U.S. patent application number 10/350042 was filed with the patent office on 2003-12-25 for multiparametric fluorescence in situ hybridization.
Invention is credited to Ballard, Stephen Gwyn, Speicher, Michael, Ward, David C..
Application Number | 20030235840 10/350042 |
Document ID | / |
Family ID | 27492191 |
Filed Date | 2003-12-25 |
United States Patent
Application |
20030235840 |
Kind Code |
A1 |
Ward, David C. ; et
al. |
December 25, 2003 |
Multiparametric fluorescence in situ hybridization
Abstract
The invention relates to a set of combinatorially labeled
oligonucleotide probes each member thereof: (i) having a
predetermined label distinguishable from the label of any other
member of said set, and (ii) being capable of specifically
hybridizing with a predetermined chromosome or nucleic acid
molecule, and to the use of such molecules, alone or in concert
with nucleic acid amplification methods.
Inventors: |
Ward, David C.; (Madson,
CT) ; Speicher, Michael; (Riemerling, DE) ;
Ballard, Stephen Gwyn; (Hamden, CT) |
Correspondence
Address: |
Alan J. Grant, Esq.
c/o Carella, Byrne, Bain, Gilfillan, Cecchi,
Stewart & Olstein
6 Becker Farm Road
Roseland
NJ
07068
US
|
Family ID: |
27492191 |
Appl. No.: |
10/350042 |
Filed: |
January 24, 2003 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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10350042 |
Jan 24, 2003 |
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09988584 |
Nov 20, 2001 |
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6548259 |
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10350042 |
Jan 24, 2003 |
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09468823 |
Dec 22, 1999 |
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6506563 |
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10350042 |
Jan 24, 2003 |
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09088845 |
Jun 2, 1998 |
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6007994 |
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10350042 |
Jan 24, 2003 |
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09088087 |
Jun 1, 1998 |
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10350042 |
Jan 24, 2003 |
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08640657 |
May 1, 1996 |
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5759781 |
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10350042 |
Jan 24, 2003 |
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08580717 |
Dec 29, 1995 |
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10350042 |
Jan 24, 2003 |
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08577622 |
Dec 22, 1995 |
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Current U.S.
Class: |
435/6.14 ;
536/24.3 |
Current CPC
Class: |
C12Q 1/6841 20130101;
C12Q 1/6816 20130101; C12Q 1/6841 20130101; C12Q 2537/143 20130101;
C12Q 2565/102 20130101; C12Q 2563/107 20130101; C12Q 2561/119
20130101; C12Q 2565/1025 20130101; C12Q 2563/131 20130101; C12Q
2565/601 20130101; C12Q 2565/102 20130101; C12Q 1/6841 20130101;
C07H 21/00 20130101; C12Q 1/6841 20130101; C12Q 1/6841
20130101 |
Class at
Publication: |
435/6 ;
536/24.3 |
International
Class: |
C12Q 001/68; C07H
021/04 |
Claims
What is claimed is:
1. A set of combinatorially labeled oligonucleotide probes
comprised of a first and a second subset of probes, wherein: (A)
each member of said first subset of probes comprises a plurality of
an oligonucleotide: (i) being linked or coupled to a predetermined
label distinguishable from the label of any other member of said
first or second subsets of probes, and (ii) being capable of
specifically hybridizing with one predetermined autosomal or sex
chromosome of a human karyotype; said first subset of probes set
having sufficient members to be capable of specifically hybridizing
each autosomal or sex chromosome of said human karyotype to at
least one member; and (B) each member of said second subset of
probes comprises a plurality of an oligonucleotide: (i) being
linked or coupled to a predetermined label distinguishable from the
label of any other member of said first or second subset, and (ii)
being capable of specifically hybridizing with one
extra-chromosomal polynucleotide copy of a predetermined region of
an autosomal or sex chromosome of said human karyotype.
2. The set of combinatorially labeled oligonucleotide probes of
claim 1, wherein said extra-chromosomal polynucleotide copy of a
predetermined region of an autosomal or sex chromosome of said
human karyotype is an RNA molecule.
3. The set of combinatorially labeled oligonucleotide probes of
claim 1, wherein said extra-chromosomal polynucleotide copy of a
predetermined region of an autosomal or sex chromosome of said
human karyotype is a DNA molecule.
4. The set of combinatorially labeled oligonucleotide probes of
claim 1, wherein said set contains at least one member probe
specific for a sub-chromosomal fragment of an autosomal or sex
chromosome of said human karyotype.
5. A set of combinatorially labeled oligonucleotide probes
comprised of a first subset of genotypic probes and a second subset
of phenotypic probes, wherein: (A) each member of said first subset
of genotypic probes comprises a plurality of an oligonucleotide:
(i) being linked or coupled to a predetermined label
distinguishable from the label of any other member of said first or
second subsets of probes, and (ii) being capable of specifically
hybridizing with a region of a nucleic acid of a preselected
bacterium, virus or lower eukaryote; said first subset of probes
set having sufficient members to be capable of distinguishing said
preselected bacterium, virus, or lower eukaryote from other
bacteria, viruses, or lower eukaryotes; and (B) each member of said
second subset of phenotypic probes comprises a plurality of an
oligonucleotide: (i) being linked or coupled to a predetermined
label distinguishable from the label of any other member of said
first or second subset, and (ii) being capable of specifically
hybridizing with a predetermined polynucleotide region of said
chromosome of said preselected bacterium, virus, or lower
eukaryote, or an extra-chromosomal copy thereof so as to permit the
determination of whether said preselected bacterium, virus, or
lower eukaryote exhibits a preselected phenotype.
6. The set of combinatorially labeled oligonucleotide probes of
claim 5, wherein said extra-chromosomal polynucleotide copy of a
predetermined region of said chromosome is an RNA molecule.
7. The set of combinatorially labeled oligonucleotide probes of
claim 5, wherein said extra-chromosomal polynucleotide copy of a
predetermined region of said chromosome is a DNA molecule.
8. The set of combinatorially labeled oligonucleotide probes of
claim 5, wherein said predetermined region of said chromosome of
said bacterium, virus or lower eukaryote encodes an antibiotic
resistance deterninant.
9. The set of combinatorially labeled oligonucleotide probes of
claim 5, wherein said predetermined region of said chromosome of
said bacterium, virus or lower eukaryote encodes a toxin.
10. The set of combinatorially labeled oligonucleotide probes of
claim 5, wherein said predetermined region of said chromosome of
said bacterium, virus or lower eukaryote encodes a protein that is
determinative of the species of said bacterium, virus or lower
eukaryote.
11. The set of combinatorially labeled oligonucleotide probes of
claim 10, wherein said predetermined region of said chromosome of
said bacterium, virus or lower eukaryote encodes a protein that is
determinative of the subspecies of said species of bacterium, virus
or lower eukaryote.
12. The set of combinatorially labeled oligonucleotide probes of
any of claims 3 and 7, wherein said DNA molecule is produced
through in situ nucleic acid amplification.
13. The set of combinatorially labeled oligonucleotide probes of
any of claims 3 and 7, wherein said in situ nucleic acid
amplification is an in situ polymerase chain reaction.
14. The set of combinatorially labeled oligonucleotide probes of
any of claims 3 and 7, wherein said in situ nucleic acid
amplification is an in situ rolling circle amplification reaction.
,
15. The set of combinatorially labeled oligonucleotide probes of
any of claims 3 and 7, wherein said probes contain nucleotide
residues that are labeled with a biotin moiety.
16. The set of combinatorially labeled oligonucleotide probes of
claim 15, wherein said probes are additionally labeled with a
labeled biotin-binding ligand.
17. The set of combinatorially labeled oligonucleotide probes of
claim 16, wherein at least one of said probes is labeled with a
biotin-binding ligand that comprises one or more fluorophores.
18. The set of combinatorially labeled oligonucleotide probes of
claim 17, wherein at least one of said fluorophores is selected
from the group consisting of the fluorophores FITC, Cy3, Cy3.5,
Cy5, Cy5.5 and Cy7.
19. The set of combinatorially labeled oligonucleotide probes of
claim 17, wherein at least one of said probes is labeled with a
biotin-binding ligand that comprises more than one fluorophore.
20. The set of combinatorially labeled oligonucleotide probes of
claim 19, wherein one of the fluorophores of said biotin-binding
ligand that comprises more than one fluorophore is selected from
said group of fluorophores FITC, Cy3, Cy3.5, Cy5, Cy5.5 and
Cy7.
21. The set of combinatorially-labeled oligonucleotide probes of
claim 20, wherein all of the fluorophores of said biotin-binding
ligand that comprises more than one fluorophore are selected from
said group of fluorophores FITC Cy3, Cy3.5, Cy5, Cy5.5 and Cy7.
22. The set of combinatorially labeled oligonucleotide probes of
claim 17, wherein at least one member of said set is labeled with
more than one fluorophore selected from the group consisting of the
fluorophores FITC, Cy3, Cy3.5, Cy5, Cy5.5 and Cy7, and wherein each
member of said set is labeled with at least one fluorophore
selected from said fluorophore group.
23. The set of combinatorially labeled oligonucleotide probes of
claim 22, wherein at least one member of said set is labeled with
two fluorophores, each selected from the group consisting of the
fluorophores FITC, Cy3, Cy3.5, Cy5, Cy5.5 and Cy7, and wherein each
member of said set is labeled with at least one fluorophore
selected from said fluorophore group.
24. The set of combinatorially labeled oligonucleotide probes of
claim 22, wherein at least one member of said set is labeled with
three fluorophores, each selected from the group consisting of the
fluorophores FITC, Cy3, Cy3.5, Cy5, Cy5.5 and Cy7, and wherein each
member of said set is labeled with at least one fluorophore
selected from said fluorophore group.
25. A method of simultaneously identifying and distinguishing the
individual autosomal and sex chromosomes of a human karyotype which
comprises the steps: (I) contacting a preparation of said
chromosomes, in single-stranded form, under conditions sufficient
to permit nucleic acid hybridization to occur with a set of
combinatorially labeled oligonucleotide probes comprised of a first
and a second subset of probes, wherein: (A) each member of said
first subset of probes comprises a plurality of an oligonucleotide:
(i) being linked or coupled to a predetermined label
distinguishable from the label of any other member of said first or
second subsets of probes, and (ii) being capable of specifically
hybridizing with one predetermined autosomal or sex chromosome of a
human karyotype; said first subset of probes set having sufficient
members to be capable of specifically hybridizing each autosomal or
sex chromosome of said human karyotype to at least one member; and
(B) each member of said second subset of probes comprises a
plurality of an oligonucleotide: (i) being linked or coupled to a
predetermined label distinguishable from the label of any other
member of said first or second subset, and (ii) being capable of
specifically hybridizing with an a predetermined extra-chromosomal
polynucleotide copy of a region of an autosomal or sex chromosome
of said human karyotype. (II) for each chromosome of said
preparation hybridized to a member of said first subset of probes,
detecting and identifying the predetermined label of that member
and correlating the identity of the label of that member with the
identity of the autosomal or sex chromosome of said human karyotype
with which that member specifically hybridizes, to thereby identify
the chromosome hybridized to said member; (III) repeating step (II)
until each autosomal and sex chromosome of said human karyotype has
been identified in said preparation (IV) for each member of said
second subset of probes hybridized to a predetermined
extra-chromosomal polynucleotide copy of a region of an autosomal
or sex chromosome detecting and identifying the predetermined label
of that member and correlating the identity of the label of that
member with the identity of the region of the autosomal or sex
chromosome of said human karyotype with which that member
specifically hybridizes, to thereby identify the region of said
autosomal or sex chromosome hybridized to said member; (V)
repeating step (IV) for each member of said second subset of
probes.
26. The method of claim 25, wherein said extra-chromosomal
polynucleotide copy of said predetermined region of an autosomal or
sex chromosome of said human karyotype is an RNA molecule.
27. The method of claim 25, wherein said extra-chromosomal
polynucleotide copy of said predetermined region of an autosomal or
sex chromosome of said human karyotype is a DNA molecule.
28. The method of claim 25, wherein said set of oligonucleotide
probes contains at least one member probe specific for a
sub-chromosomal fragment of an autosomal or sex chromosome of said
human karyotype.
29. A method of simultaneously identifying and distinguishing a
preselected bacterium, virus, or lower eukaryote from other
bacteria, viruses or lower eukaryotes that may be present in a
sample which comprises the steps: (I) contacting a preparation
suspected to contain said preselected bacterium, virus, or lower
eukaryote, under conditions sufficient to permit in situ nucleic
acid hybridization to occur, with a set of combinatorially labeled
oligonucleotide probes comprised of a first subset of genotypic
probes and a second subset of phenotypic probes, wherein: (A) each
member of said first subset of genotypic probes comprises a
plurality of an oligonucleotide: (i) being linked or coupled to a
predetermined label distinguishable from the label of any other
member of said first or second subsets of probes, and (ii) being
capable of specifically hybridizing with a region of a nucleic acid
of said preselected bacterium, virus or lower eukaryote; said first
subset of probes set having sufficient members to be capable of
distinguishing said preselected bacterium, virus, or lower
eukaryote from other bacteria, viruses, or lower eukaryotes present
in said preparation; and (B) each member of said second subset of
probes comprises a plurality of an oligonucleotide: (i) being
linked or coupled -to a predetermined label distinguishable from
the label of any other member of said first or second subset, and
(ii) being capable of specifically hybridizing with a predetermined
polynucleotide region of said nucleic acid of said preselected
bacterium, virus, or lower eukaryote, or an extra-chromosomal copy
thereof so as to permit the determination of whether said
preselected bacterium, virus, or lower eukaryote exhibits a
preselected phenotype. (II) for each member of said first subset of
probes hybridized to a region of a chromosome of a preselected
bacterium, virus or lower eukaryote, detecting and identifying the
predetermined label of that member and correlating the identity of
the label of that member with the identity of the bacterium, virus
or lower eukaryote with which that member specifically hybridizes,
to thereby identify said bacterium, virus or lower eukaryote
hybridized to said member; (III) repeating step (II) for each
member of said first subset of probes; (IV) for each member of said
second subset of probes hybridized to a predetermined
polynucleotide region of said chromosome of said preselected
bacterium, virus, or lower eukaryote, or an extra-chromosomal copy
thereof, detecting and identifying the predetermined label of that
member and correlating the identity of the label of that member
with the identity of said predetermined region, to thereby identify
the presence of said predetermined region on a chromosome of said
preselected bacteria, virus or lower eukaryote; (V) repeating step
(IV) for each member of said second subset of probes.
30. The method of claim 29, wherein said extra-chromosomal
polynucleotide copy of said predetermined region of said chromosome
of said bacterium, virus or lower eukaryote is an RNA molecule.
31. The method of claim 29, wherein said extra-chromosomal
polynucleotide copy of said predetermined region of said chromosome
of said bacterium, virus or lower eukaryote is a DNA molecule.
32. The method of claim 29, wherein said predetermined region of
said chromosome of said bacterium, virus or lower eukaryote encodes
an antibiotic resistance determinant.
33. The method of claim 29, wherein said predetermined region of
said chromosome of said bacterium, virus or lower eukaryote encodes
a toxin.
34. The method of claim 29, wherein said predetermined region of
said chromosome of said bacterium, virus or lower eukaryote encodes
a protein that is determinative of the species of said bacterium,
virus or lower eukaryote.
35. The method of claim 34, wherein said predetermined region of
said chromosome of said bacterium, virus or lower eukaryote encodes
a protein that is determinative of the subspecies of said species
of bacterium, virus or lower eukaryote.
36. The method of any of claims 27 and 31, wherein said in situ
nucleic acid amplification is an in situ polymerase chain
reaction.
37. The method of claim 36, wherein said in situ nucleic acid
amplification is an in situ rolling circle amplification
reaction.
38. The method of claim 36, wherein said probes contain nucleotide
residues that are labeled with a biotin moiety.
39. The method of claim 36, wherein said probes are additionally
labeled with a labeled biotin-binding ligand.
40. The method of claim 39, wherein at least one of said probes is
labeled with a biotin-binding ligand that comprises one or more
fluorophores.
41. The method of claim 40, wherein at least one of said
fluorophores is selected from the group consisting of the
fluorophores FITC, Cy3, Cy3.5, Cy5, Cy5.5 and Cy7.
42. The method of claim 40, wherein at least one of said probes is
labeled with a biotin-binding ligand that comprises more than one
fluorophore.
43. The method of claim 42, wherein one of the fluorophores of said
biotin-binding ligand that comprises more than one fluorophore is
selected from said group of fluorophores FITC, Cy3, Cy3.5, Cy5,
Cy5.5 and Cy7.
44. The method of claim 43, wherein all of the fluorophores of said
biotin-binding ligand that comprises more than one fluorophore are
selected from said group of fluorophores FlTC, Cy3, Cy3.5, Cy5,
Cy5.5 and Cy7.
45. The method of claim 40, wherein at least one member of said set
is labeled with more than one fluorophore selected from the group
consisting of the fluorophores FITC, Cy3, Cy3.5, Cy5, Cy5.5 and
Cy7, and wherein each member of said set is labeled with at least
one fluorophore selected from said fluorophore group.
46. The method of claim 45, wherein at least one member of said set
is labeled with two fluorophores, each selected from the group
consisting of the fluorophores FITC, Cy3, Cy3.5, Cy5, Cy5.5 and
Cy7, and wherein each member of said set is labeled with at least
one fluorophore selected from said fluorophore group.
47. The method of claim 45, wherein at least one member of said set
is labeled with three fluorophores, each selected from the group
consisting of the fluorophores FITC, Cy3, Cy3.5, Cy5, Cy5.5 and
Cy7, and wherein each member of said set is labeled with at least
one fluorophore selected from said fluorophore group.
48. The method of any of claims 25 and 29, wherein an optical
optical comb is employed in step (b) to detect the predetermined
label of said hybridized probe member.
49. The method of claims 25 and 29, wherein an optical filter is
employed in step (b) to detect the predetermined label of said
hybridized probe member.
50. The method of claim 49, wherein a plurality of optical filters
is sequentially employed in step (b) to detect the predetermined
label of said hybridized probe member.
51. The method of claim 48, wherein at least one member of said set
is labeled with more than one fluorophore selected from the group
consisting of the fluorophores FlTC, Cy3, Cy3.5, Cy5, Cy5.5 and
Cy7, and wherein each member of said set is labeled with at least
one fluorophore selected from said fluorophore group.
52. The method of claim 49, wherein at least one member of said set
is labeled with two fluorophores, each selected from the group
consisting of the fluorophores FITC, Cy3, Cy3.5, Cy5, Cy5.5 and
Cy7, and wherein each member of said set is labeled with at least
one fluorophore selected from said fluorophore group.
53. The method of claim 49, wherein at least one member of said set
is labeled with three fluorophores, each selected from the group
consisting of the fluorophores FITC, Cy3, Cy3.5, Cy5, Cy5.5 and
Cy7, and wherein each member of said set is labeled with at least
one fluorophore selected from said fluorophore group.
54. A set of combinatorially labeled oligonucleotide probes, each
member thereof: (i) having a predetermined label distinguishable
from the label of any other member of said set, and (ii) being
capable of specifically hybridizing with a telomeric region of one
predetermined autosomal or sex chromosome of a human karyotype;
said set having sufficient members to be capable of specifically
hybridizing each autosomal or sex chromosome of said human
karyotype to at least one member.
55. The set of combinatorially labeled oligonucleotide probes of
claim 54, wherein at least one of said labels is a fluorophore
selected from the group consisting of the fluorophores FITC, Cy3,
Cy3.5, Cy5, Cy5.5 and Cy7.
56. A method of simultaneously identifying and distinguishing the
individual autosomal and sex chromosomes of a human karyotype which
comprises the steps: (a) contacting a preparation of said
chromosomes, in single-stranded form, under conditions sufficient
to permit nucleic acid hybridization to occur with a set of
combinatorially labeled oligonucleotide probes, each member
thereof: (i) having a predetermined label distinguishable from the
label of any other member of said set, and (ii) being capable of
specifically hybridizing with a telomeric region of one
predetermined autosomal or sex chromosome of a human karyotype;
said set having sufficient members to be capable of specifically
hybridizing each autosomal or sex chromosome of said human
karyotype to at least one member; wherein said contacting thereby
causes at least one of each autosomal or sex chromosome of said
preparation to become hybridized to at least one member of said set
of probes; (b) for each chromosome of said preparation hybridized
to a member of said set of probes, detecting and identifying the
predetermined label of that member and correlating the identity of
the label of that member with the identity of the autosomal or sex
chromosome of said human karyotype with which that member
specifically hybridizes, to thereby identify the chromosome
hybridized to said member; and (c) repeating step (b) until each
autosomal and sex chromosome of said human karyotype has been
identified in said preparation.
57. The method of claim 56, wherein at least one of said labels is
a fluorophore selected from the group consisting of the
fluorophores FITC, Cy3, Cy3.5, Cy5, Cy5.5 and Cy7.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S. patent
application Ser. No. 08/640,657 (filed May 1, 1996), which is a
continuation in part of U.S. patent application Ser. No. 08/580,717
(filed Dec. 29, 1995), which is a continuation in part of U.S.
patent application Ser. No. 08/577,622 (filed Dec. 22, 1995), all
of which applications are herein incorporated by reference in their
entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to nucleic acid chemistry, and
more specifically to reagents and methods for accomplishing
multiplex image analysis of chromosomes and chromosomal fragments.
The invention may be used to diagnose chromosomal abnormalities,
infectious agents, etc. This invention was made in part using
Government funds. The Government has certain rights in this
invention.
BACKGROUND OF THE INVENTION
[0003] The determination of the presence and condition of
chromosomes and chromosomal fragments in a biological sample is of
immense importance in the diagnosis of disease. Traditionally, such
determinations have been done manually by inspecting metaphase
chromosomal preparations that have been treated with specialized
stains to reveal characteristic banding patterns. Unfortunately,
the interpretation of such banding patterns requires substantial
skill and is technically difficult. Hence, alternate methods of
analyzing chromosomal presence and arrangement have been
sought.
[0004] One alternative approach to the problem of chromosome
identification has involved the use of labeled chromosome-specific
oligonucleotide probes to label repetitive sequences of interphase
chromosomes (Cremer, T. et al., Hum. Genet. 74:346-352 (1986);
Cremer, T. et al., Exper. Cell Res. 176:119-220 (1988)). Such
methods have been shown to be useful in the prenatal diagnosis of
Down's Syndrome, as well as in the detection of chromosomal
abnormalities associated with tumor cell lines. Chromosome-specific
probes of repetitive DNA that localize to discrete sub-regions of a
chromosome are, however, unsuitable for analyses of many types of
chromosomal abnormalities (e.g., translocations or deletions).
[0005] Ward, D. C. et al. (PCT Application WO/05789, herein
incorporated by reference) discloses a chromosomal in situ
suppression ("CISS") hybridization method for specifically labeling
selected mammalian chromosomes in a manner that permits the
recognition of chromosomal aberrations. In that method, sample DNA
is denatured and permitted to hybridize with a mixture of
fluorescently labeled chromosome-specific probes having high
genetic complexity and unlabeled non-specific competitor probes.
Chromosomal images were obtained as described by Manuelidis, L. et
al. (Chromosoma 96:397-410 (1988), herein incorporated by
reference). The method provides a rapid and highly specific
assessment of individual mammalian chromosomes. The method permits,
by judicious selection of appropriate probes and/or labels, the
visualization of sub-regions of some or all of the chromosomes in a
preparation. For example, by using more than one probe, each
specific for a sub-region of a target chromosome, the method
permits the simultaneous analysis of several sub-regions on that
chromosome. The number of available fluorophores limits the number
of chromosomes or chromosomal sub-regions that can be
simultaneously visualized.
[0006] As described in PCT Application WO/05789, a "combinatorial"
variation of the CISS method can be employed. In the simplest case,
two fluors permit three different chromosomes or chromosomal
sub-regions to be simultaneously visualized. In this variation, a
hybridization probe mixture is made from a single set of probe
sequences composed of two halves, each separately labeled with a
different fluorophore. Upon hybridization, the two fluorophores
produce a third fluorescence signal that is optically
distinguishable from the color of the individual fluorophores.
Extension of this approach to Boolean combinations of n
fluorophores permits the labeling of 2.sup.n-1 chromosomes.
[0007] Ried, T. et al. (Proc. Natl. Acad. Sci. (U.S.A.)
89:1388-1392 (1992), herein incorporated by reference) describes
the use of an epi-fluorescent microscope equipped with a digital
imaging camera and computer software to "pseudocolor" the
fluorescence patterns obtained from simultaneous in situ
hybridization with seven probes using three fluorophores. The use
of wavelength-selective filters allows one to isolate and collect
separate gray scale images of each fluorophore. These images can be
subsequently merged via appropriate software. The sensitivity and
linearity of CCD cameras surmounts the technical difficulties
inherent in color film-based photomicroscopy.
[0008] Although such efforts have increased the number of
chromosomes that can be simultaneously detected and analyzed using
in situ hybridization methods, it would be highly desirable to
define a set of fluorophores having distinguishable emission
spectra to permit the simultaneous detection and analysis of large
numbers of different chromosomes and chromosomal sub-regions. The
present invention provides such reagents as well as methods and
apparatus for their use.
SUMMARY OF THE INVENTION
[0009] The invention concerns reagents and methods for
combinatorial labeling of nucleic acid probes sufficient to permit
the visualization and simultaneous identification of all 22
autosomal human chromosomes and the human X and Y chromosomes, or
defined sub-regions thereof. Such specific labeling of entire
chromosomes or defined sub-regions thereof is referred to as
"painting." The invention further concerns reagents and methods for
combinatorial labeling of nucleic acid probes sufficient to permit
the characterization of bacteria, viruses and/or lower eukaryotes
that may be present in a clinical or non-clinical preparation.
[0010] In detail, the invention concerns a set of combinatorially
labeled oligonucleotide probes comprised of a first and a second
subset of probes, wherein:
[0011] (A) each member of the first subset of probes comprises a
plurality of an oligonucleotide: (i) being linked or coupled to a
predetermined label distinguishable from the label of any other
member of the first or second subsets of probes, and (ii) being
capable of specifically hybridizing with one predetermined
autosomal or sex chromosome of a human karyotype;
[0012] the first subset of probes set having sufficient members to
be capable of specifically hybridizing each autosomal or sex
chromosome of the human karyotype to at least one member, and
[0013] (B) each member of the second subset of probes comprises a
plurality of an oligonucleotide: (i) being linked or coupled to a
predetermined label distinguishable from the label of any other
member of the first or second subset, and (ii) being capable of
specifically hybridizing with one extra-chromosomal polynucleotide
copy of a predetermined region of an autosomal or sex chromosome of
the human karyotype.
[0014] The invention further concerns a set of combinatorially
labeled oligonucleotide probes comprised of a first subset of
genotypic probes and a second subset of phenotypic probes,
wherein:
[0015] (A) each member of the first subset of genotypic probes
comprises a plurality of an oligonucleotide: (i) being linked or
coupled to a predetermined label distinguishable from the label of
any other member of the first or second subsets of probes, and (ii)
being capable of specifically hybridizing with a region of a
nucleic acid of a preselected bacterium, virus or lower
eukaryote;
[0016] the first subset of probes set having sufficient members to
be capable of distinguishing the preselected bacterium, virus, or
lower eukaryote from other bacteria, viruses, or lower eukaryotes;
and
[0017] (B) each member of the second subset of phenotypic probes
comprises a plurality of an oligonucleotide: (i) being linked or
coupled to a predetermined label distinguishable from the label of
any other member of the first or second subset, and (ii) being
capable of specifically hybridizing with a predetermined
polynucleotide region of the chromosome of the preselected
bacterium, virus, or lower eukaryote, or an extra-chromosomal copy
thereof so as to permit the determination of whether the
preselected bacterium, virus, or lower eukaryote exhibits a
preselected phenotype.
[0018] The invention additionally concerns a method of
simultaneously identifying and distinguishing the individual
autosomal and sex chromosomes of a human karyotype which comprises
the steps:
[0019] (I) contacting a preparation of the chromosomes, in
single-stranded form, under conditions sufficient to permit nucleic
acid hybridization to occur with a set of combinatorially labeled
oligonucleotide probes comprised of a first and a second subset of
probes, wherein:
[0020] (A) each member of the first subset of probes comprises a
plurality of an oligonucleotide: (i) being linked or coupled to a
predetermined label distinguishable from the label of any other
member of the first or second subsets of probes, and (ii) being
capable of specifically hybridizing with one predetermined
autosomal or sex chromosome of a human karyotype;
[0021] the first subset of probes set having sufficient members to
be capable of specifically hybridizing each autosomal or sex
chromosome of the human karyotype to at least one member; and
[0022] (B) each member of the second subset of probes comprises a
plurality of an oligonucleotide: (i) being linked or coupled to a
predetermined label distinguishable from the label of any other
member of the first or second subset, and (ii) being capable of
specifically hybridizing with an a predetermined extra-chromosomal
polynucleotide copy of a region of an autosomal or sex chromosome
of the human karyotype.
[0023] (II) for each chromosome of the preparation hybridized to a
member of the first subset of probes, detecting and identifying the
predetermined label of that member and correlating the identity of
the label of that member with the identity of the autosomal or sex
chromosome of the human karyotype with which that member
specifically hybridizes, to thereby identify the chromosome
hybridized to the member,
[0024] (III) repeating step (II) until each autosomal and sex
chromosome of the human karyotype has been identified in the
preparation
[0025] (IV) for each member of the second subset of probes
hybridized to a predetermined extra-chromosomal polynucleotide copy
of a region of an autosomal or sex chromosome detecting and
identifying the predetermined label of that member and correlating
the identity of the label of that member with the identity of the
region of the autosomal or sex chromosome of the human karyotype
with which that member specifically hybridizes, to thereby identify
the region of the autosomal or sex chromosome hybridized to the
member;
[0026] (V) repeating step (IV) for each member of the second subset
of probes.
[0027] The invention additionally concerns a method of
simultaneously identifying and distinguishing a preselected
bacterium, virus, or lower eukaryote from other bacteria, viruses
or lower eukaryotes that may be present in a sample which comprises
the steps:
[0028] (I) contacting a preparation suspected to contain the
preselected bacterium, virus, or lower eukaryote, under conditions
sufficient to permit in situ nucleic acid hybridization to occur,
with a set of combinatorially labeled oligonucleotide probes
comprised of a first subset of genotypic probes and a second subset
of phenotypic probes, wherein:
[0029] (A) each member of the first subset of genotypic probes
comprises a plurality of an oligonucleotide: (i) being linked or
coupled to a predetermined label distinguishable from the label of
any other member of the first or second subsets of probes, and (ii)
being capable of specifically hybridizing with a region of a
nucleic acid of the preselected bacterium, virus or lower
eukaryote;
[0030] the first subset of probes set having sufficient members to
be capable of distinguishing the preselected bacterium, virus, or
lower eukaryote from other bacteria, viruses, or lower eukaryotes
present in the preparation; and
[0031] (B) each member of the second subset of probes comprises a
plurality of an oligonucleotide: (i) being linked or coupled to a
predetermined label distinguishable from the label of any other
member of the first or second subset, and (ii) being capable of
specifically hybridizing with a predetermined polynucleotide region
of the nucleic acid of the preselected bacterium, virus, or lower
eukaryote, or an extra-chromosomal copy thereof so as to permit the
determination of whether the preselected bacterium, virus, or lower
eukaryote exhibits a preselected phenotype.
[0032] (II) for each member of the first subset of probes
hybridized to a region of a chromosome of a preselected bacterium,
virus or lower eukaryote, detecting and identifying the
predetermined label of that member and correlating the identity of
the label of that member with the identity of the bacterium, virus
or lower eukaryote with which that member specifically hybridizes,
to thereby identify the bacterium, virus or lower eukaryote
hybridized to the member;
[0033] (III) repeating step (II) for each member of the first
subset of probes;
[0034] (IV) for each member of the second subset of probes
hybridized to a predetermined polynucleotide region of the
chromosome of the preselected bacterium, virus, or lower eukaryote,
or an extra-chromosomal copy thereof, detecting and identifying the
predetermined label of that member and correlating the identity of
the label of that member with the identity of the predetermined
region, to thereby identify the presence of the predetermined
region on a chromosome of the preselected bacteria, virus or lower
eukaryote;
[0035] (V) repeating step (IV) for each member of the second subset
of probes.
[0036] The invention particularly contemplates the embodiments in
which the members of the above sets of probes are detectably
labeled with fluorophores, and, wherein at least one member of the
set is combinatorially labeled with either one, two, three, four or
five fluorophores selected from the group consisting of the
fluorophores FITC, Cy3, Cy3.5, Cy5, Cy5.5 and Cy7, and wherein each
member of the set is labeled with at least one fluorophore selected
from the fluorophore group.
[0037] The invention additionally provides a set of combinatorially
labeled oligonucleotide probes, each member thereof: (i) having a
predetermined label distinguishable from the label of any other
member of the set, and (ii) being capable of specifically
hybridizing with a telomeric region of one predetermined autosomal
or sex chromosome of a human karyotype; the set having sufficient
members to be capable of specifically hybridizing each autosomal or
sex chromosome of the human karyotype to at least one member.
[0038] The invention additionally provides a method of
simultaneously identifying and distinguishing the individual
autosomal and sex chromosomes of a human karyotype which comprises
the steps:
[0039] (a) contacting a preparation of the chromosomes, in
single-stranded form, under conditions sufficient to permit nucleic
acid hybridization to occur with a set of combinatorially labeled
oligonucleotide probes, each member thereof: (i) having a
predetermined label distinguishable from the label of any other
member of the set, and (ii) being capable of specifically
hybridizing with a telomeric region of one predetermined autosomal
or sex chromosome of a human karyotype; the set having sufficient
members to be capable of specifically hybridizing each autosomal or
sex chromosome of the human karyotype to at least one member,
wherein the contacting thereby causes at least one of each
autosomal or sex chromosome of the preparation to become hybridized
to at least one member of the set of probes;
[0040] (b) for each chromosome of the preparation hybridized to a
member of the set of probes, detecting and identifying the
predetermined label of that member and correlating the identity of
the label of that member with the identity of the autosomal or sex
chromosome of the human karyotype with which that member
specifically hybridizes, to thereby identify the chromosome
hybridized to the member, and
[0041] (c) repeating step (b) until each autosomal and sex
chromosome of the human karyotype has been identified in the
preparation.
BRIEF DESCRIPTION OF THE FIGURES
[0042] FIG. 1 provides a schematic illustration of a CCD camera and
microscope employed in accordance with the present methods.
[0043] FIG. 2 shows the raw data from a karyotypic analysis of
chromosomes from a bone marrow patient (BM2486). Adjacent to each
source image is a chromosome "mask" generated by the software
program. In FIG. 2, panels A and B are the DAPI image and mask;
panels C and D are FITC image and mask; panels E and F are Cy3
image and mask; panels G and H are Cy3.5 image and mask; panels I
and J are Cy5 image and mask; and panels K and L are Cy7 image and
mask.
[0044] FIGS. 3A and 3B show the identification of individual
chromosomes by spectral signature of patient BM2486. FIG. 2 is the
same photograph as FIG. 3A, except that it is gray scale
pseudocolored. FIG. 3B displays the karyotypic array of the
chromosomes.
[0045] FIG. 4 shows the differentiation of bacteria by in situ
hybridization. Panels A-E represent in situ hybridization assay
results on a laboratory derived mixture of three bacteria (F.
nucleatum, A. actinomycetemcomitans and E. corrodens) using a
mixture of genomic DNA probes for each organism. Panel A shows all
the bacteria present in the microscope field detected by (DAPI), a
general DNA binding fluorophore. Panel B shows F. nucleatum using
Cascade Blue detection. Panel C shows A. actinomycetemcomitans
using FITC detection. Panel D shows E. corrodens using Rhodamine
detection. Panel E is a computer-merged composite of panels B-D
showing the differentiation of each bacteria in the sample. Panel F
is a similar analysis of a mixture of C. gingivalis (using
rhodamine detection) and P. intermedia (using FITC detection) when
hybridized with a combination of genomic DNA probes for those
organisms. Panel G shows an in situ hybridization assay for a
combination of seven different bacteria hybridized with a mixture
of seven genomic DNA probes. In panel G, the numbers (1)-(7)
identify the bacteria. F. nucleatum (1), E. corrodens (2) and A.
actinomycetemcomitans (3) are shown in blue (Cascade Blue
detection). P. gingivalis (4) and C. ochracea (5) are shown in red
(Rhodamine detection), whereas P. intermedia (6) and C. gingivalis
(7) are seen in yellow (FITC detection). Panel H represents an in
situ hybridization assay for the presence of A.
actinomycetemcomitans (using FITC detection) in a plaque sample
obtained from a patient with localized juvenile periodontist.
[0046] FIG. 5A shows a normal male metaphase chromosomal spread
after hybridization with a 24-color set of telomere-specific
probes, shown as a pseudocolorized image. FIG. 5B shows the final
karyotype generated on the basis of the boolean spectral signature
of the telomere-specific probes.
[0047] FIG. 6A shows the hybridization pattern of the chromosome 8
subtelomeric YACs (telomere-specific probes) on a normal metaphase
chromosomal pread. FIG. 6B shows the hybridization pattern of the
same probes (as those used in FIG. 6A) on a metaphase chromosomal
spread from a patient with a myeloproliferative disorder. Previous
cytogenetic analysis of this patient using G-banding revealed a
trisomy 8 as the only change; the M-FISH telomere-specific probes
show a split telomere signal on the short arms of two of the three
chromosome 8, indicating an additional change: an inversion in this
region.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0048] A. Overview of the Invention
[0049] Fluorescence in situ hybridization (FISH) is used in a
variety of areas of research and clinical diagnostics (Gray, J. W.
et al., Curr Opin Biotech 3:623-631 (1992); Xing, Y. et al., In:
The Causes and Consequences of Chromosomal Aberrations. I. R.
Kirsch Ed. CRC Press, Boca Raton, pages 3-28 (1993)). For the study
of the chromosomal and suprachromosomal organization of the cell
nucleus it is an indispensable tool (Cremer, T. et al., In: Cold
Spring Harbor Symposia on Quantitative Biology, Volume LVIII, pp.
777-792, Cold Spring Harbor Laboratory Press, NY (1994)). Most
importantly FISH offers the capacity for multiparameter
discrimination. This allows the simultaneous visualization of
several DNA probes using either a combinatorial (Nederlof, P. M. et
al., Cytometry 10:20-27 (1989); Nederlof, P. M. et al., Cytometry
11:126-131 (1990); Ried, T. et al., Proc Natl Acad Sci (U.S.A.)
89:1388-1392 (1992a); Ried, T. et al., Hum Mol Genet 1:307-313
(1992b); Lengauer, C. et al., Hum Mol Genet 2:505-512 (1993); Popp,
S. et al., Human Genetics 92:527-532 (1993); Wiegant, J. et al.,
Cytogenet Cell Genet 63:73-76 (1993)) or a ratio labeling
(Dauwerse, J. G. et al., Hum Mol Genet 1:593-598(1992); Nederlof,
P. M. et al., Cytometry 13:839-845 (1992); du Manoir, S. et al.,
Hum Genet 90:590-610 (1993)) strategy. Up to twelve DNA probes have
been visualized (Dauwerse, J. G. et al., Hum Mol Genet 1:593-598
(1992)). Consequently, the goal of 24 different colors has long
been sought (Ledbetter, D. H., Hum Mol Genet 5:297-299 (1992)).
Twenty-four different colors are an important threshold because
they would allow the simultaneous visualization of the 22 autosomes
and both sex chromosomes. Beside improved karyotyping, the
possibility of simultaneously hybridizing 24 different and
distinguishable DNA probes would allow the addressing of a large
number of important biological questions. However, the previously
published multicolor systems lacked the versatility for an
extension to 24 colors and only proof-of-principle experiments were
ever published.
[0050] The present invention results, in part, from the realization
of multiparametric fluorescence in situ hybridization to achieve
the simultaneous visualization of 24 different genetic targets with
a combinatorial labeling strategy. This strategy permits
discrimination between many more target sequences than there are
spectrally distinguishable labels. The simplest way to implement
such labeling is using a simple "Boolean" combination, i.e., a
fluor is either completely absent (i.e. the value of "0" will be
assigned) or present in unit amount (value of 1). For a single
fluor A, there is only one useful combination (A=1) and for two
fluors A and B, there are 3 useful combinations (A=1/B=0; A=0/B=1;
A=1/B=1). There are 7 combinations of 3 fluors, 15 combinations of
4 fluors, 31 combinations of 5 fluors, 63 combinations of 6 fluors,
and so on (n fluorophores permitting the labeling of 2.sup.n-1
chromosomes). Thus, to uniquely identify all 24 chromosome types in
the human genome using chromosome painting probe sets, only 5
distinguishable fluors are needed (31 total combinations). If each
probe set is labeled with one or more of five spectrally distinct
fluorophores in a combinatorial fashion, simple Boolean combination
can be used to identify each DNA probe by a spectral signature
dictated by its fluorophore composition (Speicher, M. R. et al,
Nature Genet 12:368-375 (1996), which reference is herein
incorporated by reference).
[0051] B. Terminology of the Invention
[0052] The invention concerns a set of combinatorially labeled
oligonucleotide probes, each member thereof: (i) having a
predetermined label distinguishable from the label of any other
member of the set, and (ii) being capable of specifically
hybridizing with one predetermined autosomal or sex chromosome of a
human karyotype. In the most preferred embodiment, the set will
have a sufficient number of members to be capable of specifically
and distinguishably hybridizing each autosomal or sex chromosome of
said human karyotype to at least one member. As used herein, the
term "karyotype" denotes the compliment of chromosomes found in a
normal or aberrant cell. In a normal cells, the number of
chromosomes is 46, comprising 22 pairs of autosomal chromosomes and
2 sex chromosomes (either 2 X chromosomes (if female) or an X and Y
chromosome (if male)). The labels are said to be distinguishable in
that the particular label of any one member of the set (and the
identity of that member) differ from the particular label and
identity of any other member of the set. Since each probe member is
capable of specifically hybridizing to only one chromosome (or
sub-chromosomal region) and since the identity of the label and
probe are known in advance, the detection of a particular label
associated with an unidentified chromosomal region means that the
probe bearing that label has become hybridized to the unidentified
chromosomal region. Since the chromosome to which that probe
specifically hybridizes is known, the detection of a
distinguishable label permits the identification of the chromosomal
region.
[0053] More specifically, the invention concerns fluors that can be
used to label oligonucleotide probes so that such probes may be
used in multiparametric fluorescence in situ hybridization. As used
herein, a "fluor" or "fluorophore" is a reagent capable of emitting
a detectable fluorescent signal upon excitation. Most preferably,
the fluor is coupled directly to the pyrimidine or purine ring of
the nucleotides of the probe (Ried, T. et al. (Proc. Natl. Acad.
Sci. (U.S.A.) 89:1388-1392 (1992), herein incorporated by
reference; U.S. Pat. Nos. 4,687,732; 4,711,955; 5,328,824; and
5,449,767, each herein incorporated by reference. Alternatively,
the fluor may be indirectly coupled to the nucleotide, as for
example, by conjugating the fluor to a ligand capable of binding to
a modified nucleotide residue. The most preferred ligands for this
purpose are avidin, streptavidin, biotin-binding antibodies and
digoxigenin-binding antibodies. Methods for performing such
conjugation are described by Pinkel, D. et al., Proc. Nat'l. Acad.
Sci. (U.S.A.) 83:2934-2938 (1986), herein incorporated by
reference).
[0054] The term "multiparametric fluorescence" denotes the
combinatorial use of multiple fluors to simultaneously label the
same chromosome or sub-chromosomal fragment, and their detection
and characterization. Chromosomes or sub-chromosomal fragments are
said to be simultaneously labeled if they are exposed to more than
a single chromosome-specific probe under conditions sufficient to
permit each chromosome-specific probe to independently hybridize to
its target chromosome. As used herein, it is thus unnecessary for
all such hybridization reactions to commence and conclude at the
same instant. The simultaneous labeling permitted by the present
invention is thus in contrast to protocols in which chromosomes are
exposed to only a single chromosome-specific probe at a time.
[0055] The simultaneous detection and characterization permitted by
the present invention denotes an ability to detect multiple (and
most preferably all) of the autosomal and/or sex chromosomes in a
sample, without any need to add further reagent, or probe after the
detection of the first chromosome.
[0056] In the simplest embodiment, digital images of the
chromosomes are obtained for each fluorophore employed, thereby
providing a series of gray scale fluorescence intensities
associated with each fluorophore and each chromosome. The final
image is obtained by pseudocoloring the blended gray scale
intensities for each chromosome.
[0057] The invention thus provides a method of simultaneously
identifying and distinguishing the individual autosomal and sex
chromosomes of a human karyotype which comprises contacting a
preparation of chromosomes, that has been previously treated to
render it in single-stranded form, with the above-described set of
combinatorially labeled oligonucleotide probes, under conditions
sufficient to permit nucleic acid hybridization to occur.
[0058] Such treatment causes at least one of each autosomal or sex
chromosome of the preparation to become hybridized to at least one
member of said set of probes. For each chromosome of the
preparation hybridized to a member of the set of probes one next
detects and identifies the predetermined label of that member and
correlates the identity of the label of that member with the
identity of the autosomal or sex chromosome of said human karyotype
with which that member specifically hybridizes. This process
identifies the chromosome hybridized to the member. This last step
is repeated until each or a desired number of autosomal and sex
chromosome of the human karyotype has been identified in the
preparation.
[0059] The oligonucleotide probes used in accordance with the
methods of the present invention are of either of two general
characteristics. In one embodiment, such probes are chromosome or
sub-chromosome specific (i.e., they hybridize to DNA of a
particular chromosome at lower c.sub.Ot.sup.1/2 than with DNA of
other chromosomes; c.sub.Ot.sup.1/2 being the time required for one
half of an initial concentration (c.sub.O) of probe to hybridize to
its complement). Alternatively, such probes are feature (e.g.,
telomere, centromere, etc.) specific. Such probes, being proximal
to the telomere, are capable of defining and identifying
translocations that may be so close to the chromosomal termini as
to be otherwise cryptic.
[0060] Both types of probes may be used if desired. Sources of such
probes are available from the American Type Culture Collection, and
similar depositories.
[0061] The oligonucleotide probes used in accordance with the
methods of the present invention are of a size sufficient to permit
probe penetration and to optimize reannealing hybridization. In
general, labeled DNA fragments smaller than 500 nucleotides in
length, and more preferably of approximately 150-250 nucleotides in
length, probes are employed. Probes of such length can be made by
synthetic or semi-synthetic means, or can be obtained from longer
polynucleotides using restriction endonucleases or other techniques
suitable for fragmenting DNA molecules. Alternatively, longer
probes (such as polynucleotides) may be employed.
[0062] Most preferably, the oligonucleotide probes are synthesized
so as to contain biotinylated or otherwise modified nucleotide
residues. Methods for accomplishing such biotinylation or
modification are described in U.S. Pat. Nos. 4,687,732; 4,711,955;
5,328,824; and 5,449,767, each herein incorporated by reference.
Biotinylated nucleotides and probes are obtainable from Enzo
Biochem, Boehringer Mannheim, Amersham and other companies. In
brief, such biotinylated or otherwise modified nucleotides are
produced by reacting a nucleoside or nucleotide with a mercuric
salt under conditions sufficient to form a mercurated nucleoside or
nucleotide derivative. The mercurated product is then reacted in
the presence of a palladium catalyst with a moiety (e.g., a biotin
group) having a reactive terminal group and comprising three or
more carbon atoms. This reaction adds the moiety to the purine or
pyrimidine ring of the nucleoside or nucleotide.
[0063] In a highly preferred embodiment, such modified probes are
used in conjunction with competitor DNA in the manner described by
Ward et al. (WO90/05789), herein incorporated by reference.
Competitor DNA is DNA that acts to suppress hybridization signals
from ubiquitous repeated sequences present in human and other
mammalian DNAs. In the case of human DNA, alu or kpn fragments can
be employed, as described by Ward et al. (WO90/05789). Initially,
probe DNA bearing a detectable label and competitor DNA are
combined under conditions sufficient to permit hybridization to
occur between molecules having complementary sequences. As used
herein, two sequences are said to be able to hybridize to one
another if they are complementary and are thus capable of forming a
stable anti-parallel double-stranded nucleic acid structure.
Conditions of nucleic acid hybridization suitable for forming such
double stranded structures are described by Maniatis, T., et al.
(In: Molecular Cloning, A Laboratory Manual, Cold Spring Harbor
Laboratories, Cold Spring Harbor, N.Y. (1982)), by Haymes, B. D.,
et al. (In: Nucleic Acid Hybridization, A Practical Approach, IRL
Press, Washington, D.C. (1985), and by Ried, T. et al. (Proc. Natl.
Acad. Sci. (U.S.A.) 89:1388-1392 (1992)). For the purpose of the
present invention, the sequences need not exhibit precise
complementarity, but need only be sufficiently complementary in
sequence to be able to form a stable double-stranded structure.
Thus, departures from complete complementarity are permissible, so
long as such departures are not sufficient to completely preclude
hybridization and formation of a double-stranded structure.
[0064] The quantity of probe DNA combined with competitor DNA is
adjusted to reflect the relative DNA content of the chromosome
target. For example, as disclosed by Ward et al. (WO90/05789),
chromosome 1 contains approximately 5.3 times as much DNA as is
present in chromosome 21. Thus, a proportionally higher probe
concentration would be employed when using chromosome 1 specific
probes.
[0065] The resulting hybridization mixture is then treated (e.g.,
by heating) to denature the DNA present and is incubated at
approximately 37.degree. C. for a time sufficient to promote
partial reannealing. The sample containing chromosomal DNA to be
identified is also heated to render it susceptible to being
hybridized to the probe. The hybridization mixture and the sample
are then combined, under conditions sufficient to permit
hybridization to occur. Thereafter, the detection and analysis of
the hybridized product is conducted by detecting the fluorophore
label of the probe in any of the methods described below.
[0066] In an alternative embodiment, a modification of the method
of Ried T. et al. (Proc. Natl. Acad. Sci. (U.S.A.) 89:1388-1392
(1992), herein incorporated by reference) is employed. Thus, probes
are labeled either directly (e.g., with fluorescein) or indirectly
(e.g., with biotinylated nucleotides or other types of labels), and
permitted to hybridize to chromosomal DNA. After hybridization, the
hybridized complexes are incubated in the presence of streptavidin,
that had been conjugated to one or more fluors. The streptavidin
binds to the biotinylated probe of the hybridized complex thereby
permitting detection of the complex, as described below.
[0067] C. The Preferred Fluorophores of the Invention
[0068] By labeling with two or more fluors in combination, it is
possible to discriminate between many more objects than there are
available fluors. The simplest way to implement such labeling is by
Boolean combination, i.e., a fluor is either completely absent (0)
or present in unit amount (1). For a single fluor A, there is only
one useful combination (A=1). For two fluors A, B there are 3
useful combinations (A=1, B=0; A=0, B=1; A=1, B=1). For three
fluors A, B, C, there are 7 combinations (A=1, B=0, C=0; A=0, B=1,
C=0; A=0, B=0, C=1; A=1, B=1, C=0; A=1, B=0, C=1; A=0, B=1, C=1;
A=1, B=1, C=1). There are 15 combinations of 4 fluors, 31
combinations of 5 fluors, 63 combinations of 6 fluors, and so
on.
[0069] To uniquely code all 24 chromosome types in the human
genome, 5 distinguishable combinatorial fluors are needed. With a
5-fluor set, 15 chromosomes can be distinguished using combinations
of 4 of the 5 fluors. The labeling of the remaining 9 chromosomes
requires all five fluors to be used combinatorially. Seven of the
available 5-fluor combinations are not required. Thus, there is a
certain amount of latitude available to avoid any 5-fluor
combination that might prove particularly hard to resolve. In
particular, quaternary or quinternary combinations may be
avoided.
[0070] One aspect of the present invention concerns the
identification of a set of seven fluors that are be well resolvable
by the excitation-emission contrast (EEC) method.
[0071] As indicated above, multi-fluor combinatorial labeling
depends in general on acquiring and analyzing the spectral
signature of each object i.e., obtaining the relative weighting
coefficients of the component fluors. Because full spectroscopic
analysis, of mixed fluor spectra (e.g., by interferometry) is not
yet sufficiently developed, the method chosen was conventional
bandwidth-restricted widefield imaging using epi-fluorescence
triplets, viz. excitation filter, dichroic reflector and emission
bandpass filter. The limited spectral bandwidth available for
imaging (roughly 380-750 nm), and the extensive overlap between the
spectra of organic fluors, makes separating multiple fluors
spectroscopically during the imaging step a significant technical
challenge.
[0072] To make software segmentation of the source images as
straightforward as practicably possible, a target figure of <10%
crosstalk between any given fluor and the two adjacent channels was
set. Computer modeling indicated that for DAPI plus the five
combinatorial fluors FITC, Cy3, Cy3.5, Cy5, Cy5.5, this level of
contrast cannot be attained using either excitation selection or
emission selection alone, no matter how narrow the filter
bandwidths. Thus, both excitation selection and emission selection
must be invoked simultaneously. This is referred to as
excitation-emission contrast (EEC).
[0073] Contrast ratio plots were first computed for each of the
fluors vs. its two neighbors. These plots indicate regions where
pairwise contrast is high enough to be useful. A constraint on the
practically attainable contrast is that regions of high contrast
generally lie far down the flanks of at least one of the spectra
i.e., where excitation and/or emission are strongly sub-optimum.
Further, to attain the required degree of selectivity it is
necessary to use filters of bandwidths in the range 5-15 nm (cf.
approx. 50 nm for `standard` filter sets). Together, these impose a
severe sensitivity penalty. The goal of 10% maximum crosstalk
represents an acceptable, practical compromise between sensitivity
and selectivity.
[0074] A fundamental asymmetry exists between excitation contrast
and emission contrast. For low-noise detectors such as the cooled
CCD, restricting the excitation bandwidth has little effect on
attainable image S/N ratio; the only penalty is the need for longer
exposure times. Restricting the emission bandwidth is very
undesirable, however, since every fluorescence photon blocked by
the filter represents irreversible photochemical bleaching of the
fluor. For this reason, the highest practicable contrast was
invoked on the excitation side. A suitable emission filter was then
found to give the necessary EEC ratios. Filter selection is
additionally constrained by the fact that each channel must
adequately reject both adjacent channels simultaneously: improving
one may significantly degrade the other. Good contrast was
attainable in practice for all fluors except the Cy5.5/Cy5 pair,
which is marginal. For this reason, Cy7 was later substituted for
Cy5.5. Other considerations relating to choice of filters
include:
[0075] 1. Commercial narrow-band interference filters may have a
large amount of wedging i.e., non-parallelism between the top and
bottom faces. This results in large image shifts (up to several
microns equivalent). The shift is a vector characteristic of each
filter and its orientation in the epicube. Thus, automatic
compensation for image displacement is a necessary part of the
processing software.
[0076] 2. Manufacturing variations of a few nm in peak wavelength
and FWHM specifications can have significant effects on the EEC
ratios. Filter errors to long wavelength may be fine-tuned by
tilting, but this option is severely curtailed in the case of
emission filters because of increased image aberrations and
worsened pixel shifts. There is no equivalent way to compensate
short-wavelength errors.
[0077] 3. the need to prevent infra-red light emitted by the arc
lamp from reaching the detector. Silicon CCD's are extremely
sensitive in this region. Filter sets for the blue and midvisible
fluors were found not to need additional IR blocking, but loss of
image contrast due to spurious IR was found to be a serious problem
for the red--far red--near IR fluors. Heat filters routinely used
in microscopy (e.g., Schott BG-38 glass) are completely inadequate
to alleviate this problem. Thus, extensive additional blocking was
required. However, available commercial interference filters for
infra-red blocking filters also transmit poorly in the near UV, and
thus cannot be inserted in the excitation path. Instead, it was
found necessary to put the IR blocking filters into the emission
path. To minimize loss of image quality by insertion of these
filters in the image path, they are placed inside the CCD camera,
immediately in front of the window. In practice, two
interchangeable filters were chosen, one for use with Cy5, Cy5.5
(Oriel #58893; 740 nm cutoff) and one for use with Cy7 (Oriel
58895; 790 nm cutoff).
[0078] The first member of the set of fluors is the counterstain
DAPI, which gives a weak G-like banding pattern. Five of the
remaining six fluors may be used combinatorially to paint the
entire human chromosome set. All are available as avidin conjugates
(for secondary detection of biotinylated probe libraries) or
directly linked to dUTP (for direct labeling).
[0079] Thus, a set of six fluors and corresponding optical filters
spaced across the spectral interval 350-750 nm was identified that
achieve a high discrimination between all possible fluor pairs.
These fluors comprise the preferred fluors of the present invention
and are: 4'-6-diamidino 2-phenyl indole (DAPI), fluorescein (FITC),
and the new generation cyanine dyes Cy3, Cy3.5, Cy5, Cy5.5 and Cy7.
Of these Cy3, Cy3.5, Cy5 and Cy7 are particularly preferred, The
absorption and emission maxima for the respective fluors are: DAPI
(Absorption maximum: 350 nm; Emission maximum: 456 nm), FITC
(Absorption maximum: 490 nm; Emission maximum: 520 nm), Cy3
(Absorption maximum: 554 nm; Emission maximum: 568 nm), Cy3.5
(Absorption maximum: 581 nm; Emission maximum: 588 nm), Cy5
(Absorption maximum: 652 nm; Emission maximum: 672 nm), Cy7
(Absorption maximum: 755 nm; Emission maximum: 778 nm). Complete
properties of selected fluorescent labeling reagents are provided
by Waggoner, A. (Methods in Enzymology 246:362-373 (1995) herein
incorporated by reference). In light of the above, it is readily
apparent that other fluorophores and filter combinations having
adequate spectral resolution can alternatively be employed in
accordance with the methods of the present invention.
[0080] D. Methods for the Detection of Fluorescent In situ
Hybridization
[0081] 1. The Theory of Fluorescence Detection
[0082] Of the various methods for contrast generation in
site-specific labeling, fluorescence is arguably the most powerful,
because of its high absolute sensitivity and multiparameter
discrimination capability. Modem electronic cameras used in
combination with high numerical aperture microscope objectives and
state of the art optical filters are capable of imaging structures
labeled with as little as a 10-100 fluor molecules per pixel. Thus,
fluor-tagged single-copy DNA sequences as small as a few hundred
bases in size are detectable under favorable conditions. The
availability of families of spectrally distinguishable fluors makes
simultaneous imaging of several different targets in the same
specimen possible, either directly or through combinatorial or
analog multiplex methods. In principle, multi-fluor discrimination
may be based on differential excitation of the fluors, differential
emission, fluorescence lifetime differences, or on more complex but
still analyzable observables such as fluorescence anisotropy. This
discussion assumes an epi-imaging geometry. Table 1 describes the
symbols and operators relevant to the theoretical considerations of
fluorescence.
1 TABLE 1 Units Definition Instrument Parameter
.psi..sub.s(.lambda.) photon s.sup.-1 nm.sup.-1 spectral
distribution of source, assumed to be an isotropic radiator over
4.pi. steradians .phi..sub.s dimensionless collection of efficiency
of condenser optics .psi.1(.lambda.) photon s.sup.-1 m.sup.-2
nm.sup.-1 spectral distribution of photons in collimated beam of
excitation light impinging on excitation filter F1 f1(.lambda.)
dimensionless transmittance function of excitation filter F1
.psi.2(.lambda.) photon s.sup.-1 cm.sup.-2 nm.sup.-1 spectral
distribution of photons in collimated beam of excitation light
emerging from F1 and impinging on dichroic beamsplitter DB1
R(.lambda.) dimensionless reflectance function of dichroic
beamsplitter DB1 T(.lambda.) dimensionless transmittance function
of dichroic beamsplitter DB1 .psi.3(.lambda.) photon s.sup.-1
cm.sup.-2 nm.sup.-1 spectral distribution of photons in collimated
beam of fluorescence emerging from DB1 and impinging on emission
filter F2 f2(.lambda.) dimensionless transmittance function of
emission filter F2 .psi.4(.lambda.) photon s.sup.-1 cm.sup.-2
nm.sup.-1 spectral distribution of photons in collimated beam of
excitation light emerging from F2 and entering the entrance pupil
of the objective lens .mu. dimensionless linear magnification
factor of objective lens .psi.5(.lambda.) photon s.sup.-1 cm.sup.-2
nm.sup.-1 spectral distribution of photons in focused beam of
excitation light traversing the specimen plane,
.phi..sub.d(.lambda.) dimensionless quantum efficiency of detector
.OMEGA. pixel..mu..sup.-1 magnification factor of final image at
detector Fluor Parameter .epsilon.(.lambda.) M.sup.-1 cm.sup.-1
molar decadic extinction coefficient of fluor F.sub.a. .tau. s
decay lifetime of excited state of fluor F.sub.a.
.sigma..sub.Fa(.lambda.) cm.sup.-1 photon absorption cross-section
of fluor F.sub.a. .phi.Fa dimensionless fluorescence quantum
efficiency of fluor F.sub.a. .function. .sub.a(.lambda.)
dimensionless normalized spectral distribution of fluorescence from
fluor F.sub.a.
[0083] Whether a given excitation rate at pixel location p will
give an acceptable fluorescence signal/noise ratio (defined as
S/N=[signal mean]/[variance due to all noise sources]) in a given
integration period depends on the number of fluor molecules within
p, their quantum yield and photochemical stability, and the quantum
efficiency and noise performance of the detector.
[0084] In the limit of weak excitation
(.psi..sup.(.lambda.)..sigma..sub.F-
.sup.(.lambda.)<<.tau..sup.-1), the rate of excitation of the
N molecules of fluor F within pixel p in object space is: 1 R ( p )
= N i 5 ( , p ) F ( ) s - 1
[0085] .psi..sup.5(.lambda.) is the spectral distribution of
exciting light passing through the focal plane of the microscope
(photon nm.sup.-1.cm.sup.-2.s.sup.-1). It is approximately given by
.psi..sub.s.sup.(80)..phi..sub.s.f1(.lambda.).R(.lambda.)..alpha..sup.2..-
mu., where .alpha.=diameter of objective lens entry pupil/diameter
of collimated beam from condenser. The integral is taken over the
bandwidth (i) for which the fluor has non-zero absorption cross
section .pi..sub.F.sup.(.lambda.), which is related to the molar
decadic extinction coefficient .epsilon..sub.F(.lambda.) by the
expression:
.sigma..sub.F.sup.(.lambda.)=3.825.times.10.sup.-21.epsilon..sub.F(.lambda-
.)cm.sup.2.
[0086] In practice, .intg..sub.i.psi..sup.5(.lambda.,p).d.lambda.
can be measured with a bolometric detector such as a calibrated
micro-thermopile placed at or near the focal plane of the objective
lens.
[0087] For a perfectly noiseless detector and a non-bleachable
fluor, the S/N of each pixel increases indefinitely as (photons
detected).sup.1/2. How rapidly S/N increases depends on excitation
strength, but the relationship between S/N and dose does not. The
effect of non-zero bleaching constant is to change the t.sup.1/2
function to an asymptotic function, the form of which depends on
the bleaching mechanism. However, because the bleaching rate and
the signal strength are linearly related, the asymptote once again
does not depend at all on excitation rate, although the speed of
approach to the asymptote does. If finite camera noise is added to
photobleaching, it is found that the S/N climbs to a maximum value,
then falls as the fluor is exhausted. Now both the kinetics and the
peak S/N depend of excitation rate, in general the faster the
excitation the higher the maximum attainable S/N. However, for
contemporary cooled CCD cameras the dark noise is so low that it
can be virtually ignored on the timescale of bleaching (typically a
few minutes); a more important factor in determining ultimate S/N
is the stray light background (esp. from nonspecific luminescences
and leakage of excitation light).
[0088] Note that although the microscope objective compresses the
excitation beam, in a nonconfocal microscope it does not focus it
to a point and so has no bearing on the fluorescence image
resolving power (incoherent emitter).
[0089] The commonest excitation source for fluorescence microscopy
is the high pressure short mercury arc, whose spectrum consists of
pressure-broadened lines from the UV to the middle region of the
visible spectrum (principal wavelengths are 334.1 nm, 365.6 nm,
404.7 mm, 435.8 nm, 546.1 nm. 577.9 nm), superimposed on a weaker
thermal continuum. Many fluorophores have excitation spectra that
overlap one or other of the mercury lines to an acceptable extent.
Others (of which the best known is FITC) do not, but may be
adequately excited by the continuum if a wide enough excitation
bandwidth is employed. Another common source is the high pressure
xenon short arc, which produces an almost uniform continuum from
ca. 300 nm: to beyond 900 nm. However, the power nm.sup.-1, is
almost everywhere less than the mercury continuum for the same arc
wattage. If high intensity light with no structure is required
(e.g., for fluorescence ratio imaging) a high CW power or pulsed
quartz halogen lamp outperforms the xenon beyond about 450 nm.
Certain fluors are well matched to laser excitation (e.g., Ar+@488
nm for FITC, He--Ne @632.8 nm for Cy5, semiconductor diode--pumped
YAG @680 nm for Cy5.5).
[0090] In single-fluor imaging, use of the available spectral
bandwidth is rarely stringent. The excitation filter Fl and
dichroic beamsplitter DBl can usually be chosen to give adequate
overlap between the source spectrum and the fluor excitation
spectrum. If an arc line is available, the Fl bandpass need be no
wider than the line. If not, and part of the thermal continuum must
be used, the wider the Fl bandpass the greater will be the
available excitation flux. However, with low noise integrating
detectors the goal of high excitation efficiency is generally
secondary to the need to exclude excitation light from the emission
path. This limits how close the excitation and emission bandpasses
can be placed to one another, and hence constrains the excitation
bandwidth. For fluors with small Stokes' shifts, high quality
filters with very steep skirts are required. The excitation filter
must be rigorously `blocked` on the long wavelength side, and have
no pinholes, scratches, or light leaks around the edge.
[0091] Dichroic beamsplitters are currently much less `evolved`
than bandpass interference filters, meaning that the slopes of
their transmit <-> reflect transitions are far less than the
skirt slopes of premium notch filters, and there may be large
spectral intervals where they oscillate between intermediate states
of partial reflectance and transmittance. The main purpose of a
dichroic beamsplitter is to improve the combined efficiency of
excitation and emission, rather than to define the wavelength
response of the instrument.
[0092] The resolvability of overlapping fluors in imaging
microscopy may depend critically on the degree of excitation
contrast that can be achieved (see C, below). The variation with
wavelength of the ratio of the extinction coefficient of two fluors
is the excitation contrast spectrum. It can readily be calculated
from the digitized absorption spectra. Depending on the overlap of
the absorptions, their ratio spectrum may either show a distinct
peak or may grow indefinitely large. In either case, it is usually
possible to choose an excitation wavelength that favors one fluor
over another to a useful extent (from a factor of 3-4 fold up to a
hundredfold or more). Some difficulties in obtaining high contrast
multi-fluor images include:
[0093] a. The excitation wavelengths required for high contrast
imaging are often far from the absorbance peaks. Thus, there may be
a high degree of intensity trade-off to obtain high signal contrast
vs. other fluors.
[0094] b. From the above, standard filter sets cannot be used.
[0095] c. Arc source spectral lines that are useful for exciting
single fluors may not give high contrast discrimination against
adjacent fluors.
[0096] d. When using a broadband source or the continuum spectrum
of an arc source, the need for narrow excitation bandwidth may
reduce the excitation flux to problematically low levels. It is
sometimes possible to relax the constraints on excitation
wavelength for the sake of more efficient excitation.
[0097] Collecting the fluorescence of F and imaging it onto the
detector with high efficiency is the principal design goal of the
emission optics. Operationally, it is even more important to have
an efficient emission path than an efficient excitation path. The
reason is that inefficient excitation causes inefficient
photobleaching; the only penalty is a long image integration time
(assuming a low-noise detector). On the other hand, any
fluorescence lost on its way to the detector represents
photobleaching without concomitant increase in the information
content of the image.
[0098] The detector pixel p accumulates signal (detected photons)
at a rate: 2 F ( p ) = G R ( p ) F ii T ( ) f2 ( ) d ( ) s - 1
[0099] The integral is over the bandwidth (ii) for which the
detector quantum efficiency .phi..sub.d(.lambda.)>0. G
represents the efficiency with which the optics gather the
fluorescence and transmit it to the detector; it may be assumed to
be wavelength independent to first order. The principal factor in G
is the numerical aperture (NA) of the objective lens, which
determines the fraction of the isotropically radiated fluorescence
collected by the imaging system (.sctn.=1/.pi..
sin.sup.-1(.theta.)=1/.pi.. sin.sup.-1(NA/n), where .theta. is the
half-angle subtended by the objective lens from its focal point.
For an oil immersion lens with NA=1.3; n=1.515; .sctn.=0.328). NA
additionally determines the spatial resolving power of the
microscope, because it scales the dimensions of the Frauenhofer
diffraction pattern produced in the image plane by a point source
in the specimen plane. Several `rules` are in use for specifying
the resolving power of a lens, depending on how much overlap of the
Airy discs of two adjacent objects is deemed to constitute the
threshold of resolution. The commonly used Rayleigh criterion is
r=1.22.lambda./2NA (e.g., for the above NA=1.3 lens working at 500
nm, r=0.24 .mu.).
[0100] For a noise-free detector, image `noise` at p is determined
by the statistical variance in the number S(p) of fluorescent
photons detected in time interval .DELTA.t. The only detector
characteristic that has any bearing on this is its quantum
efficiency .PHI.d(.lambda.).
[0101] In the absence of photobleaching (probability of destruction
per hit .PI..sub.b=0), S(p)=F(p)..DELTA.t, with variance
S(P).sup.1/2, i.e., S/N=S(p).sup.1/2. Image quality will therefore
increase indefinitely with .DELTA.t, though at an ever decreasing
rate.
[0102] In the presence of photobleaching (.PI..sub.b.noteq.0), S(p)
is an integral of the form: 3 S ( p ) = F ( p ) t f ( t ) t
[0103] where f(t) is the photobleaching decay function. S/N rises
along a more or less complex path to an asymptotic value that
corresponds to total exhaustion of the fluor. For the case of a
unimolecular photobleaching process this would be an exponential
function. i.e., N(p,t)=N.sub.0(1-exp-[k.sub.b/.SIGMA.k].t) where
k.sub.b=.PI..sub.b.sup.-- 1 is the photobleaching constant and
.SIGMA.k represents all other processes by which the excited state
of F is deactivated. Note that the normalized asymptote in this
first-order system depends only on k.sub.b/.SIGMA.k, and is
independent of the strength of the excitation. Thus, the extent of
bleaching is exponentially related to the accumulated excitation
dose, but is independent of the path. In reality, however,
bleaching of fluors in solution may be mechanistically and
kinetically much more complex. A common mechanism involves ring
opening following peroxidation of the fluor excited state, e.g., by
.sup.1O.sub.2 or O.sub.2.sup.2-. This type of bleaching may be
considerably slowed by rigorous deoxygenation or by the use of
oxygen radical scavengers (i.e., antifade agents) such as tertiary
amines (p-phenylene diamine or DABCO). Nevertheless, other (as yet
poorly characterized) irreversible processes are not excluded,
including reactions with impurities.
[0104] A nonideal detector contributes noise of many kinds,
detailed analysis of which may be intractable. The simplest noise
component is fluctuation in the so-called `dark current,` i.e., the
flow of thermally excited carriers within the detector. If this
noise is assumed to be random, it adds to the photon shot noise in
RMS fashion. Thus, if the mean photogenerated signal is F s.sup.-1
and the mean dark count is D s.sup.-1, the S/N after time .DELTA.t
is F.(.DELTA.t).sup.1/2/(F+D).sup.1- /2; S/N still increases as
(.DELTA.t).sup.1/2, but more slowly than for a noiseless detector.
When photobleaching is present, however, the situation is entirely
different. In this case, S/N rises initially as (.DELTA.t).sup.1/2,
but at some point reaches a shallow maximum and then begins to fall
again, as the fluorescent signal declines but the thermal noise
power remains constant. In this case, it is in principle desirable
to continuously monitor the S/N of the image, and terminate the
exposure when the peak is reached. Most commercial digital imaging
systems make no provision for this. Fortunately, state of the art
cooled CCD cameras have so little dark noise per pixel (typically
<0.01 electron s.sup.-1 in inverted clock mode) that S/N would
not begin to fall until almost complete exhaustion of the fluor. In
practice, autofluorescences and stray light dominate system
performance long before the noise threshold of the CCD is
reached.
[0105] The principal design goals for a single-fluor imaging system
are:
[0106] 1. To achieve an adequate rate of excitation of the fluor
(F).
[0107] 2. To collect the fluorescence of F and image it onto the
detector with high efficiency and with the necessary spatial
resolution.
[0108] 3. To prevent reflected and/or scattered excitation light
from reaching the detector.
[0109] Design of the emission channel for a single fluor is
straightforward. The dichroic beamsplitter transition wavelength is
specified at for example, 20 mn to the red of the excitation
passband. This ensures a high level of rejection of exciting light
reflected and/or scattered from the specimen and/or microscope
optics. The emission filter cut-on is usually considerably steeper
than the dichroic edge, and so can be placed practically coincident
with it. The most efficient emission filter is a long-pass element.
The preferred filter of this type is Schott glass, which transmits
upwards of 90% of all fluorescence to the red of its cut-on, while
rejecting other light (especially any excitation light that gets
through the dichroic beamsplitter) to very high order--typically
>10.sup.5. However, it is usually inadvisable to leave the
emission channel `wide open` into the near infrared, especially
with silicon detectors which have high sensitivity there.
[0110] The multiparametric imaging of the present invention not
only increases the throughput of information about the system under
observation and makes more efficient use of the biological
material, but also can reveal spatial and temporal correlations
that might otherwise be difficult to establish reliably. When a
large number of different objects must be visualized, two or more
labels can be used combinatorially, which permits discrimination
between many more object types than there are spectrally
distinguishable labels. Some examples of multi-fluor imaging
are:
[0111] a. The co-distribution of proteins in structures such as
microtubule networks may readily be visualized using immunolabels
linked to different fluors.
[0112] b. Multiple genes may be simultaneously mapped by
fluorescence in situ hybridization (FISH) to a single metaphase
chromosome spread. Such signals cannot usually be discriminated
reliably on the basis of intensity alone, and are usually
morphologically identical (diffraction-limited points). However,
they are readily discriminated by discrete or combinatorial
multi-fluor labeling.
[0113] c. Identification of small chromosomal translocations is
most readily done by painting with chromosome specific DNA probe
libraries linked to separable fluors, used either singly or
combinatorially.
[0114] d. Analysis of mixed populations of morphologically
identical bacteria can also be achieved using species-specific DNA
or ribosomal RNA probes coupled to separable fluors.
[0115] The primary design goal of a multi-fluor imager (in addition
to those for of a single fluor imager) is to spectrally resolve the
fluorescence at any pixel location into components corresponding to
each fluor. Methods for spectrally resolving complex signals in
fluorescent microscope images are outlined below.
[0116] There are several ways to resolve spectrally complex
signals, i.e., to determine which fluors contribute to the
fluorescence at a given pixel location. The most general method in
principle is to spectrally disperse the 2D image along a third
axis, orthogonal to the x,y axes. This amounts to imaging through
an optical system with a very large amount of chromatic aberration,
such that at each position on the z axis there is an image x,y that
contains only a small spectral bandwidth .lambda.+.DELTA..lambda..
An area detector with very small depth of field (such as a
spatially filtered confocal imager) could then be moved
incrementally along the z axis to obtain a family of images, each
containing its own small spectral interval. A trace through the
images at given x,y would constitute an emission spectrum for that
pixel. Unfortunately, implementing such a scheme is technically
very difficult.
[0117] If the spectrum of only a small number of objects within the
image is required (such as individual stars in a telescope image),
a solution is to extract the light corresponding to each object
with a probe (e.g., a fiber optic) and disperse it with an imaging
spectrograph onto a 1-dimensional array detector. To be useful in
microscopy, such a device would have to be arbitrarily positionable
in the field, and have an adjustable acceptance area.
[0118] The most reasonable method for full spectral analysis in
microscopy is to image through a variable narrow band filter. An
image is recorded at each wavelength; intensity values at a given
pixel location through the series represent a weighted emission
spectrum that can be fit to a linear combination of the known
spectra of the component fluors. The coefficients are products of
the relative molar amounts of the fluors with their extinction
coefficients at the exciting wavelength and their fluorescence
quantum yields. If the last two are known, the first is obtainable
from the fit. In general, it is necessary to take several such
image sets, at several excitation wavelengths, to get a unique fit.
With enough iterations, this process generates a 3D surface of
intensity values as a function of both excitation and emission
wavelength. This comprises a complete spectral signature of the
pixel, giving a very highly constrained solution for the relative
amounts of its component fluors. The mole ratios could be mapped
back onto the x,y coordinates of the image, with appropriate
pseudocolor coding, to give a `composition map`. This general (and
quantitative) method has a number of technical difficulties,
although none of them is insurmountable. The first is that a large
number of images are required to evaluate a single microscope
field. Imaging time is long, and extensive differential
photobleaching of the fluors would make it impossible to achieve a
self consistent "fit" to the spectral data. Instrument stability is
also an issue, particularly with arc sources, the output spectra of
which change throughout their life. Finally, the amount of
computation required to generate a composition map would
realistically limit the analysis to small image regions only.
[0119] For most applications, there is no need of a full-blown
spectral analysis capability because the fluors to be analyzed are
known ahead of time. Thus, it is only necessary to be able to
identify which fluor is which, and, for multiplex imaging, to
ascertain the relative amount of each present. A preferred approach
to this category of problems involves the use filter sets that
achieve a high degree of selective excitation and visualization of
each component fluor. The ideal system of this type would perfectly
isolate each fluorophore, i.e., "one image--one fluor" and all
off-diagonal elements in the matrix of intensity coefficients would
be zero. However, the facts that the spectra of typical
fluorophores are 10-30% of the total available bandwidth, and
emission filters must have significant bandwidths to pass usable
amounts of light place severe limitations on the attainable degree
of spectral isolation. Nevertheless, it is not difficult to achieve
useful levels of contrast between suitably chosen fluors, such that
residual crosstalk can be removed numerically.
[0120] The excitation-emission contrast (EEC) approach is in
principle applicable to analysis of images involving multiple
fluors with fine-grained distributions of mole fraction (e.g.,
fluorescence ratio imaging), subject to image S/N and the
limitations of differential bleaching rates and source instability.
However, it is particularly simple for the limiting case of
combinatorial labeling, in which fluors are multiplexed in a
strictly Boolean fashion (present=1, or absent=0). In this case, it
is necessary only to be able to reliably discriminate between a 1
and a 0, for which a 10:1 intensity ratio between any fluor and its
neighbors would be sufficient. However, for many useful fluors this
is not achievable on the basis of excitation contrast or emission
contrast alone. Simultaneous selection on the basis of both
excitation and emission are required.
[0121] In some circumstances it is required to excite and image
several fluors simultaneously, e.g., for direct viewing and/or
color video recording. Excitation and emission optics that have no
wavelength selectivity cannot be used, because the excitation light
scattered into the detector would overwhelm the fluorescence by
several orders of magnitude. One solution is to use
multiple-bandpass filters designed for the specific set of fluors
to be used. The excitation filter defines narrow passbands that
overlap the fluor excitation spectra. The emission filter defines
similar passbands that interdigitate between the excitation bands
and overlap the fluor emission spectra (the reddest fluor could use
a long-pass filter). The dichroic beamsplitter alternates between
reflect (overlapping the excitation passbands) and transmit
(overlapping the emission passbands).
[0122] Use of a multi-pass filter set also has an advantage even
when multicolor visualization is not required, viz. the absence of
geometric displacement (pixel shifts) between signals passing
through the several emission passbands. However, when used with a
greyscale imaging system, it is necessary to make either the
excitation filter or the emission filter switchable, in order to
determine which greyscale signal corresponds to which fluor. The
preferred choice is to switch the excitation filter, because this
causes no image displacement.
[0123] A second theoretical solution would be to excite at a
wavelength where all the fluors absorb. This is often possible
because many fluors are excitable to states higher than S 1 using
photons in the middle UV, but because of internal relaxation
processes give `normal` fluorescence. For example, many laser dyes
can be excited at the nitrogen laser wavelength, 337 nm, far to the
blue of their visible absorbances. It would be straightforward to
block such exciting light from the emission path, using a long-pass
filter (e.g., 380 nm), while allowing all fluorescences to
simultaneously reach the detector. Drawbacks to the use of UV
excitation include increased rates of photochemical decomposition
of the fluor, and the expense of suitable UV optics. Thus, the
method has not found widespread use.
[0124] The multi-bandpass method has the limitation that
construction of multiple bandpass elements giving adequate contrast
between more than 3 fluors is extremely difficult. A generally more
powerful approach is to construct optimized filter sets for each
fluor, and switch them as needed. In the case of a single fluor,
the primary goal on the excitation side is high excitation flux, to
give a bright image. When imaging multiple fluors, however, this
becomes secondary to the goal of achieving high contrast between
fluors. In the weak overlap limit, fluors may be imaged by
sequentially switching filters that are designed using the same
criteria as single-fluor sets (except that long-pass emission
filters are proscribed for all but the longest-wavelength fluor).
Crosstalk of a few percent is usually allowable, and can be
compensated numerically if necessary. More generally, though,
generation of adequate contrast between fluors requires both
strongly selective excitation and emission. The optimum wavelengths
can be found by ratioing the spectra of adjacent fluors. These
wavelengths may be far from the excitation maxima, implying a
significant tradeoff between signal brightness and contrast.
Standard filter sets cannot be used. The need for precisely placed
excitation makes it less likely that a strong excitation line will
be available, and also reduces the flux available from a continuum
source. It may be necessary to pulse the broadband source (arc or
filament lamp) to transiently very high output levels, or to
supplement it with laser sources.
[0125] The principal technical problem with serial imaging is image
displacement when filters are changed i.e., the coordinate systems
of the individual members of an image series are not in precise
registration. This problem arises from nonidealities in the
emission channel optics. Two displacement components can be
identified:
[0126] i. a reproducible offset that is unique to each filter set.
This component is a fixed vector, and arises mainly from imperfect
parallelism (i.e., wedging) between the top and bottom faces of the
emission bandpass filter. There is also a small component due to
wedging in the dichroic beamsplitter, but since this element is
very thin the effect is minor. Since the wedging vector is a
constant for each filter set, it can be automatically removed in
the computer. The size of the offset can also be reduced to very
small values (<0.1 .mu.) by selecting emission filters for a
high degree of parallelism e.g., by measurement in a laser
autocollimator.
[0127] ii. a random component due to vibration and hysteresis in
the filter switching mechanism. The magnitude of the noise depends
on the filter switching mechanism. The worst are manual push-pull
sliders, particularly when the operator actuates them using
uncompensated forces. The best are motorized filter cassettes, in
which all mechanical torques act against the microscope body.
[0128] Note that both the constant and random components of the
image offset noise are minimized by using an objective lens of the
highest possible magnification, and the minimum amount of
magnification in the camera projection optics. Image displacements
encountered in optical microscopy are linear vectors only; there is
no evidence for rotation or significant changes in scale
(magnification).
[0129] Design considerations for high contrast imaging of multiple
overlapping fluors are similar to those already outlined for the
excitation channel. By calculating the ratio of each fluor's
emission spectrum to that of its neighbors, it is possible to
identify spectral regions of high contrast that can be defined with
narrowband filters. High contrast is often achievable only at the
cost of throwing away significant amounts of the fluorescence (90%
or more). Inefficiency in the fluorescence channel is much more
damaging to image S/N than excitation Inefficiency. Thus, where
possible, selective excitation is the preferred method of achieving
contrast between multiple fluors. In difficult cases, however, both
excitation and emission contrast are required.
[0130] 2. Optical Filter
[0131] As indicated, in a preferred embodiment of the invention,
the detection of fluor is accomplished using optical filters, in a
modification of the method of Ried, T. et al. (Proc. Natl. Acad.
Sci. (U.S.A.) 89:1388-1392 (1992), herein incorporated by
reference).
[0132] Imaging DAPI
[0133] 4',6-diamidino-2-phenylindole (DAPI) is a commonly used DNA
counterstain that intercalates preferentially into AT-rich regions
of chromosomes and so gives rise to a weak G-type banding pattern.
It is a very bright fluor (.epsilon.=3.3.times.10.sup.4 M.sup.-1
cm.sup.-1 at 347 nm, with approx. 20-fold enhancement of
fluorescence quantum yield when intercalated into the minor groove
of double-stranded DNA), and is relatively resistant to
photobleaching. The following points are relevant to imaging DAPI
with high contrast against its neighbors:
[0134] a. DAPI has very broad excitation and emission spectra, and
a very large Stoke's shift. Thus, although the DAPI excitation
maximum (347 nm) is to the blue of Cascade Blue (CB), the
fluorescence of DAPI peaks to the red of Cascade Blue, and actually
overlaps quite strongly with FITC.
[0135] b. The usual excitation wavelength for DAPI (Hg 366 nm line)
cannot be used in this application, because it is almost isosbestic
for DAPI and CB and thus gives no excitation contrast.
[0136] c. The peak of the DAPI/CB excitation contrast spectrum is
at 320 nm, which is too far into the UV to pass through the
microscope optics. An acceptable compromise is to excite with the
Hg 334.1 nm line, which the microscope transmits with about 30%
efficiency. The Ealing 35-2989 interference filter has an
appropriate bandpass. The excitation contrast ratio for DAPI/CB at
334.1 nm has an absolute value of 4.0.
[0137] d. To further increase the DAPI/CB selectivity, emission
contrast must be used in addition to excitation contrast. Note that
DAPI emits the bulk of its fluorescence to the red of CB. The
wavelength of maximum emission contrast for DAPI vs. both CB and
FITC is 490 nm. A suitable imaging-grade filter is the Omega
485DF22. There are no mercury lines within its bandpass, so good
DAPI images with low flare are expected. The calculated emission
contrast between DAPI and both Cascade Blue and FITC is 6.8. Hence,
the overall contrast achievable for DAPI vs. Cascade Blue is
approximately 27-fold. The overall contrast of DAPI vs. FITC is
very much higher than this, because at the DAPI excitation
wavelength the excitation of FITC is close to zero.
[0138] e. The Omega 450DRLP02 dichroic beamsplitter is very well
matched to the proposed excitation and emission filters.
[0139] Imaging Cascade Blue
[0140] Cascade Blue (CB) has a broad, two-peak excitation spectrum
that overlaps DAPI extensively, though not its neighbor on the red
side (FITC). The Stokes shift for CB is very small, i.e., there is
very extensive overlap between its excitation and emission spectra.
These factors combine to make imaging CB in the presence of DAPI
problematical. To summarize:
[0141] a. The peak of the CB/DAPI excitation contrast spectrum is
at 396-404 nm. However, because of the very small Stokes' shift of
CB this is very close to the emission contrast peak (408 nm). Since
it is more important for image S/N to collect the emission with
high efficiency than to excite with high efficiency, the 400-410 nm
region is reserved for the CB fluorescence. The best compromise for
CB excitation is the Omega 380HT15, which overlaps with the Hg 366
nm line enough to provide good excitation strength.
[0142] b. To achieve any emission contrast at all vs. DAPI, the
emission filter must be narrow and very carefully placed. A
suitable filter is the Omega 405DF10, but the theoretical maximum
contrast is only 2.5. Thus, imaging CB while excluding DAPI is
expected to be marginal.
[0143] c. Cascade Blue exhibits high excitation contrast vs. FITC
(contrast ratio with the Omega 380HT15 filter=6).
[0144] d. The emission contrast ratio for CB/FITC goes to very
large values below 490 nm, and is essentially infinite at the
position of the 405DF10 filter.
[0145] The above considerations indicate that it will be possible
to use either DAPI or Cascade Blue, but not both together unless
the DAPI counterstain is weak. Cascade Blue is well separable from
FITC and the other five combinatorial fluors considered herein.
[0146] Imaging FITC
[0147] The following points are relevant to high-contrast imaging
of FITC:
[0148] a. The excitation spectrum of FITC has insignificant overlap
with that of Cascade Blue (contrast parameter Rb for FITC/CB
becomes extremely large beyond 420 nm). This makes it possible to
excite FITC on the high frequency shoulder of its absorbance, so
avoiding appreciable excitation of Cy3.
[0149] b. There is no mercury line that overlaps well with the FITC
excitation spectrum. Thus, it is necessary to use the continuum to
excite this fluor, as with single-fluor imaging. The gap between
FITC and CB makes it possible to use a very wide excitation filter;
the Omega 455DF70 is well suited to this function.
[0150] c. The Omega 455DF70 bandpass also corresponds fortuitously
to the maximum in the excitation contrast ratio for FITC/Cy3 (460
nm; absolute extinction ratio R.sub.a=8.8).
[0151] d. The emission spectrum of FITC overlaps that of Cy3 (on
the red side) and both DAPI and CB (on the blue side) to an
appreciable extent. However, because the conditions defined for
FITC excitation do not excite DAPI or Cascade Blue at all, they are
not relevant. The FITC/Cy3 emission contrast ratio goes to very
high values below 540 nm. Thus, the Omega 530DF30 filter gives very
high emission contrast, which compounds the high excitation
contrast ratio for FITC vs. Cy3 (8.8) given by the 455DF70. It
therefore appears possible to image FITC very cleanly.
[0152] Imaging Cy3
[0153] The following points are relevant to high-contrast imaging
of Cy3:
[0154] a. The absorbance peak of Cy3 is at 551 nm, at which
wavelength the excitation of FITC is essentially zero (contrast
parameter Rb jumps to extremely high values to the red of 525
nm).
[0155] b. The Cy3 extinction peak overlaps strongly with the Hg
546.1 nm line.
[0156] c. The excitation contrast ratio for Cy3/Cy3.5 is everywhere
small, and varies weakly with wavelength. At 551 nm, the absolute
value of the excitation contrast for Cy3 vs. Cy3.5 is less than 2,
and it only rises significantly far to the blue where the Cy3
absorbance is very low and FITC absorbance is high. In fact, the
apparent rise in R.sub.a around 460-470 nm may be an artifact of
the low absolute precision of the spectra in that region. From the
above discussion, it is clear that the excitation contrast
available for Cy3 vs. Cy3.5 is too low to be useful.
[0157] d. The emission contrast ratio for Cy3/Cy3.5 rises abruptly
below 570 nm. At the 567 nm peak of the Cy3 fluorescence, the
absolute value of S.sub.a is approximately 6. This, combined with
the factor 2 in the excitation contrast parameter, should just
about meet the goal of a 10-fold discrimination between Cy3 and
Cy3.5. The Ealing 35-3722 narrowband interference filter is
suitable, although it does overlap the Hg 577/579 line
significantly. Any stray light from this line getting through the
546DF10 filter is, however, expected to be well blocked by the
chosen dichroic, Omega 560DRLP02.
[0158] e. The inability to differentially excite Cy3 vs. Cy3.5
means wasteful bleaching of Cy3.5 during imaging of Cy3.
[0159] Imaging Cy3.5
[0160] The following points summarize high contrast imaging with
this dye:
[0161] a. The excitation contrast ratio between Cy3.5 and Cy3 rises
to very high values beyond approximately 565 nm. This region
includes the peak of the Cy3.5 excitation spectrum.
[0162] b. The mercury arc line at 577/579 nm is almost exactly
coincident with the peak of the Cy3.5 absorbance. At this
wavelength the excitation contrast ratio is approximately 25, i.e.,
very strong selective excitation of Cy3.5 relative to Cy3 is
possible. An ideal filter for this purpose is the Ealing 35-3763.
Note that the high contrast is mainly a consequence of the Hg line
position, not the filter bandwidth.
[0163] c. At the Hg 577/579 excitation wavelength, the excitation
contrast ratio for Cy3.5 relative to Cy5 is also quite large
(absolute value approximately 8.0).
[0164] d. The emission contrast parameter for Cy3.5 vs. Cy3 is
small at all wavelengths where the Cy3.5 emission is usefully
strong, i.e., isolation of Cy3.5 from Cy3 must rely mainly on
excitation contrast.
[0165] e. The emission contrast for Cy3.5 vs. Cy5 is also large
over a considerable spectral interval (and rises to very high
values below 640 nm). This permits a fairly broadband filter to be
used to image Cy3.5; a suitable element is the Omega 615DF45.
Almost no bleadthrough of either Cy3 or Cy5 into the Cy3.5 channel
is expected with the above combination of excitation and emission
conditions.
[0166] f. The Omega 590DRLP02 is a suitable dichroic for this
channel.
[0167] Imaging Cy5
[0168] The following points are relevant to imaging with this
dye:
[0169] a. Beyond 600 nm, the excitation contrast ratio for Cy5 vs.
Cy3.5 gets very large, i.e., very high excitation contrast relative
to Cy3.5 is possible.
[0170] b. The placement of the Cy5 excitation is determined by the
extinction ratio relative to Cy5.5 rather than Cy3.5. The Cy5/Cy5.5
excitation contrast parameter peaks at 650 nm with a numerical
value of 2.25. Thus, analogous with the Cy3/Cy3.5 pair, excitation
contrast for Cy5/Cy5.5 is poor.
[0171] c. There is no Hg line available for exciting Cy5. Thus,
with an arc source, the continuum must be used, analogously with
FITC excitation. The "official" filter for exciting Cy5 in this way
is the Omega 640DF20, which will give an excitation contrast with
Cy5 of about 1.8.
[0172] d. A much brighter source for exciting Cy5 is the He--Ne
laser (632.8 nm). It does not, however, improve excitation contrast
vs. Cy5.5.
[0173] e. The emission contrast for Cy5 vs. Cy3.5 peaks at 673 nm,
just to the red of the fluorescence intensity peak. The closest
available filter is the Omega 660DF32 where the emission contrast
ratio is approximately 3.1. This compounds the very high excitation
contrast for Cy5 vs. Cy3.
[0174] f. The emission contrast for Cy5 vs. Cy5.5 goes to very
large values' at wavelengths shorter than 675 nm. The Omega 660DF32
filter is ideally set up to take full advantage of this.
Unfortunately, it is uncomfortably close to the 640DF20 exciter, so
some flare from reflected/scattered excitation light is to be
expected. Use of the He--Ne laser would remove this problem.
[0175] g. The best available dichroic beamsplitter for Cy5 imaging
is the Omega 645DRLP02, particularly if a He--Ne is used as the
excitation source.
[0176] Imaging Cy5.5
[0177] CY5.5 is the penultimate dye of the set, and is very well
separated from Cy7. Thus, only its contrast relative to Cy5 need be
considered in detail:
[0178] a. The contrast parameter Rb for the Cy5.5/Cy5 pair rises to
large values to the red of 670 nm. Thus, it is possible to achieve
very high excitation contrast for this pair of fluors (analogously
to Cy3.5/Cy3).
[0179] b. As with Cy5, the Hg arc is a poor source for exciting
Cy5.5. The best available source is a 680 nm diode-pumped frequency
doubled YAG microlaser (Amoco), which coincides with the peak of
the Cy5.5 absorbance. At 680 nm, the excitation contrast ratio for
CY5.5/Cy5 is 5.1. A suitable excitation filter is the Ealing
35-4068.
[0180] c. The emission contrast ratio between Cy5.5 and Cy5 peaks
at 705 nm, approximately 3 nm to the red of the Cy5.5 intensity
curve. The numerical value for S.sub.b at this point is 4. If the
Omega 700EFLP longpass emission filter is used, a contrast ratio of
approximately 3 (averaged out to 800 nm) is expected. This,
combined with the high excitation contrast, makes imaging Cy5.5
very clean.
[0181] d. At the expense of slightly lower emission contrast (this
would not be significant) and some loss of intensity, a bandpass
filter such as the Ealing 35-6345 could be substituted for the
Omega 700EFLP. The potential advantage would be reduction of the
infra-red background. i.e., overall improved image contrast.
[0182] Imaging Cy7
[0183] Cy7 is the reddest dye of the set. The excitation and
emission spectra are well separated from Cy5.5, and are well
matched to the Omega 740DF25/770DRLPO2/780EFLP triplet. The Oriel
58895 is an appropriate IR blocker for Cy7.
[0184] Filters selected for imaging the DAPI, FITC, Cy3, Cy3.5,
Cy5, Cy5.5, Cy7 fluor set are summarized in the Table 2 below. None
of these filter sets correspond to the filter sets supplied by
manufacturers of conventional fluorescence microscopes as narrow
band excitation and fluorescence detection is mandatory to achieve
sufficient contrast.
2 TABLE 2 Epicube Filter Configuration (for 75 W Xe Arc Source
Excitation Emission Bandpass Dichroic Bandpass Flour Filter
Beamsplitter Filter IR Blocking DAPI Zeiss Zeiss Zeiss None 365 nm
395 nm >397 nm FITC Omega Omega Omega BG38 455DF70 505DRLP02
530DF30 Cy3 Omega Omega Ealing BG38 546DF10 560DRLP02 35-3722 Cy3.5
Ealing Omega Zeiss BG38 35-3763 590DRLP02 630/30 Cy5 Omega Omega
Omega Oriel 58893 640DF20 645DRLP02 670DF32 Cy5.5 Ealing Omega
Omega Oriel 58893 35-4068 DRLP02 700EFLP Cy7 Omega Omega Omega
Oriel 58895 740DF25 770DRLP02 780EFLP
[0185] Characteristics of the microscope system are described in
detail by Ballard S. G. et al. (J. Histochem. Cytochem.
41:1755-1759 (1993), herein incorporated by reference). A high
pressure 75W DC xenon arc (XBO) was used as an excitation source
because of its approximately constant spectral power distribution.
A Zeiss Axioskop microscope workstation equipped with a cooled CCD
camera (Photometrics CH250) was employed. The objective lens was a
63.times.1.25 NA Plan Neofluar which should be "plan" and
apochromatic with a high numerical aperture. The filter sets
selected were able to discriminate between the six fluors with a
maximum contrast (Table 2). To minimize the crosstalk between
fluorophores, the filter sets were selected on the basis of maximum
spectral discrimination rather than maximum photon throughput.
Image exposure times were varied to adjust for photon flux
differences and flux excitation cross-sections. Narrow band
excitation and fluorescence detection is mandatory to achieve
sufficient contrast. Appropriate excitation and emission filter
sets were used to optically discriminated these fluors (Table
2).
[0186] The combinatorial labeling strategy relies on accurate
measurements of intensity values for each fluorophore. Critical
features are accurate alignment of the different images, correction
of chromatic aberrations, and specific quantitation of each
fluorophore. Because simple manual image manipulation could not
realize these demands new software was developed in our lab. This
program comprises the following steps in sequential order: (1)
correction of the geometric image shift; (2) calculation of a DAPI
segmentation mask; (3) for each combinatorial fluor, calculation
and subtraction of background intensity values, calculation of a
threshold value and creation of a segmentation mask; (4) use of
this segmentation mask of each fluor to establish a "Boolean"
signature of each probe; (5) for each chromosome, display of the
chromosome material next to the DAPI image; (6) create a composite
gray value image, where each labeled object is encoded with a
unique gray value; (7) final presentation of the results using a
look-up-table (LUT) that assigns each gray value a particular
color
[0187] The above-described program was developed on the basis of an
image analysis package (BDS-Image) implemented on a Macintosh
Quadra 900. Image shifts caused by optical and mechanical
imperfections were corrected by the alignment of the gravity center
(center of mass) of a single chromosome in each image according to
a procedure described by Waggoner, A. et al. (Methods Cell Biol
30:449-478 (1989)) and modified (du Manoir, S. et al., Cytometry
9:4-9 (1995); du Manoir, S. et al., Cytometry 9:21-49 (1995), all
herein incorporated by reference). The DAPI image was used to
define the morphological boundary of each chromosome. Accurate
chromosome segmentation was achieved by pre-filtering the images
through a top-hat filter (Smith, T. G., et al., J. Neurosci Methods
26:75-81 (1988); modified in du Manoir, S. et al., Cytometry 9:4-9
(1995); du Manoir, S. et al., Cytometry 9:21-49 (1995)). The mode
of the gray level histogram of the top-hat filtered DAPI image was
used as the threshold. For each fluor background was eliminated by
subtracting the mean interchrombsomal fluorescence intensity from
the image. The mean of the chromosomal fluorescence intensities was
used to calculate the threshold for the individual segmentation
mask of each fluor. Individual DNA targets were assigned distinct
gray values depending on the "Boolean" signature of each probe,
i.e. combination of fluors used to label this DNA probe. In a final
step a look-up table was used to assign each DNA target a
pseudocolor depending on this gray value, for display.
[0188] Switching the filter sets in excitation and emission path as
well as the dichroic mirror was done manually, but a
computer-controlled electro-mechanical solution will allow
automation of the procedure.
[0189] 3. Optical Combs
[0190] In an alternative embodiment, selective excitation of
multiple fluors and analysis of fluorescence spectral signatures
can be carried out using dispersion optics rather than
wavelength-selective transmission filters. Such optics may be used
to create filters of any passband characteristic, including
short-pass, long-pass, single bandpass and multiple bandpass
functions. In this method, a dispersion element (prism or grating)
is used in conjunction with a wavelength-selective spatial filter
to create the desired spectral response. The combination is
referred to herein as a "comb filter." Using a comb filter, the
spectral distribution of the exciting light may be tailored for
optimum simultaneous excitation of multiple fluors. The inverse
comb filter may also be used to selectively block from the CCD
camera only the wavelengths used for excitation: the remaining
wavelength intervals (corresponding to the gaps between teeth of
the comb) are available for spectral analysis of the fluorescence
emitted by the fluors. This analysis constitutes the spectral
signature.
[0191] 4. Interferometers
[0192] In lieu of using the optical filters described above, an
interferometer may be used in conjunction with an epi-fluorescent
microscope. A light source for excitation of fluorescence that is
either coherent (e.g. an Argon laser) or incoherent (e.g. a Mercury
arc lamp) may be used. A Mercury-Xenon mixed gas arc lamp is
preferred due to its intense Mercury lines and broad Xenon visible
and near-infrared continuum.
[0193] Although any of a variety of interferometer designs (such as
Michelson interferometer) may be employed, the use of a Sagnac
interferometer is preferred. The Sagnac interferometer has a larger
acceptance angle, greater entendue, and is less sensitive to
alignment, vibration, and temperature variations than a similar
Michelson interferometer.
[0194] The Sagnac interferometer is a common path interferometer.
An interferometer consists of two or more interfering beams of
light. In a common path interferometer there are two beams each
traveling the same path but in opposite directions. The optical
paths are produced by reflecting light through beamsplitters, for
example.
[0195] Multiple beam interferometers operate by dividing the
optical energy from a light source into two substantially equal
beams of light. The two beams of light are combined after one is
permitted to pass through a sample and the interference pattern
(the changes in intensity of the combined light caused by the
interference of two beams) is detected.
[0196] In the Sagnac interferometer, the light source is also
divided into two substantially equal parts. Changing the angle of
incidence of light on the beamsplitter, (by rotation of the
interferometer, or rotation of an optic, such as a galvanometer
driven mirror within the interferometer) causes the optical path
length to be changed along one optical axis of the interferometer.
This produces a fringe pattern along one axis of the detector, for
example a CCD detector. The other axis of the detector can sample
gray scale. As the optical path length is scanned, by rotating the
interferometer or a mirror, the fringe pattern produces an
interferogram at each pixel. The Fourier Transform of this
interferogram yields the spectrum of light falling on that pixel of
the CCD. Thus, an advantage of the Sagnac interferometer is that it
produces an optical path difference across an entire field of view,
rather than at a single point.
[0197] Disturbances, such as a small shift of one of the optical
elements, effect both beams in the same way, and hence have no
effect on the measurement. This mechanical stability also makes the
interferometer relatively insensitive to temperature changes as
well. Thus, another advantage of the common path interferometer is
its intrinsic stability. Sagnac interferometers and their use are
well known (see, for example, U.S. Pat. Nos. 3,924,952; 4,410,275;
4,529,312; 4,637,722; 4,671,658; 4,687,330; 4,836,676 and
5,108,183).
[0198] In one implementation of a Sagnac interferometer (J. Bruce
Rafert et al., "Monolithic Fourier-Transform imaging spectrometer",
Applied Optics, November 1995), the acceptance angle of the
interferometer is determined to be:
q=2n tan.sup.-1(w/8a)
[0199] where w/a=tan 30.degree., w is the aperture width of the
interferometer, a is the length of each leg, and n is the index of
refraction of the interferometer glass. The input beam to the
interferometer need not be collimated.
[0200] The interference pattern or interferogram is most preferably
detected with a CCD camera (such as a Princeton Instruments frame
transfer CCD camera) capable of 512.times.512 pixels or larger.
Since the interferogram in a Sagnac interferometer has an angular
dependence, each pixel of the CCD detector measures a small
interval of the interferogram. The fringe spacing of the
interferogram is set such that a pixel on the CCD detector can
adequately sample the interferogram. The Optical Path Difference
(OPD) that a pixel can span, in order to properly sample the
interferogram is given by the relation:
OPD.sub.pixel=.lambda..sub.min/4
[0201] where .lambda..sub.min is the shortest wavelength in the
spectrum to be measured by the interferometer. This OPD.sub.pixel
determines the theoretical limit of the resolving power of the
interferometer.
[0202] As the OPD is being changed by the rotating mirror, the
interferogram is being moved across the CCD detector, such that the
maximum optical path difference is then given by the relation,
OPD.sub.max=N(OPD.sub.pixel)
[0203] where N is the linear dimension of the CCD detector in
pixels. Each angular displacement of the light incident on the
interferometer beamsplitter may then correspond to one or several
OPD.sub.pixel's. And, in the case of a CCD detector, one frame of
CCD data is required to sample this angular displacement.
[0204] Finally then, each pixel comprises an interferogram which
contains within it information about the spectrum of light falling
on the pixel, the intensity of light falling on that pixel, and the
x and y coordinates of the pixel. The spectrum of light may be
recovered from the interferogram by the use of a computational
Fourier Transform algorithm.
[0205] In practice, because of the limited dynamic range of CCD's,
typically about 10,000:1, the light used to excite the fluorescence
must be blocked from entering the interferometer. This excitation
light is often 10.sup.8 to 10.sup.12 more intense than the
fluorescence that is emitted from the sample. Without blocking by
using optical filtering, this excitation light would saturate the
CCD. However, this filter need only be fabricated so as to block
the excitation, all other wavelengths may be allowed to pass.
[0206] In one embodiment, ultra violet (UV) light is used to excite
the fluorescent probes. The UV light may be easily blocked with a
long pass interference filter allowing the visible and
near-infrared colors to pass through to the interferometer. This
embodiment has the advantage that UV will excite many of the
fluorescent dyes currently in use. This embodiment also has the
advantage that it will allow better than 90% transmittance of the
visible fluorescence to the interferometer. The disadvantage of UV
is that it photobleaches the dyes faster than visible light.
[0207] Both the input and the output lens of the interferometer are
preferably very high efficiency camera lenses, and do not
significantly effect the efficiency of imaging. The focus of the
image within the interferometer is most preferably adjusted so as
to be constant for the variable powers of the zoom eyepiece, and
thus a microscope having the characteristic of infinite image
distance (such as Olympus AX70 microscope) are preferred.
[0208] The above-described interferometer possesses certain
advantages over optical filters. One key advantage is that all the
light emitted by fluorescence is theoretically available for
detection, whereas the transmittance of an interference filter is
limited. Another advantage is that since the filters do not have to
be changed, there is no image shift due to the non-parallelism of
filters.
[0209] E. Uses of the Present Invention
[0210] The capacity of the FISH (Fluorescent In situ Hybridization)
methods and reagents of the present invention to detect and analyze
chromosomal abnormalities, such as translocations, inversions,
duplications, etc. can be used for a large number of
applications.
[0211] 1. Applications Relating to the Cytogenetic Diagnosis of
Genetic Disease
[0212] Among the primary applications of the present invention is
the cytogenetic diagnosis of genetic disease, such as the pre- or
post-natal diagnosis of disease, complex tumor karyotyping, the
analysis of cryptic translocations (especailly through the use of
telomere-specific probes). It provides a novel method for automated
chromosome identification and analysis. A large number of diseases
(prenatal disease, cancers (especially BRCA1 or BRCA2 associated
breast cancer), leukemias, Down's Syndrome, etc.) are characterized
by rearrangements and other chromosomal abnormalities that can be
discerned using the methods of the invention.
[0213] Chromosome karyotyping by conventional cytogenetic banding
methods is both time consuming, expensive and not easily automated.
The detection of recurring genetic changes in solid tumor tissues
by karyotyping are particularly problematic because of the
difficulty in routinely preparing metaphase spreads of sufficient
quality and quantity and the complex nature of many of the
chromosomal changes, which make marker chromosome identification
based solely on banding patterns extremely difficult. Indeed,
attempts to automate karyotype analysis over the past twenty years
(e.g., pattern matching, eigen analysis) have failed because robust
computer algorithms could not be developed to reliably decipher
complex banding patterns, particularly those of extensively
rearranged chromosomes.
[0214] It has been proposed that the next generation of cytogenetic
techniques would be far superior by using bands that are defined
molecularly by hybridization of probes or probe sets each labeled
with a different color (Nederlof, P. M. et al., Cytometry
11:126-131 (1990); Nederlof, P. M. et al., Cytometry 13:839-845
(1992); Lengauer, C. et al., Hum Mol Genet 2:505-512 (1993)). This
would provide a high versatility and would constitute a quantum
leap well comparable to the introduction of chromosome banding and
high resolution analysis of chromosomes in prometaphase. Advantages
of the FISH karyotype are the instant identification of the
chromosomal origin of marker chromosomes, double-minutes and
homology staining regions ("HSRs"). Even "poor quality" chromosome
spreads can be evaluated. If desired, one could design a probe set
for particular applications or for particular clinical
applications, e.g. hematologic diseases, pre- or postnatal
diagnosis. The development of specific probe sets that stain
particular regions of chromosomes (e.g. telomeric regions) for the
identification of cryptic translocations would overcome limitations
of the whole chromosome painting probes. Similarly, such probes
could be used to generate multicolor "barcodes" on individual
chromosomes thereby facilitating the automated analysis of
karyotype. Probes can also be designed that would be specific for a
particular arm of a chromosome, thereby permitting a molecular
characterization of translocation breakpoints, hot spots of
recombination, etc. Other applications would include rapid
evolutionary studies, provided that the protocols for multicolor
FISH on human chromosomes can be adjusted, as expected, for
applications on other species.
[0215] 2. Applications Relating to the Detection of Infectious
Agents
[0216] The methods of the invention may also be used to assess the
presence or absence of infectious agents (treponema pallidum,
rickettsia, borrelia, hepatitis virus, HIV, influenza virus,
herpes, Group B streptococcus, diarrhea-causing agents, pathogens
causing acute meningitis, etc.) in tissue, or in blood or blood
products. This can be accomplished by employing labeled probes
specific for such agents. Moreover, by employing serotype-specific
probes, the methods of the present invention permit the rapid
serotyping of such agents, or the determination of whether any such
agents carry drug resistance determinants. The methods of the
present invention may be used to assess chromosomal abnormalities
caused by exposure to radiation (such as personnel exposed to the
radioactivity of nuclear power plants).
[0217] The methods of the present invention may be used to
quantitate microorganisms that are difficult to propagate (such as
anaerobic microorganism is involved in periodontal disease). The
methods of the present invention provide a means for the rapid
diagnosis of acute bacterial meningitis. Just as one can employ
serotype-specific probes to perform serological analysis, one can
employ probes that are specific to particular drug resistance
determinants, and thereby rapidly determine not only the presence
and identity of an infectious agent, but also its susceptibility or
resistance to particular antibiotics.
[0218] 3. Additional Applications
[0219] The methods of the present invention further permit
simultaneous mapping of a large number of different DNA probes.
With this technique the analysis of chromosomal number and
architecture in individual intact cells becomes accessible.
Interphase cytogenetics is already possible with small region
specific probes, e.g. YAC-clones. The accuracy of such analysis
could be increased by a three dimensional analysis using a laser
scanning microscope, or more preferably, a CCD camera system with a
Z-axis stepping motor coupled with 3-dimensional (3-D) image
deconvolution software. Suitable 3-D image deconvolution software
is obtainable from Imstar Corp. (Paris, France) or Scanalytics,
Inc. (Boston). In addition, the use of such scanning microscope
systems would ultimately allow one to visualize all
whole-chromosome painting probes, and telomere- or
centromere-specific probes sets, in interphase nuclei and questions
relating to intranuclear chromosomal organization as a function of
developmental status, cell cycle or disease state could be
addressed. Different models about the chromosomal organization in
interphase nuclei could finally be explored. Although conventional
laser scanning microscopes currently do not allow the excitation of
some of the fluorophores used, other, more appropriate fluors or
devices may be employed including 3 dimensional deconvoluting
imaging systems..
[0220] Extended to non-mitotic cells, the methods of the present
invention enable one to examine chromosome architecture or
quantitate the chromosome contents of nuclei in single
hybridization experiments. Questions relating to intranuclear
chromosomal organization as a function of developmental status,
cell cycle or disease state can accordingly be addressed. In
addition, the ability to quantitatively assess the levels of
multiple mRNAs or proteins in a single cell or to determine if they
exhibit different intracellular distributions could prove extremely
useful in addressing a myriad of interesting biological questions.
The multiparametric imaging of the present invention does not
merely increases the throughput of information, it also makes more
efficient use of the biological material. Thus, it can reveal
spatial and temporal correlations as well as mosaicisms that might
otherwise be difficult to establish reliably. Since the
intracellular distribution of mRNAs and protein is not known a
priori to be spatially distinct, as in the case for the
intra-nuclear chromosome domains, it will not be possible to use
the combinatorial labeling strategies in these experiments.
However, many mRNA and protein antigens can be spectrally resolved
and detected using a multiplex format with n fluors. Thus, for the
first time the intracellular distribution of oncoproteins or tumor
suppressor proteins can be determined within the same cell
simultaneously.
[0221] F. Automated Karyotypic Analyses
[0222] One aspect of the present invention relates to automated,
preferably computer-facilitated, karyotypic analyses. As described
above, in one embodiment of the invention, the chromosomes of a
particular karyotype are pseudo-colored to thereby facilitate the
assignment of the chromosomes, or the recognition of
translocations, deletions, etc. In one sub-embodiment thereof, the
digitized images of the chromosomes may be stored in a
computer-readable storage device (such as a magnetic or optical
disk) to facilitate their comparison with other chromosomal images
or their transmission and study. In this regard, probes may be
employed that are translocation specific or specific to
sub-chromosomal elements or regions, such that the pseudocolorized
chromosomes -or chromosomal elements are displayed to the
investigator as chromosomal images depicting a cytogenetic banding
pattern (for example, the cytogenetic banding pattern of the
metaphase chromosomes of the patient). The position and sizes of
individual bands is preferably digitized and stored so that an
image of the chromosome may be stored on a computer. Similarly, the
precise position of any translocation or other karyotypic
abnormality can be discerned and stored.
[0223] The methods of the present invention thus permit karyotypic
analyses to be conducted more widely and more accurately than was
previously feasible. The present invention may thus be used to
systematically correlate karyotypic abnormalities with disease or
conditions. For example, karyotypes of asymptomatic individuals can
be obtained and evaluated in light of any subsequent illness (e.g.,
cancer, Alzheimer's disease, etc.) or condition (e.g.,
hypertension, atherosclerosis, etc.) in order to permit a
correlation to be made between a patient's karyotype and his or her
predisposition to different diseases and conditions. Similarly,
karyotypes of individuals having diagnosed diseases or conditions
can be obtained and evaluated in light of the extent of any
subsequent progression or remission of the disease or condition so
as to permit a correlation to be made between a particular
karyotype and the future course of a disease or condition.
[0224] In a further sub-embodiment, a computer or other digital
signal analyzer may be employed to orient and arrange the
chromosomal images as well as assigning and identifying the
chromosomes of the karyotype. Thus, a computer or other data
processor will, upon assigning a particular chromosome to a
particular designation (for example, upon assigning that a
particular chromosomal image is the image of the chromosome 7 of
the karyotype being evaluated), group the assigned chromosome with
its homologue (e.g., the second chromosome 7 of the patient's
karyotype) and generate, via a printer, monitor, or other output
means, an ordered array of chromosomal images (such as one in which
each autosomal chromosome is paired with its homologue, and in
which the sex chromosomes X and Y are paired together).
[0225] In one sub-embodiment, the chromosomal images of such arrays
will be the pseudocolorized images discussed above. Alternatively,
such psudocoloring may be internal to the process of assigning
chromosomal identity, and not displayed in the output of the
computer or digital signal analyzer. Rather, in this
sub-embodiment, the output generated will be the light-microscope
visible cytogenetic banding pattern of the metaphase chromosomes of
the patient whose karyotype is being evaluated. In a further
sub-embodiment, a scale (in Morgans or other suitable units) will
be superimposed upon the chromosomal images.
[0226] Having now generally described the invention, the same will
be more readily understood through reference to the following
examples which are provided by way of illustration, and are not
intended to be limiting of the present invention, unless
specified.
EXAMPLE 1
[0227] Combinatorial Labeling of Chromosomes
[0228] In order to test the feasibility to produce 24 colors
chromosome painting probes representing the 22 autosomes and the
two sex chromosomes were used. The DNA probes used were generated
by microdissection.
[0229] Microdissected probes (National Center for Human Genome
Research, Bethesda, Md.) give a very uniform labeling of the target
region. The detailed protocols for microdissection and PCR
amplification are described by Telenius et al. (Telenius, H. et
al., Genes, Chromosomes & Cancer 4:257-263 (1992); Telenius, H.
et al., Genomics 13:718-725 (1992); Meltzer, P. S. et al., Nature
Genetics 1:24-28 (1992); Guan, X. Y. et al., Hum Mol Genet
2:1117-1121 (1993); Guan, X. Y. et al., Genomics 22:101-107 (1994);
Guan, X. Y. et al., Hum Genetics 95:637-640 (1995), all herein
incorporated by reference). For some chromosomes different
DNA-probes for the p- and the q-arms were available, namely 2, 4,
5, 10, 11, 16, 18, and Y. For all other chromosomes microdissected
probes painting the entire chromosome were used.
[0230] The first member of the set of preferred fluors, DAPI, was
used as a general DNA counterstain. The remaining fluors:
fluorescein, Cy3, Cy3.5 (emission and excitation spectra are
between Cy3 and Cy5), Cy5 (Mujumdar, R. B. et al., Cytometry 10:
11-19 (1989), and Cy7 (emitting to the red of Cy5 (Ernst, L. A., et
al., Cytometry 10:3-10 (1989)), were used to combinatorially label
different probes (Table 3). Distinctive features of these dyes are
high extinction coefficients, quantum yields, and photostabilities.
Fluorescein is a xanthene dye with an extinction coefficient around
70,000 L/mol cm and quantum yields in optimal buffers around 0.7.
The respective values for cyanines are 1-200,000 and 0.3 (Waggoner,
A., Methods in Enzymology 246:362-373 (1995)).
[0231] After microdisection, the probes were subjected to a PCR
amplification and labeled by nick translation. Fluorescein
(Wiegant, J. et al., Nuc Acids Res 19:3237-3241 (1991)), Cy3, and
Cy5 were directly linked to DUTP for direct labeling. Cy3.5 and Cy7
were available as avidin or anti-digoxin conjugates for secondary
detection of biotinylated or digoxinigated probes. They were
synthesized using conventional N-succinamide ester coupling
chemistry. For each probe one to three separate nick translation
reactions were necessary, each with a single labeled fluor-labeled
triphosphate or biotin or digoxigenin (Table 3).
3TABLE 3 Fluor Chromosome 1 2 3 4 5 6 7 8 9 10 11 12 FITC X X X X
Cy3 X X X X X Cy3.5 X X X X X Cy5 X X X X Cy7 X X X X 13 14 15 16
17 18 19 20 21 22 X Y FITC X X X X X X X Cy3 X X X X X Cy3.5 X X X
X X X Cy5 X X X X X X Cy7 X X X X X X
[0232] As expected probes labeled with equal amounts of different
fluors did not give equivalent signal intensities for each fluor
reflecting the fact that the filter sets were selected to maximize
spectral discrimination rather than photon throughput. In order to
diminish signal intensity-differentials, probe concentrations for
the hybridization mix had to be established carefully in a large
number of control experiments. Hybridization conditions were
optimized for these multiplex probes. Thus, probes were denatured
and hybridized for two to three nights at 37.degree. C. to
metaphase chromosome spreads in a conventional 50% formamide
hybridization cocktail. 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 to remove excess probe. After a
blocking step in 4.times.SSC/3% bovine serum albumin for 30 min at
37.degree. C. the biotinylated probes were detected with avidin
Cy3.5 and the dig-labeled probes with anti-dig-Cy7.
Fluorescein-dUTP, Cy3-dUT'P, and Cy5-dUTP did not require any
immunological detection step. After final washes at 45.degree. C.
with 4.times.SSC/0.1% Tween 20 three times, mounting medium and a
coverslip were applied and the hybridization signals from each
fluor imaged using the filters sets listed in Table 3.
[0233] FIG. 1 provides a schematic illustration of the CCD camera
and microscope employed in accordance with the present methods.
[0234] FIG. 2 shows the raw data from a karyotypic analysis of
chromosomes from a bone marrow patient (BM2486). Adjacent to each
source image is a chromosome "mask" generated by the software
program. In FIG. 2, panels A and B are the DAPI image and mask;
panels C and D are FITC image and mask; panels E and F are Cy3
image and mask; panels G and H are Cy3.5 image and mask; panels I
and J are Cy5 image and mask; and panels K and L are Cy7 image and
mask.
[0235] FIGS. 3A and 3B show the identification of individual
chromosomes by spectral signature. FIG. 3A is the same photograph
as FIG. 2, except that it is gray scale pseudocolored. FIG. 3B
displays the karyotypic array of the chromosomes. The exceptional
power of the methods of the present invention are illustrated by
the ease with which the translocation of chromosomes 5 and 8 are
identified in FIGS. 3A and 3B, relative to conventional
non-chromosome specific karyotype analysis.
[0236] The above experiment demonstrates that five fluors can be
spectrally discriminated to produce at least twenty four different
colors. The combinatorial labeling schemes need not be as complex
as previously thought, because using 5 fluors for probe labeling,
only 9 painting probes need to be labeled with as many as three
fluors. A sixth fluor for probe labeling would allow up to 63
possible fluor combinations. Such a high number of different
targets will not be required for most applications, but would allow
the selection of combinations with the best spectral signature.
These above-described protocols allow highly reliable and
reproducible multicolor FISH.
EXAMPLE 2
[0237] Rapid Analysis of Multiple Oral Bacteria in Mixed Cell
Populations Using Multiparametric Fluorescence In situ
Hybridization (M-FISH)
[0238] In situ hybridization has been recognized as having
potential application as a means of analyzing individual bacterial
cells in the absence of culture. DeLong, E. F. et al. (Science
243:1360-1363 (1989)), for example, used oligonucleotide probes
complementary to the 16s ribosomal RNA (rRNA) sequences to
differentiate an archaebacterium (Methanosarcina acetivorans) from
the eubacteria Bacillus megaterium and Proteus vulgaris. Van Den
Berg, F. M. et al. (J. Clin. Pathol. 42:995-1000 (1989)) used total
genomic DNA to detect Campylobacter pylori in stomach tissue.
Amann, R. et al. (Nature 351:161-164 (1991)) detected Holospora
obtusa in the macronucleus of the protozoan Paramecium
caudatum.
[0239] Prior to the present invention, however, the use of in situ
hybridization to differentiate microorganisms in clinical specimens
with mixed populations, especially in a quantitative fashion, was
difficult and time consuming. This is particularly true of bacteria
with fastidious growth requirements, in which culture methods may
result in the selective enrichment of a subset of the organisms
present in the specimen.
[0240] A. Analysis of Non-Cross Hybridizing Microbial Species
[0241] To further illustrate the present invention, the M-FISH
methods of the present invention are used used to speciate bacteria
involved in the microbial etiology of periodontal disease.
[0242] Several methods have previously been used to establish the
microbial etiology of periodontal disease. Well over 200 bacterial
species can be identified in one sublingual plaque sample using
labor-intensive cultural methods (Dzink, J. L. et al., J. Clin.
Periodont. 12:648-659 (1985); Moore, W. E. C.; J. Periodont. Res.
22:335-341 (1987); Moore, W. E. C., Infect. Immunity 38:651-667
(1987)). This diversity has hampered the association of specific
microbes in the etiology of the various forms and stages of
inflammatory periodontal diseases. Direct assessment, both
qualitatively and quantitatively, of plaque bacteria would obviate
the need for culturing and its attendant difficulties. DNA probes
(French, C. K. et al., Oral Microbiol. Immunol. 2:58-62 (1986))
have been widely used in a blot hybridization format as a direct
method for bacterial assessment. However, this method does not
allow for the direct visualization of the bacterial or provide
information on the relative abundance of a particular bacterial
within a complex mixture.
[0243] In order to more simply illustrate the capacity of the
invention to speciate microbial strains, a test for in situ
hybridization specificity is conducted using a mixture of bacteria
that are morphologically distinct and non-cross hybridizing. Using
such a mixture, one can immediately differentiate positive
hybridization from that of non-specific binding by correlating the
correct morphology with the correct probe.
[0244] Thus, Fusobacterium nucleatum (25586), Porphyromonas
gingivalis (33277), Eikenella corrodens (23834), Prevotella
intermedia (25611), and Actinobacillus actinomycetemcomitants
(29522) were obtained from the American Type Culture Collection,
Bethesda, Md., and Capnocytophaga species, C. ochracea (C25) and C.
gingivalis (DR2001) were obtained from The National Institute of
Dental Research, Bethesda, Md.
[0245] Total genomic DNAs were isolated from F. nucleatum, P.
gingivalis, E. corrodens, P. intermedia, A. actinomycetemcomitans,
C. gingivalis, and C. ochracea. using the procedure of Chassy, B.
M. et al., Appl. Environ Microbiol. 39:153-158 (1980)) as modified
by Donkersloot, J. A. et al., J. Bacteriol. 162:1075-1078 (1985)).
Each DNA was labeled with .sup.32P and tested for
crosshybridization with each of the other DNAs by dot blot
hybridization. In all cases, the total genomic DNA probes show
specificity for the bacteria from which it was derived; no
hybridization with any of the other bacterial DNAs was detected.
Since the genomic DNA probes were highly specific in the dot blot
assays, they were used directly for the in situ identification of
these bacteria in reconstructed mixtures.
[0246] Total genomic bacterial DNAs are labeled by nick translation
using biotin-11-dUTP (BIO), digoxigenin-11-dUTP (DIG) or
dinitrophenol-11-dUTP (DNP) as described by Lichter, P. et al.,
Hum. Genet. 80:224-234 (1988), herein incorporated by reference).
Unincorporated nucleotides are removed using a Sephadex G-50 spin
column equilibrated with 10 mM Tris-HCI/1 mM EDTA/0.1% SDS, pH 8.0.
Labeled DNAs (2-6 .mu.g) are ethanol precipitated and redissolved
in 100% deionized formamide. F. nucleatum DNA is labeled with
BIO-11-dUTP (BIO) and detected with Cascade Blue avidin; A.
actinomycetemcomitans DNA is labeled with DNP-11-UTP (DNP) and
detected indirectly with an FITC conjugated antibody; and E.
corrodens DNA is labeled with DIG-11-dUTP (DIG) and detected with a
Rhodamine labeled anti-DIG antibody.
[0247] To prepare slides for in situ hybridization, small aliquots
(1-2 ml) of cultured bacteria are centrifuged at 1,200.times.g in
an HBI microcentrifuge. The cell pellet is suspended in phosphate
buffered saline (PBS) and adjusted to approximately 10.sup.3
bacterial per .mu.l. Aliquots of both pure cultures and synthetic
mixtures are covalently bound to activated glass slides prepared as
described by Maples, J. A. (Amer. J. Clin. Pathol. 83:356-363
(1985), herein incorporated by reference). Alternatively, bacterial
are spotted directly onto positively (+) charged slides and air
dried. Suitable slides are commercially available from Fisher
Scientific, Pittsburgh, Pa. (cat. #12-550-15), and retain all the
bacterial strains listed above throughout the in situ hybridization
procedure with high efficiency. All slides are then fixed in
Carnoy's B Fixative (Ethanol: Chloroform: Acetic Acid, 6:3:1) for 5
minutes.
[0248] The bacteria are hybridized simultaneously with the three
differentially labeled genomic DNAs. Each sample is hybridized
using 15 .mu.l of hybridization solution which contains 50 ng of
labeled probe and DNAse-treated salmon sperm DNA (15 .mu.g) in 50%
(vol/vol) deionized formamide/2.times.SSC (0.3 M sodium
chloride/0.03 M sodium citrate, pH 7.0)/5% dextran sulfate. The
solution is applied to the sample, covered with a coverslip and
sealed with rubber cement. Both bacterial DNA and labeled DNA probe
are denatured by heating at 80.degree. C. for 5-8 minutes in a
oven. These steps are sufficient to permeabilize the bacteria for
probe assessability.
[0249] The DNAs were allowed to reassociate by incubating the
slides at 37.degree. C. for 18 hrs in a moist chamber.
Posthybridization washings, blocking and detection were as
described by Lichter et al. (7). Briefly, the slides were washed
3.times. in 50% formamide/2.times.SSC at 42.degree. C. for 5
minutes, and then washed 3.times. in 0.1.times.SSC at 60.degree. C.
for 5 minutes. The slides were then incubated in a solution of 3%
bovine serum albumin/4.times.SSC (blocking solution) for 30 minutes
at 37.degree. C.
[0250] Biotinylated probes were detected using fluorescein
isothiocyanate-(FITC)-avidin DCS (Vector Laboratories, Burlingame,
Calif., 5 .mu.g/ml) Cascade Blue-avidin (Molecular Probes Inc.,
Eugene, Oreg., 10 .mu.g/ml)) or Rhodamine-avidin (Boehringer
Mannheim, Indianapolis, Ind., 10 .mu.g/ml).). Digoxigenin-labeled
probes were detected using FITC or Rhodamine conjugated sheep
anti-digoxigenin Fab fragments (Boehringer Mannheim, 2 .mu.g/ml).
Dinitrophenol labeled probe was detected by incubating with rat
anti-DNP antibodies (Novagen, Madison, Wis., 1:500 dilution)) and
then with goat anti-rat FITC-conjugated antibodies (Sigma, St.
Louis, Mo., 1 .mu.g/ml). In some experiments, bacterial DNA was
counterstained with 4,6-diamidino-2-phenylindole (DAPI) at a
concentration of 200 ng/ml.
[0251] For detection, the fluorochrome-conjugated antibodies or
avidin are diluted into a solution of 4.times.SSC/1% BSA and 0.1%
tween-20 (200 .mu.l/slide) and incubated with the sample at
37.degree. C. for 30 minutes in the dark. The slides are then
washed 3 times in 4.times.SSC/0.1% tween-20 at 42.degree. C. prior
to viewing via epifluorescence microscopy.
[0252] Epifluorescence microscopy is conducted using a Zeiss
Axioskop-20 wide-field microscope with a 63.times.NA 1.25 Plan
Neofluar oil immersion objective and a 50W high pressure mercury
arc lamp. Images are projected with a Zeiss SFL-10 photo-eyepiece
onto a cooled charged-coupled device (CCD) camera (Photometrics
CH220; 512.times.512 pixel array). Effective magnification is set
by changing the microscope-camera distance, using a bellows. The
8-bit greyscale images are recorded sequentially using DAPI, FITC
and Rhodamine filter sets (manufactured by C. Zeiss, Inc., Germany)
to minimize image offsets. Camera control, greyscale image
acquisition and image pre-processing are done using an Apple
Macintosh 11.times. computer preferably running custom software
(Dr. Marshall Long; Yale University). Images are pseudocolored,
aligned and merged using a Macintoch 11 ci computer equipped with
an accelerated 24-bit graphics system (SuperMac Spectrum 24 PDQ).
The merging software, "Gene Join Max Pix" (Yale University) is
preferably employed. This software assigns a user definable
pseudocolor and greyscale value to each of the source images, and
then generates an output ("merged") image based on the most intense
source image at each pixel location. Thus, objects with higher
scaled brightness (e.g., probe signals) override the DAPI
counterstain signals in the corresponding image location. Objects
displayed in merged image retain the color assigned to them and
therefore appear distinct and easily identifiable. Merged images
were photographed directly from the computer monitor.
[0253] A mixed sample of F. nucleatum, E. corrodens and A.
actinomycetemcomitans, in a 1:1:1 ratio was used as a test sample.
As can be seen in FIG. 4, the three bacteria are distinguishable on
the basis of morphology when stained with DAPI.
[0254] FIG. 4 shows the differentiation of bacteria by in situ
hybridization. Panels A-E represent in situ hybridization assay
results on a laboratory derived mixture of three bacteria (F.
nucleatum, A. actinomycetemcomitans and E. corrodens) using a
mixture of genomic DNA probes for each organism. Panel A shows all
the bacteria present in the microscope field detected by (DAPI), a
general DNA binding fluorophore. F. nucleatum is seen using Cascade
Blue detection (panel B), A. actinomycetemcomitans with FITC
detection (panel C) and E. corrodens using Rhodamine detection
(panel D). Panel E is a computer-merged composite of panels B-D
showing the differentiation of each bacteria in the sample. Note
that a single A. actinomyetemcomitans cell is detected in a clump
of E. corrodens (panel E) that could not be identified by DAPI
staining (panel A). Panel F is a similar analysis of a mixture of
C. gingivalis (Rhodamine) and P. intermedia (FITC) when hybridized
with a combination of genomic DNA probes for those organisms. Panel
G shows an in situ hybridization assay for a combination of seven
different bacteria hybridized with a mixture of seven genomic DNA
probes. F. nucleatum (1), E. corrodens (2) and A.
actinomycetemcomitans (3) are shown in blue (Cascade Blue
detection). C. gingivalis (4) and C. ochracea (5) are shown in red
(Rhodamine detection), whereas P. intermedia (6) and C. gingivalis
(7) are seen in yellow (FITC detection). Phase contrast microscopy
was used to assist in determining morphological differences between
E. corrodens and A. actinomycetemcomitans which were not readily
evident through the hybridization generated signals. Panel H
represents an in situ hybridization assay for the presence of A.
actinomycetemcomitans (FITC detection) in a plaque sample obtained
from a patient with localized juvenile periodontist.
[0255] When assaying for Cascade Blue, only the F. nucleatum
bacteria (FIG. 4, panel B) were seen, when assaying for FITC only
A. actinomycetemcomitans bacteria (FIG. 4, panel C) were visible,
and when assaying for Rhodamine, only the E. corrodens bacteria
(FIG. 4, panel D) were positive. The three separate fluorophore
images were then merged to generate the composite image that is
shown in FIG. 4, panel E. Each of the morphologically distinct
bacteria exhibit the correct fluorescence specificity.
[0256] The four additional bacterial species are included in
subsequent experiments. These bacteria are combined pairwise such
that each mixture again would differ morphologically. FIG. 4, panel
F shows a combined analysis of P. intermedia (BIO label and FITC
detection) and C. gingivalis (DIG label and Rhodamine detection).
An analysis of C. ochracea and P. gingivalis, whose morphology is
similar to the P. intermedia and C. ochracea and P. gingivalis,
whose morphology is similar to the P. intermedia and C. gingivalis
pair, is also performed. In both cases, absolute specificity of the
probe for its cognate bacteria is demonstrated.
[0257] Since the above experiments indicate that none of the
bacteria cross-hybridized and that each species of bacteria, as
grouped, could be distinguished by morphology as well as by
hybridization via the binding of a specific fluorochrome, it was
reasoned that all seven bacteria could be detected in a single
mixed sample. The experimental design was to label the DNA from the
bacteria within each of the three morphologically distinct groups
with the same "reporter" (BIO, DIG or DNP). The three groups would
be initially differentiated by hybridization and the specificity of
the fluorescence signal. The bacteria within a group could then be
differentiated on the basis of morphology.
[0258] To demonstrate this capacity of the present invention, P.
intermedia and C. gingivalis DNAs were labeled with DIG, F.
nucleatum, A. actinomycetemcomitans, and E. corrodens DNAs were
labeled with BIO whereas P. gingivalis and C. ochracea DNAs were
labeled with DNP. All seven probes were then combined and used for
in situ hybridization on mixed bacterial samples containing all
organisms (FIG. 4, panel G). In FIG. 4, panel G, the F. nucleatum,
E. corodens, A. actinomycetemcomitans group is shown using Cascade
Blue (BIO detection) and its members are numbered 1, 2 and 3
respectively; the P. gingivalis and C. ochracea group is seen with
Rhodamine (DIG detection) and these bacteria are numbered 4 and 5
respectively; the C. gingivalis, P. intermedia group is identified
with FITC (DNP detection) and they are numbered 6 and 7
respectively. All seven bacteria are clearly differentiated using
the combination of hybridization fluorescence and morphology. While
morphological identification could be made simply on the basis of
size, shape and color of the hybridization signal, more definitive
morphological characterization is made by examination of the sample
using phase-contrast optics.
[0259] To determine how few hybridization-positive bacteria could
be detected in a sample, 4.0 .mu.l of E. corrodens containing
10.sup.5 bacteria/.mu.l is mixed with 1.0 .mu.l of serial dilutions
of A. actinomycetemcomitans and the 5 .mu.l mixture applied to
slides. The sample is then tested for the presence of A.
actinomycetemcomitans using a BIO-labeled probe for that bacterium.
Following the hybridization, the total bacteria on the slide are
visualized using Propidium Iodide and the hybridization-positive
bacteria are detected using Avidin-FITC. The results of this
experiment show that single A. actinomycetemcomitans cells could be
detected in the presence of an excess of at least 10.sup.4E.
corrodens cells. Thus, it is apparent that hybridization positive
bacteria can be identified readily even when they constitute only a
minor fraction of a mixed cell population.
[0260] Clinical samples are not usually as free of contaminating
debris as laboratory derived mixtures of bacteria. To test the
potential utility of M-FISH for the analysis of clinical specimens,
a dental plaque sample from a patient with localized juvenile
periodontitis, in which prior studies (Slots, J., Dent. Res. 84:1
(1976); Slots, J. et al., J. Clin Periodontal. 13:570-577 (1986);
Zambon, J. et al., J. Periodontal. 54:707 (1983)) had suggested the
involvement of A. actinomycetemcomitans as the microbial pathogen
is employed. FIG. 4, panel H shows the results of an in situ
hybridization experiment with this patient's sample using
BIO-labeled A. actinomycetemcomitans DNA and detecting with
FITC-conjugated avidin. As can be seen, a large number of A.
actinomycetemcomitans or other closely related organisms were
detected. Quantitative studies indicate that more than 50% of the
individually identifiable bacteria seen in the specimen hybridized
with the A. actinomycetemcomitans DNA probe. Cultural studies on
this sample showed the presence of A. actinomycetemcomitans.
[0261] All of the above experiments employ a standard overnight
hybridization time (.about.18 hours) to differentiate the bacteria
of interest. Even though such analyses can often be done faster
than the traditional methods of culturing and biochemical testing,
a number of experimental parameters could be adjusted to give a
more rapid assay. It is well known that hybridization time is
directly related to probe concentration. Thus, by increasing probe
concentration, hybridization time can be proportionately reduced. A
mixture of F. nucleatum and A. actinomycetemcomitans is therefore
analyzed using a 10-fold higher concentration of probe than
previously used (from 50 ng to 0.5 .mu.g per slide). At the higher
probe concentration no specific or non-specific hybridization is
observed with undenatured probe and bacterial DNA. However, when
the probe and bacterial DNA are denatured simultaneously, and the
washing cycles begun immediately, some of the DNAs hybridized
during the time necessary for subsequent sample processing.
[0262] In this experiment, in which 0.5 .mu.g of A.
actinomycetemcomitans probe is introduced to a sample containing
both A. actinomycetemcomitans and F. nucleatum bacteria using a 2.5
minute incubation at 37.degree. C. prior to initiating the washing
cycles, the hybridization signal is as strong and as specific as
that observable in experiments utilizing 50 ng of probe and
hybridizing for 18 hours. Furthermore, by reducing the blocking and
antibody detection times to 20 minutes each, a total assay time of
1.5 hours is achieved. Further reduction in assay times clearly can
be obtained using probe DNA directly labeled with the fluorophore
detector.
[0263] The above experiments provide a relatively rapid and simple
method for identifying individual bacteria in mixed microbial
populations using M-FISH. The method provides a quantitative
assessment of specific microorganisms without the necessity of
culturing. The technique can be applied to samples containing only
a limited number of organisms and a single hybridization-positive
bacteria can be detected quite easily in a 10.sup.4- fold excess of
hybridization-negative bacteria. In addition, as exemplified by the
overlapping and "hidden" A. actinomycetemcomitans cell among the
group of E. corrodens cells (FIG. 4, panel E), bacterial specified
that are not clearly identified by a non-specific analysis (i.e.,
DAPI staining of DNA, FIG. 4, panel A) can be detected by the
hybridization-specific assay. Since both the hybridization
specificity and the morphological characteristics of bacteria can
be assessed simultaneously by this procedure, the number of
different bacterial species can be increased still further by
judicious selection of probe labeling and fluorophore detector
combinations.
[0264] However, morphological characteristics are not always a
reliable indicator of bacterial identity since many different
bacterial species appear morphologically similar and some are
pleomorphic depending on the culturing methods. For example, A.
actinomycetemcomitans are generally coccobacillary in clinical
specimens but can assume rod and even filamentous forms upon in
vitro passage and in clinical specimens. Thus, additional
fluorescence colors would preferably be employed to expand the
number of different bacteria that can be analyzed simultaneously
and identified unequivocally.
[0265] The intensity of the hybridization signal is extremely
strong using either whole genomic DNA or subtracted (suppressed)
probes. Although data is collected by digital imaging,
hybridization signals can be detected readily by eye and recorded
by conventional photographic methods. However, the sensitivity and
photon counting capabilities of CCD-camera based imaging system
offer considerable advantages for further refining microbial
analysis by in situ hybridization. Detection of single copy
sequences for specific toxin or drug resistance genes in individual
bacteria should be feasible; individual human and murine genes have
been visualized, both in metaphase chromosomes and interphase
nuclei by FISH (Lichter, P. et al., Hum. Genet. 80:224-234 (1988)).
The level of cross-homology between bacterial strains also can be
rapidly assessed by quantitating the photon output of the
hybridization signal from individual bacteria in a specimen.
Finally, and most importantly, digital imaging is essential to
fully exploit the potential of combinatorial fluorescence.
[0266] As exemplified here, total genomic DNA can often provide
adequately specific probes, thus eliminating the usual cloning and
screening efforts necessary for probe production. Although not
required in these studies because of the absence of cross
hybridization between the bacterial genomes, suppression
hybridization techniques (Lichter, P. et al., Hum. Genet.
80:224-234 (1988)) can be used to squelch cross-hybridization
between organisms that share partial sequence homology. For
example, C. orchracea and C. sputigena exhibit 22-23% sequence
homology (Zambon, J. et al., J. Periodontal. 54:707 (1983)) yet
specific hybridization to C. orchracea can be obtained with genomic
DNA by preannealing the C. ochracea probe with an excess of C.
sputigena DNA just prior to the in situ hybridization reaction.
[0267] In sum, total genomic DNAs from seven anaerobic and
facultative oral bacteria recognized as pathogens in periodontal
disease (Fusobacterium nucleatum, Porphyromonas (Bacteroides)
gingivalis, Prevotella intermedia, Eikenella corrodens,
Actinobacillus actinomycetemcomitans, Capnocytophaga gingivalis and
Capnocytophaga ochracea) are labeled non-isotopically and
hybridized in situ to reconstructed bacterial mixtures. Each probe
is hybridized uniquely to the bacterium from which it was derived.
Three bacterial strains are differentiated simultaneously by
labeling DNA with different reporter groups and visualizing the
hybridization signal with reporter-specific detector proteins
labeled with different fluorophores. By assessing bacterial
morphology in conjunction with the hybridization signal each
bacteria in a mixture of all seven organisms can be identified. The
total assay time is as short as 1.5 hours. A dental plaque sample
from a patient with localized juvenile periodontitis, an oral mixed
microbial infection, reveals numerous hybridization positive
bacteria when probed with DNA from A. actinomycetemcomitans, the
putative microbial pathogen for this disorder. This example
demonstrates the usefulness of the in situ hybridization methods of
the present invention for the identification of individual
non-cross hybridizing bacteria in mixed cell populations.
[0268] The seven bacterial species analyzed above are facultative
or obligate anaerobes that are often found in association with
inflammatory periodontal disease. The role of these microorganisms
is uncertain due to the lack of rapid and reliable methods to
identify specifies. Direct analysis of oral microbial specimens
circumvents biases imposed by culture methods and may allow a
better understanding of the role of these bacteria in the
pathogenisis of periodontal disease. Data on the detection of A.
actinomycetemcomitans in a specimen from a patient with localized
juvenile periodontitis indicates that the above-described in situ
hybridization procedure facilitates such studies. It also
complements traditional culture methods for the identification of a
broad spectrum of microorganisms, especially notoriously slow
growing bacterial such as anaerobes or mycobacterium. For example,
swabs taken from early culture plates may be streaked on a slide
and assayed by in situ hybridization using probes for suspected
pathogens of interest.
[0269] The present invention permits one to increase further the
number of simultaneously detectable bacteria without increasing the
number of available fluorophores by using combinatorial
fluorescence imaging (Nederlof, P. M. et al., Cytometry 11:126-131
(1990)). By labeling a single probe with two (or more) different
reporter molecules, the resultant hybridized probe will be detected
by more than one of the fluorescent detectors and the signal will
appear in more than one of the separate flurorescence images. When
these separate pseudocolored images are merged into a composite
image (as in FIG. 4 panels E, F and G), fluorescence signals
appearing at the same site on two images will be "blended" and
generate a new pseudocolor that is distinguishable from either of
the originals. In this fashion, three fluorophores have been used
combinationally to visualize seven probes simultaneously, each
appearing as a distinct color (Ried, T. et al., Proc Natl Acad Sci
(USA) 89:1388-1392 (1992)). By applying this combinatorial
fluorescence imaging strategy to bacterial identification, one can
obtain a distinct pseudocolor for each of the seven bacterial
species analyzed above.
[0270] The above example demonstrates the feasibility of
differentiating multiple bacterial species (such as those found in
the periodontal microflora) using multiparametric fluorescence in
situ hybridization (M-FISH) and digital imaging microscopy. By
monitoring both hybridization signal specificity and bacterial
morphology, seven different bacteria are readily and simultaneously
distinguished in a mixed population using only three distinct
fluorophores. Such methods are readily applicable to the detection
of bacteria (as well as viruses, and/or lower. eukaryotes) that may
be present in other clinical samples (including those that contain
complex mixtures of microflora (e.g., stool, saliva, throat swabs,
sputum, vaginal secretions, etc.) and those that are normally
(e.g., central spinal fluid (meningitis), blood (sepsis), etc.) or
non-clinical samples (food products, water reservoirs, ecosytems,
etc.). Furthermore, by expanding the number of spectrally
resolvable fluors used in combinatorial fashion, the number of
different types of bacteria, viruses and/or lower eukaryotes that
can be analyzed in a mixed population can be markedly
increased.
[0271] B. Analysis of Cross Hybridizing Microbial Species
[0272] As indicated above, using differentially labeled total
genomic DNA probes, the in situ hybridization and digital imaging
fluorescence microscopy methods of the present invention can
simultaneously differentiate seven microorganisms in a laboratory
derived mixed sample. This technique provides both single cell
detection sensitivity and the ability to correlate hybridization
signal specificity with microbial morphology. The relative
abundance of a specific organism in a complex mixture or bacteria,
can be readily assessed and a single hybridization positive
bacteria can be detected in an excess of 10.sup.4 other bacteria.
Moreover, the assays can be completed in less than 1.5 hours.
However, none of the genomic DNAs from the seven bacteria used in
the above study exhibited cross-hybridization.
[0273] To illustrate the capacity of the present invention to
differentiate among related (i.e., cross-hybridizing) microflora, a
study is performed using Capnocytophaga DF 1 strains: C. gingivalis
(DR2001), C. sputigena (D4), and C. ochracea (C25) Such strains
exhibit cross-hybridization (Rubin, S. J., Eur. J. Clin. Microbiol.
3:253-257 (1984)). The strains are obtained from the NIH Institute
of Dental Research, Bethesda, Md. Bacterial DNA is isolated as
described above. Probes are made as follows: total genomic
bacterial DNAs is labeled by nick translation using biotin-11-dUTP
(BIO), digoxigenin-11-dUTP (DIG) or dinitrophenol-11-dUTP (DNP) as
described by Lichter, P. et al. (Hum. Genet. 80:224-234 (1988)).
Unincorporated nucleotides are removed using a Sephadex G-50 spin
column equilibrated with 10 mM Tris-HCl/1 mM EDTA/0.1% SDS, pH 8.0.
Labeled DNAs (2-6 .mu.g) are ethanol precipitated and redesolved in
100% deionized formamide.
[0274] The genus Capnocytophaga encompasses a group of fusiform,
gram-negative, capnophilic, fermatative, gliding bacteria
(Ledbetter, E. R. et al., Arch. Microbiol. 122:17-27 (1979)) that
are common inhabitants of the oropharyngeal flora (Holdeman, L. V.
et al., J. Periodon. Res. 20:475-483 (1985)). C. gingivalis, C
ochracea and C. sputigena have been implicated in gingivitis in
children (Moore, W. E. C. et al., Infect. Immun. 46:1-6 (1984)) and
periodontitis in juvenile diabetics and adults (Dzink, J. L et al.,
J. Clin. Periodont. 12:648-659 (1985); Mashimo, P. A. et al., J.
Periodont. 54:420-430 (1983); Slots, J. et al., J. Dent. Res.
63:414-421 (1984)). These three bacteria are members of the first
of three groups of "Dysgonic Fermenters" (DF-1) defined by the
Center for Disease Control (Speck, H. et al., Zbl. Bakt. Hyg. A.
266:390-402 (1987)). The DF-2 group contain species designated as
C. canimorsus and C. cynodeami, although these bacteria appear both
phenotypically and genetically distinct from the Capnocytophaga
species of the DF-1 group (Brenner, D. J. et al., J. Clin.
Microbiol. 27:231-235 (1989)). Few of the microbes comprising the
DF-3 group have been characterized, however it is thought that they
are closely related to the Capnocytophaga DF-1 species (Gill, V. J.
et al., J. Clin. Microbiol. 29:1589-1592 (1991)). Members from all
three DF groups have been identified as the etiologic agents found
in some cases of septicemia, joint infections and endocarditis
(Arlet, G. et al., Ann. Biol. Clin. 44: 373-379. (1986); Juhl, G.
et al., J. Infect. Dis. 149:654 (1984); Matlow, A. et al., J.
Infect. Dis. 152:223-234 (1985); Mosher, C. B. et al., J. Clin.
Microb. 24:161-162 (1986); Parenti, D. M. et al., J. Infect. Dis.
151:140-147 (1985); Ratner, H., Clin Microbiol 6:10 (1984);
Rummens, J. L. et al., J. Clin. Microbiol. 75:376 (1985); von
Graevenitz, A., Eur. J. Clin. Microbiol. 3:223-224 (1984)). The
increased spectrum of oral and intra-oral infections caused by
Capnocytophaga DF-1 species shows the pathogenic potential of this
genus and may reflect differences in clinical significance of the
respective species in medically important infections and
periodontal disease (von Graevenitz, A., Eur. J. Clin. Microbiol.
3:223-224 (1984)). Conventional methods of identifying bacteria
from oral sources requires first, isolation of single bacterial
colonies from the oral microbial milieu, and second, the assignment
of isolates to genus and species based upon cellular. morphology,
gram stain, fermentation of carbohydrates and hydrolysis of various
substrates. These procedures require considerable time and
expertise, taking upwards of 2 weeks to identify single species
from clinical samples. Such conventional methods often fail to
isolate Capnocytophaga from oral samples because these bacteria are
slow growing, fastidious as to growth conditions and are therefore
overgrown by other oral flora (Mashimo, P. A. et al., J. Periodont.
54:420-430 (1983); Rummens, J. L. et al., J. Clin. Microbiol.
75:376 (1985)). Moreover, such methods may fail to discriminate C
ochracea and C. sputigena unambiguously due to phenotypic
similarities (Gill, V. J. et al., J. Clin. Microbiol. 29:1589-1592
(1991)).
[0275] Recent methods of microbial identification using enzymatic
tests (Heltsberg, O. et al., Eur. J. Clin. Microbiol. 3:236-240
(1984); Kristiansen, J. E. et al., Eur. J. Clin.
Microbiol.3:236-240 (1984)), specific immunologic or molecular
probes (Murray, P. A. et al., Oral Microbiol. Immunol. 6:34-40
(1991); Smith, G. L. F. et al., Oral Microbiol. Immunol. 4:41-46
(1989)) have been used with some success for identifying bacteria
in clinical oral samples. With the exception of enzymatic
techniques, these methods have not been applied to the
identification of Capnocytophaga spp. Enzymatic tests still require
the isolation of pure colonies of bacteria, whereas immunologic and
molecular methods can be performed directly on an oral sample,
thereby overcoming the difficulties of isolating pure colonies of
fastidious microorganisms. Generally, molecular methods rely on the
use of DNA probes, formatted as a dot-blot hybridization assay, for
the identification of a particular microorganism in a clinical
sample (Parenti, D. M. et al., J. Infect. Dis. 151:140-147 (1985)).
A major drawback of this method is that DNA from 10.sup.3 to
10.sup.6 microorganisms of interest are required for such assays,
therefore bacteria in low abundance in a mixed population are
likely not to be detected. In addition, cross-hybridization, which
is common in closely related species, can impede identification to
the species level.
[0276] It had been previously reported (Williams, B. L. et al.,
Arch. Microbiol. 122:35-39 (1979)) that C. gingivalis, C. ochracea,
and C., sputigena DNAs exhibit up to 22% sequence homology, based
on reassociation kinetics. Using conventional in situ hybridization
methods, such extensive homology would appear to preclude the use
of total genome DNA probes for the speciation of Capnocytophaga
strains and necessitate the production and characterization of
species-specific subgenomic clones. However, one of the attributes
of the computerized digital imaging camera system of the present
invention is the ability to collect hybridization data in the form
of quantitative fluorescence images and to process such image data
with appropriate computer software.
[0277] The intensity of the hybridization signal (i.e.,
fluorescence photon output) from a bacteria ("bacteria A") A will
be greatest with a genomic DNA derived from that bacteria. Related
strains which share some DNA sequences in common with a DNA probe
of genomic DNA for bacteria A (genoric DNA probe A) will give less
intense hybridization signals, which directly reflect the extent of
sequence homology. Unrelated bacteria that share little or no
sequence homology to bacteria A will yield virtually no
fluorescence signal at all. Once the relative extent of DNA
sequence homology between stains has been established,
cross-hybridization noise can be electronically suppressed
(preferably via appropriate computer software and image
thresholding techniques) thus directly leading to bacterial
identification.
[0278] Computer thresholding of fluorescence images is used to
establish the extent of cross-hybridization (i.e., sequence
homology) between the three Cagnocytophaga species. In this
experiment, pure samples of each bacteria are separately hybridized
with biotinylated DNA probes from all three species. For example,
three slides are prepared that contain only C. ochracea cells. The
first slide is hybridized in situ using BIO-labeled C. ochracea
genomic DNA, the second slide is hybridized in situ using
BIO-labeled C. gingivalis genomic DNA and the third slide is
hybridized in situ using BIO-labeled C. sputigena genomic DNA. The
slides are then washed and hybridization positive bacteria are
identified using FITC-avidin.
[0279] C. ochracea and C. gingivalis did not crosshybridize with
one another, however C. sputigena cross-hybridized with both C.
gingivalis and C. ochracea. A computer analysis of the intensity of
the fluorescent hybridization signals indicates that C. sputigena
had significant sequence homology with C. ochracea (.about.22%) but
much less (.about.5%) with C. gingivalis.
[0280] To demonstrate how image thresholding can be used for
bacterial speciation, a slide with a mixture of C. sputigena and C.
orchracea is hybridized with Bio-labeled genomic DNA from C.
sputigena. To prepare such slides for in situ hybridization, small
aliquots (1-2 ml) of cultured bacteria are centrifuged at
1,200.times.g in a HBI microcentrifuge. The cell pellet is
suspended in phosphate buffered saline (PBS) and adjusted to
approximately 10.sup.3 bacteria per .mu.l. Aliquots of both pure
cultures and synthetic mixtures are spotted directly onto
commercially available (Fisher Scientific, cat #12-550-15)
positively (+) charged slides and air dried. All slides are then
fixed in Camoy's B Fixative (Ethanol: Chloroform: Acetic Acid,
6:3:1) for 5 minutes.
[0281] Hybridization is accomplished using 15 .mu.l of
hybridization solution which contained 50 ng of labeled probe and
DNAse treated salmon sperm DNA (15 .mu.g) in 50% (vol/vol)
deionized formamide/2.times.SSC (0.3 M sodium chloride/0.03 M
sodium citrate, pH 7.0)/5% dextran sulfate. The solution is applied
to the sample, covered with a coverslip and sealed with rubber
cement. Both bacterial DNA and labeled DNA probe are denatured by
heating at 80.degree. C. for 5-8 minutes in an oven. These steps
are sufficient to permeabilize the bacteria for probe
assessability.
[0282] The DNAs are allowed to reassociate by incubating the slides
at 37.degree. C. for 2.5 min. in a moist chamber. Posthybridization
washings, blocking and detection are conducted as described by
Lichter, P. et al. (Hum. Genet. 80:224-234 (1988)). Briefly, the
slides are washed 3 times in 50% formamide/2.times.SSC at
42.degree. C. for 5 minutes and then washed 3 times in
0.1.times.SSC at 60.degree. C. for 5 minutes. The slides are then
incubated in a solution of 3% bovine serum albumin/4.times.SSC
(blocking solution) for 30 minutes at 37.degree. C.
[0283] Biotinylated probes are detected using fluorescein
isothiocyanate-(FITC)-avidin DCS (Vector Laboratories, 5 .mu.g/ml),
Cascade Blue-avidin (Molecular Probes Inc., 10 .mu.g/ml) or
Rhodamine-avidin (Boehringer Mannheim, 10 .mu.g/ml).
Digoxigenin-labeled probes are detected using FITC or Rhodamine
conjugated sheep anti-digoxigenin Fab fragments (Boehringer
Mannheim, 2 .mu.g/ml). Dinitrophenol labeled probe is detected by
incubating with rat anti-DNP antibodies (Novagen, 1:500 dilution))
and then with goat anti-rat FITC-conjugated antibody (Sigma, 1
.mu.g/ml). In some experiments, bacterial DNA was counterstained
with 4,6-diamidino-2-phenylindole (DAPI) at a concentration of 200
ng/ml. For detection, fluorochrome-conjugated antibodies or avidin
are diluted into a solution of 4.times.SSC/1% BSA and 0.1% tween-20
(200 .mu.l/slide) and incubated with the sample at 37.degree. C.
for 30 minutes in the dark. The slides are then washed 3 times in
4.times.SSC/0.1% tween-20 at 42.degree. C. prior to viewing.
[0284] Hybridization is visualized via epifluorescence microscopy
using a Zeiss Axioskop-20 wide-field microscope with a 63.times.NA
1.25 Plan Neofluar oil immersion objective and a 50W high pressure
mercury arc lamp. Images are projected with a Zeiss SFL-10
photo-eyepiece onto a cooled charged-coupled device (CCD) camera
(Photometries CH220; 512.times.512 pixel array). Effective
magnification is set by changing the microscope-camera distance,
using a bellows. The 8-bit greyscale images are recorded
sequentially using DAPI, FITC and Rhodamine filter sets,
manufactured by C. Zeiss, Inc., Germany to minimize image offsets.
Camera control, greyscale image acquisition and image
pre-processing are done as described above.
[0285] All bacteria are found to be hybridization positive in this
experiment, although some appear to fluorescence more intensely
than others. After the fluorescence image had been thresholded
(using the Gene Join Max Pix software; Yale University) to remove
components of the image that exhibited less than 25% of the maximum
fluorescence intensity, only a subset of the original bacteria
appear hybridization positive. These bacteria are all C. sputigena
because only they exhibit 100% homology to the probe. This general
strategy can be used to speciate multiple related microorganisms,
provided the level of their sequence relatedness is known so that
appropriate thresholding parameters can be applied. Bacteria
exhibiting up to 40% sequence homology can be discriminated with
genomic DNA probes using this technique.
[0286] The Gene Join Max Pix software can be used in a second way
to achieve bacterial speciation with genornic DNA probes. This
embodiment exploits the fact that when separate fluorescence images
(e.g., fluorescein and rhodamine) are merged to form a composite
image; the source image with the greatest fluorescent intensity
(i.e., photon output) at each pixel location will be color-dominant
in the merged image. This attribute permits the use of multiple
differentially labeled genornic DNA probes simultaneously to probe
a single or mixed bacterial population. Since each bacteria in a
mixture will exhibit the greatest fluorescence signal with the
genomic probe that is 100% homologous, the image merging process
results in effective speciation.
[0287] This is demonstrated by the following experiment. A mixture
of the three Capnocytophaga strains is hybridized using a 1:1:1 of
all three genomic DNA probes, each labeled with a different
reporter. C. ochracea DNA was labeled with DNP-11-dUTP for
detection with FITC (yellow green), C. gingivalis DNA was labeled
with biotin-11-dUTP for detection with Cascade Blue (blue) and C.
sputigena DNA was labeled with DIG-11-dUTP for detection with
Rhodamine (red). The image generated by Cascade Blue shows intense
signals for some bacteria present in the sample, presumably C.
gingivalis cells and weak signals for other bacteria. The FITC
fluorescence image produced by the C. ochracea probe and the
Rhodamine fluorescence imge produced by the C. sputigena probe also
show both intense signals for some bacteria and weak signals for
others, reflecting interspecies hybridization. However, each
bacterium shows an intense signal in one of the three images and a
weaker (or absent) signal in the other images (reflecting
interspecies hybridization). Accordingly, through the use of
thresholding (i.e., using the signal generated to determine which
probe gave the more dominant signal with which bacteria), the
method enables one to determine the species of each bacterium.
[0288] Thus, in contrast to traditional methods of culturing and
testing biochemical products for the differentiation and/or
speciation of a bacteria, that are often difficult and time
consuming, the in situ hybridization methods of the present
invention provide a rapid and simple method for the speciation of
cross-hybridizing rnicroorganisms, such as the oral Capnocytophaga.
The basis of this method relies on the finding that all
microorganisms within different genera and species have unique DNA
sequences within their genome. Closely related organisms will have
some DNA sequences in common, and the amount of such
cross-hybridizing sequences will directly affect their ability to
be differentiated by in situ hybridization.
[0289] In this analysis, the fluorescence images obtained from in
situ hybridization with each detector (e.g., Rhodamine, FITC or
Cascade Blue), which reflect the amount of hybridization of a
bacterium to each of the genomic DNA probes, are digitalized via
computer. If a bacteria has no cross-hybridizing sequence with any
of the other bacteria it will hybridize only to its homologous DNA
and thereby be detected by only a single detector and its presence
will be found in only the image corresponding to that detector.
However, if a bacteria does have cross-hybridizing sequences with
one of the other bacteria, then it will be detected by the DNA
probes from each bacteria and its presence will be seen in the
corresponding images for each probe (detectors). However, the
intensity of the signal generated in each image will be directly
proportional to the amount of hybridization of a bacterium with a
DNA probe. The corresponding images from each detector are
pseudocolored and overlaid (merged) to form a composite image.
Since any given pixel on the monitor can only be a single color,
only the most intense signal at each pixel is shown, and displayed
as the corresponding pseudocolor.
[0290] Whereas two of the Capnocytophaga species (C gingivalis and
C ochracea) of the above experiment show no cross-hybridization,
the third species (C sputigena) cross-hybridized with both of the
preceding organisms. The amount of cross-hybridization as estimated
by a computer analysis of the intensity of the hybridization signal
following in situ hybridization is in good agreement with that
found in the literature (22-23% for C. sputigena and C. ochracea
and 9-11% for C. sputigena and C. gingivalis (Heltberg, O. et al.,
Eur. J. Clin. Microbiol. 3:236-240 (1984)). The 22-23%
crosshybridization of C. sputigena and C. ochracea is evident in
the composite image within some of the C. sputigena cells, but not
evident in the C. ochracea cells. This may reflect differences in
how the cross-hybridizing sequences are arranged within those
genomes.
[0291] This example further demonstrates that for an in situ
hybridization analysis, the DNA probe need not be totally specific.
Cross-hybridizing sequences must only be reduced below the level
necessary for showing specificity. The level of unique sequences
necessary for specificity would desirably be ascertained for each
bacteria of interest, since it would probably be affected by not
only the total number of cross-hybridizing sequences, but also how
those sequences are arranged within a genome. However, some
microorganisms, such as Neisseria meningitis and N. gonorrhoea show
greater than 80% sequence homology (Kingsbury, D. T., J. Bacteriol.
94:870-874 (1967)). Such large amounts of cross-hybridizing
sequences may result in an ambiguous analysis using in situ
hybridization with total genomic DNA probes that can be addressed
by the methods of the present invention.
[0292] Thus, in sum, multiparametric fluorescence in situ
hybridization (M-FISH) and computer assisted digital imaging
microscopy have been used to develop a rapid method for
differentiating C. gingivalis, C ochracea and C. Sputigena with
total genomic DNA probes. Although these bacteria exhibit up to 22%
sequence homology, cross-hybridization signals can be completely
suppressed. Speciation of these three Capnocytophaga species can be
achieved in as little as 30-90 minutes, markedly faster than the
2-3 weeks required using conventional microbiological methods. The
general discrimination strategy reported here is applicable to the
broad spectrum of microbial pathogens for which an extent of
sequence homology is known.
EXAMPLE 3
[0293] Synergistic Use of Nucleic Acid Amplification Technology in
Concert with M-FISH Analysis
[0294] Nucleic acid amplification technology has greatly increased
the ability to investigate detailed questions about the genotype or
the transcriptional phenotype in small biological samples, and has
provided the impetus for many significant advances in biology,
especially in the field of genetics.
[0295] One aspect of the present invention concerns the use of the
above-described multiparametric fluorescence in situ hybridization
(M-FISH) methods in concert with one or more methods of nucleic
acid amplification to facilitate the detection and characterization
of mutations, chromosomal elements, chromosomal rearrangements, and
infectious agents (e.g., viruses, proviruses, etc.) that may be
present in small amount or proportion in a sample undergoing
analysis. For example, through the use of fluorophore combinations
designed for multiparametric color coding, a 6-fluor tagging
approach permits the simultaneous identification of 63 different
types of signals by virtue of their unique spectral signatures.
[0296] As described above, the M-FISH method of the present
invention can distinguish and identify all of the chromosomes of a
human karyotype. However, by amplifying a specific gene, allele or
chromosomal element, and employing one or more labeled probes that
are capable of specifically hybridizing to the amplified sequences,
the invention permits the determination of whether a particular
karyotype contains the gene, allele or chromosomal element in
question. By way of illustration, the use of a probe specific for a
deletion, rearrangement, insertion or other mutation characteristic
of a genetic disease (e.g., hemophilia, cystic fibrosis, breast or
other cancer, etc.) it is possible to diagnose whether a patient
exhibits the genetic disease (e.g., to diagnose cystic fibrosis,
etc.). Similarly, the invention permits one to determine whether a
patient's chromosomes carry a recessive allele associated with
genetic disease. Likewise, the invention permits one to determine
whether a patient is predisposed to a disease by virtue of the
presence of a genetic lesion (e.g., a mutation in the apoE, p53,
rb, or brcal1/2 genes) associated with a future disease state (such
as Alzheimer's Disease, heart disease, cancer, etc.).
[0297] Similarly, as applied to the investigation of microbes
(e.g., bacteria, viruses, and lower eukaryotes), the M-FISH method
of the present invention can distinguish and identify the species
of microorganisms present in a clinical or non-clinical sample.
However, by amplifying a specific gene, allele or chromosomal
element, and employing one or more labeled probes that are capable
of specifically hybridizing to the amplified sequences, the
invention permits the determination of whether a particular
microorganism contains the specific gene, allele or chromosomal
element. Thus, for example, by using a probe specific for an
antibiotic resistance determinant, a cellular antigen, a toxin,
etc., the combined use of M-FISH and nucleic acid amplification
permits a determination of whether a particular microbial or viral
strain is resistant to an antibiotic, or is pathogenic, or
expresses a toxin. Such combination of methodologies thus permits
the serotyping and subspeciation of pathogens without any
requirement of culturing and/or purification.
[0298] The M-FISH methods of the present invention particularly
concern probe sets and methods that are sufficient to characterize
both the genotypic and phenotypic charateristics of micobes present
in a preparation. A genotypic probe is one capable of specifically
hybridizing to phylogenetically related species of microbes;
hybridization of such a probe to nucleic acid of a preparation thus
reveals whether a preselected microbe, or its cross-hybridizing
(i.e., phylogenetically) related microbes are present in such a
preparation. A phenotypic probe is one capable of specifically
hybridizing to genes, or genetic elements associated with a
particular phenotype (e.g., antibiotic resistance, toxin
production, antigen presentation, etc.).
[0299] The invention particularly contemplates the multiparametric
use of multiple fluors, and particularly concerns the embodiments
in which 2, 3, 4, 5, 6 or more fluorophores are employed so as to
permit the detection and/or characterization of 3, 7, 15, 31, 63 or
more combinations of genotypic or phenotypic elements. Thus, for
example, if five fluors are employed, 31 genotypic/phenotypic
elements can be probed. Any combination of such elements can be
employed. For example, 25 of such elements can be genotypic
elements (thus permitting the identification of 25 different
species of microbes), whereas 6 of such elements can be genotypic
(for example permitting the determination of whether any of the 25
identified species are resistant to any of 4 antibiotics or present
any of 2 toxins). Similarly, 10 of such elements can be genotypic
elements (thus permitting the identification of 10 different
species of microbes), whereas 21 of such elements can be genotypic
(for example permitting the determination of whether any of the 10
identified species. are resistant to any of 9 antibiotics, present
any of 3 toxins, present any of 4 surface antigens, and express any
of 5 genes).
[0300] Any of a wide group of bacteria (e.g., E. coli strains and
strains of other enterics (e.g., Salmonella), Clostridria, Vibrio,
Corynebacteria, Listeria, Bacilli (especially B. anthracis),
Staphylococcus Streptococci (especially beta-hemolytic Streptococci
and S. pneumoniae), Borrelia, Mycobacterium (especially M.
tuberculosi); Neisseria (especially N. gonorrhoeae), Trepanoma,
bacteria implicated in periodontal disease (e.g., Fusobacteria,
Porphyromonas, Eikenella, Prevotella, Actinobacillus, and
Capnocytophaga species), etc.), viruses (e.g., parvoviruses,
papoviruses, herpesviruses, togaviruses, retroviruses (especially
HIV), rhabdoviruses, influenza viruses, etc.), and lower eukaryotes
(fungi (e.g., Dermatophytes; Pneumocystis, Trypanosoma; etc.),
yeast, helminths, nematodes, etc.) can be detected and
characterized using such a method.
[0301] Significantly, the combined use of a nucleic acid
amplification method and the multiparametric fluorescence in situ
hybridization methods of the present invention can be used to
explore the quiescence or expression state of cells. Thus, by
employing probes specific to RNA produced by, for example, growing
cells, cells expressing tumor antigens or hormones, etc., the
methods of the present invention can determine not merely the
presence of tumor cells, but the extent of their malignancy.. Such
mRNA profiling may be conducted even in circumstances in which
standard cDNA hybridization approaches may not be sensitive enough
to detect changes in the concentration of low abundancy gene
products.
[0302] While the combined use of a nucleic acid amplification
method and the multiparametric fluorescence in situ hybridization
methods of the present invention can employ any suitable
amplification method or methods (e.g., Polymerase Chain Reaction
(Mullis, K. et al., Cold Spring Harbor Symp. Quant. Biol.
51:263-273 (1986); Erlich H. et al., EP 50,424; EP 84,796, EP
258,017, EP 237,362; Mullis, K., EP 201,184; Mullis K. et al., U.S.
Pat. No. 4,683,202; Erlich, H., U.S. Pat. No. 4,582,788; Saiki, R.
et al., U.S. Pat. No. 4,683,194 and Higuchi, R. "PCR Technology,"
Ehrlich, H. (ed.), Stockton Press, NY, 1989, pp 61-68), Strand
Displacement Amplification (Walker, G. T. et al., Proc. Natl. Acad.
Sci. (U.S.A.) 89:392-396 (1992); Walker et al., U.S. Pat. No.
5,270,184; Walker, U.S. Pat. No. 5,455,166); Ligase Chain Reaction
(Segev, WO90/01069; Birkenmeyer, WO93/00447), etc.), the preferred
method of amplification is the Rolling Circle Amplification (RCA)
method (Caplan, M. et al., PCT Patent Application Publication No.
WO 97/19193; herein incorporated by reference in its entirety).
[0303] Rolling Circle Amplification (RCA) is an amplification
strategy in which nucleic acid amplification is driven by a DNA
polymerase that can replicate circularized oligonucleotide probes
with either linear or geometric kinetics, under isothermal
conditions. In detail, RCA involves incubating at least one one
rolling circle replication primer (RCRP) with at least one
amplification target circle (ATC). The ATC comprises a single
stranded circular DNA molecule that contains a region complementary
to the RCRP, such that the RCRP can hybridize to the ATC and
mediate amplification in the presence of a DNA polymerase.
[0304] In the presence of two primers, one hybridizing to the "+"
strand, the other to the "-" strand of DNA, a complex pattern of
DNA strand displacement ensues that generates 10.sup.9 or more
copies of each circle in 90 minutes, enabling detection of point
mutations in human genomic DNA. This expanding cascade of strand
displacement and fragrnent-generation events is termed "DNA
Hyperbranching," and the special rolling circle amplification
driven by two primers is termed "Hyperbranched-RCA" or "HRCA."
Using a single primer, RCA generates hundreds of tandemly linked
copies of a covalently closed circle in a few minutes.
[0305] It is preferred to conduct HRCA using exo(-) Vent DNA
polyrnerase, or the large fragment of Bst DNA polymerase (Aliotta,
J. M. et al., Genet. Anal. 12:185-195 (1996); Thomas, D. C. et al.,
Clin. Chem. 43:2219 Abs 38 (1997); both herein incorporated by
reference), and by the Sequenase 2.0 variant of T7 DNA polymerase.
It had been shown that Sequenase supports rolling circle
amplification of circular oligonucleotides, albeit relatively
slowly (Fire, A. et al., Proc. NatL Acad. Sci. (USA) 92:4641-4645
(1995); Liu, D. et al., J. Amer. Chem. Soc. 118:1587-1594 (1996)).
The efficiency of RCA and HRCA reactions is increased by the
addition of proteins that bind single-stranded DNA. The addition of
E. coli single-strand binding protein (SSB) stimulates
Sequenase-catalyzed HRCA, while phage T4 gene-32 protein stimulates
Vent exo(-) catalyzed HRCA. In contrast, the Bst polymerase does
not require such ssingle-strand binding proteins for maximal
activity.
[0306] If matrix-associated, the DNA product remains bound at the
site of synthesis, where it may be tagged, condensed, and imaged as
a point light source. Linear oligonucleotide probes bound
covalently on glass surfaces can generate RCA signals, whose color
indicates the allele status of the target, depending on the outcome
of specific, target-directed ligation events. Single molecule
counting by RCA provides a powerful mutation detection method for
studies using oligonucleotide arrays, and is particularly amenable
for the analysis of rare somatic mutations.
[0307] In a further embodiment, the combined M-FISH/nucleic acid
amplification methods of the invention (especially RCA/HRCA) may be
used to detect circularizable oligonucleotides, called "padlock
probes" (Landegren, U. et al., Methods 9:84-90 (1996); Landegren,
U. et al., Ann Med. 29:585-590 (1997); (Nilsson, M. et al., Science
265:2085-2088 (1994); Nilsson, M. et al., Nat. Genet. 16: 252-255
(1997), all herein incorporated by reference) bound to single copy
genes in cytological preparations.
[0308] Multiple applications exist for the above-described gap-fill
reaction, in which target-complementary sequences are incorporated
into circles by copying and covalent closure. In principle, any DNA
sequence thus captured into a circular DNA may be amplified by RCA
or HRCA. The reaction may thus be advantageously used in situations
where it is desirable to interrogate the sequence incorporated into
a padlock probe at some point after RCA. Longer sequences, such as
microsatellite repeats, should also be capable of being copied into
circularizable probes for amplification and analysis. Subsequent to
such copying, since there is little likelihood that rolling circle
amplification will modify the number of repeats incorporated into a
circularized probe (Fire, A. et al., Proc. Natl. Acad Sci. (USA)
92:4641-4645 (1995)), direct measurement of repeat size would be
feasible.
[0309] In particular, any of three alternative HRCAIRCA strategies
for allele discrimination utilizing ligation of circularizable DNA
probes and rolling circle replication--"gap-probe RCA,"
"polymerase-mediated gap-fill RCA," and "ligase-mediated extension
of immobilized probe RCA"--may be employed in concert with the
M-FISH methods of the present invention. The polymerase mediated
"gap-fill" reaction is preferred over the "gap probe" ligation
reaction for solution studies of complex genomes. This is because
the gap oligonucleotides, which cannot be washed away in a solution
assay, are preferably used at relatively high concentrations for
the ligation step, and may therefore interfere with the HRCA
reaction, inducing the formation of amplicon artefacts. In
contrast, the gap ligation reaction is considered preferable for
allele analysis of DNA in cytological specimens. Here, the
specificity of ligation should be enhanced relative to probes
without a gap because three different sequence recognition events
and two independent ligation events must occur before "padlock"
closure. The third assay method, ligase-mediated extension of an
oligonucleotide linked to a solid surface, provides a totally novel
approach to quantify individual hybridization/ligation events and
to score rare somatic mutations.
[0310] In a highly preferred embodiment of HRCA/RCA, the DNA
generated by RCA is labeled with combinatorially labeled
fluorescent DNP-oligonucleotide tags that hybridize at multiple
sites in the tandem DNA sequence. The "decorated" DNA, labeled by
specific encoding tags, is then condensed into a small object by
cross-linking with a multivalent antibody (e.g., anti-DNP IgM). The
wild-type specific primer generates RCA products which can
hybridize to fluorescein-labeled DNP-oligonucleotide tags, while
the mutant RCA products hybridize to Cy3-labeled
DNP-oligonucleotides. The process of Condensation of Amplified
Circles after Hybridization of Encoding Tags is termed "CACHET."
The use of CACHET, in concert with the M-FISH methods of the
present invention is particularly preferred.
EXAMPLE 4
[0311] Use of M-FISH Analysis to Evaluate Telomere Integrity
[0312] Conventional analyses of chromosomes using, for example,
Giemsa banding of metaphase chromosomes have several salient
deficiencies. In particular, karyotypic changes involving
chromosome fragments that have very similar banding patterns can be
impossible to resolve using this method (Saccone, E. et al., Proc.
Natl. Acad. Sci. (USA) 89:4913-4917 (1992)). Such a finding is
particularly true of the telomeric bands which are virtually all
Giemsa negative and which are also the most gene-rich bands within
the genome. The correct identification of telomeric regions is very
important because telomeres may be involved in breakage and healing
events that can result in terminal deletion, gene amplification or
cryptic translocations.
[0313] Indeed, there is an increasing number of clinical cases that
have been found to be the result of cryptic translocations such as
hemoglobin H (Lamb, J et al., Lancet 2:819-824 (1989)), Cri-du-Chat
(Overhauser, J. et al., Amer. J. Hum. Genet. 45:296-303 (1989)),
Wolf-Hirschhorn (Altherr, M. R. et al, Amer. J. Hum. Genet.
49:1235-12342 (1991)), and Miller-Dieker lissencephaly syndrome
(Kuwano, A. et al., Amer. J. Hum. Genet. 49:707-714 (1991)). In
addition, cryptic translocations occur in up to 6% of the
population with mild to moderate mental retardation who have no
detectable chromosomal changes upon karyotyping. Many of these
genetic changes go undetected using standard cytogenetic
analysis.
[0314] As indicated above, the present invention provides an
ability to simultaneously identify the twenty-four different human
chromosomes in a metaphase spread by hybridizing a complete set of
chromosome-specific DNA probes, each labeled with a different
combination of dyes. One aspect of the present invention is the
recognition that, through the use of telomere-specific probes, the
methods of the present invention may be used to identify
translocations that would be non-identifiable (i.e., cryptic)
through the use of conventional methods. As such, the present
invention provides, for the first time, a simple screening test to
assess the integrity of telomeric regions using a single
hybridization reaction. Such multiplex hybridization assays
significantly improve the ability to detect terminal deletions and
cryptic chromosomal rearrangements, and thereby facilitate the
identification of structural abnormalities that elude detection
with conventional cytogenetic banding or multicolor
whole-chromosome painting methods. Thus, this aspect of the present
invention extends prior efforts in both karyotyping technology
(see, e.g., Speicher, M. R. et al., Nature Genet 12:368-375 (1996);
Speicher, M. R. et al., Bioimaging 4:52-64 (1996); Schrock, E. et
al., Science 273:494-497 (1996)) and telomere integrity assay
technology (see, e.g., Ledbetter, D. H., Amer. J. Hum. Genet.
51:451-456 (1992)); all herein incorporated by reference).
[0315] To illustrate this capacity of the invention, a set of YAC
clones containing human DNA sequences specific for the
sub-telomeric region of each chromosome arm is assembled. This
probe set employed a microdissected probe for the short arm of the
Y chromosome (Guan, X. Y., Nature Genet. 12:10-11 (1996), herein
incorporated by reference) because no suitable sub-telomeric YAC
for the short arm could be identified. Similarly, because of
extensive cross-hybridization, the probe set did not include probes
for the p arms of the acrocentric chromosomes 13, 14, 15, 21 and
22. The probes are combinatorially labeled such that the telomeric
regions of these chromosomes are visualized in different
pseudocolors based upon their unique fluorophore composition.
[0316] The p arm and q arm telomere proximal probes for each
chromosome are labeled with the same "color code," for example both
chromosome 1 telomeres are labeled in color "a," while the
chromosome 2 telomeres are labeled in color "b," etc. Thus, any
cytogenetically cryptic chromosome translocation occurring on a
non-acrocentric chromosome are visualized in the metaphase spreads
as a chromosome harboring 2 colors rather than a single color per
chromosome. Although the 24 color telomere integrity assays utilize
a complex probe mixture, such probe cocktails can be hybridized
with high reproducibility. Such hybridization is visualized, as
described above, with, for example, an epifluorescence microscope
equipped with an appropriate filter set and a cooled CCD-camera,
etc. The fluors and high contrast filters are the same as those
described above. In brief, fluorescein (FLU), Cy3, and Cy5 are
linked to dUTP for direct labelling; Cy3.5 and Cy7 are available as
avidin or antidigoxigenin conjugates for secondary detection of
biotin- or digoxigenin-labelled probes. For each probe, 1-3
separate nick translations are performed.
[0317] The telomeric probe set may be composed of a mixture of
YACs, Half-Yacs (Vocero-Akbani, A. et al., Genomics 36:492-506
(1996); Macini, R. A. et al., Genomic Res. 5:225-232 (1995)), and
CEPH-YACs (Chumakov, I. M. et al., Nature 377:175-297 (1995)),
chosen to contain the most telomeric genetic markers. Half-YACs are
generated from specialized YAC libraries enriched for telomeric
sequences. The term "half-YACs" refers to the fact that one of the
vector telomere sequences of the molecule is provided by human DNA
rather than yeast DNA. Half-YACs contain sub-telomere repeats that
are known to reside on different chromosomes thus, in some
instances, they hybridize to multiple telomeres. However, specific
M-FISH signals can be obtained from half-YACs, for example through
the use of DNA amplification (using, for example, PCR with Alu
primers to reduce the relative representation of repeat sequences);
addition of Cot-1 DNA to the hybridization cocktail will suppress
hybridization of any residual repetitive sequences. Suitable YACS,
half-YACs and/or CePH-YACs are described by Vocero-Akhani, A. et
al. (Genomics 36:492-506 (1996)), by the National Intitutes of
Health and Institute of Molecular Medicine Collaboration (Nature
Genet 14:86-89 (1996)) and by Bray-Ward, P. et al. (Genomics
36:1-14 (1996)); all of which references are herein incorporated by
reference. The fluors used to label telomeric regions are shown in
Table 4. In Table 4, the references are (1) Vocero-Akbani, A. et
al., Genomics 36:492-506 (1996); (2) National Intitutes of Health
and Institute of Molecular Medicine Collaboration (Nature Genet
14:86-89 (1996)); (3) Bray-Ward, P. et al. (Genomics 36:1-14
(1996)); (4) Bray-Ward, P. et al. (Genomics 36:104-111 (1996));
(5)Guan, X. Y., Nature Genet. 12:10-11 (1996)), all herein
incorporated by reference.
4TABLE 4 Evidence for Most Distal Telomeric Insert STS on
Localization/ FISH Library Size Integrated Flpter Telomere Probe
Label Source (kb) Mapl (min-max) Reference 1p TYAC179 Flu/Cy3
HDK-TYACL 200 sJCW15 Half-YAC 1 (HTY3222) 1q yRM2123 Flu/Cy3
HR-TYACL 270 Half-YAC 2 2p TYAC47 CY5/Cy7 HDK-TYACL 350 sAVA22
Half-YAC 1 (HTY3047) CEPH-L 1350 D2S359 0.01-0.06 3 695h7 CEPH-L
1280 D2S304 0.01-0.06 3 955c11 2q yRM2112 CY5/Cy7 HR-TYACL 240
sSMMI Half-YAK 2 TYAC75 HDK-TYACL 75 Half-YAC 1 933c7 CEPH-L 1690
D2S159 0.93-0.95 3 3p 852b3 Flu/Cy7 CEPH-L 1100 D3S1270 0.00-0.05 3
3q TYAC162 Flu/Cy7 HDK-TYACL 250 Sava54 Half-YAC 1 (HTY3205) 4p
764h1 Cy3.5/Cy5/Cy7 CEPH-L 400 3104 3 4q 946a3 Cy3.5/Cy5/Cy7 CEPH-L
1120 d4s2930 0.97-1.00 3 5p 789d2 Cy3/Cy7 CEPH-L 1630 D5S676 3 5q
TYAC139 Cy3/Cy7 HDK-TYACL 75 Half-YAC 1 (HTY3182) CEPH-L 945 D5S498
0.91-1.00 3 887f10 6p 870d6 Flu/Cy3/Cy7 CEPH-L 1260 D6S344 0.00 4
6q 820f9 Flu/Cy3/Cy7 CEPH-L 1050 D6S281 0.92-0.95 3 7p 626g11
Cy3.5/Cy7 CEPH-L 290 D7S531 3 7q yRM2000 Cy3.5/Cy7 HR-TYACL 240
Half-YAC 2 TYAC109 HDK-TYACL Half-YAC 1 (HTY3152) 8p yRM2205
Cy3/Cy5 HR-TYACL 250 D8S264 Half-YAC 2 931b2 CEPH-L 0.00-0.04 3 8q
yRM2053 Cy3/Cy5 HR-TYACL 170 2058VI Half-YAC 2 TYAC48 HDK-TYACL 200
Half-YAC I (HTY3048) 9p 822e7 Flu/Cy3/Cy5 CEPH-L 1640 D9S178 3 9q
908a4 Flu/Cy3/Cy5 CEPH-L 900 D9S1872 0.86-0.91 3 10p TYAC95 Flu/Cy5
HDK-TYACL Half-YAC 1 (HTY3138) 10q TYAC93 Flu/Cy5 HDK-TYACL 250
Half-YAC 1 (HTY3136) 11p yRM2209 Cy3.5/Cy5 HR-TYACL 125 D11S2071
Half-YAC 2 11q 790g9 Cy3.5/Cy5 CEPH-L 1060 D11S968 0.95-1.00 3 12p
922c8 Cy3/Cy3.5 CEPH-L 1390 D12S1455 0.01-0.05 3 12q TYAC175
Cy3/Cy3.5 HR-TYACL 250 Half-YAC 1 (HTY3218) 13q yRM2067
Cy3/Cy3.5/Cy5 HR-TYACL Half-YAC 2 14q yRM2006 Cy3.5 HR-TYACL 200
Half-YAC 2 15q 877h4 Flu/Cy3.5/Cy7 CEPH-L 610 WI-5214 3 16p 927a8
Cy7 CEPH-L 120 D16S423 0.02-0.06 3 16q TYAC135 Cy7 HDK-TYACL 100
Sava47 Half-YAC 1 (HTY3178) 17P 898a10 Flu CEPH-L 570 wi-5436 3 17q
965f1 Flu CEPH-L 1470 CHCLGATA4 3 FOR 18p yRM2102 Flu/Cy3/Cy3.5
HR-TYACL 220 Half-YAC 2 18q yRM2050 Flu/Cy3/Cy3.5 HR-TYACL 290
Half-YAC 2 19p 872g3 Cy3 CEPH-L 590 WI-9127 3 19q 926g5 Cy3 CEPH-L
1440 D19S887 3 20p 761d12 Flu/Cy3 CEPH-L 1330 AFMA049Y 3 D1 20q
793a9 Flu/Cy3 CEPH-L 1620 D20S173 3 21q yRM2208 Flu/Cy3.5/Cy5
HR-TYACL 290 Half-YAC 2 22q TYAC104 Cy3/Cy3.5/Cy7 HDK-TYACL 75
Sava39 Half-YAC 1 (HTY31476) Xp 827e10 Cy5 CEPH-L 920 wi-3553 3 Xq
883h10 Cy5 CEPH-L 1240 wi-7878 3 Yq MDP Flu/Cy5/Cy7 NIH 5
[0318] In order to test the reproducibility of specific signals
with half YAC probes, each is carefully tested in single color
experiments on normal metaphase spreads from at least five
different donors. These studies permit the assessment of
polymorphisms as well as the frequency or cross-hybridizations to
other sub-telomeric repeats. Suitable half-YACs perform very
reproducibly; in a few cases where occasional cross-hybridization
occur, the nonspecific signals usually had very low fluorescence
intensities and thus can be easily discriminated from a real
signal.
[0319] CEPH-YACs were selected to contain the most telomeric
genetic markers. Because the distance from the true telomeric end
of the chromosome is unknown, these probes were tested by FISH for
their distal band location. For six telomeric regions (2p, 2q, 5q,
7q, 8p, 8q) the hybridization of a single sub-telomeric YAC yielded
low fluorescence intensities. Therefore two (5q, 7q, 8p, 8q) or
three (2p, 2q) subtelomeric YACs were pooled in order to increase
the signal. Alu-fingerprinting analysis was used to confirm that
the YACs overlap.
[0320] A modified version of the previously published M-FISH
software for whole chromosome painting probes was used for probe
discrimination (Speicher, M. R. et al., Bioimaging 4:52-64 (1996);
Schrock, E. et al., Science 273:494-497 (1996)); Yale University).
In brief, to correct image shifts caused by optical and mechanical
imperfections, whole chromosome painting probes, one for each fluor
used in the experiment, are hybridized simultaneously with the pool
of sub-telomeric YACs. Pixel shift correction of the chromosome
painting probes is done as described above, however the chromosome
paints are not shown on the final images to facilitate the color
discrimination of the sub-telomeric probes. Instead of calculating
a specific threshold for each fluor, based on the whole chromosome
paint signals, the terminal regions of the chromosomes are
specifically analyzed. Chromosome ends are identified and
segmentation masks with different thresholds are calculated and
checked for regions with increased fluorescence intensities. This
is necessary because the simultaneous hybridization of multiple
small region-specific probes results in signals that are often in
different focal planes. In the absence of Z-axis optical sectioning
and image merging, this results in some YAC probes giving
significantly reduced fluorescence intensity values. If a specific
telomeric region is not labeled with a particular fluor, no
increased fluorescence intensity peaks is observed in at least one
of the segmentation masks. The calculated fluor segmentation masks
were not overlayed with the DAPI segmentation mask that delineates
the chromosomal boundaries (since some of the telomeric FISH
signals can lie outside the DAPI segment mask). Individual
YAC-clones first are assigned distinct gray values depending on the
boolean signature of each probe or the combination of fluors used
to label it (Speicher, M. R. et al., Bioimaging. 4:52-64 (1996);
Schrock, E. et al., Science 273:494-497 (1996)). A look-up-table
then is used to assign each DNA target a psuedocolor depending on
this gray value. Finally, this pseudocolored image was overlayed
onto the DAPI-stained chromosome image that was assigned a light
blue color.
[0321] In order to optimize the experimental parameters for
hybridizing such a large number of combinatorially labeled YAC
clones simultaneously, the multiplex telomere probe set is
hybridized to normal metaphase spreads from peripheral blood
lymphocytes. Similar to the results described above obtained with
whole chromosome painting probes, YAC probes that are labeled with
equal amounts of different fluors do not always give equal signal
intensities for each fluor. To diminish signal intensity
differentials, probe concentrations for the hybridization mix and a
reliable combinatorial labeling scheme are therefore established by
control experiments. YACs yielding large fluorescence signals are
preferentially labeled with three different fluors while YACs
yielding rather weak signals were labeled with one fluor only.
[0322] Using such probes, a 24 color telomere integrity assay is
conducted on karyotypes of 10 normal male and female donors. No
indication of polymorphisms are detected thereby suggesting that
cryptic translocations are extremely infrequent in normal
populations. A typical metaphase spread is observed (FIG. 5A), and
a normal karyotype based on the boolean signature of our
subtelomere probes is attained as expected (FIG. 5B).
[0323] In the process of testing the 24 color telomere integrity
assay, the probe set is hybridized on metaphase spreads from a
patient with a myeloproliferative disorder. Karyotyping using
G-bands reveals trisomy 8 as the only detectable cytogenetic
change. This is verified by M-FISH using chromosome specific
painting probes. The telomere integrity assay, however, yields an
intriguing hybridization pattern on the chromosomes 8. The trisomy
8 is verified in all 10 metaphase spreads analyzed. However, on 8
of these 10 metaphase spreads a split telomere signal is observed
on two chromosomes 8, indicating an inversion in this region (FIG.
6). This analysis demonstrates the power of this new technique to
detect structural abnormalities that are undetectable by standard
cytogenetic methods or 24-color techniques using chromosome
specific painting probes.
[0324] Cases where the application of the telomere-integrity assay
may be very rewarding include evaluating karyotypes of patients
with mental retardation, a combination of mental retardation and
dysmorphic features, and cancer cells that are known to have a high
genomic instability. The latter cells should be highly predisposed
to sub-telomeric translocation events. Screening for cryptic
translocations can not be done efficiently with chromosome specific
painting probes because they were not designed to detect subtle
deletion or rearrangment events.
[0325] In a further embodiment of the above methods, the
telomere-specific probe sets can be improved using PCR-assisted
chromatography (Craig, J. et al., Hum. Genet 100:472-476 (1997),
herein incorporated by reference). This tool removes repetitive
DNA, including the polymorphic repetitive sequences from half-YACs
or other M-FISH probes, and thereby facilitates the generation of
probes that will hybridize specifically in the absence of Cot-1
suppressor DNA.
[0326] While the invention has been described in connection with
specific embodiments thereof, it will be understood that it is
capable of further modifications and this application is intended
to cover any variations, uses, or adaptations of the invention
following, in general, the principles of the invention and
including such departures from the present disclosure as come
within known or customary practice within the art to which the
invention pertains and as may be applied to the essential features
hereinbefore set forth and as follows in the scope of the appended
claims.
* * * * *