by another layer of fluorescein-
avidin DCS. Fluorescein-avidin DCS, goat antiavidin and goat serum are all
available commercially, e.g., Vector Laboratories (Burlingame, CA). After
washing in BN, a fluorescence antifade solution, p-phenylenediamine (1.5
microliter/cmz of coverslip) is added before observation. It is important to
keep this layer thin for optimum microscopic imaging. This antifade
significantly reduced fluorescein fading and allows continuous microscopic
observation for up to 5 minutes. The DNA counterstains (DAPI or
propidium iodide) are included in the antifade at 0.25-0.5 microgram/ml.
~~~-
CA 02449414 2003-12-11 i!!
- 122 -
The red-fluorescing DNA-specific dye propidium iodide (PI) is used to allow
simultaneous observation of hybridized probe and total DNA. The fluorescein
and
PI are, excited at 450-490 nm (Zeiss fitter combination 487709). Increasing
the
excitation wavelength to 546 nm (Zeiss filter combination 487715) allows
observation of, the PI only. DAPI, a blue fluorescent DNA-specific stain
excited in
the ultraviolet (Zeiss filter combination 487701 ), is used as the
counterstain when
biotin-Labeled and total DNA are observed separately. Metaphase chromosomes
21.s are detected by randomly located spots of yellow distributed over the
body of
the chromosome.
The descriptions of the foregoing embodiments of the invention have been
presented for purpose of illustration and description. They are not intended
to be
exhaustive or to limit the invention to the precise form disclosed, and
obviously
many modifications and variations are possible in light of the above
teachings.
The embodiments were chosen and described in order to best explain the
principles of the invention and its practical application to thereby enable
others
skilled in the art to best utilize the invention in various embodiments and
with
various modifications as are suited to the particular use contemplated. It is
intended that the scope of the invention be defined by the claims appended
hereto.
CA 02449414 2003-12-11 ',
