U.S. patent application number 10/330089 was filed with the patent office on 2003-06-12 for methods and compositions for chromosome-specific staining.
Invention is credited to Gray, Joe W., Pinkel, Daniel.
Application Number | 20030108943 10/330089 |
Document ID | / |
Family ID | 27124357 |
Filed Date | 2003-06-12 |
United States Patent
Application |
20030108943 |
Kind Code |
A1 |
Gray, Joe W. ; et
al. |
June 12, 2003 |
Methods and compositions for chromosome-specific staining
Abstract
Methods and compositions for chromosome-specific staining are
provided. Compositions comprise heterogenous mixtures of labeled
nucleic acid fragments having substantially complementary base
sequences to unique sequence regions of the chromosomal DNA for
which their associated staining reagent is specific. Methods
include methods for making the chromosome-specific staining
compositions of the invention, and methods for applying the
staining compositions to chromosomes.
Inventors: |
Gray, Joe W.; (Livermore,
CA) ; Pinkel, Daniel; (Walnut Creek, CA) |
Correspondence
Address: |
BURNS, DOANE, SWECKER & MATHIS, L.L.P.
P.O. Box 1404
Alexandria
VA
22313-1404
US
|
Family ID: |
27124357 |
Appl. No.: |
10/330089 |
Filed: |
December 30, 2002 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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10330089 |
Dec 30, 2002 |
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08472312 |
Jun 7, 1995 |
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6500612 |
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08472312 |
Jun 7, 1995 |
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07627707 |
Dec 14, 1990 |
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5447841 |
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07627707 |
Dec 14, 1990 |
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06937793 |
Dec 4, 1986 |
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06937793 |
Dec 4, 1986 |
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06819314 |
Jan 16, 1986 |
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Current U.S.
Class: |
435/6.11 ;
536/24.3 |
Current CPC
Class: |
C12Q 1/6886 20130101;
C12Q 2525/151 20130101; C12Q 2563/131 20130101; C12Q 2525/151
20130101; C12Q 2525/186 20130101; C12Q 2563/107 20130101; C12Q
2525/151 20130101; C12Q 2600/156 20130101; C12Q 2525/186 20130101;
C12Q 1/6832 20130101; C12Q 1/6879 20130101; Y10S 435/805 20130101;
Y10S 435/82 20130101; C12Q 1/6811 20130101; C12Q 1/6841 20130101;
C12Q 1/6841 20130101; C12Q 1/6832 20130101; C12Q 1/6853 20130101;
C12Q 1/6841 20130101 |
Class at
Publication: |
435/6 ;
536/24.3 |
International
Class: |
C12Q 001/68; C07H
021/04 |
Goverment Interests
[0002] The United States Government has rights in this invention
pursuant to Contract No. W-7405-ENG-48 between the U.S. Department
of Energy and the University of California, for the operation of
Lawrence Livermore National Laboratory.
Claims
1. A chromosome-specific staining reagent comprising a
heterogeneous mixture of labeled nucleic acid fragments, the
labeled nucleic acid fragments being derived from the same
chromosome type, and being substantially free of repetitive nucleic
acid sequences having hybridization capacity.
2. The chromosome-specific staining reagent according to claim 1
wherein said chromosome type is an abnormal human chromosome
type.
3. The chromosome-specific staining-reagent according to claim 1
wherein said chromosome type is selected from the group consisting
of normal human chromosomes 1 through 22, X, and Y.
4. The chromosome-specific staining reagent of claim 3 wherein said
heterogeneous mixture comprises labeled nucleic acid fragments
derived from substantially equal amounts of between about 10-1000
distinct cloned inserts.
5. The chromosome-specific staining reagent of claim 4 wherein said
labeled nucleic acid fragments are derived from substantially equal
amounts of between about 10-1000 distinct cloned inserts each
having a length within the range of between about 20-45
kilobases.
6. The chromosome-specific staining reagent of claim 5 wherein said
distinct cloned inserts form said labeled nucleic acid fragments,
each fragment having a length within the range of between about
50-500 bases.
7. The chromosome-specific staining reagent of claim 6 wherein said
labeled nucleic acid fragments are single stranded.
8. The chromosome-specific staining reagent of claim 7 wherein said
labeled nucleic acid fragments are biotinylated or modified with
N-acetoxy-N-2-acetylamino-fluorene.
9. The chromosome-specific staining reagent of claim 4 wherein said
labeled nucleic acid fragments are derived from substantially equal
amounts of between about 100-400 distinct cloned unique sequence
inserts.
10. The chromosome-specific staining reagent of claim 9 wherein
said distinct cloned inserts form said labeled nucleic acid
fragments, each fragment having a length within the range of
between about 50-5000 bases.
11. The chromosome-specific staining reagent of claim 10 wherein
said distinct cloned inserts form said labeled nucleic acid
fragments, each fragment having a length within the range of
between about 50-500 bases.
12. The chromosome-specific staining reagent of claim 11 wherein
said labeled nucleic acid fragments are single stranded.
13. The chromosome-specific staining reagent of claim 12 wherein
said labeled nucleic acid fragments are biotinylated or modified
with N-acetoxy-N-2-acetylamino-fluorene.
14. A chromosome-specific staining reagent produced by the process
of: isolating chromosome-specific DNA; cloning pieces of the
isolated chromosome-specific DNA; disabling the hybridization
capacity of repeated sequences contained in the cloned pieces of
the isolated chromosome-specific DNA to form a collection of
nucleic acid fragments which hybridize to unique chromosomal DNA
sequences; labeling the nucleic acid fragments of the collection to
form a heterogeneous mixture of nucleic acid fragments.
15. The chromosome-specific staining reagent of claim 14, wherein
said step of disabling said hybridization capacity includes
selecting cloned pieces of said isolated chromosome-specific DNA
which have substantially unique base sequences.
16. The chromosome-specific staining reagent of claim 14 wherein
said step of disabling said hybridization capacity includes
prereassociating said cloned pieces of said isolated
chromosome-specific DNA with unlabeled repetitive sequence nucleic
acid fragments.
17. A method of staining chromosomal DNA of a particular chromosome
type or portion thereof, or a particular group of chromosome types,
the method comprising the steps of: providing a heterogeneous
mixture of labeled nucleic acid fragments, substantial portions of
each labeled nucleic acid fragment in the heterogeneous mixture
having base sequences substantially complementary to base sequences
of the chromosomal DNA; and reacting the heterogeneous mixture with
the chromosomal DNA by in situ hybridization.
Description
RELATED APPLICATION
[0001] This application is a continuation in part of U.S. Ser. No.
819,314, filed Jan. 16, 1986 by the named inventors hereof and
assigned to the same assignee and claims priority in said prior
filed application.
BACKGROUND OF THE INVENTION
[0003] The invention relates generally to the field of
cytogenetics, and more particularly, to methods for identifying and
classifying chromosomes.
[0004] Chromosome abnormalities are associated with genetic
disorders, degenerative diseases, and exposure to agents known to
cause degenerative diseases, particularly cancer, German, "Studying
Human Chromosomes Today," American Scientist, Vol. 58, pgs. 182-201
(1970); Yunis, "The Chromosomal Basis of Human Neoplasia," Science,
Vol. 221, pgs. 227-236 (1983); and German, "Clinical Implication of
Chromosome Breakage," in Genetic Damage in Man Caused by
Environmental Agents, Berg, Ed., pgs. 65-86 (Academic Press, New
York, 1979). Chromosomal abnormalities can be of three general
types: extra or missing individual chromosomes, extra or missing
portions of a chromosome, or chromosomal rearrangements. The third
category includes translocations (transfer of a piece from one
chromosome onto another chromosome), and inversions (reversal in
polarity of a chromosomal segment).
[0005] Detectable chromosomal abnormalities occur with a frequency
of one in every 250 human births. Abnormalities that involve
deletions or additions of chromosomal material alter the gene
balance of an organism and generally lead to fetal death or to
serious mental physical defects. Down's syndrome is caused by
having three copies of chromosome 21 instead of the normal 2. This
syndrome is an example of a condition caused by abnormal chromosome
number, or aneuploidy. Chronic myelogeneous leukemia is associated
with the exchange of chromosomal material between chromosome 9 and
chromosome 22. The transfer of chromosomal material in this
leukemia is an example of a translocation. Clearly, a sensitive
method for detecting chromosomal abnormalities would be a highly
useful tool for genetic screening.
[0006] Measures of chromosome breakage and other aberrations caused
by ionizing radiation or chemical mutagens are widely used as
quantitative indicators of genetic damage caused by such agents,
Biochemical Indicators of Radiation Injury in Man (International
Atomic Energy Agency, Vienna, 1971); and Berg, Ed. Genetic Damage
in Man Caused by Environmental Agents (Academic Press, New York,
1979). A host of potentially carcinogenic and teratogenic chemicals
are widely distributed in the environment because of industrial and
agricultural activity. These chemicals include pesticides, and a
range of industrial wastes and by-products, such as halogenated
hydrocarbons, vinyl chloride, benzene, arsenic, and the like,
Kraybill et al., Eds., Environmental Cancer (Hemisphere Publishing
Corporation, New York, 1977). Sensitive measures of chromosomal
breaks and other abnormalities could form the basis of improved
dosimetric and risk assessment methodologies for evaluating the
consequences of exposure to such occupational and environmental
agents.
[0007] Current procedures for genetic screening and biological
dosimetry involve the analysis of karyotypes. A karyotype is a
collection of indices which characterize the state of an organism's
chromosomal complement. It includes such things as total chromosome
number, copy number of individual chromosome types (e.g., the
number of copies of chromosome X), and chromosomal morphology,
e.g., as measured by length, centromeric index, connectedness, or
the like. Chromosomal abnormalities can be detected by examination
of karyotypes. Karyotypes are determined by staining an organism's
metaphase, or condensed, chromosomes. Metaphase chromosomes are
used because, until recently, it has not been possible to visualize
nonmetaphase, or interphase chromosomes due to their dispersed
condition in the cell nucleus.
[0008] The metaphase chromosomes can be stained by a number of
cytological techniques to reveal a longitudinal segmentation into
entities generally referred to as bands. The banding pattern of
each chromosome within an organism is unique, permitting
unambiguous chromosome identification regardless of morphological
similarity, Latt, "Optical Studies of Metaphase Chromosome
Organization," Annual Review of Biophysics and Bioengineering, Vol.
5, pgs. 1-37 (1976). Adequate karyotyping for detecting some
important chromosomal abnormalities, such as translocations and
inversions requires banding analysis. Unfortunately, such analysis
requires cell culturing and preparation of high quality metaphase
spreads, which is extremely difficult and time consuming, and
almost impossible for tumor cells.
[0009] The sensitivity and resolving power of current methods of
karyotyping, are limited by the lack of stains that can readily
distinguish different chromosomes having highly similar staining
characteristics because of similarities in such gross features as
size, morphology, and/or DNA base composition.
[0010] In recent years rapid advances have taken place in the study
of chromosome structure and its relation to genetic content and DNA
composition. In part, the progress has come in the form of improved
methods of gene mapping based on the availability of large
quantities of pure DNA and RNA fragments for probes produced by
genetic engineering techniques, e.g., Kao, "Somatic Cell Genetics
and Gene Mappings," International Review of Cytology, Vol. 85, pgs.
109-146 (1983), and D'Eustachio et al., "Somatic Cell Genetics in
Gene Families," Science, Vol. 220, pgs. 9, 19-924 (1983). The
probes for gene mapping comprise labeled fragments of single
stranded or double stranded DNA or RNA which are hybridized to
complementary sites on chromosomal DNA. The following references
are representative of studies utilizing gene probes for mapping:
Harper et al. "Localization of the Human Insulin Gene to the Distal
End of the Snort Arm of Chromosome 11," Proc. Natl. Acad. Sci.,
Vol. 78, pgs. 4458-4460; Kao et al., "Assignment of the Structural
Gene Coding for Albumin to Chromosome 4," Human Genetics, Vol. 62,
pgs. 337-341 (1982); Willard et al., "Isolation and
Characterization of a Major Tandem Repeat Family from the Human X
Chromosome," Nucleic Acids Research, Vol. 11, pgs. 2017-2033
(1983); and Falkow et al., U.S. Pat. No. 4,358,535, issued Nov. 9,
1982, entitled "Specific DNA Probes in Diagnostic Microbiology."
The hybridization process involves unraveling, or melting, the
double stranded nucleic acids by heating, or other means. This step
in the hybridization process is sometimes referred to as denaturing
the nucleic acid. When the mixture of probe and target nucleic
acids cool, strands having complementary bases recombine, or
anneal. When a probe anneals with a target nucleic acid, the
probe's location on the target can be detected by a label carried
by the probe. When the target nucleic acid remains in its natural
biological setting, e.g., DNA in chromosomes or cell nuclei (albeit
fixed or altered by preparative techniques) the hybridization
process is referred as in situ hybridization.
[0011] Use of hybridization probes has been limited to identifying
the location of genes or known DNA sequences on chromosomes. To
this end it has been crucially important to produce pure, or
homogeneous, probes to minimize hybridizations at locations other
than at the site of interest, Henderson, "Cytological Hybridization
to Mammalian Chromosomes," International Review of Cytology, Vol.
76, pgs. 1-46 (1982).
[0012] Manuelidis et al., in "Chromosomal and Nuclear Distribution
of the Hind III 1.9-KB Human DNA Repeat Segment," Chromosoma, Vol.
91, pgs. 28-38 (1984), disclose the construction of a single kind
of DNA probe for detecting multiple loci on chromosomes
corresponding to members of a family of repeated DNA sequences.
[0013] Wallace et al., in "The Use of Synthetic Oligonucleotides as
Hybridization Probes. II. Hybridization of Oligonucleotides of
Mixed Sequence to Rabbit Beta-Globin DNA, "Nucleic Acids Research,
Vol. 9, pgs. 879-894 (1981), disclose the construction of synthetic
oligonucleotide probes having mixed base sequences for detecting a
single locus corresponding to a structural gene. The mixture of
base sequences was determined by considering all possible
nucleotide sequences which could code for a selected sequence of
amino acids in the protein to which the structural gene
corresponded.
[0014] Olsen et al., in "Isolation of Unique Sequence Human X
Chromosomal Deoxyribonucleic Acid," Biochemistry, Vol. 19, pgs.
2419-2428 (1980), disclose a method for isolating labeled unique
sequence human X chromosomal DNA by successive hybridizations:
first, total genomic human DNA against itself so that a unique
sequence DNA fraction can be isolated; second, the isolated unique
sequence human DNA fraction against mouse DNA so that homologous
mouse/human sequences are removed; and finally, the unique sequence
human DNA not homologous to mouse against the total genomic DNA of
a human/mouse hybrid whose only human chromosome is chromosome X,
so that a fraction of unique sequence X chromosomal DNA is
isolated.
SUMMARY OF THE INVENTION
[0015] The invention includes methods and compositions for staining
chromosomes. In particular, chromosome specific staining reagents
are provided which comprise heterogeneous mixtures of labeled
nucleic acid fragments having substantial portions of substantially
complementary base sequences to the chromosomal DNA for which
specific staining is desired. The nucleic acid fragments of the
heterogenous mixtures include double stranded or single stranded
RNA or DNA. Heterogeneous in reference to the mixture of labeled
nucleic acid fragments means that the staining reagents comprise
many copies each of fragments having different base compositions
and/or sizes, such that application of the staining reagent to a
chromosome results in a substantially uniform distribution of
fragments hybridized to the chromosomal DNA.
[0016] "Substantial proportions" in reference to the basic
sequences of nucleic acid fragments that are complementary to
chromosomal DNA means that the complementarity is extensive enough
so that the fragments form stable hybrids with the chromosomal DNA
under standard hybridization conditions for the size and complexity
of the fragment. In particular, the term comprehends the situation
where the nucleic acid fragments of the heterogeneous mixture-
possess regions having non-complementary base sequences.
[0017] As discussed more fully below, preferably the heterogeneous
mixtures are substantially free from so-called repetitive
sequences, both the tandem variety and the interspersed variety
(see Hood et al., Molecular Biology of Eucaryotic Cells
(Benjamin/Cummings Publishing Company, Menlo Park, Calif., 1975)
for an explanation of repetitive sequences). Tandem repeats are so
named because they are clustered or contiguous on the DNA molecule
which forms the backbone of a chromosome. Members of this class of
repeats are also associated with well-defined regions of the
chromosome, e.g., the centromeric region. Thus, if these repeats
form a sizable fraction of a chromosome, and are removed from the
heterogeneous mixture of fragments employed in the invention,
perfect uniformity of staining may not be possible. This situation
is comprehended by the use of the term "substantially uniform" in
reference to the heterogeneous mixture of labeled nucleic acid
fragments of the invention.
[0018] It is desirable to disable the hybridization capacity of
repetitive sequences because copies occur on all the chromosomes of
a particular organism; thus, their presence reduces the chromosome
specificity of the staining reagents of the invention. As discussed
more fully below, hybridization capacity can be disabled in several
ways, e.g., selective removal or screening of repetitive sequences
from chromosome specific DNA, selective blocking of repetitive
sequences by pre-reassociation with complementary fragments, or the
like.
[0019] Preferably, the staining reagents of the invention are
applied to interphase or metaphase chromosomal DNA by in situ
hybridization, and the chromosomes are identified or classified,
i.e., karyotyped, by detecting the presence of the label on the
nucleic acid fragments comprising the staining reagent.
[0020] The invention includes chromosome staining reagents for the
total genomic complement of chromosomes, staining reagents specific
to single chromosomes, staining reagents specific to subsets of
chromosomes, and staining reagents specific to subregions within a
single chromosome. The term "chromosome-specific," is understood to
encompass all of these embodiments of the staining reagents of the
invention. The term is also understood to encompass staining
reagents made from both normal and abnormal chromosome types.
[0021] A preferred method of making the staining reagents of the
invention includes isolating chromosome-specific DNA, cloning
fragments of the isolated chromosome-specific DNA to form a
heterogeneous mixture of nucleic acid fragments, disabling the
hybridization capacity of repeated sequences in the nucleic acid
fragments, and labeling the nucleic acid fragments to form a
heterogeneous mixture of labeled nucleic acid fragments. As
described more fully below, the ordering of the steps for
particular embodiments varies according to the particular means
adopted for carrying out the steps.
[0022] The preferred method of isolating chromosome-specific DNA
for cloning includes isolating specific chromosome types by
fluorescence-activated sorting.
[0023] The present invention addresses problems associated with
karyotyping chromosomes, especially for diagnostic and dosimetric
applications. In particular, the invention overcomes problems which
arise because of the lack of stains that are sufficiently
chromosome-specific by providing reagents comprising heterogeneous
mixtures of labeled nucleic acid fragments that can be hybridized
to the DNA of specific chromosomes, specific subsets of
chromosomes, or specific subregions of specific chromosomes. The
staining technique of the invention opens up the possibility of
rapid and highly sensitive detection of chromosomal abnormalities
in both metaphase and interphase cells using standard clinical and
laboratory equipment. It has direct application in genetic
screening, cancer diagnosis, and biological dosimetry.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIG. 1a is a binary image of a DAPI stained human metaphase
spread.
[0025] FIG. 1b is a binary image of fluorescein staining of the
same human metaphase spread as FIG. 1a.
[0026] FIG. 1c is a binary image of the chromosome 21s alone,
nonspecifically stained objects having been removed by standard
image processing techniques.
DETAILED DESCRIPTION OF THE INVENTION
[0027] The present invention includes compositions for staining
individual chromosome types and methods for making and using the
compositions. The compositions comprise heterogeneous mixtures of
labeled nucleic acid fragments. The individual labeled nucleic acid
fragments making up the heterogeneous mixture are essentially
standard hybridization probes. That is, each chromosome-specific
staining composition of the invention can be viewed as a large
collection of hybridization probes to unique sequence regions of a
specific chromosome. In fact, the preferred method of making the
compositions of the invention entails generating a heterogeneous
mixture on a fragment-by-fragment basis by isolating, cloning
chromosomal DNA, and selecting from the clones hybridization probes
to unique sequence regions of a particular chromosome. The precise
number of distinct labeled nucleic acid fragments, or probes,
comprising a heterogeneous mixture is not a critical feature of the
invention. However, in particular applications, a trade-off may
have to be established between the number of distinct fragments in
a heterogeneous mixture and the degree of nonspecific background
staining: Where the tendency for nonspecific background staining is
high, giving rise to low signal to noise ratios, it may be
necessary to reduce the number of distinct fragments comprising the
heterogeneous mixture. On the other hand, where nonspecific
background staining is low, the number of distinct fragments may be
increased. Preferably, the numbers of distinct fragments in a
heterogeneous mixture is as high as possible (subject to acceptable
signal to noise ratios) so that staining appears continuous over
the body of the chromosomes.
[0028] Another constraint on the number of distinct fragments in
the heterogeneous mixture is solubility. Upper bounds exist with
respect to the fragment concentration, i.e., unit length of nucleic
acid per unit volume, that can be maintained in solution. Thus, if
fragments of a given average length are used at a given
concentration (fragment per volume), then the number of different
such fragments that can comprise the heterogeneous mixture is
limited.
[0029] In one preferred embodiment where the heterogeneous mixture
is generated on a fragment-by-fragment basis, the chromosomal DNA
is initially cloned in long sequences, e.g., 35-45 kilobases in
cosmids, or like vector. After amplification the inserts are cut
into smaller fragments and labeled for formation to a heterogeneous
mixture. In this embodiment, the chromosomal binding sites of the
fragments are clustered in the chromosomal regions complementary to
the cloned "parent" nucleic acid sequence. Fluorescent signals from
Such clusters are more readily detected than signals from an
equivalent amount of label dispersed over the entire chromosome. In
this embodiment, the clusters are substantially uniformly
distributed over the chromosome.
[0030] Repetitive sequences, repeated sequences, and repeats are
used interchangeably throughout.
[0031] I. Isolation of Chromosome-Specific DNA and Formation of DNA
Fragment Libraries.
[0032] The first step in the preferred method of making the
compositions of the invention is isolating chromosome-specific DNA.
This step includes first isolating a sufficient quantity of the
particular chromosome type to which the staining composition is
directed, then extracting the DNA from the isolated chromosomes.
Here "sufficient quantity" means sufficient for carrying out
subsequent steps of the method. Preferably, the extracted DNA is
used to create a chromosome-specific library of DNA inserts which
can be cloned using standard genetic engineering techniques. The
cloned inserts are then isolated and treated to disable the
hybridization capacity of repeated sequences. In this case,
"sufficient quantity" means enough for the particular method used
in constructing the DNA insert library.
[0033] Several methods are available for isolating particular
chromosome types. For example, a technique for isolating human
chromosome types involves forming hybrid cell lines from human
cells and rodent cells, particularly mouse or hamster cells, e.g.,
see Kao, "Somatic Cell Genetics and Gene Mapping," International
Review of Cytology. Vol. 85, pgs. 109-146 (1983), for a review.
Human chromosomes are preferentially lost by the hybrid cells so
that hybrid cell lines containing a full complement of rodent
chromosomes and a single human chromosome can be selected and
propagated, e.g., Gusella et al., "Isolation and Localization of
DNA Segments from Specific Human Chromosomes," Proc. Natl. Acad.
Sci., Vol. 77, pgs. 2829-2833 (1980). Chromosome specific DNA can
then be isolated by techniques disclosed by Schmeckpeper et al.,
"Partial Purification and Characterization of DNA from Human X
Chromosome," Proc. Natl. Acad. Sci., Vol. 76, pgs. 6525-6528
(1979); or Olsen et al., "Isolation of Unique Sequence Human X
Chromosomal Deoxyribonucleic Acid," Biochemistry, Vol. 19, pgs.
2419-2428 (1980). Accordingly, these references are incorporated by
reference. Briefly, sheared total human DNA is hybridized against
itself on hydroxyapatite under conditions that allow elution of
substantially pure unique sequence total human DNA from the
hydroxyapatite. The unique sequence total human DNA is then
reassociated and nick translated to add a label (see Maniatis et
al., Molecular Cloning A Laboratory Manual, Cold Spring Harbor
Laboratory, 1982, pgs. 109-112, for a description of the nick
translation technique to add radioactive labels; and Brigato et
al., "Detection of Viral Genomes in Cultured Cells and
Paraffin-Embedded Tissue Sections Using Biotin-Labeled
Hybridization Probes," Virology, Vol. 126, pgs. 32-50 (1982) for a
description of the nick translation technique to add a biotin
label). Poly(dA) tails are then synthesized to the 3' hydroxyl
termini of the unique sequence DNA by incubating with terminal
transferase under appropriate conditions. The poly(dA) unique human
DNA is then hybridized against rodent DNA to remove homologous
sequences. The nonhomologous poly(dA) unique human DNA is then
hybridized against the total DNA of the human/rodent hybrid on
hydroxyapatite; double stranded DNA is isolated. (This is the
chromosome-specific unique sequence human DNA.) The poly(dA) tail
is used to separate the labeled unique sequence DNA from the
unlabeled unique sequences by binding the poly(dA) tails to
oligo(dT) cellulose.
[0034] The preferred means for isolating particular chromosome
types is by direct flow sorting of metaphase chromosomes with or
without the use of interspecific hybrid cell systems. Direct
sorting is preferred because there is considerable DNA sequence
homology between rodent and human DNA, which necessitates
additional hybridization steps (e.g., see Olsen et al., cited
above), and the hybrid cell lines tend to be unstable with respect
to retention of the human chromosomes.
[0035] For some species, every chromosome can be isolated by
currently available sorting techniques. Most, but not all, human
chromosomes are currently isolatable by flow sorting, Carrano et
al., "Measurement and Purification of Human Chromosomes by Flow
Cytometry and Sorting," Proc. Natl. Acad. Sci., Vol. 76, pgs.
1382-1384 (1979). Thus, for isolation of some human chromosomes,
use of the human/rodent hybrid cell system may be necessary.
Chromosome sorting can be done by commercially available
fluorescence-activated sorting machines, e.g., Becton Dickinson
FACS-II or like instrument.
[0036] DNA is extracted from the isolated chromosomes by standard
techniques, e.g., Marmur, "A Procedure for the Isolation of
Deoxyribonucleic Acid from Micro-Organisms," J. Mol. Biol., Vol. 3,
pgs. 208-218 (1961); or Maniatis et al., Molecular Cloning: A
Laboratory Manual (Cold Spring Harbor Laboratory, 1982) pgs.
280-281. These references are incorporated by reference for their
descriptions of DNA isolation techniques.
[0037] Generation of insert libraries from the isolated
chromosome-specific DNA is carried out using standard genetic
engineering techniques, e.g., Davies et al., "Cloning of a
Representative Genomic Library of the Human X Chromosome After
Sorting by Flow Cytometry," Nature, Vol. 293, pgs. 374-376 (1981);
Krumlauf et al., "Construction and Characterization of Genomic
Libraries from Specific Human Chromosomes," Proc. Natl. Acad. Sci.,
Vol. 79, pgs. 2971-2975 (1982); Lawn et al., "The Isolation and
Characterization of Linked Delta-and-Beta-Globin Genes from a
Cloned Library of Human DNA." Cell, Vol. 15, pgs. 1157-1174 (1978);
and Maniatis et al., "Molecular Cloning: A Laboratory Manual,"
(Cold Springs Harbor Laboratory, 1982), pgs. 256-308, the cited
pages of Maniatis et al. and Lawn et al. being incorporated by
reference.
[0038] In some cases, it is preferable that the nucleic acid
fragments of the heterogeneous mixture consist of single-stranded
RNA or DNA. The binding efficiency of single stranded nucleic acid
probes has been found to be higher during in situ hybridization,
e.g., Cox et al., "Detection of mRNAs in Sea Urchin Embryos by In
Situ Hybridization Using Asymmetric RNA Probes," Developmental
Biology, Vol. 101, pgs. 485-502 (1984). Standard methods are used
to generate RNA fragments from isolated DNA fragments. For example,
a method developed by Green et al., described in Cell, Vol. 32,
pgs. 681-694 (1983), is commercially available from Promega Biotec
(Madison, Wis.) under the tradename "Riboprobe." Other
transcription kits suitable for use with the present invention are
available from United States Biochemical Corporation (Cleveland,
Ohio) under the tradename "Genescribe." Single stranded DNA probes
can be produced with the single stranded bacteriophage M13, also
available in kit form, e.g. Bethesda Research Laboratories
(Gaithersburg, Md.).
[0039] II. Disabling the Hybridization Capacity of Repetitive
Sequences.
[0040] As mentioned above, it is desirable to disable the
hybridization capacity of repetitive sequences by removal, block,
or like means. Repetitive sequences are distributed throughout the
genome; most are not chromosome-specific. Consequently, in spite of
the fact that the nucleic acid fragments of the invention are
derived from isolated chromosomes, the presence of repeats greatly
reduces the degree of chromosome-specificity of the staining
reagents of the invention, particularly in genomes containing a
significant fraction of repetitive sequences, such as the human
genome.
[0041] Several techniques are available for disabling the
hybridization capacity of repetitive sequences. Highly repetitive
DNA sequences can be removed from the extracted chromosome-specific
DNA by denaturing and incubating the extracted DNA against itself
or against repetitive sequence-enriched total genomic DNA on
hydroxyapatite, or like absorbent, at low C.sub.ot values, followed
by fractionation of double stranded DNA from single stranded
DNA.
[0042] Hydroxyapatite chromatography is a standard technique for
fractionating DNA on the basis of reassociation conditions such as
temperature, salt concentration, or the like. It is also useful for
fractionating DNA on the basis of reassociation rate at fixed
reassociative conditions, or stringencies. Materials for
hydroxyapatite chromatography are available commercially, e.g.,
Bio-Rad Laboratories (Richmond, Calif.).
[0043] Fractionation based on resistance to S.sub.1 nuclease can
also be used to separate single stranded from double stranded DNA
after incubation to particular C.sub.ot values. See Britten et al.
"Analysis of Repeating DNA Sequences by Reassociation," in Methods
in Enzymology, Vol. 29, pgs. 363-418 (1974), for an explanation of
C.sub.ot values. Preferably, this initial reassociation step is
carried out after the extracted chromosome-specific DNA has been
broken into pieces and amplified by cloning.
[0044] One embodiment of the invention is obtained by labeling the
fragments of the single stranded fraction eluted from the
hydroxyapatite in the initial reassociation step. The particular
C.sub.ot values required to separate middle repetitive and highly
repetitive sequences from the chromosome-specific DNA may vary from
species to species because of inter-specific differences in the
fraction of the genomic DNA comprising middle and highly repetitive
sequences. Most preferably in this embodiment, the initial
reassociation step is to a C.sub.ot value in the range of about a
few hundred to a few thousand.
[0045] In addition to self hybridization or hybridization against
repetitive-sequence-enriched total genomic DNA, removal of repeats
from fragment mixtures can also be accomplished by hybridization
against immobilized high molecular weight total genomic DNA,
following a procedure described by Brison et al., "General Method
for Cloning Amplified DNA by Differential Screening with Generic
Probes," Molecular and Cellular Biology, Vol. 2, pgs. 578-587
(1982). Accordingly, this reference is incorporated by reference.
Briefly, the procedure removed repeats from fragment mixtures in
the size range of a few tens of bases to a few hundred bases.
Minimally sheared total genomic DNA is bound to diazonium
cellulose, or like support. The fragment mixture is then hybridized
against the immobilized DNA to C.sub.ot values in the range of
about 1 to 100. The preferred stringency of the hybridization
conditions may vary depending on the base composition of the
DNA.
[0046] The preferred means for disabling hybridization capacity is
selecting unique sequence nucleic acid inserts from a
chromosome-specific DNA library. For example, following Benton and
Davis, "Screening Lambda gt Recombinant Clones by Hybridization to
Single Plaques in situ," Science, Vol. 196, pgs. 180-182 (1977),
pieces of chromosome-specific DNA are inserted into lambda gt
bacteriophage or like vector. The phages are plated on agar plates
containing a suitable host bacteria. DNA from the resulting phage
plaques is then transferred to a nitrocellulose filter by
contacting the filter to the agar plate. The filter is then treated
with labeled repetitive DNA so that phage plaques containing
repetitive sequence DNA can be identified. Those plaques that do
not correspond to labeled spots on the nitrocellulose filter
comprise clones which may contain unique sequence DNA. Clones from
these plaques are selected and amplified, radioactively labeled,
and hybridized to Southern blots of genomic DNA which has been
digested with the same enzyme used to generate the inserted
chromosome-specific DNA. Clones carrying unique sequence inserts
are recognized as those that produce a single band during Southern
analysis.
[0047] Another method of disabling the hybridization capacity of
repetitive DNA sequences within nucleic acid fragments involves
blocking the repetitive sequences by pre-reassociation of fragments
with fragments of repetitive-sequence-enriched DNA, by
pre-reassociation of the target DNA with fragments of
repetitive-sequence-enriched DNA, or pre-reassociation of both the
fragments of the heterogeneous mixture and the target DNA with
repetitive-sequence-enriched DNA. The method is generally described
by Sealy et al., "Removal of Repeated Sequences from Hybridization
Probes," Nucleic Acid Research, Vol. 13, pgs. 1905-1922 (1985),
which reference is incorporated by reference.
[0048] The term pre-reassociation refers to a hybridization step
involving the reassociation of unlabeled, repetitive DNA or RNA
with the nucleic acid fragments of the heterogeneous mixture just
prior to the in situ hybridization step, or with the target DNA
either just prior to or during the in situ hybridization step. This
treatment results in nucleic acid fragments whose repetitive
sequences are blocked by complementary fragments such that
sufficient unique sequence regions remain free for attachment to
chromosomal DNA during the in situ hybridization step.
[0049] III. Labeling the Nucleic Acid Fragments of the
Heterogeneous Mixture.
[0050] Several standard techniques are available for labeling
single and double stranded nucleic acid fragments of the
heterogeneous mixture. They include incorporation of radioactive
labels, e.g. Harper et al. Chromosoma, Vol 83, pgs. 431-439 (1984);
direct attachment of fluorescent labels, e.g. Smith et al., Nucleic
Acids Research, Vol. 13, pgs. 2399-2412 (1985), and Connolly et
al., Nucleic Acids Research, Vol. 13, pgs. 4485-4502 (1985); and
various chemical modifications of the nucleic acid fragments that
render them detectable immunochemically or by other affinity
reactions, e.g. Tchen et al., "Chemically Modified Nucleic Acids as
Immunodetectable Probes in Hybridization Experiments," Proc. Natl.
Acad. Sci., Vol 81, pgs. 3466-3470 (1984); Richardson et al.,
"Biotin and Fluorescent Labeling of RNA Using T4 RNA Ligase,"
Nucleic Acids Research, Vol. 11, pgs. 6167-6184 (1983); Langer et
al., "Enzymatic Synthesis of Biotin-Labeled Polynucleotides: Novel
Nucleic Acid Affinity Probes," Proc. Natl. Acad. Sci., Vol. 78,
pgs. 6633-6637 (1981); Brigati et al., "Detection of Viral Genomes
in Cultured Cells and Paraffin-Embedded Tissue Sections Using
Biotin-Labeled Hybridization Probes," Virology, Vol. 126, pgs.
32-50 (1983); Broker et al., "Electron Microscopic Visualization of
tRNA Genes with Ferritin-Avidin: Biotin Labels," Nucleic Acids
Research, Vol. 5, pgs. 363-384 (1978); Bayer et al., "The Use of
the AvidinBiotin Complex as a Tool in Molecular Biology," Methods
of Biochemical Analysis, Vol. 26, pgs. 1-45 (1980) and Kuhlmann,
Immunoenzyme Techniques in Cytochemistry (Weinheim, Basel,
1984).
[0051] All of the labeling techniques disclosed in the above
references may be preferred under particular circumstances.
Accordingly, the above-cited references are incorporated by
reference. Several factors govern the choice of labeling means,
including the effect of the label on the rate of hybridization and
binding of the nucleic acid fragments to the chromosomal DNA, the
accessibility of the bound probe to labeling moieties applied-after
initial hybridization, the nature and intensity of the signal
generated by the label, the expense and ease in which the label is
applied, and the like.
[0052] The term labeled nucleic acid fragment as used herein
comprehends labeling means which include chemical modification of
nucleic acid fragment by substituting derivatized bases, by forming
adducts, or the like, which after hybridization render the nucleic
acid fragments detectable by immunochemical stains or affinity
labels, such as biotin-avid labeling systems,
N-acetoxy-N-2-acetylaminofluorene (AFF) labeling systems, or the
like.
[0053] For most applications, labeling means which generate
fluorescent signals are preferred.
[0054] IV. In Situ Hybridization.
[0055] Application of the heterogeneous mixture of the invention to
chromosomes is accomplished by standard in situ hybridization
techniques. Several excellent guides to the technique are
available, e.g., Gall and Pardue, "Nucleic Acid Hybridization in
Cytological Preparations," Methods in Enzymology, Vol. 21, pgs.
470-480 (1981); Henderson, "Cytological Hybridization to Mammalian
Chromosomes," International Review of Cytology, Vol. 76, pgs. 1-46
(1982); and Angerer, et al., "In Situ Hybridization to Cellular
RNAs," in Genetic Engineering: Principles and Methods, Setlow and
Hollaender, Eds., Vol. 7, pgs. 43-65 (Plenum Press, New York,
1985). Accordingly, these references are incorporated by
references.
[0056] Three factors influence the staining sensitivity of a
heterogeneous mixture of hybridization probes: (1) efficiency of
hybridization (fraction of target DNA that can be hybridized by
probe), (2) detection efficiency (i.e., the amount of visible
signal that can be obtained from a given amount of hybridization
probe), and (3) level of noise produced by nonspecific binding of
probe or components of the detection system.
[0057] Generally in situ hybridization comprises the following
major steps: (1) fixation of tissue or biological structure to be
examined, (2) prehybridization treatment of the biological
structure to increase accessibility of target DNA, and to reduce
nonspecific binding, (3) hybridization of the heterogeneous mixture
of probe to the DNA in the biological structure or tissue; (4)
posthybridization washes to remove probe not bound in specific
hybrids, and (5) detection of the hybridized probes of the
heterogeneous mixture. The reagents used in each of these steps and
their conditions of use vary depending on the particular
chromosomes being stained.
[0058] The following comments are meant to serve as a guide for
applying the general steps listed above. Some experimentation may
be required to establish optimal staining conditions for particular
applications.
[0059] Fixatives include acid alcohol solutions, acid acetone
solutions, Petrunkewitsch's reagent, and various aldehydes such as
formaldehyde, paraformaldehyde, glutaraldehyde, or the like.
Preferably, ethanol-acetic acid or methanol-acetic acid solutions
in about 3:1 proportions are used to fix the chromosomes. For cells
or chromosomes in suspension, the fixation procedure disclosed by
Trask, et al., in Science, Vol. 230, pgs. 1401-1402 (1985), is
preferred. Accordingly, Trask, et al., is incorporated by
reference. Briefly, K.sub.2CO.sub.3 and dimethylsulferimidate (DMS)
are added (from a 5.times. concentrated stock solution, mixed
immediately before use) to a suspension containing about
5.times.10.sup.6 nuclei/ml. Final K.sub.2CO.sub.3 and DMS
concentrations are 20 mM and 3 mM, respectively. After 15 minutes
at 25.degree. C., the pH is adjusted from 10.0 to 8.0 by the
addition of 50 microliters of 100 mM citric acid per milliliter of
suspension. Nuclei are washed once by centrifugation (300 g, 10
minutes, 4.degree. C. in 50 mM kCl, 5 mM Hepes buffer, at pH 9.0,
and 10 mM MgSO.sub.4).
[0060] Preferably, before application of the stain, chromosomes are
treated with agents to remove proteins. Such agents include enzymes
or mild acids. Pronase or proteinase K are the preferred enzymes.
Optimization of deproteinization requires a combination of protease
concentration and digestion time that maximize hybridization, but
does not cause unacceptable loss of morphological detail. Optimum
conditions vary according to chromosome types and method of
fixation. Thus, for particular applications, some experimentation
may be required to optimize protease treatment.
[0061] Proteins can also be removed by mild acid extraction, e.g.,
0.02-0.2 N HCl, followed by high temperature (e.g., 70.degree. C.)
washes.
[0062] In some cases pretreatment with RNase may be desirable to
remove residual RNA from the fixed chromosomes. Such removal can be
accomplished by incubation of the fixed chromosomes in 50-100
microgram/milliliter RNase in 2.times. SSC (where SSC is a solution
of 0.15M NaCL and 0.015M sodium citrate) for a period of 1-2 hours
at room temperature.
[0063] The step of hybridizing the probes of the heterogeneous
mixture to the chromosomal DNA involves (1) denaturing the
chromosomal DNA so that probes can gain access to complementary
single stranded regions, and (2) applying the heterogeneous mixture
under conditions which allow the probes to anneal to complementary
sites. Methods for denaturation include incubation in the presence
of high pH, low pH, high temperature, or organic solvents such as
formamide, tetraalkylammonium halides, or the like, at various
combinations of concentration and temperature. The preferred
denaturing procedure is incubation for between about 1-10 minutes
in formamide at a concentration between about 35-95 percent in
2.times. SSC and at a temperature between about 25-70.degree. C.
Determination of the optimal incubation time, concentration, and
temperature within these ranges depends on several variables,
including the method of fixation and chromosome type.
[0064] After the chromosomal DNA is denatured, the denaturing
agents are removed before application of the heterogeneous mixture.
Where formamide and neat are the primary denaturing agents, removal
is conveniently accomplished by plunging the substrate or vessel
containing the denatured chromosomes into an ice bath, and/or by
several washes by an ice-cold solvent, such as a 70%, 80%, 95% cold
ethanol series.
[0065] The ambient physiochemical conditions of the chromosomal DNA
at the time the heterogeneous mixture is applied is referred to
herein as the hybridization conditions, or annealing conditions.
Optimal hybridization conditions for particular applications depend
on several factors, including salt concentration, incubation time
of chromosomes in the heterogeneous mixture, and the
concentrations, complexities, and lengths of the probes making up
the heterogeneous mixture. Roughly, the hybridization conditions
must be sufficiently denaturing to minimize nonspecific binding and
hybridizations with excessive numbers of base mismatches. On the
other hand, the conditions cannot be so stringent as to reduce
hybridizations below detectable levels or to require excessively
long incubation times. Generally, the hybridization conditions are
much less stringent than the conditions for denaturing the
chromosomal DNA.
[0066] The concentrations of probes in the heterogeneous mixture is
an important variable. The concentrations must be high enough so
that the respective chromosomal binding sites are saturated in a
reasonable time (e.g., within about 18 hours), yet concentrations
higher than that just necessary to achieve saturation should be
avoided so that nonspecific binding is minimized. A preferred
concentration range of nucleic acid fragments in the heterogeneous
mixture is between about 1-10 nanograms per kilobase of complexity
per milliliter.
[0067] The fixed chromosomes can be treated in several ways either
during or after the hybridization step to reduce nonspecific
binding of probe DNA. Such treatments include adding a large
concentration of nonprobe, or "carrier", DNA to the heterogeneous
mixture, using coating solutions, such as Denhardt's solution
(Biochem. Biophys. Res. Commun., Vol. 23, pgs. 641-645 (1966), with
the heterogeneous mixture, incubating for several minutes, e.g.,
5-20, in denaturing solvents at a temperature 5-10.degree. C. above
the hybridization temperature, and in the case of RNA probes, mild
treatment with single strand RNase (e.g., 5-10 micrograms per
milliliter RNase) in 2.times. SSC at room temperature for 1
hour).
[0068] V. Making and Using a Staining Reagent Specific to Human
Chromosome 21.
[0069] A. Isolation of Chromosome 21 and Construction of a
Chromosome 21-Specific Library
[0070] DNA fragments from human chromosome-specific libraries are
available from the National Laboratory Gene Library Project through
the American Type Culture Collection (ATCC), Rockville, Md.
Chromosome 21-specific DNA fragments were generated by the
procedure described by Fuscoe et al., in "Construction of Fifteen
Human Chromosome-Specific DNA Libraries from Flow-Purified
Chromosomes," Nucleic Acids Research, Vol. ______, pgs. _______
(1986), which reference is incorporated by reference. Briefly, a
human diploid fibroblast culture was established from newborn
foreskin tissue. Chromosomes of the cells were isolated by the
MGSO.sub.4 method for van den Engh et al., Cytometry, Vol. 5, pgs.
108-123 (1984), and stained with the fluorescent dyes. Hoechst
33258 and Chromomycin A3. Chromosome 21 was purified on the
Lawrence Livermore National Laboratory high speed sorter, described
by Peters et al., Cytometry, Vol. 6, pgs. 290-301 (1985).
[0071] After sorting, chromosome concentrations were approximately
4.times.10.sup.5/ml. Therefore, prior to DNA extraction, the
chromosomes (0.2- 1.0.times.10.sup.6) were concentrated by
centrifugation at 40,000.times.g for 30 minutes at 4.degree. C. The
pellet was then resuspended in 100 microliters of DNA isolation
buffer (15 mM NaCl, 10 mM EDTA, 10 mM Tris HCl pH 8.0) containing
0.5% SDS and 100 micrograms/ml proteinase K. After overnight
incubation at 37.degree. C., the proteins were extracted twice with
phenol:chloroform:isoamyl alcohol (25:24:1) and once with
chloroform:isoamyl alcohol (24:1). Because of the small amounts of
DNA, each organic phase was reextracted with a small amount of 10
mM Tris pH 8.0, 1 mM EDTA (TE). Aqueous layers were combined and
transferred to a Schleicher and Schuell mini-collodion membrane
(#UH020/25) and dialyzed at room temperature against TE for 6-8
hours. The purified DNA solution was then digested with 50 units of
Hind III (Bethesda Research Laboratories, Inc.) in 50 mM NaCl, 10
mM Tris HCl pH 7.5, 10 mM MgCl.sub.2, 1 mM dithiothreitol. After 4
hours at 37.degree., the reaction was stopped by extractions with
phenol and chloroform as described above. The aqueous phase was
dialyzed against water overnight at 4.degree. C. in a minicollodion
bag and then 2 micrograms of Charon 21A arms cleaved with Hind III
and treated with calf alkaline phosphatase (Boehringer Mannheim)
were added. This solution was concentrated under vacuum to a volume
of 50-100 microliters and transferred to a 0.5 ml microfuge tube
where the DNA was precipitated with one-tenth volume 3M sodium
acetate pH 5.0 and 2 volumes ethanol. The precipitate was collected
by centrifugation, washed with cold 70% ethanol, and dissolved in
10 microliters of TE.
[0072] After allowing several hours for the DNA to dissolve, 1
microliter of 10.times. ligase buffer (0.5M Tris HCl pH 7.4, 0.1 M
MgCl.sub.2, 0.1M dithiothreitol, 10 mM ATP, 1 mg/ml bovine serum
albumin) and 1 unit of T4 ligase (Bethesda Research Laboratory,
Inc.) were added. The ligation reaction was incubated at 10.degree.
C. for 16-20 hours and 3 microliters aliquots were packaged into
phage particles using in vitro extracts prepared from E. coli
strains BHB 2688 and BHB 2690, described by Hohn in Methods in
Enzymology, Vol. 68, pgs. 299-309 (1979) Molecular Cloning: A
Laboratory Manual, (Cold Spring Harbor Laboratory, New York, 1982).
Briefly, both extracts were prepared by sonication and combined at
the time of in vivo packaging. These extracts packaged wild-type
lambda DNA at an efficiency of 1-5.times.10.sup.8 plaque forming
units (pfu) per microgram. The resultant phage were amplified on E.
coli LE392 at a density of approximately 10.sup.4 pfu/150 mm dish
for 8 hours to prevent plaques from growing together and to
minimize differences in growth rates of different recombinants. The
phage were eluted from the agar in 10 ml SM buffer (50 mM Tris HCl
pH 7.5, 10 mM MgSO.sub.4, 100 mM NaCl, 0.01% gelatin) per plate by
gentle shaking at 4.degree. C. for 12 hours. The plates were then
rinsed with an additional 4 ml of SM. After pelleting cellular
debris, the phage suspension was stored over chloroform at
4.degree. C.
[0073] B. Construction and Use of Chromosome 21-Specific Stain for
Staining Chromosome 21 of Human Lymphocytes
[0074] Clones having unique sequence inserts are isolated by the
method of Benton and Davis, Science, Vol. 196, pgs. 180-182 (1977).
Briefly, about 1000 recombinant phage are isolated at random from
the chromosome 21-specific library. These are transferred to
nitrocellulose and probed with nick translated total genomic human
DNA.
[0075] Of the clones which do not show strong hybridization,
approximately 300 are picked which contain apparent unique sequence
DNA. After the selected clones are amplified, the chromosome 21
insert in each clone is .sup.32P labeled and hybridized to Southern
blots of human genomic DNA digested with the same enzyme used to
construct the chromosome 21 library, i.e., Hind III. Unique
sequence containing clones are recognized as those that produce a
single band during Southern analysis. Roughly, 100 such clones are
selected for the heterogeneous mixture. The unique sequence clones
are amplified, the inserts are removed by Hind III digestions, and
the inserts are separated from the phage arms by gel
electrophoresis. The probe DNA fragments (i.e., the unique sequence
inserts) are removed from the gel and biotinylated by nick
translation (e.g., by a kit available from Bethesda Research
Laboratories). Labeled DNA fragments are separated from the nick
translation reaction using small spin columns made in 0.5 ml
eppendorph tubes filled with Sephadex G-50 (medium) swollen in 50
mM Tris, 1 mM EDTA, 0.1% SDS, at pH 7.5. Human lymphocyte
chromosomes are prepared following Harper et al, Proc. Natl. Acad.
Sci., Vol. 78, pgs. 4458-4460 (1981). Metaphase and interphase
cells were washed 3 times in phosphate buffered saline, fixed in
methanol-acetic acid (3:1) and dropped onto cleaned microscope
slides. Slides are stored in a nitrogen atmosphere at -20.degree.
C.
[0076] Slides carrying interphase cells and/or metaphase spreads
are removed from the nitrogen, heated to 65.degree. C. for 4 hours
in air, treated with RNase (100 micrograms/ml for 1 hour at
37.degree. C.), and dehydrated in an ethanol series. They are then
treated with proteinase K (60 ng/ml at 37.degree. C. for 7.5
minutes) and dehydrated. The proteinase K concentration is adjusted
depending on the cell type and enzyme lot so that almost no phase
microscopic image of the chromosomes remains on the dry slide. The
hybridization mix consists of (final concentrations) 50 percent
formamide, 2.times. SSC, 10 percent dextran sulfate, 500
micrograms/ml carrier DNA (sonicated herring sperm DNA), and 2.0
microgram/ml biotin-labeled chromosome 21-specific DNA. This
mixture is applied to the slides at a density of 3
microliters/cm.sup.2 under a glass coverslip and sealed with rubber
cement. After overnight incubation at 37.degree. C., the slides are
washed at 45.degree. C. (50% formamide-2.times.SS pH 7, 3 times 3
minutes; followed by 2.times.SSC pH 7, 5 times 2 minutes) and
immersed in BN buffer (0.1 M Na bicarbonate, 0.05 percent NP-40, pH
8). The slides are never allowed to dry after this point.
[0077] The slides are removed from the BN Duffer and blocked for 5
minutes at room temperature with BN buffer containing 5% non-fat
dry milk (Carnation) and 0.02% Na Azide (5 microliter/cm.sup.2
under plastic coverslips). Tne coverslips are removed, and excess
liquid briefly drained and fluorescein-avidin DCS (3 microgram/ml
in BN buffer with 5% milk and 0.02% NaAzide) is applied (5
microliter/cm.sup.2). The same coverslips are replaced and the
slides incubated 20 minutes at 37.degree. C. The slides are then
washed 3 times for 2 minutes each in BN buffer at 45.degree. C. The
intensity of biotin-linked fluorescence is amplified by adding a
layer of biotinylated goat anti-avidin antibody (5 microgram/ml in
BN buffer with 5% goat serum and 0.02% NaAzide), followed, after
washing as above, by another layer of fluorescein-avidin DCS.
Fluorescein-avidin DCS, goat antiavidin and goat serum are all
available commercially, e.g., Vector Laboratories (Burlingame,
Calif.). After washing in BN, a fluorescence antifade solution,
p-phenylenediamine (1.5 microliter/cm.sup.2 of coverslip) is added
before observation. It is important to keep this layer thin for
optimum microscopic imaging. This antifade significantly reduced
fluorescein fading and allows continuous microscopic observation
for up to 5 minutes. The DNA counterstains (DAPI or propidium
iodide) are included in the antifade at 0.25-0.5 microgram/ml.
[0078] The red-fluorescing DNA-specific dye propidium iodide (PI)
is used to allow simultaneous observation of hybridized probe and
total DNA. The fluorescein and PI are excited at 450-490 nm (Zeiss
filter combination 487709). Increasing the excitation wavelength to
546 nm (Zeiss filter combination 487715) allows observation of the
PI only. DAPI, a blue fluorescent DNA-specific stain excited in the
ultraviolet (Zeiss filter combination 487701), is used as the
counterstain when biotin-labeled and total DNA are observed
separately. Metaphase chromosome 21s are detected by randomly
located spots of yellow distributed over the body of the
chromosome.
[0079] VI. Chromosome 21-Specific Staining by Blocking Repetitive
Probe and Chromosomal DNA
[0080] High concentrations of unlabeled human genomic DNA and
lambda phage DNA were used to inhibit the binding of repetitive and
vector DNA sequences to the target chromosomes. Heavy proteinase
digestion and subsequent fixation improved access of probes to
target DNA.
[0081] Human metaphase spreads were prepared on microscope slides
with standard techniques and stored immediately in a nitrogen
atmosphere at -20.degree. C.
[0082] Slides were removed from the freezer and allowed to warm to
room temperature in a nitrogen atmosphere before beginning the
staining procedure. The warmed slides were first treated with 0.6
microgram/ml proteinase K in P buffer (20 mM Tris, 2 mM CaCl.sub.2
at pH 7.5) for 7.5 minutes, and washed once in P buffer. The amount
of proteinase K used needs to be adjusted for the particular enzyme
lot and cell type. Next the slides were washed once in
paraformaldehyde buffer (phosphate buffered saline (PBS) plus 50 mM
MgCl.sub.2, at pH 7.5), immersed for 10 minutes in 4%
paraformaldehyde in paraformaldehyde buffer, and washed once in
2.times.SSC (0.3 M NaCl, 0.03 M sodium citrate at pH 7). DNA on the
slides was denatured by immersing in 70% formamide and 2.times.SSC
at 70.degree. C. for 2 minutes. After denaturing the slides were
stored in 2.times.SSC. A hybridization mix was prepared which
consisted of 50% formamide, 10% dextran sulfate, 1% Tween 20,
2.times.SSC, 0.5 mg/ml human genomic DNA, 0.03 mg/ml lambda DNA,
and 3 microgram/ml biotin labeled probe DNA. The probe DNA
consisted of the highest density fraction of phage from the
chromosome 21 Hind III fragment library (ATCC accession number
57713), as determined by a cesium chloride gradient. (Both insert
and phage DNA of the probe were labeled by nick translation.) The
average insert size (amount of chromosome 21 DNA), as determined by
gel electrophoresis is about 5 kilobases. No attempt was made to
remove repetitive sequences from the inserts or to isolate the
inserts from the lambda phage vector. The hybridization mix was
denatured by heating to 70.degree. C. for 5 minutes followed by
incubation at 37.degree. C. for 1 hour. The incubation allows the
human genomic DNA and unlabeled lambda DNA in the hybridization mix
to block the human repetitive sequences and vector sequences in the
probe.
[0083] The slide containing the human metaphase spread was removed
from the 2.times.SSC and blotted dry with lens paper. The
hybridization mix was immediately applied to the slide, a glass
cover slip was placed on the slide with rubber cement, and the
slide was incubated overnight at 37.degree. C. Afterwards
preparation of the slides proceeded as described in Section V
(wherein chromosome 21 DNA was stained with fluorescein and total
chromosomal DNA counterstained with DAPI). FIGS. 1a-c illustrate
the results. FIG. 1a is a DAPI image of the human metaphase spread
obtained with a computerized image analysis system. It is a binary
image showing everything above threshold as white, and the rest as
black. The primary data was recorded as a gray level image with 256
intensity levels. (Small arrows indicate the locations of the
chromosome 21s.) FIG. 1b is a fluorescein image of the same spread
as in FIG. 1a, again in binary form. (Again, small arrows indicate
the locations of the chromosome 21s.) FIG. 1c illustrates the
positions of the chromosome 21s with other less densely stained
objects removed standard by image processing techniques.
[0084] The descriptions of the foregoing embodiments of the
invention have been presented for purpose of illustration and
description. They are not intended to be exhaustive or to limit the
invention to the precise form disclosed, and obviously many
modifications and variations are possible in light of the above
teaching. The embodiments were chosen and described in order to
best explain the principles of the invention and its practical
application to thereby enable others skilled in the art to best
utilize the invention in various embodiments and with various
modifications as are suited to the particular use contemplated. It
is intended that the scope of the invention be defined by the
claims appended hereto.
* * * * *