Multiparametric fluorescence in situ hybridization

Claim 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.

Claim 27

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.

Claim 28

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.

Claim 32

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.

Claim 33

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.

Claim 40

The method of any of claims 25 and 29, wherein an optical comb is employed in step (b) to detect the predetermined label of a member of said second subset of probes.

Claim 42

The method of claims 25 and 29, wherein an optical filter is employed in step (b) to detect the predetermined label of a member of said second subset of probes.

Claim 49

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.

Claim 50

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.

Claim 52

The method of any of claims 25 and 29, wherein at least one probe of said set of combinatorially labeled probes is hybridized to a DNA molecule that is produced through in situ nucleic acid amplification.