U.S. patent application number 10/913960 was filed with the patent office on 2005-09-01 for delineation of individual human chromosomes in metaphase and interphase cells by in situ suppression hybridization.
This patent application is currently assigned to YALE UNIVERSITY. Invention is credited to Baldini, Antonio, Cremer, Thomas, Lichter, Peter, Manuelidis, Laura, Ried, Thomas, Ward, David C..
Application Number | 20050191642 10/913960 |
Document ID | / |
Family ID | 26955026 |
Filed Date | 2005-09-01 |
United States Patent
Application |
20050191642 |
Kind Code |
A1 |
Ward, David C. ; et
al. |
September 1, 2005 |
Delineation of individual human chromosomes in metaphase and
interphase cells by in situ suppression hybridization
Abstract
This disclosure relates to a method of specifically decorating
selected mammalian chromosomes and of detecting, identifying and
and/or quantitating selected individual chromosomes, by means of
chromosomal in situ suppression (CISS) hybridization. The method is
useful in analyzing cells for the occurrence of chromosomes,
chromosome fragments or chromosome aberrations.
Inventors: |
Ward, David C.; (New Haven,
CT) ; Lichter, Peter; (Heidelberg, DE) ;
Cremer, Thomas; (Heidelberg, DE) ; Manuelidis,
Laura; (New Haven, CT) ; Ried, Thomas;
(Heidelberg, DE) ; Baldini, Antonio; (London,
GB) |
Correspondence
Address: |
LAHIVE & COCKFIELD, LLP.
28 STATE STREET
BOSTON
MA
02109
US
|
Assignee: |
YALE UNIVERSITY
New Haven
CT
|
Family ID: |
26955026 |
Appl. No.: |
10/913960 |
Filed: |
August 5, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10913960 |
Aug 5, 2004 |
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09335956 |
Jun 18, 1999 |
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09335956 |
Jun 18, 1999 |
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08312429 |
Sep 26, 1994 |
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6203977 |
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08312429 |
Sep 26, 1994 |
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07837664 |
Feb 14, 1992 |
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07837664 |
Feb 14, 1992 |
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07271609 |
Nov 15, 1988 |
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Current U.S.
Class: |
435/6.14 ;
435/455 |
Current CPC
Class: |
C12Q 1/6809 20130101;
C12Q 1/6841 20130101 |
Class at
Publication: |
435/006 ;
435/455 |
International
Class: |
C12Q 001/68; C12N
015/85 |
Goverment Interests
[0001] Work described herein was supported by Grant Numbers
GM-32156 and CA-15044 awarded by the National Institutes of Health.
The United States Government has certain rights in the invention.
Claims
1. A method of labeling individual mammalian chromosomes in mitotic
cells or in interphase cells by in situ hybridization with
chromosome-specific probes, to produce a chromosome-specific
signal.
2. A method of labeling individual human chromosomes in mitotic
cells or in interphase cells by in situ hybridization with
chromosome-specific probes, to produce a chromosome-specific
signal.
3. A method of producing highly specific decoration of an
individual human target chromosome, comprising in situ suppression
hybridization of labeled DNA probes, which are chromosome specific,
to DNA in human mitotic cells or in human interphase cell.
4. A method of claim 3, wherein the DNA probes are selected from
the group consisting of total recombinant library DNA, DNA inserts
purified from a chromosome-derived recombinant DNA library, and
specific DNA fragments derived from chromosomes.
5. A method of claim 4, wherein the labeled DNA probes are selected
from the group consisting of: DNA probes labeled with at least one
fluorochrome; DNA probes labeled with at least one member of a
specific binding pair; and DNA probes labeled with an enzyme.
6. A method of-Claim 5, wherein the fluorochrome is selected from
the group consisting of fluorescein, rhodamine, Texas red, Lucifer
yellow, phycobiliproteins and cyanin dyes and the member of a
specific binding pair is biotin.
7. A method of assessing chromosome abberations in human cells by
chromosomal in situ suppression hybridization.
8. A method of claim 7, wherein the human cells are selected from
the group consisting of metaphase cells, prophase cells and
interphase cells.
9. A method of detecting chromosome aberrations in human aneuploid
cells, comprising a) combining 1) the human aneuploid cells,
treated so as to render nucleic acid sequences present available
for hybridization with complementary nucleic acid sequences; and 2)
a hybridization mixture comprising labeled human DNA derived from a
specific chromosome; competitor DNA; and nonhuman genomic DNA,
under conditions appropriate for hybridization of complementary
nucleic acid sequences to occur; and b) detecting labeled human DNA
derived from the specific chromosome hybridized to nucleic acid
sequences from the aneuploid cells.
10. A method of claim 9, wherein the aneuploid cells are human
tumor cells.
11. A method of claim 10, in which the human tumor cells are
selected from the group consisting of: metaphase cells, prophase
cells and interphase cells.
12. A method of claim 10, wherein the human tumor cell is a human
glioma cell.
13. A method of detecting in a sample numerical alterations in a
human chromosome present in the sample, comprising: a) combining 1)
the sample; treated so as to render nucleic acid sequences present
in the sample available for hybridization with complementary
nucleic acid sequences; and 2) a hybridization mixture comprising
labeled human DNA derived from the selected chromosome; competitor
DNA; and nonhuman genomic DNA, under conditions appropriate for
hybridization of complementary nucleic occur; and b) detecting
labeled DNA de-selected chromosome hybridization of complementary
nucleic acid sequences to occur; and b) detecting labeled DNA
derived from the selected chromosome hybridized to nucleic acid
sequences present in the sample.
14. A method of claim 13, wherein the selected human chromosome is
selected from the group consisting of the following chromosomes:
13, 18, 21, X and Y.
15. A method of claim 13, wherein the selected human chromosome is
chromosome number 21 and the labeled human DNA derived from the
selected chromosome is DNA inserts purified from a
chromosome-derived recombinant DNA library.
16. A method of determining over-representation or
under-representation of a selected chromosome or a portion thereof
in human tumor cells comprising the steps of: a) combining 1) human
tumor cells, treated so as to render nucleic acid sequences present
in the cells available for hybridization with complementary nucleic
acid sequences; and 2) a hybridization mixture comprising labeled
DNA fragments derived from a selected chromosome; competitor DNA;
and nonhuman genomic DNA, under conditions appropriate for
hybridization of complementary nucleic acid sequences to occur; and
b) detecting labeled human chromosome-specific DNA fragments
hybridized to nucleic acid sequences from the tumor cells.
17. A method of identifying chromosome-specific DNA present in a
selected mammalian chromosome, comprising: a) combining the
following substances: 1) the selected mammalian chromosome; 2) DNA
fragments derived from the selected mammalian chromosome bearing a
detectable label; 3) competitor DNA; and 4) carrier DNA, under
conditions appropriate for hybridization of complementary nucleic
acid sequences to occur; to form a complex of the DNA fragments
bearing a detectable label with the selected mammalian chromosome;
and b) detecting complexes formed in step (a).
18. A method of claim 17, further comprising isolation of
chromosome-specific DNA in a selected mammalian chromosome by
separating the selected complexes formed from the remaining
substances combined in step (a).
Description
BACKGROUND
[0002] Chromosome banding techniques have facilitated the
identification of specific human chromosomes and presently provide
the major basis upon which chromosomal aberrations are diagnosed.
The interpretation of chromosome banding patterns requires skilled
personnel and is often technically difficult, especially with
respect to detecting minor structural changes and when analyzing
complex karyotypes, such as those of highly aneuploid tumor cells.
An additional complexity is that readable metaphase chromosome
spreads are sometimes very difficult or impossible to prepare from
certain cell types or tissues. Alternative methods for identifying
chromosomal aberrations would be valuable because they could
augment current methods of cytogenic analysis, particularly if such
alternative methods were applicable to both mitotic and interphase
cell populations.
[0003] Over the past few years, a considerable body of evidence has
been obtained which indicates that the DNA of individual
chromosomes occupy focal territories, or spatially cohesive
domains, within mammalian interphase nuclei. Cremer, T. et al.,
Hum. Genet., 60:46-56 (1982); Hens, L. et al., Exp. Cell Res., 149;
257 269 (1983); Schwardin, M. et al., Hum. Genet., 71:281-287
(1985); Manuelidis, L., Hum. Genet., 71:288-293 (1985); and Pinkel,
D. et al., Proc. Natl. Acad. Sci. USA, 83:2934-2938 (1986). These
observations suggest that chromosome-specific probe sets could be
used to detect numerical or structural aberrations of chromosomal
domains in non-mitotic cells, an approach termed "interphase
cytogenics". Cremer, T. et al., Hum. Genet., 74:346-352 (1986).
Indeed, recent in situ hybridization studies have demonstrated the
prenatal diagnosis of trisomy-18 with interphase cells and the
detection of numerical chromosomal abnormalities in tumor cells
lines using chromosome-specific repetitive DNAs as probes. Cremer,
T. et al., Hum. Genet., 74:346-352 (1986) and Cremer, T. et al.,
Exp. Cell Res., 176:119 220 (1988). All chromosome-specific
repetitive DNAs reported to date are localized to discrete
subregions of each chromosome and, thus, such DNA probes are
unsuitable for analyses of many types of chromosomal aberrations
(e.g., translocations and deletions). If it were possible to detect
uniquely the spectrum of sequences comprising a specific
chromosome, analysis of aberrations of chromosomal domains in
non-mitotic cells would be possible. Furthermore, such a general
labeling technique would make it possible to address fundamental
questions concerning the spatial organization of chromosomal DNA
within interphase nuclei.
DISCLOSURE OF THE INVENTION
[0004] The subject invention relates to a method of detecting,
identifying and/or quantitating selected individual chromosomes in
mammalian mitotic or interphase cells, by means of chromosomal in
situ suppression (CISS) hybridization and its use in analyzing
cells for the occurrence of chromosomes, chromosome fragments, or
chromosome aberrations, such as those associated with a condition
or disease. In the method of the present invention,
chroniosome-specific probes (DNA or RNA) are combined with a sample
to be analyzed, in such a manner that an individual chromosome(s)
of interest is labeled and the complex spectrum of sequences which
comprise the chromosome can be detected. The probes used in the
present method are of high genetic complexity and can be
appropriately-selected cloned DNA or RNA fragments, used
individually or in pools, or chromosome library DNA.
[0005] The method of the present invention, referred to as CISS
hybridization, is particularly useful because it can be used to
specifically stain individual mammalian chromosomes at any point in
the cell cycle. It can be used to assess chromosomal content,
particularly chromosome aberrations (e.g., deletions,
rearrangements, change in chromosome number) which, until the
present invention, have been time-consuming and/or difficult, if
not impossible, to detect. The method is useful in providing a
rapid and highly specific assessment of individual mammalian
chromosomes in any context (e.g., diagnosis and/or monitoring of a
genetic condition or a disease state) in which such an assessment
is desired.
BRIEF DESCRIPTION OF THE DRAWINGS
[0006] FIG. 1 presents an outline of CISS hybridization for
specific staining of human chromosomes.
[0007] FIGS. 2A-2F show suppression from cross-reacting sequences
within a chromosome 7-derived DNA library by different
concentrations of human competitor DNA. Biotin-labeled chromosome 7
DNA inserts (20 .mu.g/ml ) were prehybridized for 20 minutes with
human genomic DNA prior to hybridization with metaphase chromosome
spreads and detection with FITC-labeled avidin. Genomic salmon DNA
was added to each sample to adjust the final DNA concentration to
1.0 mg/ml (see the text for details). The arrows mark the target
chromosome 7 and the arrowheads mark additional strong signals on
non-7 chromosomes. All negatives printed were exposed and developed
under identical photographic conditions.
[0008] FIG. 2A shows prehybridization with 0 .mu.g/ml human
competitor DNA.
[0009] FIG. 2B shows prehybridization with 50 .mu.g/ml of human
competitor DNA.
[0010] FIG. 2C shows prehybridization with 100 .mu.g/ml of human
competitor DNA.
[0011] FIG. 2D shows prehybridization with 200 .mu.g/ml of human
competitor DNA.
[0012] FIG. 2E shows prehybridization with 1000 .mu.g/ml of human
competitor DNA.
[0013] FIG. 2F is the same as FIG. 2E except that the metaphase
spread is post-stained with DAPI.
[0014] FIGS. 3A-3D show the effect of pre-annealing time on the
specificity and strength of the hybridization signal.
Biotin-labeled chromosome 7 DNA inserts (20 .mu.g/ml) were
preannealed with 200 .mu.g/ml human competitor DNA for different
times prior to hybridization to metaphase chromosomes.
[0015] FIG. 3A shows preannealing for 0 minutes.
[0016] FIG. 3B shows preannealing for 2 minutes.
[0017] FIG. 3C shows preannealing for 5 minutes.
[0018] FIG. 3D shows preannealing for 20 minutes.
[0019] FIG. 4A show decoration of chromosome 1 in normal human
lymphocytes. The signal of chromosome I was amplified by the
sandwich technique of Pinkel et al. (1986).
[0020] FIG. 4B shows decoration of chromosome 7 in normal human
lymphocytes.
[0021] FIG. 4C shows decoration of chromosome 4 in normal human
lymphocytes.
[0022] FIG. 4D shows decoration of chromosome 18 in normal human
lymphocytes.
[0023] FIG. 4E shows decoration of chromosome 13 in normal human
lymphocytes. Only the chromosome 13 insert DNA pool shows
significant cross-hybridization to other chromosomes after the
prehybridization suppression step.
[0024] FIG. 4F shows decoration of chromosome 20 in normal human
lymphocytes. The detection of chromosome 20 was done with the
entire chromosome library (including .lambda. phage arms) and
detected with avidin-alkaline phosphatase using nitro blue
tetrazolium/5-bromo-4-chloro- -3-indolyl phosphate (NBT-BCIP) as
the enzyme substrate mixture.
[0025] FIGS. 5A-5F show chromosome domains in human lymphocyte
nuclei delineated by preannealed chromosome library DNA inserts.
FIG. 5A and FIG. 5B show domains for chromosome 1. Hybridization to
acetic acid-methanol fixed nuclei was detected by fluorescein
isothiocyanate (FITC)-conjugated avidin.
[0026] FIG. 5C and FIG. 5D show domains for chromosome 7.
Hybridization to acetic acid-methanol fixed nuclei was detected by
fluorescein isothiocyanate (FITC)-conjugated avidin. A predominant
staining of the centromere region is seen within the chromosome 7
domains, reflecting preferential hybridization of the chromosome
7-specific alphoid DNA repeat; a similar signal distribution on
metaphase chromosomes was also observed in the particular
experiment.
[0027] FIG. 5E shows domains for chromosome 18. Hybridization to
acetic acid-methanol fixed nuclei was detected by fluorescein
isothiocyanate (FITC)-conjugated avidin.
[0028] FIG. 5F shows domains for chromosome 18. Hybridization to
acetic acid-methanol fixed nuclei was detected by alkaline
phosphatase-conjugated avidin.
[0029] FIG. 6A shows chromosomal in situ suppression (CISS)
hybridization of chromosome 1 inserts to metaphase spreads of the
TC 620 glioma cell line detected with FITC-avidin. FIG. 6B is the
same as FIG. 6A except that the metaphase spreads are post-stained
with 4,6-diamidino-2-phenylin- dole dihydrochloride (DAPI). TC 620
show two apparently complete 1 chromosomes (small arrows in B) and
two marker translocation chromosomes (arrowheads) specifically
decorated by these inserts. One of the two marker chromosomes
contains a 1p (lower left), the other a 1q arm (lower right); the
1p terminal (relatively GC rich region) in the two normal
chromosomes and submetacentric marker is less completely
delineated. Also, the 1q12 regions here show little decoration in
contrast to most experiments. X950.
[0030] FIG. 6C shows chromosomal in situ suppression (CISS)
hybridization of chromosome 1 inserts to metaphase spreads of the
TC 593 glioma cell line detected with FITC-avidin.
[0031] FIG. 6D is the same as FIG. 6C except that the metaphase
spreads are post-stained with 4,6-diamidino-2-phenylindole
dihydrochloride (DAPI). Typical TC 593 metaphase spreads show six
specifically decorated chromosomes. Three acrocentric marker
chromosomes all with truncation of 1p show particularly intense
fluorescence of repeats that localize to 1q12 (arrows in C). In two
of these, 1q arms appear to be complete, while a major deletion is
obvious in the third (arrow in D). A fourth decorated chromosome
(small arrowhead in C, D) again shows a major deletion of the
distal part or 1q, but has retained an apparently complete 1p arm.
A fifth submetacentric chromosome (large arrowhead) contains an
apparently complete 1p arm; the DNA of its short arm is not
identified. Note the similarity of this marker to one of the marker
chromosomes of TC 620 (1p) described above. The sixth entirely
decorated chromosome is an iso (1p) as demonstrated by DAPI-binding
(open arrows). X 1200.
[0032] FIGS. 7A-7J show CISS hybridization of chromosome 4 library
inserts detected with FITC-avidin.
[0033] FIG. 7A shows interphase nuclei of TC 593. Note that the two
apparently complete interphase domains are widely separated.
[0034] FIG. 7B shows interphase nuclei of TC 593. Note that the two
apparently complete interphase domains are close to each other.
[0035] FIG. 7C shows interphase nuclei of TC 593. Note that the two
apparently complete interphase domains are widely separated.
[0036] FIG. 7D shows interphase nuclei of TC 620 showing four
chromosome 4 interphase domains of largely different sizes.
[0037] FIG. 7E shows metaphase spread of TC 593 showing two
apparently complete 4 chromosomes, and a small, decorated region
(arrow) in a submetacentric chromosome. This marker with
translocated 4 sequences was observed in about 30% of the
spreads.
[0038] FIG. 7F shows metaphase spread of TC 620 showing one
apparently complete chromosome 4 and three translocation markers
(t) containing different amounts of chromosome 4 material.
[0039] FIG. 7G shows double hybridization of biotinylated
chromosome 7 inserts and an aminoacetylfluorene (AAF)-modified
7-specific alphoid repeat. Chromosome 7 inserts depict five
entirely decorated metaphase chromosomes. Four of them are complete
7 chromosomes, the fifth (arrow) is an iso (7p) (see FIG. 3E).
[0040] FIG. 7H shows the same field as G showing AAF-7 alphoid
signals on only four decorated chromosomes; no signal is detected
on the iso (7p).
[0041] FIG. 7I shows an interphase nucleus of TC 593 showing five
domains delineated by chromosome 7 inserts. The arrow represents
the iso (7p) marker in interphase.
[0042] FIG. 7J shows the same field as I showing that four of the
domains are labeled by 7 alphoid probes.
[0043] FIG. 8A shows CISS hybridization of library inserts of
chromosome 7 to metaphase spreads of TC 620 glioma cells detected
with FITC-avidin. x 875. Three apparently normal 7 chromosomes and
an additional translocation chromosome containing 7 sequences are
indicated by large arrowhead.
[0044] FIG. 8B is the same as for FIG. 8A except counter-stained
with DAPI. DAPI-stained complete chromosomes are indicated by small
arrowheads. Other studies (see the text) indicated a translocation
of 7pter-q11 in the marker chromosome (large arrowhead).
[0045] FIG. 8C shows CISS hybridization of library inserts of
chromosome 18 to metaphase spreads of TC 620 glioma cells detected
with FITC-avidin. Two apparently complete 18 chromosomes and a
truncated minute chromosome (large arrowhead) are shown.
[0046] FIG. 8D is the same as for FIG. 8C except counter-stained
with DAPI.
[0047] FIG. 8E shows metaphase spread from pseudotetraploid TC 593
cells showing five chromosomes highlighted by 7 library inserts.
The metacentric chromosome (m) represents the iso (7p) marker
typical for this line (see also FIG. 2G). Insert chromosomes (small
arrows) show DAPI-stained normal and metacentric 7 chromosomes. The
landmark band 7q21 and a block of constitutive heterochromatin at
7q11 are both prominent on the normal chromosome 7 insert (arrows)
but not present on the marker chromosome. Instead both arms of the
latter show a mirror-like staining pattern with a fafint distal
band at 7p21.
[0048] FIG. 8F shows CISS hybridization of library inserts of
chromosome 18 to metaphase spreads of TC 593 glioma cells detected
with FITC-avidin. Four decorated 18 chromosomes are shown.
[0049] FIG. 8G is the same as for FIG. 8F except counter-stained
with DAPI. Three decorated 18 chromosomes are clearly
translocated.
[0050] FIG. 9 is a summary chromosome idiogram of complete and
aberrant chromosomes detected by CISS hybridization of library
inserts of chromosome 1, 4, 7, 18 and 20 in glioma cell lines TC
620 (left) and TC 593 (right). G-bands (black) are shown with
approximate breakpoints suggested by our data; the shaded areas
with a wavy pattern are from other chromosomes that constitute part
of the marker translocation chromosomes. The black dot beside two
of the TC 620 translocated 4 segments indicates that the assignment
of the chromosome 4 material is based on circumstantial evidence
(e.g., size measurements). A small translocation of chromosome 18
material in ca. 20% of TC 593 metaphase spreads (+) also could not
be further identified. Note the over-representation of 7p in both
cell lines.
[0051] FIGS. 10A-10H show representative nuclei reflecting
metaphase abnormalities in glioma cell lines (cf. FIGS. 1-3).
[0052] FIG. 10A: Detection of the 7p translocation (t) in a
prophase TC 620 nucleus. X 1,000.
[0053] FIG. 10B: Detection of five well-separated chromosome 7
domains in interphase TC,593. X 1,240.
[0054] FIG. 10C: Detection of two large and one very small 18
domains (indicated by arrowheads) in interphase TC 6. X 1,450.
[0055] FIG. 10D: Detection of four chromosome 18 domains in
interphase TC 593; one of these signals (arrows) appears smaller. X
1,450.
[0056] FIG. 10E: Detection of four chromosomal 1 domains detected
in interphase TC 620 (cf. FIG. 1A, 1B). X 1,200.
[0057] FIG. 10F: Detection of at least five chromosome 1 domains in
interphase TC 593; one (arrow head) is appreciably smaller than the
others. X 1,250.
[0058] FIG. 10G: CISS hybridization of a metaphase spread of TC 593
with chromosome 18.
[0059] FIG. 10H: The same as for 10G except counter-stained with
DAPI. The technically poor metaphase spread still highlights four
distinct chromosomes bearing 18 sequences. X 1,000.
[0060] FIGS. 11A-11 E are graphic representations of the interphase
and/or metaphase counts of chromosomes 7, 22 and 4 by CISS
hybridization. Interphase counts were performed on 150 nuclei of
well-hybridized preparations. For metaphase counts >25 complete
DAPI-stained spreads were evaluated.
[0061] FIG. 11A: Counts of 7 specific alphoid repeats (white
columns) compared to 7 library inserts (shaded columns) from
interphase nuclei of phytohemagglutinin-stimulated human
lymphocytes (46, XY).
[0062] FIG. 11B: The same as for FIG. 11A except from TC 620
interphase nuclei (7-specific alphoid repeats) and metaphase
spreads (7 library inserts).
[0063] FIG. 11C: The same as for FIG. 11A except from TC 593
interphase nuclei (7-specific alphoid repeats) and metaphase
spreads (7 library inserts).
[0064] For FIGS. 11A-C: High stringency hybridization (see
Materials and Methods) of 7 alphoid repeat was used to avoid
cross-hybridization to other chromosomes. In cases of double
hybridization with both 7 library inserts and alphoid repeat (shown
in FIG. 2 G-I) standard conditions with 50% formamide were
sufficient to avoid cross-hybridization, possibly due to the
presence of human competitor DNA.
[0065] FIG. 11D: Counts of chromosome 22 (library inserts) in
metaphase spreads of TC 620 (black columns) and TC 593 (shaded
columns). For comparison, CISS hybridization was simultaneously
performed with 7 library inserts in these experiments as an
internal control (see C, D and FIG. 7).
[0066] FIG. 11E: Interphase counts (white columns) and metaphase
counts (shaded columns) compared in TC 593 hybridized with
chromosome 4 inserts. Note the ratio of signal preparations 2:3 are
the same in metaphase and interphase.
[0067] FIG. 12 shows a TC 620 metaphase spread after double
hybridization with inserts from chromosome 7 and 22 (both labeled
with biotin and detected with avidin-FITC). Two strongly decorated
22 chromosomes (arrows), three complete 7 chromosomes and the
metacentric marker chromosome containing 7pter-q11 are also
seen.
[0068] FIG. 13 is a graphic representation of the relative size of
decorated normal and aberrant chromosomes 4, 7 and 18 in typical
metaphase spreads (n=24) from glioma cell lines TC 593 and TC 620.
Individual areas were normalized so that a complete chromosome is
represented by an area of 1 (see legend to Table 1). The total
added signals reflect the number of specific chromosome equivalents
present. The white regions correspond to apparently normal
chromosomes, the black regions indicate small free chromosome
segments entirely decorated by specific library inserts, and
translocated segments are shade. One of the three translocated 18
chromosomes in TC 593 represents a complete chromosome by this
measurement (indicated by the black dot), while the two other
translocations are slightly smaller, possibly due to the small
sample size.
[0069] FIGS. 14A-14O show specific labeling of human chromosome 21
by CISS hybridization with biotinylated DNA probe sets.
[0070] FIG. 14A: Hybridization of plasmid pPW519-1R (6 kb insert)
to a normal lymphocyte metaphase spread. Signals are located on the
termini of 21q (see DAPI-stained chromosomes in Inset) as verified
by DAPI banding (not shown).
[0071] FIG. 14B: Hybridization of the 94 kb plasmid pool probe set
to normal human lymphocyte metaphase spread. The terminal band
21q22.3 is specifically labeled.
[0072] FIG. 14C: The same as for FIG. 14B except that hybridization
was to normal human lymphocyte nuclei.
[0073] FIG. 14D: Hybridization of the 94 kb probe set to trisomy 21
(47, +21) lymphocyte metaphase spreads.
[0074] FIG. 14E: Hybridization of chromosome 21 library DNA inserts
to trisomy 21 (47, +21) lymphocyte metaphase spreads. Three
chromosomes 21 are entirely delineated by the library inserts;
additional minor signals (see the text) are indicated by arrowheads
(also in FIG. 14G).
[0075] FIG. 14F: Hybridization of chromosome 21 library DNA inserts
to trisomy 21 (47, +21) lymphocyte interphase nuclei (compare with
14E).
[0076] FIG. 14G: Hybridization of chromosome 21 library DNA inserts
to trisomy 21 (47, +21) lymphocyte interphase nuclei.
[0077] FIG. 14H: Hybridization of chromosome 21 library DNA inserts
to trisomy 21 (47, +21) lymphocyte interphase nuclei.
[0078] FIG. 14I: Hybridization of chromosome 21 library DNA inserts
to trisomy 21 (47, +21) lymphocyte interphase nuclei.
[0079] FIG. 14J: Hybridization of chromosome 21 library DNA inserts
to trisomy 21 (47, +21) lymphocyte interphase nuclei.
[0080] FIG. 14K: Hybridization of the 94 kb probe set to chorionic
villi (CV) cell interphase nuclei.
[0081] FIG. 14L: Hybridization of the 94 kb probe set to CV cell
metaphase spreads.
[0082] FIG. 14M: Hybridization of the 94 kb probe set to CV cell
metaphase spreads.
[0083] FIG. 14N: Hybridization of chromosome 21 library DNA inserts
to TC 620 metaphase spreads.
[0084] FIG. 14O: Hybridization of chromosome 21 library DNA inserts
to TC 620 metaphase spreads.
[0085] FIG. 15(A-C) shows hybridization signals from centromeric
repeat probes on metaphase chromosomes from a normal male. The
labeling combinations used are given in Table 5. The images were
taken separately with the appropriate filters and pseudocolored.
(A) Image taken with the fluorescein filter, displaying the
fluorescein-1-dUTP-labeled probes for the centromeres of
chromosomes 8, 11, 12 and 18. The arrowheads indicate the
centromere for chromosome 12, which was singly labeled with
fluorescein-dUTP. The arrows show the centromere of chromosome 8,
which was labeled with a triple combination. (B) Detection of the
dig-labeled probes with the rhodamine-specific filter. The
centromeres of chromosomes 7 (arrowheads), 8 (arrows), 9 and 18
reveal hybridization signals. (C) Using the infrared filter
combination, the biotinylated probes that were detected with
streptavidin conjugated to the infrared dye Ultralite 680 are
shown. Chromosomes 3 (arrowheads), 8 (arrows), 9 and 11 were
detected. (D,E) Independently acquired gray scale images were
merged and pseudocolored, resulting in seven differentially colored
centromeric sequences on metaphase chromosomes (D) and in an
interphase nucleus (E). DAPI was used as a DNA counterstain. (F)
Example of combinatorial labeling of chromosome specific libraries
with PCR. The libraries for chromosomes 1, 2, 4, 8, 14 and X were
labeled singly or combinatorially (see Table 2) and pseudocolored
in green, pink, yellow, white, orange and red respectively. (G)
PCR-labeled chromosome-specific libraries were used for detection
of a t(2;14) translocation. The library for chromosome 2 was
labeled with biotin and detected with avidin fluorescein; the
chromosome 14 library was labeled with dig and detected with
anti-dig rhodamine. Both translocation chromosomes are clearly
visible (arrowheads). (H) Combinatorial labeling of cosmid and
phage clones by nick-translation. Single gene probes for six
different chromosomes were hybridized simultaneously. The probes
and the labeling combinations are described in Materials and
Methods. Chromosomes 5, 6, 8, 11, 21 and X show hybridization
signals. (I) Combinatorial labeling of six cosmid clones specific
for chromosome 5. The differentially pseudocolored probes label six
loci on this chromosome simultaneously. (J) Hybridization of the
chromosome 5 specific probes to an interphase nucleus. The order of
the cosmid clone is maintained.
DETAILED DESCRIPTION OF THE INVENTION
[0086] The present invention is based on a hybridization strategy
in which suppression of hybridization signals from ubiquitous
repeated DNA sequences is achieved by using total DNA in a
reannealing procedure which is based on rapid reassociation
kinetics. The hybridization method of the present invention
referred to as chromosomal in situ suppression (CISS) hybridization
because of the selective suppression of such signals, has been
shown to result in specific cyto-staining of one or more selected
individual chromosomes, particularly human chromosomes, at any
point in the cell cycle and has been used to detect, identify and
quantitate chromosomal aberrations in both mitotic cells and
interphase cells (i.e., interphase nuclei).
[0087] Described below and in greater detail in the Examples, are
the following:
[0088] 1. specific staining in mitotic and interphase cells of
individual human chromosomes, by the method of the present
invention (CISS hybridization), using chromosome-specific probe
sets which are of high genetic complexity (i.e., chromosome library
DNA, cloned DNA fragments);
[0089] 2. specific staining of metaphase and interphase tumor cells
by CISS, using chromosome-specific library probes; and
[0090] 3. rapid detection in mitotic and interphase cells from a
variety of sources of aberrations in a human chromosome (chromosome
21) which associated with a genetic condition (Down syndrome),
using CISS hybridization.
[0091] 4. demonstration that a nested set of chromosome specific
unique sequence probes used to identify chromosome aberrations and
to detect genetic disease (e.g., Down Syndrome).
[0092] Specific Staining of Individual Human Chromosomes
[0093] By use of the CISS hybridization method, individual (such as
the X and Y chromosomes or homolog pairs of chromosomes 1-22) human
chromosomes have been specifically stained in both mitotic and
interphase cells. This has been carried out in both metaphase
spreads and interphase nuclei and has been used to stain or label
one selected (individual) chromosome and to stain or label multiple
selected (individual) chromosomes simultaneously, using,
respectively, signal-probe CISS hybridization and multi-probe CISS
hybridization in conjunction with an appropriate detection method.
The method is represented schematically in FIG. 1.
[0094] Specific Chromosome Staining Using Genomic DNA Libraries and
Cloned DNA
[0095] CISS hybridization was carried out as follows, to produce
specific staining of individual human chromosomes, using
commercially-available genomic DNA libraries that originated from
flow-cytometry sorted human chromosomes and cloned DNA fragments.
Van Dilla, M. A. et al., Biotechnology, 4:537-552 (1986).
Suppression of hybridization signals from ubiquitous repeated
sequences, such as the Alu and KpnI elements, was achieved using
total human DNA in a reannealing procedure that is based on rapid
reassociation kinetics. Similar principles have been used by others
to facilitate the selective hybridization of unique sequence
subsets from cosmid DNA clones for Southern blotting and in situ
hybridization experiments. Sealey, P. G. et al., Nucleic Acids,
13:1905-1922 (1985); and Landegent, J. E. et al., Hum. Genet.,
77:366-370 (1987). Specific labeling of individual chromosomes in
both metaphase spreads and interphase nuclei, is carried out (and
shown to have occurred) in the following manner, which is described
in detail in the Examples. The feasibility of using
computer-assisted optical sectioning for 3-D reconstruction of
chromosomal domains for the analysis of nuclear topography was also
demonstrated in conjunction with CISS hybridization.
[0096] Initially, genomic DNA from a selected chromosome or
selected chromosomes is prepared for use as probe DNA. Genomic DNA
is available from several sources. For example, one or more genomic
DNA libraries, each containing the chromosome of interest (a
chromosome-derived library), is used to produce the necessary DNA
probes. Such libraries can be commercially-available genomic DNA
libraries that originated from flow-cytometry sorted human
chromosomes. These are available from the American Type Culture
Collection (Rockville, Md.). Such DNA libraries for human
chromosomes 1, 4, 7, 8, 9, 12, 13, 14, 16, 17, 18, 20, 21, 22 and
chromosome X have been used in the present method, as described in
the Examples. Other commercially available genomic DNA libraries or
genomic DNA libraries from noncommercial sources can also be used.
Alternatively, individual plasmid, phage, yeast artificial
chromosomes with non-yeast DNA inserts, and cosmid DNA clones can
be used as a source of DNA probes for a selected individual
chromosome or multiple selected chromosomes. In the case of DNA
from a genomic library, the DNA can be separated as a pool from the
vector containing it, prior to labeling with a detectable signal,
or can be used without separation from the vector.
[0097] Probes are labeled with a detectable signal, which can be a
fluorescent reporter, one member of a specific binding pair (e.g.,
biotin-avidin or ligand-antibody), or an enzyme. DNA removed from
the vector is labeled by nick translation (using, for example,
Bio-11-dUTP), by random primer extension with (e.g., 3' end
tailing), for example, the Amersham multiprime DNA labeling system,
substituting dTTP with Bio-11-dUTP, or other appropriate technique.
In the case of DNA which has not been separated from the vector,
biotin labeling is carried out directly by nick translation, using
standard techniques. Brigati et al., Virology, 126:32 50 (1983).
Other labels can be added in a similar manner (e.g., 2,4-dinitro
phenol, digoxin).
[0098] Probe size is carefully selected and controlled in order to
facilitate probe penetration and to optimize reannealing
hybridization. In general, labeled DNA fragments smaller than 500
nucleotides are used, and, more generally, the majority of the
probes are 150-250 nucleotides in length. Probes of this length are
made from longer nucleotide sequences using publicly available
restriction enzymes or known techniques for producing and
recovering appropriately-sized fragments. It is also possible, if
the nucleotide sequence of a selected chromosome is known, to
synthesize an oligonucleotide having that sequence, using known
techniques. Such oligonucleotides, once labeled, can be used to
decorate specific chromosomal regions. For example, oligonucleotide
probes which specifically hybridize to telomeric sequences of
mammalian chromosomes have been identified. Moyuif et al.,
Proceedings of the National Academy of Sciences, USA, September
1988.
[0099] Competitor DNA, which is DNA which acts to suppress
hybridization signals from ubiquitous repeated sequences, will be
selected as needed (e.g., based on the mammal whose chromosomes are
being analyzed). In the case of analysis of human chromosomes,
competitor DNA is total human DNA which acts to suppress
hybridization from ubiquitous repeated sequences, such as the Alu
and the KpnI elements. It is available from many sources. For
example, human genomic DNA from placenta or white blood cells can
be prepared using known techniques, such as that described by Davis
et al. Davis, L. G. et al. Basic methods in molecular biology,
Elsevier, N.Y./Amsterdam (1986). It is digested, using standard
methods (e.g., with DNAse), to produce competitor DNA fragments
within the same size distribution as the probe DNA.
[0100] DNA from another source, which will compete with only a
small portion of the human DNA and which is used, as necessary, to
adjust the total (final) DNA concentration of the hybridization
mixture will also be included, as needed. This DNA is referred to
as carrier DNA. This DNA is produced or treated, using standard
methods, so that it is within the same size distribution as the
probe DNA.
[0101] Initially, probe DNA bearing a detectable label and
competitor DNA are combined under conditions appropriate for
preannealing to occur. The quantity of probe DNA combined with
competitor DNA is adjusted to reflect the relative DNA content of
the chromosome target. For example, chromosome 1 contains
approximately 5.3 times as much DNA as is present in chromosome 21.
Probe concentrations were 30 .mu.g/ml and 5 .mu.g/ml, respectively.
When total genomic library DNA is used as the probe mixture
(instead of purified DNA inserts), approximately 10 times as much
labeled DNA is added to compensate for the vector sequences, which
are present in large quantities. Only twice as much labeled library
DNA is added in the case of the libraries LA1XNL01 (X chromosome)
and LA16NL02 (chromosome 16) because the human DNA inserts
constitute almost half of the total library DNA. Carrier DNA, such
as trout or salmon testis DNA, is added to bring the total DNA
concentration to a predetermined level, if necessary. As described
herein, sufficient salmon testis DNA was added to result in a final
DNA concentration of 1.0 mg/ml in the hybridization mixture (which
includes all three types of DNA: probe DNA, competitor DNA and DNA
which does not significantly compete).
[0102] The resulting hybridization mixture is treated (e.g., by
heating) to denature the DNA present and incubated at approximately
37.degree. C. for sufficient time to promote partial
reannealing.
[0103] The sample containing chromosome DNA to be identified
(specifically labeled) is also treated to render DNA present in it
available for hybridization with complementary sequences, such as
by heating to denature the DNA. The hybridization mixture and the
sample are combined, under conditions and for sufficient time
conducive to hybridization. After sufficient time, detection of
specific labeling of the chromosome target is carried out, using
standard techniques. For example, as described in the Examples, a
biotinylated probe is detected using fluorescein-labeled avidin or
avidin-alkaline complexes. For fluorochrome detection, samples are
incubated, for example, with fluorescein isothiocyanate
(FITC)-conjugated avidin DCS (see Example 1). Amplification of the
FITC signal can be effected, if necessary, by incubation with
biotin-conjugated goat anti-avidin D antibodies, washing and a
second incubation with FITC-conjugated avidin. For detection by
enzyme activity, samples are incubated, for example, with
streptavidin, washed, incubated with biotin-conjugated alkaline
phosphatase, washed again and pre-equilibrated (e.g., in AP-buffer,
as described in Example 1). The enzyme reaction is carried out in,
for example, AP buffer containing nitroblue tetrazolium and 5'
oromo 4 chloro 3 indoyl phosphate and stopped by incubation in
2.times.SSC.
[0104] Detection of Chromosome Aberrations Using CISS
Hybridization
[0105] Using the above-described steps, it is possible to
specifically stain or label any selected individual chromosome (or
chromosomes) referred to as a target chromosome, or a subregion(s)
thereof. As explained in the examples, the present method has been
shown to be useful in a variety of cells, both in mitotic (e.g.,
metaphase, prophase) and interphase cells. As described in detail
in Example 2, the CISS hybridization method of the present method
is useful for rapidly screening mitotic and interphase aneuploid
tumor cells for complex numerical and structural aberrations of
individual chromosomes (e.g., changes in number of chromosomes,
deletions and rearrangements or translocations).
[0106] In this context, biotinylated library DNA inserts were used
in the CISS hybridization method to produce hybrid molecules which
were detected using known techniques. Two glioma lines were used as
general models of aneuploid cells, particularly tumor cells. One
was an oligodendroglioma line and the other was a gliobastoma line.
These were analyzed, using the biotinylated DNA probes specific for
chromosome 1, 4, 7, 18 and 22. Specific labeling of the
chromosomes, from pter to qter, made it possible to visualize
numerical changes, deletions and rearrangements in these
chromosomes in metaphase spreads and in early prophase and
interphase nuclei. Complete chromosomes, deleted chromosomes and
segments of translocated chromosomes were rapidly delineated in the
very complex karyotypes of such cells. Additional subregional
probes were also used to further define aberrant chromosomes.
Digital image analysis was used to quantitate the total complement
of specific chromosomal interphase DNAs in individual metaphase and
cells of each line. Under-representation of chromosome 21 and
over-representation of chromosome 7 (specifically 7p) were
observed. This is in agreement with previous observations by others
using conventional cytogenetic bauding techniques. Bigner, S. H. et
al., Cancer Genet. Cytogenet., 22:121-135 (1987); Shapiro, J. R.,
Semin. Oncol., 3:4-15 (1986).
[0107] The two glioma cell lines used display several cytogenetic
features common to many glioma cells. Thus, it is reasonable to
expect that the CISS hybridization method can be used in a similar
manner to specifically decorate other chromosomes and to detect
those chromosomes in glial tumors. The two cell types analyzed are
highly aneuploid (i.e., they have 100 chromosomes, rather than the
normal 46). Therefore, it is reasonable to expect that the CISS
hybridization method can be used in assessing any type of aneuploid
(tumor) cell.
[0108] Thus, the CISS hybridization method can be used in assessing
chromosomal aberrations associated with cancer, both in diagnosis
of the disease and in monitoring its status (e.g., progression,
regression or change with treatment) in patients. In this
application, assessment of a single chromosome or of multiple
chromosomes, and subregions thereof, can be carried out. Double
hybridizations using two DNA probes, each bearing a different label
can also be carried out. That is, biotinylated chromosome 7 library
DNA inserts and a probe specific for alphoid repeats on chromosome
7 (pa7tl) which was modified with aminoacetylfluorene (AAF) were
used to assess chromosome 7 content/characteristics in both
metaphase spreads and interphase nuclei of the two types of tumor
cells (TC 593, TC 620). After hybridization, biotinylated
chromosome 7 inserts were detected using avidin-FITC and chromosome
7-specific alphoid AAF labeled sequences were detected with
tetramethylfhodamine isothiocyanate (TRITC) conjugated second
antibodies. Double CISS hybridization was used to detect
translation between chromosome 8 and 14, Burkitt lymphoma cells, a
high malignancy form of B lymphocyte tumors such were seen in both
metaphase spreads and interphase cells.
[0109] This made it possible to detect similarities and differences
in chromosome number 7 present in the two tumor cell types: only
the four complete number 7 chromosomes found in TC 593 contained a
detectable 7 centromeric signal; a smaller and metacentric number 7
chromosome lacked the 7 alphoid sequences and a small block of
heterochromatin at 7q11 (indicating that it lacked a characteristic
centromeric region). In contrast, all four chromosome number 7 of
TC 620 were labeled with the 7 alphoid probe. Double CISS
hybridization also made it possible to distinguish among number 7
chromosomes present in one cell type (TC 593) and to demonstrate
similarity (at least as to the characteristics assessed) among
number 7 chromosomes present in the other cell type (TC 620).
[0110] Double CISS hybridization was used to detect translocations
between chromosome 8 and chromosome 14 in Burkitt's lymphoma cells;
Burkitt's lymphoma is a highly malignant form of B lymphocyte
tumors. Translocations were detected in both metaphase spreads and
interphase cells.
[0111] It is possible, through the use of appropriately-selected
probes and/or labels-to increase the number of different
chromosomes, as well as the number of subregions on some or all of
those chromosomes, which can be analyzed simultaneously using
multiple CISS hybridization. For example, it is possible to use
more than one probe, each specific for a subregion of a target
chromosome, to analyze several subregions on that single chromosome
at one time. It is also possible to label each probe set (set of
DNA or RNA fragments) with a distinct fluorochrome or different
reporter molecule, which can be distinguished from one another,
after probe-target chromosome hybridization has occurred, by known
techniques (e.g., by using specific fluorescent or enzyme
reagents).
[0112] Furthermore, a "combinatorial" variant of CISS hybridization
can be used to enhance the number of chromosomes which can be
assessed simultaneously. That is, it is possible to use a
hybridization probe mixture made from a single set of probe
sequences composed of two halves, each separately labeled with a
different fluorochrome (e.g., fluorescein and rhodamine), which,
upon hybridization, produce a third fluorescence "color" or signal
optically distinguishable from each of the original individual
fluorochromes. Pairing of two different fluorochromes in this
manner makes it possible to identify three different chromosomes.
For example, a probe set labeled only with fluorescein will yield
one color upon hybridization; the same probe set labeled only with
rhodamine will yield a second (different) color upon hybridization.
When half of the probe set is labeled with one of the two, both
sequence subsets can hybridize to target with equal probability and
be perceived as a third (different) color (in a way not dissimilar
to mixing paint). It is important here that two fluorochromes are
not introduced into the same molecule, in order to minimize the
possibility of E transfer (a well-known process where light emitted
by one fluorochrome whose spectrum overlaps that of the other
fluorochrome is absorbed by the second fluorochrome. The
transferred electrons are emitted by second fluorochrome, which
leads to quenching of the first fluorochrome. Pairwise combinations
of three different fluorochromes selected for their spectral
characteristics can be used singly and in pairwise combinations to
produce in a similar manner. This can result in the production of
six different fluorescent colors or signals (e.g., three pairs plus
three single fluorochromes). Similar combinations of four different
fluorochromes results in production of 10 different fluorescent
colors or signals, of five different fluorochromes results in
production of 15 different colors or signals, etc. This principle
of combinatorial fluorescence (combining two or more fluorochromes
to label the same probe set) is applicable to metaphase and
interphase chromosome analysis because each chromosome is a
physically separate entity and is, thus, a distinct target.
Composite probe labeling in which mixtures of three different
fluorochromes are used provides even greater diversity of colors or
signals useful in simultaneous multiparameter analysis.
[0113] Another approach to enhance the number of chromosomes which
can be analyzed simultaneously involves a "time-resolved" method of
fluorescence detection. In this instance, the DNA 9 or RNA) probes
are labeled with chelating "cages" which bind specific lanthanides
(e.g., Europium, turbium). Such metal chelates can be made to
fluoresce. They exhibit excited state lifetimes that are much
longer (micro to millisec) than those of most normal fluorochromes
(whose half lives are in the nanosecond range). Both the wavelength
and the fluorescence lifetime is influenced by the nature of the
lanthanide metal ion employed. If a pulsed-gating system, which
excites the sample with light for a few nanoseconds and then shuts
off is used, it is possible to let short-lived fluorochromes decay
to their ground state, open the detector system at a defined time
after excitation, (i.e., 1-100 microseconds) and detect only
long-lived fluorochrome. This method can be used to discriminate 2
fluorescent dyes which have identical spectra but different
lifetimes, thus adding a time factor to fluorochrome
discrimination.
[0114] Another approach to increase the number of different
chromosomes that can be analyzed simultaneously is based on a
detection system which distinguishes chromosomes in terms of the
flexibility or rigidity of an attached fluorochrome. Here, two
single stranded probe sets can be labeled with the same
fluorochrome, in one probe set the fluorochrome is introduced into
the body of DNA sequences which will form hybrid molecules with the
target DNA of interest. In the second probe set, the fluorochrome
is introduced into DNA sequences, that do not hybridize with the
target DNA (e.g., by adding a 3'-tail of poly dA-fluorochrome with
deoxynucleotide terminal transferase, ligation of
fluorochrome-labeled heterologous DNA to the probe DNA or other
conventional secondary labeling techniques known in the art).
Fluorochromes within the body of the DNA which form probe-target
chromosome hybrids will become immobilized and thus will be unable
to rotate freely in solution. In contrast, fluorochromes in the
single-strand DNA that is not involved in hybrid formation are not
immobilized and can rotate much more freely in solution. By
measuring the rate of fluorochrome rotational freedom, (i.e., by
measuring how fast the fluorochromes become depolarized when
illuminated with polarized light) one can discriminate the two sets
of probes.
[0115] Use of CISS Hybridization and Regionally Defined Probe Sets
for Rapid Assessment of Chromosome Aberrations Associated with
Genetic Disorders and Chromosomal Damage
[0116] It has been demonstrated that the CISS hybridization method
is useful for the rapid assessment of chromosome aberrations (such
as numerical and structural aberrations of chromosome 21)
associated with genetic disorders (e.g., in the case of chromosome
21, Down syndrome). DNA probe sets which specifically label the
terminal band 21q22.3 or decorate the entire chromosome 21
aberrations in metaphase and interphase cells are described in
Example 3, the cloned DNA fragments from the human chromosome 21
are useful to specifically label the cognate chromosomal region in
metaphase spreads and interphase nuclei in a variety of cell types.
That is, CISS hybridization using a chromosome 21 probe set was
shown to be effective in labeling/identifying chromosome 21 DNA in
lymphocytes, embryonic chorionic villi cells and a glioma tumor
cell line (TC 620). Unique probe sets from band 21q22.3 were also
used to detect chromosome solid tissue ("normal" human brain
tissue). Thus, CISS hybridization and hybridization with pools of
unique sequence probes clearly have potential as a diagnostic for
Down syndrome and for other genetic diseases or other conditions
associated with chromosomal aberrations.
[0117] Results demonstrate that a trisomic karyotype can be
diagnosed easily in interphase cells because the majority of the
nuclei (55-65%) exhibit three distinct foci of hybridization. In
contrast, less than 0.2% of nuclei in lymphocytes with a disomic
karyotype show three nuclear signals; interestingly, the percentage
of such nuclei in normal CV cells was higher but still considerably
less than 5%. In general, as few as 20-30 cells were sufficient to
unambiguously distinguish between disomic and trisomic cell
populations. However, in view of the uncertainty of the level of
chromosome 21 mosaicism in clinical samples, the number of cells
required to make an unambiguous diagnosis will likely be higher.
Additional clinical correlations will be required to establish the
absolute number. Nevertheless, this analytical approach could allow
the diagnosis of Down syndrome without the need to culture cells or
to obtain metaphase spreads. It would also decrease the time
required to make the diagnosis, from the current 10-14 days to 1
day or less.
[0118] Although selected plasmid clones containing only unique
human DNA sequences were used here, cosmid clones containing
repetitive sequences can also be used to specifically label their
cognate genomic region in metaphase and interphase cells by
applying hybridization protocols like CISS hybridization that
suppress the signal contribution of repetitive sequence elements.
Therefore, single or nested sets of cosmids could be used as
diagnostic tools for other genetic diseases in a fashion similar to
that reported here. Trisomy of chromosomes 13, 18 and 21 and
numerical changes in chromosomes X and Y together account for the
vast majority of numerical chromosome abnormalities identified
during prenatal karyotyping. With the continued development of
multiple nonisotopic probe labeling and detection systems it should
be possible to visualize three or more chromosomes simultaneously
following in situ hybridization. The variations, described in the
previous section, of the CISS hybridization method which increase
the number of chromosomes, and/or the number of chromosome regions
which can be assessed simultaneously can also be used for detecting
chromosomal aberrations associated with genetic disorders and
chromosomal damage. Thus, the development of a rapid and automated
screening test to detect the major trisomic disorders directly in
interphase cells from amniotic fluid or chorionic villi cells is a
viable future objective. The analysis of specific human chromosomes
by in situ hybridization has already been used to complement
conventional cytogenetic studies of highly aneuploid tumor lines
(Example 2) and the extension to prenatal diagnostic applications
seem warranted.
[0119] The analysis of karyotypes with translocations of chromosome
21 shows the usefulness of a regional probe set to rapidly identify
and characterize even small translocations by unambiguous signals
on metaphase chromosomes, thus circumventing an extensive analysis
by high-resolution banding. In contrast, the library insert probe
is more suitable for defining the relative amount of chromosome 21
DNA that has been translocated. By analyzing interphase nuclei, one
can also determine if a balanced or unbalanced number of
chromosomal regions exists. However, the detection of a
translocated chromosome directly in nuclei would require
double-labeling techniques to identify the recipient chromosome to
which the chromosome 21 material was translocated. With prior
knowledge of the chromosome in question, such translocation events
could be assessed by measuring the juxtaposition of the nuclear
signals. Rappold, G. A. et al., Hum. Genet., 67:317-325 (1984).
[0120] A cosmid clone spanning the entire muscular dystrophy (MD)
locus on chromosome X has been used to identify translocation
between chromosome X and chromosome 4.
[0121] Probes containing 6 kb of sequence were localized in both
metaphase spreads and interphase cells with high efficiency. This
detection sensitivity with nonisotopic reagents is similar to that
achieved in other recent reports. The combination of nonisotopic_n
situ hybridization with DAPI or BrdUrd banding or total chromosome
decoration with library DNA probes thus provides a simple and
general approach for gene mapping. Combinatorial fluorescent
technology will also make it possible to examine several
chromosomal regions simultaneously, thus permitting genetic linkage
analysis by in situ hybridization. It also should facilitate the
use of small DNA probes to rapidly pinpoint the breakpoints on
translocation chromosomes, which could further aid in defining the
genomic segments critical for Down syndrome.
[0122] Identifying and Isolating Chromosome-Specific Sequences
Using CISS Hybridization
[0123] The CISS hybridization method of the present invention can
also be used to identify chromosome-specific sequences and,
subsequently, to separate them from repetitive sequences, using
known techniques. Such chromosome-specific sequences, separate from
the non-specific or repetitive sequences, and labeled, can be used
in hybridization assays carried out, for example, in a diagnostic
context, to identify, detect, and/or quantitate a chromosome or
chromosome region of interest (e.g., one which is associated with a
genetic disorder or causes an infectious disease). Combination of a
sample to be assayed for a selected target nucleic acid sequence or
sequences and appropriately-selected, labeled chromosome-specific
sequences separated from repetitive sequences (e.g., specific for
sequences on the chromosome(s), generally referred to as target
nucleic acid sequences, which are to be detected and/or quantitated
in the sample under appropriate conditions results in hybridization
with complementary sequences present in the sample. Hybridization
will not occur, of course, if complementary sequences are not
present in the sample.
[0124] Such separated chromosome-specific nucleic acid
sequences-can be incorporated into a kit to be used for
identification, detection and/or quantitation of chromosomes or
chromosome regions of interest, using standard hybridization
techniques. For example labeled nucleic acid sequences which are
chromosome 21 specific (or specific to a portion of chromosome 21),
identified by CISS hybridization, and separated from repetitive
sequences present on chromosome 21, can be included in a kit, along
with other reagents such as buffers, competitor DNA, carrier DNA
and substances needed for detection of labeled chromosome
21-derived nucleic acid sequences hybridized to chromosome 21
sequences present in a sample. Such kits clearly can be produced to
include chromosome-derived nucleic acid sequences from one or more
chromosome(s) of interest. Competitor DNA, carrier DNA and
substances useful for detecting hybridized sequences will be as
described above.
EXAMPLE 1
Cyto-Specific Staining of Individual Human Chromosomes Using
Genomic DNA Libraries in CISS Hybridization
[0125] DNA Libraries
[0126] The following human chromosome genomic libraries were
obtained from the American Type Culture Collection: LAO1NSO1
(chromosome 1), LL04NSO1 (chromosome 4), LA07NsO1 (chromosome 7),
LL08NS02 (chromosome 8), LA13NS03 (chromosome 13), LL14NSO1
(chromosome 14), LL19NSO1 (chromosome 18), LL20NSO1 (chromosome
20), LL21NS02 (chromosome 21), LA22NS03 (chromosome 22), LAOXNLO1
(chromosome X). Amplification of these phage libraries on agar
plates (using LE 392 cells as the bacterial host), purification of
the X phages and extraction of phage-DNA pools were carried out
according to standard protocols. Maniatis, T. et al., Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor Laborabory, Cold
Spring Harbor, N.J. (1982).
[0127] Preparation of Metaphase Spreads and Fibroblast Cells
[0128] Phytohemagglutinin-stimulated lymphocytes from a normal
adult male (46, XY) were cultured in McCoy's 5A medium (GIBCO),
arrested with Colcemid, treated with a hypotonic solution of 0.075
M KC1, fixed in acetic acid-methanol and metaphase spreads made by
standard procedures. Low-passage normal human foreskin (46, XY)
were grown on microscope slides, fixed with paraformaldehyde, and
permeabilized as described for study of preparations with a more
intact three-dimensional structure. Manuelidis, L, Ann. NY Acad.
Sci., 450:205-221 (1985).
[0129] Preparation of DNAs for in situ Hybridization
[0130] Insert DNA probes. Genomic DNA fragments from the
chromosomal DNA libraries were separated as a pool from the Charon
21 A vector arms by digestion with the appropriate restriction
enzyme [EcoRi (LA libraries) or Hind III (LL libraries)], followed
by preparative electrophoresis in 0.6% agarose gel. The insert
fragments were isolated from gel slices by electroelution into an
Elutrap (Schleicher and Schuell) and further purified by Elutip-d
column chromatography (Schleicher and Schuell). The DNA was then
extracted with phenol/chloroform (1:1) and ethanol precipitated.
This pool of DNA fragments was labeled either by nick translation
using Bio-11-dUTP or by random primer extension with the multiprime
DNA labeling system (Amersham) substituting dTTP with 0.5 mM
Bio-11-dUTP. Langer, P. R. et al., Proc. Natl. Acad. Sci., USA,
78:6633-6637 (1981) and Brigati, D. J. et al., Virology, 126:32-50
(1983). Alternatively, the DNA of the chromosome-specific libraries
was biotin-labeled directly (without separation of the vector arms)
by nick translation.
[0131] Probe size. To facilitate probe penetration and to optimize
reannealing hybridization, it is desirable to have labeled DNA
fragments smaller than 500 nucleotides; the majority of the probes
are generally 150 to 250 nucleotides in length. DNAse
concentrations were empirically established in nick-translation
reactions to yield fragments in the-desired size range and this was
verified by agarose gel electrophoresis. Random primer extensions
were also carried out under conditions which yielded a comparable
DNA size distribution.
[0132] Competitor DNA. Human genomic DNA (from placenta or white
blood cells), prepared as described, as well salmon testis genomic
DNA (Sigma) were digested with DNAse to obtain fragments with the
same size distribution as the probe DNA, then extracted with
phenol/chloroform and ethanol precipitated. Davis, L. G. et al.,
"Basic methods in molecular biology", Elsevier, New York Amsterdam
(1986). These competitor DNAs were used in varying ratios with
probe sequences, as described with reference to FIGS. 2A-2F.
[0133] Preannealing and hybridization. Under standard conditions,
from S .mu.g/ml to 30 .mu.g/ml of biotin-labeled DNA, representing
library insert fragments, and varying amounts of competitor DNAs
were combined, ethanol-precipitated and resuspended in formamide.
The probe concentration was adjusted to reflect the relative DNA
content of each chromosome target. For example, chromosome 1
contains approximately 5.3 times as much DNA as chromosome 21; thus
the probe concentrations used were 30 .mu.g/ml and 5 .mu.g/ml,
respectively. Mendelsohn, M. L. et al., Science, 179:1126 1129
(1973). When total library DNA was used as the probe mixture
instead of purified DNA inserts, 10 times as much labeled DNA was
added to compensate for the large amount of vector sequences. In
the case of the X-chromosome library, LAOXNLO1, only twice as much
labeled library DNA was used, since the human DNA inserts
constitute almost half of the total DNA. For comparative purposes,
the concentration of human competitor DNA in the hybridization
mixture was varied from 1 to 1.0 mg/ml and salmon testis DNA was
added as required to result in a final DNA concentration of 1.0
mg/ml in 50% formamide, 1.times.SSC (0.15 M sodium chloride, 0.015
M sodium citrate, pH 7.0) and 10% dextran sulfate. These solutions
were heated at 75.degree. C. for 5 min. to denature the DNA and
then incubated at 37.degree. C. for various times to promote
partial reannealing. The preannealing step was done in an Eppendorf
tube just prior to applying the hybridization mixture to the
specimen. Nuclei and chromosome spreads on glass slides were
incubated in 70% formamide, 2.times.SSC) at 70.degree. C. for 2
min. to denature chromosomal DNA and then dehydrated in a series of
ice cold ethanol (70%, 90% and 100%, each for 3 min.). After
application of the preannealed probe mixture (2.5 pl/cm ) to slides
prewarmed to 42.degree. C., a coverslip was added and sealed with
rubber cement. The samples were then immediately incubated at
37.degree. C. in a moist chamber for 10-20 h.
[0134] In those cases where paraformaldehyde fixation was used to
more optimally preserve the 3-D structure of the specimen, the
slides were equilibrated in 50% formamide, 1.times.SSC (2.times.5
min.), excess fluid was removed without permitting the sample to
dry, the probe mixture was added (5 pm/cm ), and a coverslip
mounted and sealed with rubber cement. Manuelidis, L., Ann. NY
Acad. Sci., 450:205-221 (1985). Denaturation of both probe and
cellular DNA was done at 75.degree. C. for 5 min. before
hybridization was allowed to proceed overnight at 37.degree. C.
[0135] Detection
[0136] After hybridization, the slides were washed in 50%
formamide, 2.times.SSC (3.times.5 min., 42.degree. C.) followed by
washes in 0.1.times.SSC (3.times.5 min., 60.degree. C.). Thereafter
the slides were incubated with 3% bovine serum albumin (BSA),
4.times.SSC for approximately 30 minutes at 37.degree. C. Detection
of the biotinylated probe was achieved using either
fluorescein-labeled avidin or avidin-alkaline phosphatase
complexes. All detection reagents were made up in 4.times.SSC, 0.1
% Tween 20, 1 % BSA and all washes were carried out in 4.times.SSC,
0.1 % Tween 20 (3.times.3 min., 42.degree. C.). For fluorochrome
detection, slides were incubated with 5 pg/ml fluorescein
isothiocyanate (FITC)-conjugated avidin DCS (Vector Laboratories)
at 37.degree. C. for 30 min., followed by washes. In rare cases,
the FITC signal was amplified by incubation with 5 pg/ml
biotin-conjugated goat anti-avidin D antibodies (Vector
Laboratories) at 37.degree. C. for 30 min., followed by washing, a
second incubation with 5 pg/ml FITC-conjugated avidin (37.degree.
C., 30 min.) and a final wash. Pinkel, D. et al., Proc Natl. Acad.
Sci. USA, 83:2934-2938 (1986). For detection by enzyme activity,
samples were incubated with 2.5 pg/mi streptavidin, washed,
incubated with 2 pg/ml biotin-conjugated alkaline phosphatase
(Vector Laboratories), washed again and pre-equilibrated in
Ap-buffer 9.5 (100 mM Tris-HCl, pH 9.5, 100 mM NaCl, 50 mM MgC12)
for 2.times.5 min. at room temperature. The enzyme reaction was
carried out in AP buffer 9.5 containing 330 .mu.g/ml of nitroblue
tetrazolium (NBT) and 165 pg/ml 5-bromo-4-chloro-3-indolyl
phosphate (BCIP) at 37.degree. C. for 0.5-1 hour and stopped by
incubation in 2.times.SSC. All preparations were counter-stained
with 200 ng/ml 4,6-diamidino-2-phenylindole-dihydrochlori- de
(DAPI), 2.times.SSC for 5 min. at room temperature and mounted in
20 mM Tris-HCl, pH 8.0, 90% glycerol containing 2.3% of the DAPCO
antifade, 1,4 diazabicyclo-2(2,2,2)octane. Johnson G. D. et al., J.
Immunol. Methods, 55:231-242 (1982).
[0137] Densitometry
[0138] A graphics workstation (VAX station II/GPX, Digital
Equipment Corporation) with a frame grabber (ITEX FG-101, Imaging
Technology) and a Dage-MTI-65 video camera with a Zeiss S-Planar 60
mm lens were used as described in Manuelidis, L. and J. Borden,
Chromosome, 96:397-410 (1988). Images were digitized directly from
the negatives and stored on disk. Background was removed and
polygonal regions around each chromosome were defined. Threshold
density levels were used to outline chromosome regions within the
defined polygonal areas. Means density levels within these outlined
chromosome regions, R, were determined by the total signal .intg.
I(x,Y)dR/area R, where .intg.I(x,y) is the pixel intensity (0-225)
at each point within the region R. The threshold background
intensity was substrated from the mean regional density, both for
labeled chromosome 7 and for background chromosomes. The signal to
noise ratio was calculated as mean chromosome 7 signal/mean
background chromosome signal.
[0139] The following is a description of the results of the work
described above, which clearly demonstrate specific labeling of the
individual chromosome indicated. The first sections describe use of
chromosome library inserts labeled with biotin and the second
describes use of DNA insert fragments.
[0140] FIGS. 2A-2F shows suppression of signals from cross-reacting
sequences within a chromosome 7-derived DNA library by different
concentrations, as described below.
[0141] FIG. 2A shows chromosome 7 library inserts labeled with
biotin and hybridized to metaphase spreads from normal human
lymphocytes without human competitor DNA. Prominent labeling of the
two no. 7 chromosomes is observed; additionally, a distinct
band-like patterns of hybridization is seen on most of the other
chromosomes, and two E-group chromosomes are especially brightly
stained. This general chromosomal banding pattern resembles
R-banding, and suggests that a significant portion of the
background cross-hybridization signal originates from Alu
repetitive sequences. Previous studies have shown that Alu
sequences delineate an R-banding pattern, while Giemsa
positive-banding profiles are -highlighted by KpnI interspersed
repeats. Manuelidis, L. and C. D. Ward, Chromosome, 91:28-38
(1984).
[0142] Establishment of Experimental Procedure to Eliminate the
Hybridization Signal from Repetitive Elements
[0143] A series of pilot studies were therefore undertaken to
establish experimental parameters to eliminate the hybridization
signal from such repetitive elements. The kinetics of nucleic acid
reassociation in solution are dependent on the total concentration
of nucleic acid (Co, in moles of nucleotides per liter) and the
time of renaturation (t, in seconds). When reassociation conditions
are standardized for temperature (taking into account the formamide
concentration), cation concentration and buffer system, the
reassociation kinetics are comparable with respect to Cot values.
Under defined conditions, the fast reassociating fraction of
mammalian genomes containing the highly repetitive DNA is
completely reannealed at Cot values between 1.times.10.sup.-1 and
5.times.10.sup.-1; the intermediate fraction containing the middle
repetitive DNA is completely renatured at a Cot value of
1.times.10.sup.2. Britten, R. J. and D. E. Kohne, Science, 161:529
540 (1968). Thus at a human DNA concentration of 1.0 mg/ml
(corresponding to 3.times.10.sup.-3 moles of nucleotide per liter),
the fast fraction would be renatured in approximately 10s, whereas
the middle repetitive DNA would need more than 9 h to reach
complete re-annealing. Since the fast fraction of reassociating DNA
containing most or all of the ubiquitous repetitive DNA causing
cross-hybridization signals, a total DNA concentration of 1.0 mg/ml
was used and partial reannealing of the probe mixture was allowed
prior to application to specimens. The optimal renaturation time
was determined empirically (see below). This was important because
the in situ hybridization conditions deviate from the standard
conditions under which reassociation kinetics are determined (e.g.,
hybridization in 50% formamide at 37.degree. C. corresponds to o %
formamide at about 70.degree. C.; dextran sulfate also increases
the reassociation time significantly). Furthermore, it was unclear
to what degree the middle repetitive DNA contributed to the
non-specific signal and therefore should also be prevented from
hybridization by a preannealing procedure.
[0144] The stringency for the reannealing and in situ hybridization
experiments Was determined in 50% formamide at 37.degree. C.
(adapted from standard in situ hybridization protocols) and
1.times.SSC [this cation concentration of 0.165 M comes close to
the concentration used in kinetics the study of Britten and Kohne.
Britten, R. J. and D. E. Kohne, Science, 161:529 540 (1968)].
Competitor human DNA was added in the reassociation procedure to
obtain the desired final high DNA concentration and to maintain a
high level of repetition of the DNA sequences that should
preanneal. While total human genomic DNA represents all the highly
repetitive DNA to be removed by preannealing, it also contains
sequences of the target chromosome. Thus, the addition of excessive
amounts of human DNA would be expected to diminish the
chromosome-specific signal. Therefore, the optimal concentration of
total human DNA to use as the competitor was first determined. To
keep the total DNA concentration constant at 1.0 mg/ml, genomic
salmon DNA was added as needed. Salmon DNA shares certain
repetitive DNA elements, such as poly dCdA in common with human
DNA, but lacks others, most notably the Alu and KpnI repeats.
Hamada, H. et al., Proc. Natl. Acad. Sci. USA, 79:6465-6469 (1982).
This results in a lower frequency of the latter sequences with
increasing amounts of salmon DNA in the reassociation reaction.
[0145] FIGS. 2A-2F shows typical experimental results obtained when
20 .mu.g/ml of the chromosome 7 probe set was denatured together
with 50 .mu.g/ml (FIG. 2B), 100 .mu.g/ml (FIG. 2C), 200 .mu.g/ml
(FIG. 2D) or 1000 .mu.g/ml (FIG. 2E) of DNAse-digested human
genomic DNA which was preannealed for 20 min. Hybridization and
detection using avidin-FITC were carried out as described above.
From each preparation ten black and white pictures were taken under
standardized photographic conditions for densitometric studies (see
below). In the absence of human genomic competitor (FIG. 2A) the
signal showed little chromosomal specificity. However, with 50 and
100 .mu.g/ml of human competitor DNA, as increase of label
specificity is readily apparent (FIG. 2B,C). Specific staining of
chromosome 7 was achieved with a peak of signal intensity using 100
and 200 .mu.g/ml of human competitor DNA (FIG. 2C, D). Higher
concentrations of human DNA caused an apparent decrease of signal
intensity, especially at 1000 .mu.g/ml human DNA (FIG. 2E).
However, the signal obtained under these latter conditions is still
reasonably bright to the observer, but requires a different
exposure for optimal illustration (not shown).
[0146] A computer-assisted method of quantitative densitometry (see
above) was used to establish the overall level of labeling
specificity. The ratio of fluorescence signal from the target
chromosomes of interest to the background fluorescence noise
emanating from non-target chromosomes was determined from images
digitized from multiple photographic negatives of a DNA titration
experiment, as illustrated in FIGS. 2A-2F. The signal-to-noise
ratio obtained with each concentration of human competitor DNA is
given in Table 1.
1TABLE 1 Densitometric analysis of the suppression of cross
hybridization signals by concentrations of human competitor DNA DNA
conc. .mu.g/ml) Signal-to-noise Human competitor Signal ratio
Confidence DNA conc. Pixel.sup.a .eta. Noise Pixel.sup.b .eta.
interval.sup.c 99% 0 71.48 8 54.66 26 1.31 .+-. 0.04 50 74.50 8
37.43 28 1.99 .+-. 0.07 100 162.64 8 20.06 23 8.11 .+-. 0.35 200
147.35 8 20.53 26 7.18 .+-. 0.37 500 89.78 8 18.63 21 4.82 .+-.
0.28 1000 94.37 8 30.51 17 3.09 .+-. 0.12 .sup.aMean value of pixel
intensity of target chromosome .sup.bMean value of pixel intensity
of non-target chromosomes (from the same metaphase spreads)
.sup.cThe confidence interval was calculated using Fieller's
theorem (Finney, D. J., Statistical methods in biological assay,
2nd ed., Hafner Press, N. Y., 1971) .eta. Number of chromosomes
from which the mean was determined
[0147] Optimal reannealing conditions for suppression of
nonspecific signal (using 20 pg/ml of chromosome 7 probe and
100-200 pg/ml human genomic (DNA), gave a signal-to-noise ratio of
ca. 8:1. Additional attempts to improve the signal to noise ratio
by increasing hybridization stringencies (e.g., 60% formamide or
0.2.times.SSC) gave no apparent improvement and led to an overall
decrease in signal intensity.
[0148] Since about 100-200 .mu.g/ml of human competitor DNA was
shown to give the optimal specificity, 200 .mu.g/ml was used for
another analysis of signal specificity with respect to the
renaturation time (see above). After 0, 2, 5, 10, 20, 40 and 50
min. of preannealing, aliquots were taken and used for in situ
hybridization experiments as before. As indicated in FIGS. 3A-3D,
specific labeling was obtained for all preannealing times. A small
improvement of the signal is seen with increasing renaturation
times from 0 to 20 min. Longer renaturation times up to 60 min.
(not shown) gave no significant improvement in signal strength or
chromosome specificity. The subjective impression of a signal
improvement with 20 min. of preannealing (FIG. 3D) could not be
confirmed by a densitometric analysis, carried out as described
above, since no significant differences in the signal-to-noise
ratio of the different preannealing times were observed (data not
shown). Therefore, the standard renaturation time in all subsequent
experiments was 10-20 min. Since a signal is clearly visible at
renaturation time 0, the few seconds necessary for transferring the
probe mixture to the microscope slide appear to be sufficient to
effectively preanneal many of the sequences that cause nonspecific
labeling by cross-hybridization. Furthermore, the large excess of
single-stranded competitor DNA may efficiently compete with the
biotinylated probe sequences for ubiquitous chromosomal target
sites during the hybridization reactions. These results demonstrate
that the majority of highly repetitive DNA sequences can be
sufficiently suppressed to achieve chromosome-specific labeling by
in situ hybridization.
[0149] In certain cases the signal distribution over the entire
chromosome shows some variability from experiment to experiment.
When the overall signal is decreased, some chromosomal subregions
show a brighter staining; these signal hot spots generally
constitute chromosomal sites that contain known chromosome-specific
repetitive sequences. In the experiments shown in FIGS. 2A-2F and
FIGS. 3A-3D, predominant staining of the centrometric region of
chromosome 7 is seen, which corresponds to the chromosome-specific
signal of an alphoid repetitive DNA. Waye, J. S. et al., Mol. Cell
Biol., 7:349-356 (1987) and see Example 2. Apparently, the
abundance of these repeated sequences is sufficiently low to
prevent their suppression under the conditions used here. The
unequal signal distribution can be overcome by amplifying the
overall signal using an antibody sandwich technique as described
above. Furthermore, a predominant staining of the region 1q12 that
corresponds to the chromosomal site of satellite III DNA was
frequently observed in labeling chromosome I. Cooke, H. J. and J.
Hindley, Nucleic Acids Res., 6:3177-3197 (1979) and Gosden, J. R.
et al., Cytogenet. Cell Genet., 29:32-39 (1981) and see Example 2.
An example of the balanced signal distribution seen after such an
amplification step is shown in FIG. 4A.
[0150] Several commercially available DNA libraries, each
representing a single human chromosome, were tested for their
ability to specifically label the chromosome they represented,
under the standardized reannealing conditions described above and
with the probe concentrations adjusted for chromosome size, as
described above. Some examples, for chromosomes 1, 4, 7, 13, 18 and
20, as shown in FIGS. 4A, 4B, 4C, 4D, 4E and 4F, clearly
demonstrate that specific labeling can be achieved with most
chromosome libraries. Table 2 lists the libraries tested with their
relative scores of labeling specificity. All scores are positive
because the chromosome of interest was always decorated. The
highest score (4+) is used when no significant cross-hybridization
to other chromosomes was observed and the scores decrease (3+to 1+)
with an increasing amount of cross-hybridizing sequences.
2TABLE 2 Relative quality of specific chromosome labeling in situ
using preannealed biotinylated library DNA inserts Relative
specificity Library used of in situ hybridization Chromosome (ATCC
designation) signal.sup.1 1 LA01NS01 3+ 4 LL04SN01 4+ 7 LA07NS01 4+
8 LL08NS02 4+ 13 LA13NS03 1+ 14 LL14NS01 2+ 18 LL18NS01 4+ 20
LL20NS01 4+ 21 LL21NS02 3+ 22 LA22NS03 .sup. 3+.sup.b X LA0XNL01 4+
.sup.a See the text for score definition .sup.bUnder standard
preannealing conditions the chromosome 22 library gave a score of
+1; a value of +3 was achieved only with a human competitor DNA
concentration >700 .mu.g/ml (total DNA concentration 1.0
mg/ml).
[0151] All attempts to reduce the additional signals on other
chromosomes by varying the experimental conditions failed except in
experiments with chromosome 22; in this case higher concentrations
of human competitor DNA (700 .mu.g/ml) resulted in a significant
improvement of signal specificity. The library exhibiting the
lowest chromosome specificity was the chromosome 13 library (FIG.
4E). Multiple minor binding sites on other chromosomes, as well as
an exceptionally bright staining of Yq12 were observed; the signal
on the Y chromosome was visible using either female or male human
DNA as the competitor. None of the experimental parameters tested
improved on the overall specificity of this library.
[0152] Remarkably, a weak signal or even absence of signal can be
observed at the centromeric region of some chromosomes (see
chromosomes 4 and 18, FIG. 4C, D). In contrast to chromosomes 1 and
7, which contain chromosome-specific repetitive elements, the
centromere regions of chromosomes 4 and 18 apparently contain
repetitive sequences, most likely alphoid satellite DNAs, which are
very abundant and thus are suppressed by the reannealing technique.
However, these chromosomal regions are very small and the effect
can only be observed when the corresponding chromosomes are fairly
elongated.
[0153] Biotinylated total library DNA (containing the phage vector
sequences) was also used as probes, in concentrations adjusted to
the amount of human DNA inserts. (see above). One example is shown
in FIG. 4F with the chromosome 20 library. Although good staining
of the chromosome of interest ally was achieved, significant ground
on the entire slide was Similar results were obtained with plasmid
libraries containing human DNA subcloned from the lambda phage
libraries. In contrast, there was no background problem with the
total chromosome library LA0XNL01, which contains a significantly
smaller proportion of vector sequences in the probe mixture since
the size of the human DNA inserts is much larger.
[0154] The suppression of repetitive sequences by this reannealing
technique also permits the use of flow-sorted chromosome libraries
to detect chromosomal domains within interphase nuclei. Typical
examples of results obtained after hybridization of chromosome 1,
chromosome 7 and chromosome 18 probe sets to normal human
lymphocytes after acetic acid-methanol fixation are shown in FIGS.
5A-5F. Discrete focal domains of hybridization signal are seen with
all libraries that had scores of 2+ or more (see Table 2).
[0155] Most nuclei (n.gtoreq.100 per estimate) exhibited two
domains (60%-70%); however, a significant number showed only a
single domain (20%-30%) or no hybridization signal at all (5%-10%).
Accordingly, ca. 95% of male nuclei exhibited one and ca. 5% showed
no hybridization signal when the X chromosome library DNA was used
as probe. Notably, no nuclei with three domains were found with any
of the chromosomal probe sets tested. In contrast, all metaphase
spreads showed the decoration of both chromosome homologs without
exception. This interphase variability may reflect, in part, the
close juxtaposition of two individual domains in some cells, or the
inability to resolve domains that actually occupy different areas
within the nuclear volume but are unresolved when examined by
two-dimensional imaging methods (see FIG. SD; for discussion see
also Cremer et al., Exp. Cell Res., 176:199 220 (1988). The small
number of nuclei exhibiting no hybridization signal may be a
reflection of suboptimal hybridization conditions. It is of
interest to note that the size of the intranuclear domains
correlates reasonably well with the relative size of the cognate
metaphase chromosome. These observations provide a definitive proof
that the DNA of individual chromosomes exhibits a clear territorial
organization in the interphase nucleus of a normal human cell.
[0156] Acetic acid-methanol fixed nuclear spreads, such as those
shown in FIGS. 5A-5F, clearly retain the territorial organization
for each of the chromosomes examined; however, the nuclear
structure is not optimally preserved. Additional studies with
specimens that possess better preservation of 3-D structure using
paraformaldehyde fixed human diploid fibroblasts and a
laser-scanning confocal fluorescence microscope assembly for 3-D
image reconstruction have been done. The cells were fixed and
permeabilized as described by Manuelidis and hybridized with
chromosome library probes as outlined above. Manuelidis, L., Ann.
NY Acad. Sci., 450:205-221 (1985). The probe-competitor DNA mixture
was applied directly to the slide and denatured at the same time as
the cellular DNA. Results showed the arrangement of the chromosome
7 domains in the nucleus and the frequently observed helical
structure of labeled chromatic within chromosome domains. The
degree to which this helicity reflects true domain substructure or
is an artifact reflecting preparation and fixation procedures is
currently being investigated. Nevertheless, this preliminary
observations establishes the feasibility of using chromosome
specific probes to analyze the topography of chromosomal domains in
the interphase cells.
EXAMPLE 2
Detection of Chromosome Aberrations in Tumor Cells by CISS
Hybridization Using Chromosome-Specific Library Probes
[0157] Cells
[0158] TC 593 is a pseudotetraploid cell line (modal chromosome
number, 83) established from a human glioblastoma; it grows in a
flat, spreading fashion and contains many process. TC 620 is
pseudotriploid with a modal chromosome number of 64 and was
established from a human oligodendroglioma; it grows in an
epithelial fashion. Both cell lines have been described in detail.
Manuelidis, L. and E. E. Manuelidis, In: Progress in
Neuropathology, Vol. 4, 235-266, Raven Press, N.Y. (1979). The
present experiments made use of subclones C2B (TC 593) and C2B (TC
620) at approximately 180 passages after repeated subcloning from a
single cell of the original tumor line cultured as previously
described by Manuelidis and Manuelidis (see reference above).
Standard hypotonic treatment and acid/methanol fixation of the
cells were employed. Cremer et al., Exp. Cell Res., 176:199-220
(1988).
[0159] DNA Probes and Libraries
[0160] Phage DNA libraries from sorted human chromosomes were
obtained from the American Type Culture Collection: LAO1NSO1
(chromosome 1), LL04NSO1 (chromosome 4), LA07NSO1 (chromosome 7)
LL18NSO1 (chromosome 18) and LA22NS03 (chromosome 22).
Amplification of these libraries, isolation of human DNA inserts
and biotinylation were carried out as described in Example 1. A
probe specific for alphoid repeats on chromosome 7 (pa7tl) was the
gift of H. Willard and specifically decorates pericentromeric
heterochromatin of chromosome 7 under high stringency conditions
(60% formamide). Waye et al., Mol. Cell. Biol., 7:349-356 (1987);
Cremer et al., Exp. Cell. Res., 176:199-220 (1988). Some DNA probes
were modified with aminoacetylfluorene (AAF); and detected as
described by Cremer et al. for double labeling experiments.
Landegent et al., Exp. Cell Res., 153:61 72 (1984); Cremer, R. et
al., Exp. Cell Res., 176:199-220 (1988).
[0161] In Situ Hybridization and Detection of Hybridized Probes
[0162] CISS hybridization with biotinylated library DNA inserts and
detection of hybrid molecules was generally carried out using
standard conditions, as described in detail in Example 1. In double
CISS hybridizations using biotinylated chromosome 7 library DNA
inserts and the AAF-modified 7 alphoid probe, the latter probe was
heat denatured separately and only added to the hybridization
mixture at the end of the reannealing step at a final concentration
of 10 .mu.g/ml (see Example 1).
[0163] Digital Image Analysis of Specifically Decorated Metaphase
and Interphase
[0164] Chromosome
[0165] A VAX-station II/GPX graphics workstation (Digital Equipment
Corporation) with an ITEX FG 100-Q frame grabber (Imaging
Technology) were used as previously described together with a Zeiss
S-Planar 60 mm lens and a Dage-MTI 65 video camera. Manuelidis, L.
and J. Borden, Chromosoma, 96:397-410 (1988). Images were digitized
from negatives of metaphase spreads and interphase nuclei; the
background was removed and polygonal regions were defined to
specifically decorated metaphase chromosomes or interphase domains
(see Example 1). A scan line algorithm was used to calculate
histograms within the polygonal regions. Since the value of the
histograms H(i) of a particular intensity (range 0-255) within the
defined regions is the number of pixels at that intensity i, the
area within the region falling within an intensity range
i.sub.o-.sub.1 is the integral of the histogram from
i.sub.o-i.sub.1. Similarly, the 2-D integral in the region defined
by the intensity range i.sub.o-i.sub.1 equals .SIGMA.
H(i).i.i.sub.o was chosen for each hybridization, in order to
properly outline the decorated chromatin and distinguish this area
from background regions. i.sub.1 was set to the maximum value 255
in order to capture the entire intensity range above the
threshold.
[0166] Measurements of total signal intensity versus area were
designed as a control for the potential presence of variable
chromosome domain extension within interphase nuclei. In
interphase, a more extended chromosome domain might be expected to
have a greater area (or volume) yet a lower fluorescence signal
intensity per unit area. If a constant amount of hybridized DNA
corresponds to a constant total fluorescence, the total signal
intensity is a measure of labeled DNA content. It is also possible
to measure 3-D hybridized volumes within nuclei and 3-D integrated
total hybridized signals. Manuelidis, L. and J. Borden, Chromosome,
96:397-410 (1988). The background, b, was substrated from the
discrete 2-D integral .cedilla..cedilla.I(x,y)dA within a labeled
region R, to yield the total signal:
Sig.sub.t=.cedilla..cedilla.I(x,y)dA-b.cedi- lla..cedilla.dA, where
dA is a single pixel. Similarily, the mean intensity within the
region is calculated as 2-D integral/area or .cedilla..cedilla.I(x,
y)dA/.cedilla..cedilla.dA.
[0167] The following is a description of the results, with
reference to the appropriate figures, of the work described above.
They clearly document structural and quantitative changes in the
human glioma lines, including loss and gain of entire individual
chromosomes and of chromosomal subregions. They also show that it
has been possible to characterize both minor and predominant
karyotypic features in each cell line. All chromosomes tested to
date (i.e., 1, 4, 7, 18 and 22) clearly highlighted numerical
and/or structural aberrations, some of which were subtle.
[0168] Detection of numerical and structural chromosome aberrations
in metaphase spreads.
[0169] FIGS. 6A-6D, 7A-7J and 8A-8G and 12 show typical metaphase
spreads from the malignant glioma cell lines TC 620 and 593 after
CISS hybridization with biotinylated DNA inserts from each of the
human chromosomes 1, 4, 7, 18 and 22. Hybridized inserts were
detected with avidin fluorescein isothiocyanate conjugates (FITC)
and cells were counterstained with 4,6-diamidino 2 phenylindole
dihydrochloride (DAPI). Chromosomes designated as "complete" had an
apparently normal size, centromere index and DAPI staining pattern.
Despite this designation, these complete chromosomes may contain
fine structural aberrations only detectable by additional
investigations (see below). Apparently complete chromosomes 1, 4,
7, 18 and 22 were observed in both TC 620 and TC 593. Additionally,
other homologs of these chromosomes showed significant
rearrangements and abnormalities, including translocations and
deletions. The predominant numerical and structural aberrations
delineated in each of these cell lines are described below. A
minimum of 25 good metaphase spreads were evaluated for each glioma
line and for each chromosome. These data are summarized in FIG.
9.
[0170] Chromosome 1
[0171] In TC 620, the oligodendroglioma line, chromosome 1 inserts
decorated two apparently complete 1 chromosomes and two marker
translocation chromosomes (FIGS. 6A, 6B, 9). One marker was
metacentric and contained an entirely decorated 1q arm, but its p
arm was from another chromosome (of unknown origin). The other
marker chromosome was submetacentric and showed a small segment
from another chromosome attached to the 1p arm. In both marker
chromosomes breakpoints were localized close to the centromere in
1p11 or 1q11. The identification of the 1p segment was established
by DAPI banding (FIG. 6B), by 5'-bromo-2'-deoxyuridine (BrdU)
banding, and by hybridization with a 1p36.3 probe (data not shown);
the 1p36.3 probe additionally revealed deletion of this subregion
in one of the apparently complete 1 chromosomes. The overall
picture was of a nearly trisomic representation of chromosome 1,
with a common breakpoint, and subsequent translocation.
[0172] In TC 593, the glioblastoma line, an even more complex
pattern of numerical and structural chromosome 1 aberrations was
observed. In a sample of 50 metaphase spreads, the majority (52%)
showed six aberrant chromosomes that were decorated; 14% of the
metaphases showed five aberrant chromosomes, and 34% showed higher
numbers of chromosomes 1 segments (up to 14). FIGS. 6C, D and 9
show the most typical, predominant karyotype and demonstrates the
rapid definition of chromosome 1 abnormalities in this cell line.
Aberrations included three acrocentric chromosomes with a
consistent breakpoint in 1p1, chromosomes with a deletion of the
distal park of 1q, a submetacentric translocation chromosome with a
loss of the complete 1q, and an iso (1p) marker chromosome (see
FIG. 9).
[0173] Chromosome 4
[0174] In TC 620, chromosome 4-specific inserts decorated one
apparently complete chromosome 4, and three additional chromosomes
with segments containing chromosomes 4 DNA (FIGS. 7F, 9). These
latter segments on translocation chromosomes would have been
difficult to rapidly and unambiguously define with banding
procedures alone. The smallest of the translocated chromosome 4
segments formed part of an approximately metacentric chromosome.
The two larger segments were found on submetacentric chromosomes of
different overall size. In the smaller chromosome, the short arm
and part of the long arm of 4 were present with an apparent
breakpoint at 4q2, i.e., 4pter-4q2. In the larger submetacentric
chromosome, a region that may represent the rest of 4 (4q2-qter)
appears. Thus the predominant karyotype of TC 620 showed only
slightly more than two equivalents of chromosomes 4 (see also the
area measurements described below). The non-4 regions have not been
further defined.
[0175] In TC 593, there were generally only two chromosomes
decorated by chromosomes 4 DNA inserts, and both of these were
compatible with normal 4 chromosomes. Approximately 30% of the
metaphase spreads in TC 593 showed an additional submetacentric
chromosome with chromosome 4 material (FIG. 7E). Thus, although
both 4 chromosomes were apparently normal, there was a significant
under-representation of this chromosome in this pseudotetraploid
line (FIG. 9).
[0176] Chromosome 7
[0177] Three complete 7 chromosomes, and one smaller metacentric
chromosome containing translocated 7 material were typically found
in TC 620 metaphase spreads (FIGS. 8A, 9). The translocated
chromosome 7 material included the short arm of chromosome 7 (as
shown by DAPI banding; cf. FIG. 8B) and the pericentromeric
heterochromatin with the breakpoint in 7q1 (see also below).
[0178] In TC 593, five chromosomes entirely decorated by chromosome
7. insert probes were regularly observed (FIGS. 7G, 8E). Four of
these appeared represent complete number 7 chromosomes, whereas was
smaller and metacentric. DAPI banding (FIG. 8E, insert) and size
measurement (cf. FIG. 13) were consistent with an iso(7p). This
conclusion was further supported by double in situ hybridization
experiments with biotinylated chromosome 7 inserts (detected with
avidin FITC) and chromosome 7-specific alphoid AAF labeled
sequences (detected with tetramethylrhodamine isothiocyanate
(TRITC) conjugated second antibodies). They showed that only the
four complete 7 chromosomes, contained a detectable 7 centromeric
signal (metaphase, FIG. 7G, H; interphase, FIG. 7I, J). Thus, the
iso(7p) marker chromosome did not have a characteristic to one 8E,
centromeric region as it lacked both the 7 alphoid sequences and a
small block of heterochromatin at 7q11 (see FIG. 8E, insert). In
contrast, all four chromosomes of TC 620 were labeled with the 7
alphoid probe (data not shown).
[0179] Chromosome 18
[0180] In TC 620, two apparently complete 18 chromosomes and a
truncated minute chromosome were entirely decorated (FIGS. 8C, D,
9). This truncated chromosome is 18q- (and possibly also 18p). The
rest of the chromosome 18 region(s) was never detected.
[0181] Three translocation chromosomes involving chromosome 18
material were typically detected, in addition to an apparently
normal chromosome 18 in TC 593 metaphase spreads (FIGS. 8F, G, 9).
In a minor proportion of metaphases there was a small additional
translocation observed. The exact chromosomal region from which
this translocated 18 material derived could not be resolved by DAPI
staining. The predominant karyotype for 18 is therefore close to
tetrasomic in this cell line, but is under-represented in the
pseudotriploid TC 620.
[0182] Both the 18q-marker chromosome in TC 620 and the three
translocated 18 chromosomes in TC 593 hybridized strongly to a
chromosome 18-specific alphoid repeat. Accordingly, both intact and
aberrant 18 chromosomes could also be counted after in situ
hybridization with this centromeric probe. Cremer, T. et al., Exp.
Cell Res., 176:199 200 (1988) (see also below). DAPI banding and
hybridization to 18-specific alphoid repeats indicated that these
translocation chromosomes include the entire 18q region and the
centromere, with breakpoints in 18p.
[0183] Chromosome 22
[0184] Two apparently normal 22 chromosomes were visualized in most
TC 620 and TC 593 metaphase spreads (FIG. 10E). It was difficult to
ascertain small translocations of this chromosome since
hybridization with chromosome 22 inserts resulted in some
cross-hybridization to other chromosomes. Some of this
cross-hybridization is probably due to shared sequences from the
nucleolus organizer regions (on five normal acrocentric human
chromosomes) and to shared sequence motifs at the centromeres.
McDermid, H. E. et al., Chromosome, 94:228-234 (1986) and see
Example 1. Finally, it should be noted that in contrast to
conventional banding analysis, the current experimental approach
clearly delineates numerical and structural chromosome aberrations
in metaphase spreads of very poor quality (FIG. 10G, H) or in early
prophase nuclei (FIG. 10A). These preparations are not accessible
to banding analysis, as the chromosomes extensively overlie each
other.
[0185] Evaluation of Chromosome Domains in-Interphase Nuclei
[0186] One potential advantage of in situ methods is that
individual human chromosomes may be directly visualized as-discrete
territories in interphase nuclei and thus can be of value in the
analysis of solid tumor specimen. Manuelidis, L., Hum. Genet.,
71:288-293 (1985); Schardin, M. et al., Hum. Genet., 71:281-287
(1985); Pinkel, D. et al., Proc. Natl. Acad. Sci. USA, 83:2934-2938
(1986). This feature of nuclear topography, also apparent in the
malignant cells examined here (FIGS. 7A-D, I, 10B-F), was evaluated
for its accuracy and diagnostic usefulness. FIG. 10A shows three
apparently complete 7 chromosomes and one translocated-7p arm in a
pro-phase TC 620 nucleus. FIGS. 3A-3D , and 10B show five
chromosome 7 domains in interphase nuclei of TC 593, as previously
depicted in metaphase spreads. FIG. 10C shows a TC 620 interphase
nucleus with two 18 domains of comparable sizes to those seen in
normal diploid nuclei (see Example 1). A third, appreciably
smaller, decorated 18 domain was also detected and represents the
truncated 18 chromosomes seen in metaphase spreads described above.
FIG. 10D shows four chromosome 18 domains in an interphase nucleus
of TC 593, which again is comparable to the numbers in metaphase
nuclei. FIG. 10E shows a TC 620 interphase nucleus with four
chromosomes 1 domains, while FIG. 10F shows a TC 593 nucleus with
at least five separate chromosome 1 domains (compare FIG. 6A, B and
C, D, respectively).
[0187] While the hybridization patterns of nuclei shown in FIGS.
10A-H were highly characteristic for each cell line, counts of
interphase chromosome domains have some inherent difficulties. As
an example, FIG. 11A (dark columns) presents an analysis of the
counts of labeled interphase domains in randomly selected nuclei of
diploid human lymphocytes hybridized with 7 library inserts.
Although the number and relative size of chromosome specific
domains can be accurately assessed in the majority of nuclei, not
all nuclei present a reliable index of the chromosomal
constitution, since a considerable fraction of nuclei reveal only
one decorated domain and occasional nuclei show no signals.
Furthermore all domains are not always cleared separable in these
2-D preparations.
[0188] FIGS. 11A-E show representative counts of these
preparations. In agreement with TC 593 metaphase counts of
chromosome 4, nuclear counts generally showed two clearly separated
domains (FIG. 11E). However, the percentage of two-signal
preparations was smaller in interphase than in metaphase (45.3% vs.
64%). This artifactual decrease was largely due to a corresponding
increased percentage of nuclei showing only one decorated domain or
no signal at all. Counts of zero or one domain were not present in
metaphase spreads. Significantly, 19.3% of the interphase TC 593
nuclei displayed three clearly separated chromosome 4 domains, and
these extra domains were not present in interphase nuclei of
diploid human lymphocytes hybridized to this or other libraries
under the same conditions (FIG. 10A; Example 1). Finally, the ratio
of two versus three domains was identical for both metaphase and
inter-phase cells. Thus interphase nuclei can be reliably used for
the detection of extra copies of a single chromosome or chromosomal
segment but have limited reliability for detecting the loss of
chromosome copies.
[0189] In situ hybridization of probes from subregions of
interphase chromosomes may more accurately reflect general counts
of chromosomal constitution than library probes (FIG. 11A),
provided they are done under appropriately high stringency
conditions Rappold, G. et al., Hum. Genet., 67:317-325 (1984);
Cremer, T. et al., Hum. Genet., 74:346-352 (1986); Cremer, T. et
al., Exp. Cell Res., 176:199-220 (1988). However, such regional
segment probes do not delineate translocated elements or aberrant
chromosomes that lack this segment. Therefore such probes are also
not entirely accurate. For example, counts of chromosomes 1 in TC
620 and TC 593 with a probe specific for 1q12 indicated fewer 1
chromosomes than shown here with CISS hybridization (FIG. 9).
Cremer, T. et al., Exp. Cell Res., 176:199-220 (1988). Counts of
chromosome 7 using only a centromeric sequence further emphasize
this point (see above). Double in situ hybridization with the
AAF-modified 7 alphoid probe and biotinylated chromosome 7 library
inserts typically showed interphase nuclei with five domains, of
which only four were simultaneously labeled by the centromeric
probe (FIGS. 7I, J, 11C). In TC 620, however, both probes gave
identical results (FIG. 11B).
[0190] Over Representation and Under Representation of Specific
Chromosomes
[0191] The relative chromosomal dosage in these glioma lines, was
also assessed with particular interest in chromosome 7, which has
been noted to be generally over-represented in gliomas. Bigner, S.
H. et al., Cancer Genet. Cytogenet. 29:165-170 (1986); Shapiro, J.
R., Semin. Oncol., 13:4 15 (1986). For comparison, other individual
chromosome probes were used as controls. Metaphase chromosomes
counts have shown that TC 620 is pseudotriploid with a modal number
of 64 chromosomes, while TC 593 is pseudotetraploid with a modal
number of 83. Manuelidis, L. and Manuelidis, E. E., In: Progress in
Neuropathology, Vol. 4, pp. 235-266, Raven Press, N.Y. (1979).
Accordingly, a chromosome and its segments together would be
present in a balanced state if three complete copies were present
in TC 620, and four in TC 593.
[0192] A relative over-representation is present if more than these
respective copy numbers can be demonstrated. A number lower than
the expected (trisomic or tetrasomic) value indicates that the
chromosome is relatively under-represented in the karyotype. In
cases where additional DAPI banding information was sufficient to
define the selectively decorated abnormal chromosome, the
chromosome pieces labeled by the chromosome-specific inserts were
put together for analysis (FIG. 9). In the second approach,
computer analyses were used to independently verify these results
(see below).
[0193] TC 620 analyzed by banding showed the equivalent of three 1
chromosome and thus indicated a balanced state for this chromosome.
The same was true for the 1p arm in TC 593 which was present in
four copies. However, the distal part of 1q was under-represented
in TC 593 (see the detailed description given above). In both
glioma lines, 7q appeared to be balanced, while 7p was
over-represented once in TC 620 and twice in TC 593. Additionally,
in both glioma lines chromosomes 22 was clearly under-represented.
In order to confirm this finding, double in situ hybridization with
inserts of chromosomes 7 and 22 was performed. An example of this
is shown in FIG. 12 and demonstrates over-representation of 7 DNA
and under-representation of 22 DNA in the same cell. Metaphase
counts done in both cell lines by this method of analysis are
depicted for chromosome 7 in FIG. 11B, C (dark column) and for
chromosome 22 in FIG. 11E. In summary, these two gliomas both show
relative under-representation of chromosome 22 and
over-representation of the 7p arm. The significant
under-representation of chromosome 4 in TC 593, and a portion of 4
in TC 620 is also notable.
[0194] Digitized images were also used to quantitatively measure
decorated areas-in metaphase preparations and in interphase cells
where chromosomal domains were well resolved.
[0195] Quantitative evaluation of chromosome equivalents (Table 3)
indicated highly concordant numbers for interphase versus metaphase
in 5 of 6 examples; only in TC 593 decorated with 18 inserts was
there a discrepancy. This may be due to the small sample size.
[0196] Table 3
[0197] Twenty-four metaphase spreads showing the predominant number
of chromosomes decorated with DNA inserts from libraries of
chromosomes 4, 7 and 18 were compared to twenty-eight interphase
nuclei with well-separated domains using the same probes. Images
were taken under identical (linear film) conditions and digitized.
In each metaphase spread, areas obtained for each normal and
aberrant chromosome were divided by the mean area obtained for n
apparently complete chromosomes. In interphase nuclei, domains were
compared assuming that the largest n-labeled domains represented
complete (normal) interphase chromosomes. Thus the sum these
normalized values represents a measure of number of specific
chromosomes equivalents in a single cell. The mean values of
several cells are shown for each case. The mean numbers of
chromosomes equivalents obtained for interphase and metaphase cells
show a strong overall correlation coefficient of r=+0.95. Compared
with area measurements, the mean numbers of chromosomes equivalents
determined by 2-D intensity integrals (See above) showed an overall
correlation co-efficient of r=+0.99.
3TABLE 3 Mean number of chromosome equivalents measured by digital
image analysis in malignant glioma cell lines after CISS
hybridization Chromosome Equivalents Chromosome Cell Line
Interphase Metaphase Expected 4 TC593 2.0 2.0 4.0 TC620 2.5 2.4 3.0
7 TC593 4.4 4.6 4.0 TC620 3.5 3.3 3.0 18 TC593 3.0 3.6 4.0 TC620
2.5 2.3 3.0
[0198] Chromosome equivalents derived from digital image analysis
independently confirm the relative representation of target
chromosomes noted in both glioma lines by DAPI banding. The
segments that comprise the total metaphase signal, are further
detailed graphically in FIG. 13. Computer analysis was especially
useful in cases where the breakpoints involved in translocated
segments could not be unambiguously defined. They were also of
value in a quantitative assessment of interphase-metaphase
correlations, and of normal and aberrant chromosomes with
distinctly different sizes.
EXAMPLE 3
Rapid Detection of Human Chromosome 21 Aberrations By In Situ
Hybridization
[0199] DNA Probes
[0200] All plasmids contain inserts of human chromosome 21 that
were mapped to 21q22.3. Moisan, J. P., Mattei, M. G.,
Baeteman-Volkel, M. A., Mattei, J. F., Brown, A. M. C., Garnier, J.
M., Jeltsch, J. M., Masiakowsky, P., Roberts, M. & Mandel, J.
L. (1985) Cytogenet. Cell Genet. 40, 701-702 (abstr.). Tanzi, R.,
Watkins, P., Gibons, K., Faryniarz, A., Wallace, M., Hallewell, R.,
Conneally, P. M. & Gusella, J. (1985) Cytogenet. Cell Genet.
40, 760 (abstr.). Van Keuren, M. L., Watkins, P. C., Drabkin, H.
A., Jabs, E. W., Gusella, J. F. & Patterson, D. (1986) Am. J.
Hum. Genet. 38, 793-804. Nakai, H., Byers, M. G., Watkins, P. C.,
Watkins, P. A. & Shows, T. B. (1987) Cytogenet. Cell Genet. 46,
667 (abstr.). Munke, M., Foellmer, B., Watkins, P. C., Cowan, J.
M., Carroll, A. J., Gusella, J. F. & Fracke, U. (1988) Am. J.
Humm. Genet. 42, 542-549. All inserts were either known or verified
by Southern blot analysis to be single-copy DNA: the plasmids other
than pS2 are subclones derived from a .lambda. phage library or a
cosmid library. Van Keuren, M. L., Watkins, P. C., Drabkin, H. A.,
Jabs, E. W., Gusella, J. F. & Patterson, D. (1986) Am. J. Hum.
Genet. 38, 793-804. Masiakowski, P., Breathnach, R., Bloch, J.,
Gannon, F., Krust, A. & Chambon, P. (1982) Nucleic Acids. Res.
10, 7895-7903. Watkins, P. C., Tanzi, R. E. Gibbons, K. T.,
Tricoli, J. V., Landes, G., Eddy, R., Shows, T. B. & Gusella,
J. F. (1985) Nucleic Acids Res. 13, 6075-6088. Watkins, P. C.
Watkins, P. A., Hoffman, N. & Stanislovitis, P. (1985)
Cytogenet. Cell Genet. 40, 773-774 (abstr.). The plasmids are
listed in Table 4 with the Human Gene Mapping Workshop symbols and
the approximate insert fragment length. Kaplan, J. C. & Carrit,
B. (1987) Cytogenet. Cell Genet. 46, 257-276.
4TABLE 4 Plasmids with inserts from 21q22.3 Insert length Plasmid
kb BCEI pS2 (23) 0.6 D21S3 pPW231F 0.8 pPW231G 0.7 D21S23 pPW244D
1.0 D21S53 pPW512-6B 3.0 pPW512-8B 3.8 pPW512-1H 2.9 pPW512-16P 2.7
pPW512-18P 1.6 pPW512-4R 4.7 pPW512-12R 2.0 D21S55 pPW518-4H 1.6
pPW518-1OP 2.9 pPW518-5R 5.2 D21S56 pPW520-5B 5.0 pPW520-6B 1.0
pPW520-1OR 4.6 pPW520-11R 1.8 D21S57 pPW523-1OB 6.5 pPW523-1H 7.0
pPW523-5R 2.2 pPW523-1OR 3.8 pPW523-19R 2.5 D21S64 pPW551-8P 1.9
pPW551-12P 4.2 D21S71 pPW519-1OP 0.8 pPW519-11P 3.0 pPW519-1R 6.0
pPW519-8R 2.9 pPW519-9R 1.7 pPW519-14R 4.0 pPW519-22R 1.8
[0201] Preparation of plasmid DNA was according to standard
protocols. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982)
Molecular Cloning: A Laboratory manual (Cold Spring Harbor Lab.,
Cold Spring Harbor, N.Y.). Various probe sets were obtained by
pooling plasmids (equal molar amounts), resulting in DNA probe
complexities of 95 kb (all plasmids listed). 75 kb (plasmids
labeled with an asterisk), or 29 kb (plasmids labeled with a
dagger).
[0202] The human chromosome 21 genomic library LL21NS02 was
obtained from the American Type Culture Collection and amplified on
agar plates as recommended. Phage DNA was prepared and digested
with HindIII, and the DNA inserts were separated from the vector
arms by preparative gel electrophoresis in 0.6% agarose. DNA was
isolated from gel slices by electroelution; purified by Elutip-d
chromatography. (Schleicher & Schuell); extracted with
phenol/chloroform, 1:1 (vol/vol); and precipitated with
ethanol.
[0203] Human Cells
[0204] Metaphase spreads and interphase nuclei were prepared from
(i) lymphocyte cultures of normal (46, XY) individuals, (ii)
lymphocytes of Down syndrome (47, +21) individuals, (iii) chorionic
villi samples cultured for prenatal diagnosis (ii and iii were
provided by T. Yan-Geng, Yale University Cytogenetics Laboratory),
and (iv) cultures of TC620, an oligodendroglioma-derived
pseudotriploid cell line. Manuelidis, L. & Manuelidis, E. E.
(1979) in Progress in Neuropathology, ed. Zimmerman, H. M. (Raven,
Press New York), Vol. 4, pp. 235-266. Standard techniques of
colcemid treatment, hypotonic treatment, and methanol/acetic acid
fixation were used. Biopsy material from the cortical region of a
"normal" human brain (46, XX) was fixed, sectioned, and
permeabilized as described. Manuelidis, L. & Borden, J.
Chromosome, 96:397-410 (1988).
[0205] In situ Hybridization
[0206] Various combinations of plasmid DNA, labeled with Bio11-dUTP
by nick-translation, were used for hybridization at concentrations
ranging from 2 to 15 .mu.g/ml depending on the pool size. Brigati,
D. J., Myerson, D. Leary, J. J., Spalholz, B., Travis, S. Z., Fong,
C. K. Y. Hsiung, G. D. & Ward, D. C. (1983) Virology 126,
32-50. For example, 15 .mu.g/ml was used when the probe mixture
contained 94 kilobases (kb) of insert DNA; the probe concentration
was decreased in proportion to the sequence complexity of the probe
mixture. The size of the probe DNA was adjusted to a length of
150-250 nucleotides empirically by varying the DNase concentration
in the nick-translation reaction. The hybridization cocktail also
contained 50% formamide, 0.30 M NaCl, 0.03 M sodium citrate (pH7),
10% (wt/vol) dextran sulfate, and on occasion 0.5 mg of sonicated
salmon sperm DNA per ml. Simultaneous denaturation of probe and
target DNA was carried out at 75.degree. C. for 6 min. (metaphase
spreads) or 94.degree. C. for 11 min (tissue slices). Hybridization
reactions were incubated at 37.degree. C. overnight.
[0207] Delineation of individual chromosomes with DNA probes
derived from sorted human chromosomes was done by (CISS)
hybridization as described above. Briefly, biotinylated chromosome
21 library DNA inserts (5 pg/ml), DNase-digested human genomic DNA
(200 pg/ml), and salmon sperm DNA (800 pg/ml) were combined in the
hybridization solution, heat-denatured, and partially prehybridized
for 10-30.min at 37.degree. C. before application to a separately
denatured specimen.
[0208] Posthybridization washes, detection of hybridized probe by
using either alkaline phosphate-conjugated avidin or fluorescein
isothiocyanate-conjugated avidin, and photographic conditions were
as described in Example 1. When probe sets containing 29 kb or less
of target sequence were used, the fluorescein isothiocyanate
detection was generally enhanced by one cycle of signal
amplification as described in Example 1.
[0209] All quantitative analyses of interphase signals were carried
out by using slides from several independent experiments, with more
than I 00 nuclei being analyzed per slide. Comparison of signals in
normal and trisomic samples was done in a blind-study fashion.
[0210] This work demonstrated that CISS hybridization, under the
conditions described, resulted in rapid detection of numerical and
structural aberrations of chromosome 21 in both metaphase and
interphase cells.
[0211] Use of Cloned DNA Fragments From Human Chromosome 21 to
Specifically Label Chromosomes in Lymphocyte Metaphase Spreads and
Interphase Nuclei
[0212] The maximal amount of unique sequence DNA in the probe set
was ca94 kb; this probe set resulted in a clearly visible labeling
of the terminal region of both chromatids of the chromosome 21
homologs (see FIG. 14B). These signals were seen unambiguously and
without exception in all metaphase spreads, even in spreads of poor
quality or from prophase cells (not shown). In normal interphase
cells, the majority (65-75%) of nuclei exhibited two signals (see
FIG. 14C), 25-30% showed one signal, and less than 5% showed no
signal. Nuclei with three signals were found only rarely (<0.2%)
and may reflect incomplete hybridization to a few tetraploid cells
in the sample. Similar results were obtained with probe sets
containing 29 or 75 kb of DNA. With probe sets containing fewer
than 20 kb of insert DNA, there were increased numbers of cells
with less than two signals. Thus, these probe sets were deemed
unsuitable for diagnostic purposes. However, such probes still
yielded specific signals on the majority of chromosomes 21, even
with a 6-kb single-copy DNA (see FIG. 14A), especially when signal
amplification was used.
[0213] Use of Chromosome Library DNA CISS Hybridization for
Detecting Chromosome 21
[0214] Chromosome 21 was specifically and entirely decorated in
normal lymphocyte metaphase spreads, although some additional minor
binding sites were seen at or near the centromeric region of other
acrocentric chromosomes, especially chromosome 13 (normal karyotype
not shown; FIG. 14F). Suppression with additional DNA including a
plasmid L1.26, which detects a repetitive DNA located predominantly
at the centromeric region of chromosomes 13 and 21, did not
efficiently suppress the minor non-21 chromosomal signals. Devilee,
P., Cremer, T., Slagboom, P., Bakker, E., Schoil, H. P., Hager, H.
D. Stevenson, A. F. G., Cornelisse, C. J. & Pearson, P. L.
(1986) Cytogenet. Cell Genet. 41,193-201. Quantitative evaluation
of interphase nuclei signals again showed a negligible portion of
nuclei with three signals; however, a significant increase in
nuclei with less than two signals was observed (50-60% with two
signals, 35-45% with one signal, and 5-10% without a signal). The
numerical differences observed with the two different probes can be
explained in part by the number of nuclei (up to one of three) that
were excluded from the latter analysis because they exhibited
larger and more diffuse signals, most likely from more than one
chromosome that could not be resolved unambiguously as two separate
chromosome domains in a two-dimensional representation. The minor
cross-hybridizing sites noted above presented a second experimental
complication but did not adversely influence data
interpretation.
[0215] Testing of Cells Containing Chromosome 21 Aberrations
[0216] The optimal (94 kb) plasmid pool as well as CISS
hybridization with chromosome 21 library inserts were tested
further by using cells containing chromosome 21 aberrations. Both
probe sets permitted a fast and unambiguous diagnosis of trisomy 21
in all metaphase spreads from Down syndrome lymphocyte cultures
(see examples in FIGS. 14D and E). Furthermore, the quantitative
distribution of hybridization signals in interphase nuclei of the
same preparation, analyzed as described above, was similar with
either type of probe [<5% of cells with no signal, 5-15% with
one signal, 25-35% with two signals, and 55-65% with three signals
(FIG. 14 F-J)]. Although the library DNA inserts gave up to 15% of
four-signal nuclei (compare FIGS. 14F and G), most likely due to
the minor binding sites on other chromosomes, the plasmid pool
revealed only a negligible percentage of nuclei (<0.2%) with
four signals. These results-indicate that trisomy 21 can be
detected in a diagnostically meaningful way with small populations
of nonmitotic cells.
[0217] Localization of Chromosome 21 NDA in Embyonic Chorionic
Villi Cells
[0218] Embryonic chorionic villi (CV) cells were also investigated
with the 94 kb plasmid probe sets in a case where the father had a
reciprocal t(4:21) translocation. Hybridization to metaphase
spreads of the CV cells showed that the translocated chromosome
(4pter.fwdarw.4q33::21q11.2.fwdar- w.21qter) was indeed inherited
by the fetus (see FIGS. 13L and M). The signals in the interphase
cell nuclei (see FIG. 14K) of the CV cells had a distribution that
paralleled that of cells with a normal karyotype (see above),
indicating a balanced representation of 21q22.3 and excluding Down
syndrome as a possible diagnosis. A small increase of nuclei with
three and four signals (both <5%) of normal lymphocytes was also
observed, reflecting a higher portion of tetraploid such CV
samples.
[0219] Localization of Chromosome 21 DNA in of Glioma Tumor
Cells
[0220] The diagnostic potential of the chromosome 21 probes was
further tested by using a glioma tumor cell line, TC620, known to
be pseudotriploid with a highly rearranged genome. Cremer, T. et
al., Exp. Cell Res., 176:199-220 (1988); Cremer, T. et al., Hum.
Genet., In Press, (1988); Manuelidis, L. and E. E. Manuelidis, In:
Progress in Neuropathology, 4:235-266 (ed. Zimmerman, H. M.)
(1979). The metaphase spreads revealed two apparently normal
chromosomes 21 and one translocation chromosome (see FIGS. 14N and
0). Interestingly, the chromosome 21 DNA on the translocation
chromosome labeled by the library probe has a size equivalent to a
normal 21q region, thus suggesting a Robertsonian translocation
event. However, fine structural aberrations of 21q (i.e., small
deletions, etc.) cannot be excluded by this analysis. The
interphase signals seen with both the plasmid probe set and the
library inserts were consistent with trisomy 21q22.3 and trisomy
21, respectively.
[0221] Localization of Chromosome 21 i DNA Sequences in Solid
Tissues
[0222] The ability of the 94 kb plasmid probe set to localize
chromosome 21 DNA sequences in solid tissues was also assessed.
Both chromosomes 21 were clearly labeled by the probe, and located
near the nucleolus; this nuclear location is consistent with the
fact that chromosome 21 contains a ribosomal that is usually
localized in the This observation suggests that these probes may
also prove useful for evaluating the frequency of chromosome 21
mosaicism in specific cell or tissue types. In addition, it should
be of interest to see if the various karyotypic changes associated
with the Down syndrome phenotype alter the normal nuclear
topography of chromosome 21 in neuronal tissue.
EXAMPLE 4
Simultaneous Visualization of Seven Different DNA Probes by In Situ
Hybridization Using Combinatorial Fluorescence and Digital Imaging
Microscopy
[0223] Materials and Methods
[0224] Human metaphase chromosomes were prepared by standard
procedures. Prior to in situ hybridization, slides were washed in
1.times. phosphate-buffered saline (5 min; room temperature) and
dehydrated through an ethanol series (70%, 90% and 100%; 5 min
each). Slides were stored at -70.degree. C. with Drierite
powder.
[0225] DNA Probes
[0226] The following chromosome-specific a satellite DNA clones
were used: pBS10.7AE0.6 (Baldini, unpublished), chromosome 3; p7tet
(Waye, J. S. et al. (1987) Mol. Cell. Biol. 7:349-356), chromosome
7; pMR9A (Rocchi, M. et al. (1991) Genomics 9:517-523), chromosome
9; pBR12 (Baldini, A. et al. (1990) Am J. Hum. Genet. 46:784-788),
chromosome 12. paH2 (chromosome 18) and paH5 (chromosome 8) were
cloned in our laboratory, while pRB2 (chromosome 11) was a gift of
Dr. M. Rocchi (Bari, Italy). The chromosome specific plasmid
libraries (Collins, C. et al. (1991) Genomics, in press) were a
gift of Dr. J. Gray (Livermore, Calif.). The following cosmid and
phage clones were used: cpt1, mapping to Xp21 (Ried, T. et al.
(1990) Hum. Genet. 85:581-586); c-myc, mapping to 8q24 (Ried T. et
al. (1991) Genes Chromosome Cancer, in press); c512, mapping to
21q22 (Lichter, P. et al. (1990) in Molecular Genetics of
Chromosome 21 and Down Syndrome, ed. Patterson, D., Alan R. Liss,
New York, N.Y., pp. 69-78); cosmid clone 26, mapping to 5q32
(unpublished data); cosSB1, mapping to 6p21 (Srivastava, R. et al.
(1986) Trans. Assoc. Am. Physicians, Vol. XCIX); cosmid K40,
mapping to 11p15 (Lichter, P. et al. (1990) Science 247:64-69). The
cosmid clones specific for chromosome 5 (clones 26, 29, 56, 58, 92
and 121) were provided by Dr. Greg Landes (Integrated Genetics,
Inc., Framingham, Mass.) and previously mapped by Jennifer Lu
(personal communication).
[0227] DNA was prepared according to standard techniques (Sambrook,
J. et al. (1989) Molecular cloning: a laboratory manual, Cold
Spring Harbor Lab., Cold Spring Harbor, N.Y.).
[0228] Probe Labeling
[0229] PCR's were performed using 10 ng of alphoid DNA clones or
100 ng of chromosome-specific libraries as template. Preferential
amplification of insert DNA was achieved by using primers flanking
the polylinker of each plasmid vector. T3 and T7 primers were used
for the pBS vector, M13 forward and M13 reverse primers were used
for pUC and pCR1000 vectors (all at a final concentration of 1
.mu.M). PCR was carried out in 1.5 mM MgC12/10 mM Tris-HCl/50 mM
KC1/0.001% gelatin/1.25 units of Taq polymerase (AmpliTaq;
Perkin-Elmer/Cetus) in a total volume of 50 p1 (10 p1 when
fluorescein-11-dUTP was used due to the limited amount of this
reagent). The dNTP concentrations used in the PCR-labeling
reactions are listed in Table 5. The highest concentration of
modified nucleotides used was 75 .mu.M. However, dinitrophenol
(DNP)11-dUTP at a concentration>37.5 .mu.M strongly reduced the
amplification efficiency (data not shown). When DNP-11-dUTP was
used for combinatorial labeling, the concentrations were the same
as for fluorescein-11-dUTP. The modified nucleotides were obtained
from Boehringer Mannheim (digoxigenin (dig)-11-dUTP,
fluorescein-11-dUTP), Sigma (Bio-11-dUTP) and Novagen (Madison,
Wis.) (DNP-11-dUTP). The thermocycling was performed with a
commercially available machine (Ericomp, San Diego). After an
initial denaturation at 95.degree. C. for 3 min, 32 cycles of PCR
were carried out with denaturation at 94.degree. C. for 1 min,
annealing at 55.degree. C. for 2 min and extension at 72.degree. C.
for 4 min (last cycle, 10 min). PCR products from the chromosome
libraries were treated with DNase I to obtain an average fragment
size of about 250 base pairs (bp) and were separated from free
nucleotides by Sephadex G50 spin column. Cosmid and phage clones
were labeled by standard nick-translation reactions. The final
concentration of the modified nucleotides and the DNA clones used
in these reactions were as follows: Bio, 50 .mu.M (cosSB2, clone
58); dig, 40 .mu.M (K40, clone 121); DNP, 40 .mu.M (c512, clone
92); Bio/dig, 20 .mu.M/30 .mu.M (cpt1, clone 56); Bio/DNP, 20
.mu.M/30 .mu.M (c-myc, clone 29) and dig/DNP, 20 .mu.M/30 .mu.M
(clones 28 and 26).
5TABLE 5 Labeling of Seven Centromere Probes dNTP, .mu.M AE.06
p7tet pBR12 pMR9A pRB2 p.alpha.H2 p.alpha.H5 Bio-11-dUTP 75 37.5
37.5 25 dig-11-dUTP 75 37.5 37.5 25 FITC-11-dUTP 75 37.5 37.5 25
TTP 225 225 225 225 225 225 225 dA, dC, dGTP 300 300 3003 300 300
300 300 FITC, fluorescein isothiocyanate
[0230] In Situ Hybridization and Detection
[0231] Centromeric Repeats
[0232] After PCR amplification, the probes were used without
further purification. The DNA solution was diluted 1:5 in 10 mM
Tris-HCl/1 mM EDTA. One microliter of each probe was precipitated
with 5 .mu.g of salmon sperm DNA and 5 .mu.g of yeast RNA and
resuspended in 10 p1 of 60% formamide, 2.times. standard saline
citrate (SSC) and 5% dextran sulfate. Probe DNA was denatured at
75.degree. C., 5 min, and immediately applied to the denatured
chromosome specimens; a coverslip was added and sealed with rubber
cement. The slides were denatured separately in 70%
formamide/2.times.SSC for 2 min at 80.degree. C. and dehydrated in
an ethanol series. After overnight incubation at 37.degree. C., the
coverslips were removed and the slides were washed at 45.degree. C.
in 50% formamide/2.times.SSC three times, followed by three washes
at 60.degree. C. in 0.1.times.SSC. After a blocking step (in
4.times.SSC/3% bovine serum albumin for 30 min at 37.degree. C.),
the biotinylated probes were detected using streptavidin conjugated
to the infrared dye Ultralite 680 (Ultra Diagnostic Corporation,
Seattle, Wash.; final concentration, 2 .mu.g/ml); the dig-labeled
probes were detected with a rhodamine-labeled anti-dig IgG
(Boehringer Mannheim). The fluorescein-1-dUTP (Boehringer Mannheim)
labeled probes did not require any immunological detection step.
4',6-Diamidino-2-phenylimdole (DAPI) was used as a chromosome
counterstain.
[0233] Chromosome Painting
[0234] The amplification products were treated with DNase I to an
average size of 150-500 bp. Five micrGliters of the amplification
reaction mixture (50 p1) was precipitated with 5 .mu.g salmon sperm
DNA and 5 .mu.g of yeast RNA, together with 10 .mu.g total human
competitor DNA, and then resuspended in 10 p1 50%
formamide/2.times.SSC/10% dextran sulfate. The probe was denatured
as described above and allowed to preanneal for 1 h at 37.degree.
C. Slides were denatured as described for the centromeric repeats.
Hybridization took place overnight at 37.degree. C. Slides were
washed at 42.degree. C. in 50% formamide followed by three washes
at 60.degree. C. in 0.5.times.SSC. The biotinylated sequences were
detected with streptavidin conjugated to the infrared dye Ultralite
680; the dig-labeled sequences were detected with rhodamine-labeled
anti-dig IgG (Boehringer Mannheim). DNP-labeled probes were
detected with a monoclonal rat anti-DNP antibody (Novagen) and a
secondary goat anti-rat antibody, conjugated to Fluorescein
isothiocyanate (Sigma). DAPI was used as a DNA counterstain.
[0235] Cosmid Clones
[0236] Eighty ng of each cosmid or phage was precipitated with 20
ug human competitor DNA and 5 .mu.g each of yeast RNA and salmon
sperm DNA. The detection of the differently labeled probe DNAs was
performed as described above for the chromosome specific
libraries.
[0237] Digital Imaging
[0238] Images were obtained using a Zeiss Axioskop epifluorescence
microscope coupled to a cooled charge coupled device (CCD) camera
(Photometrics, Tuscon, Ariz., PM512). Camera control and digital
image acquisition (8-bit gray scale) employed an Apple Macintosh
IIx computer. Fluorophores were selectively imaged using filter
cubes specially prepared by Zeiss (Filter 487910 for fluorescein,
filter 487915 for rhodamine and filter 487901 for DAPI) to minimize
image offsets. The infrared filter (excitation 620-658 nm;
dichroic, 650 nm, bandpass, 670-680 nm) is not a precision filter.
Images taken using the latter filter were therefore slightly
shifted. These were digitally realigned with the probe signals as
reference.
[0239] Each set of three gray scale fluorescence images revealed
probe signals that appeared in only one, in two, or in all three of
the images (i.e., the seven combinatorial possibilities). Since the
probe-positive regions were visually distinct and were relatively
few in number, their combinatorial participation was readily
identifiable by visual inspection of the image groups. As a step
toward uniquely pseudocoloring these data regions on a
combinatorial basis, the regions were isolated and segregated into
seven separate (but still spatially aligned) gray scale subimages
by using interactive graphics software. Data regions were blended
(intensity was averaged) in those cases in which probe signals
appeared in more than one of the original images.
[0240] The visual identification and manual interactive segregation
of data regions was necessary due to limitations of currently
available graphics software.
[0241] The seven intermediate gray scale images were then
separately pseudocolored, a process that converts a gray scale to a
tint scale. The pseudocolored images were then recombined through a
simulated overlay. The multicolored composite image was
simultaneously merged with a DAPI counterstain image (also
pseudocolored) using software developed in our laboratory that
combines images by picking maximum signal intensity at each pixel
position. The digital imaging technique described above can be
implemented on a general purpose computer, e.g., an Apple Macintosh
IIx, using known image processing tools, as particularly arranged
and operated in accord with the above methodology. A preferred such
implementation, termed Gene Join, can be obtained from the Office
of Cooperative Research, Yale University, Suite 401, 246 Church
St., New Haven, Conn. 06510.
[0242] Photographs were taken with a Agfa matrix procolor slide
printer using Kodak 100 HC color slide film.
[0243] Results
[0244] Combinatorial Labeling of Probes by PCR
[0245] Chromosome-specific centromeric repeats and chromosome
specific DNA libraries are frequently used as probes for
fluorescence in situ hybridization (FISH) because of their utility
in revealing chromosome aneuploidy or aberrations in interphase
cells and tissues as well as the identification of marker
chromosomes unrecognizable by conventional banding methods
(Tkachuk, D. C. et al. (1991) GATA 8:67-74; Lichter, P. et al.
(1991) GATA 8:24-35). Since clones containing such sequences
generally have relatively small inserts, ranging in size from a few
hundred nucleotides to a few kilobase pairs, we first chose to
assess vector PCR as a general method for the combinatorial
labeling of such clones. Bio, dig, DNP and fluorescein, all
conjugated to dUTP, could be efficiently incorporated during the
amplification reaction, alone or in combination, resulting in
selective enrichment of labeled chromosome-specific sequences.
Several combinations of nucleotide analogs were tested in order to
establish the appropriate concentrations necessary to give an
approximately equimolar mixture of each reporter in the probe.
These nucleotide concentrations are listed in Table 1 and Table 2.
Alphoid DNA clones specific for chromosomes 3, 7, 8, 9, 11, 12 and
18 and chromosome-specific libraries for chromosome 1, 2, 4, 8, 14
and X were then labeled combinatorially by vector PCR. Each
combination with Bio, dig and DNP (or fluorescein-dUTP) singularly
tested by in situ hybridization and each combination gave
comparable signal intensities (data not shown).
[0246] Simultaneous Detection of Seven Centromere Repeat Probes
[0247] The chromosome-specific alphoid DNA clones and the modified
nucleotides used to label them are given in Table 5. The
biotinylated probes were detected with an infrared fluorophore
emitting at 680 nm (Ultralite 680) conjugated to streptavidin. The
dig-labeled probes were detected with anti-dig antibodies coupled
to rhodamine (630 nm emission) while the probes labeled with
fluorescein-11-dUTP (580 nm emission) were detected directly. A
separate gray scale image of each fluorophore was then acquired by
using the CCD camera system. As shown in FIG. 1(A-C), four pairs of
chromosome-specific hybridization signals are seen in each image,
as expected from the experimental design. Each of the source gray
scale images have been pseudocolored to highlight the hybridization
signals. One pair of signals appears uniquely on each of the images
(see arrowheads), reflecting those probes that were labeled with
only a single reporter. Two other signal pairs appear on two images
each, while the third appears on all three images (see arrows).
Thus each probe could be selectively identified by the fluorophore
image combination on which the hybridization signal was detected.
The gray scale signal regions from the images were segregated,
pseudocolored and merged with computer software as described. FIG.
1D shows this merged image. Each of the seven centromere probes are
seen as distinct colors on the DAPI (blue) counterstained metaphase
chromosomes. The probes could also be clearly distinguished after
hybridization to fixed human lymphocyte nuclei. FIG. 1E shows a
merged image of an interphase nucleus hybridized with a mixture of
the seven centromere probes.
[0248] Simultaneous Painting of Six Chromosomes. Chromosomal
Translocation
[0249] Chromosome painting is a powerful and general approach to
study chromosomal abnormalities. Here the probes are a complex
composite of sequences cloned in plasmid or phage vectors with
flow-sorted chromosomes used as the starting DNA source (Pinkel, D.
et al. (1988) Proc. Natl. Acad. Sci. USA 85:9139-9142; Cremer, T.
et al. (1988) Hum. Genet. 80:235-246). To demonstrate that
combinatorial labeling also could be used for whole chromosome
analysis, the libraries for chromosomes 1, 2 and 4 were singly
labeled with Bio, dig and DNP, while the libraries for chromosomes
8, 14 and X were labeled combinatorially (see Table 6). Each probe
set decorated a single chromosome pair when analyzed by FISH, with
signal intensities on each fluorophore channel being of similar
intensity (data not shown). The merged image (FIG. 1F) highlights
the six target chromosomes in different pseudocolor while the
remaining chromosomes exhibit the blue DAPI counterstain.
6TABLE 6 Labeling of Chromosome-Specific Libraries dNTP, .mu.M pBS2
pBS14 pBS1 pBS4 pBSX pBS8 Bio-11-dUTP 75 37.5 37.5 dig-11-dUTP 75
37.5 37.5 DNP-11-dUTP 37.5 37.5 37.5 TTP 225 225 262.5 225 225 225
dA, dC, dGTP 300 300 300 300 300 300
[0250] The PCR-generated libraries can also be used for detection
of chromosomal translocations as exemplified in FIG. 1G. Metaphase
spreads were obtained from lymphocytes of a healthy female donor
whose karyotype (Giemsa banding) was shown to be
46,XX,t(2;14)(q37;q22). The metaphase spreads were investigated in
order to clarify the origin of an identical translocation detected
in the fetus of the donor. Using PCR-generated libraries for
chromosome 2 (Bio) and chromosome 14 (dig), the reciprocal
character of the translocation could be clearly demonstrated (see
arrowheads).
[0251] Combinatorial Labeling and Gene Mapping
[0252] The feasibility of mapping multiple genes simultaneously by
using the combinatorial labeling paradigm is demonstrated by the
data presented in FIG. 1(H-J). Six different cosmid and phage
clones, previously mapped to chromosomes 5, 6, 8, 11, 21 and X in
independent experiments, were cohybridized and separate gray scale
fluorescence images collected and processed as described above. The
merged image on a DAPI-counterstained metaphase spread is shown in
FIG. 1H. The chromosomal location of each clone, as measured by
both fractional length measurements (Lichter, P. et al. (1990)
Science 247:64-69) and Alu-PCR hybridization banding (Baldini, A.
and Ward, D. C. (1991) Genomics 9:770-774), was identical to that
obtained before (data not shown). Six cosmid clones with known
locations on chromosome 5 were also hybridized simultaneously. FIG.
1I shows the distribution of these clones on both chromosome 5
homologs in a metaphase spread while FIG. 1J demonstrates that the
relative order of the clones (i.e., the pattern of colors) is
maintained in the interphase nuclei of a T lymphocyte. It should be
noted that many of the signals appear as doublets, reflecting the
fact that these sequences have already undergone DNA replication in
this nucleus. Conversion of a probe signal from a singlet to a
doublet can be used to monitor the replication timing of DNA
segments during S phase.
[0253] Discussion
[0254] A procedure that permits the analysis of up to seven probes
simultaneously. Combinatorially labeled probes can be produced
rapidly and reproducibly by either nick translation or PCR
amplification. However, the latter approach is particularly
attractive for labeling clones with relatively small inserts (about
6 kilobases or less) since vector-derived PCR primers permit
selective amplification of insert DNA sequences with high
efficiency. For example, with the alphoid DNA clones, a typical 50
p1 amplification reaction mixture yields sufficient labeled probe
for about 250 1n situ hybridizations. Not surprisingly, the yield
for the chromosome library clone pool is lower; nevertheless, 100
ng of template gave enough amplification products to hybridize 10
slides. Reamplification of the primary PCR product pool could also
be done without any detectable loss of probe complexity (data not
shown). In contrast, using nick-translated plasmid libraries, 200
ng of DNA was required per slide. The negligible amount of labeled
vector sequences in the PCR products also reduces the potential for
vector sequence cross hybridization, a problem which was described
by Nederlof et al. (1990) Cytometry 10:20-27.
[0255] The digital imaging capabilities of the cooled CCD camera
and the computer software for pseudocoloring and merging signals
from combinatorially labeled probes will play an important role in
extending the number of simultaneously detectable probes beyond the
seven reported here. The CCD camera is sensitive to light over a
broad spectrum range. Infrared dyes, such as Ultralite 680, which
are not visible by eye, can be imaged quite readily by the CCD
camera. A series of fluorophores emitting in the 650 to 900 nm
range, have recently been reported (Ernst, L. A. et al. (1989)
Cytometry 10:3-10); this should increase the number of different
fluorophores that can be used combinatorially for probe
identification. Furthermore the infrared dyes, such as Ultralite
680, offer certain advantages over the blue fluorophores, AMCA or
Cascade Blue: i) sample autofluorescence is minimal at the longer
wavelength, ii) DAPI counterstaining of metaphase chromosomes and
interphase nuclei is possible (the emission of DAPI, AMCA and
Cascade Blue overlap) and iii) the observed bleed-through of
rhodamine signals with the DAPI filter when imaging AMCA
fluorescence is more severe than the bleed-through of rhodamine
signals using the infrared filter.
[0256] Digital imaging of combinatorially labeled probes also
circumvents a universally thorny problem in multicolor analysis,
that of precise image registration. When filter cubes are moved to
collect the fluorescence emission of a single fluorophore, optical
imperfections or mechanical motion may cause image displacement
relative to each other; these registration offsets can be as large
as 1 .mu.m. This is extremely problematic when spatial
relationships between signals are critical, such as in gene
mapping. However, when multiple probes, combinatorially labeled,
are cohybridized, signals from these probes appear on two or more
of the separate fluorophore images, thus providing internal
reference points for image registration. Provided that one
hybridization signal set is directly tied to the complete image of
a metaphase spread or interphase nucleus, i.e., by using a dual
bandpass filter (Johnson, C. V. et al. (1991) GATA B:75), all
images can be aligned, irrespective of the number of separate
images to be merged. Fluorescence in situ hybridization is becoming
an increasingly powerful experimental tool, both for basic research
and for clinical applications. The ability to visualize multiple
probes simultaneously should streamline the screening of specimens
for chromosomal aneuploidies and/or chromosomal rearrangements.
This is of particular importance in cases where clinical samples
are limited in number. In addition, by incorporating one or more
appropriate reference clones (e.g., centromere repeats or unique
sequence genes) in the experimental protocol, the assessment of
gene dosage (loss of heterozygosity, aneuploidy and mosaics) or
defining boundaries of chromosomal deletions should be more
definitive and require less statistical analysis. The generation of
physical mapping data, using either metaphase or interphase mapping
strategies should be facilitated with combinatorial fluorescence as
would studies focused on understanding the intranuclear topography
of genes and chromosomes. It should be stressed that the assessment
of the chromosomal map positions of several combinatorially labeled
clones does not necessarily require the pseudocoloring and merging
procedures. Displaying the signals separately as gray scale images,
as shown in FIG. 1(A-C), allows the physical ordering of probes,
since combinatorially labeled clones appear in several gray scale
images and can thus be identified. Manual segregation of the images
is time-consuming, which in its present format reduces the rate at
which clones can be mapped. This limitation can be eliminated by
software to automate this step.
[0257] The use of commercially available nucleotide analogs
conjugated to fluorescein is of particular value for clinical
applications since it circumvents time-consuming and sometime
troublesome immunological steps required to visualize haptenized
probes. In addition, this results in an improved signal/noise
ratio, which could enhance overall detection sensitivity,
especially if a cooled CCD camera were used for imaging. It can be
expected that other nucleotides with additional conjugated
fluorophores will be available soon, which would both simplify and
expand the combinatorial labeling strategy for multicolor
hybridization assays even more.
[0258] Equivalents
[0259] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
specifically herein. Such equivalents are intended to be
encompassed in the scope of the following claims.
* * * * *