Back to results
US 6,203,977 B1Granted
Delineation of individual human chromosomes in metaphase and interphase cells by in situ suppression hybridization
AssigneeYale University
InventorsDavid C. Ward, Peter Lichter, Thomas Cremer, Laura Manuelidis, Thomas Ried, Antonio Baldini
Life of this patent
Estimating the term…
Abstract
This disclosure relates to a method of specifically decorating selected mammalian chromosomes and of detecting, identifying and and/or quantitating selected individual chromosomes, by means of chromosomal in situ suppression (CISS) hybridization. The method is useful in analyzing cells for the occurrence of chromosomes, chromosome fragments or chromosome aberrations.
Claims32
A method of in situ hybridization to simultaneously detect multiple target sequences of interest in chromosomal DNA of interphase cells of nuclei thereof, the method comprising:
- (a) treating a sample of interphase cells or nuclei thereof to render chromosomal DNA present therein available for hybridization to sequences complementary thereto,
- (b) contacting said sample with a pool of DNA, wherein said pool comprises:
- i) different labeled DNA probes, each DNA probe comprising either DNA fragments labeled with a single fluorophores or a mixture of DNA fragments separately labeled with different fluorophores, such that each DNA probe generates an optically distinguishable signal, and each probe being specific for one human chromosome, of region thereof, suspected to be present in said sample;
- and
- ii) competitor DNA fragment, wherein said fragments comprise repetitive sequeneces complementary to sequeneces present in said sample and to sequeneces present in said probes;
- under conditions that allow hybridization of said competitor DNA fragments to said complementary repetitive sequeneces present in said sample and present in said probes sufficient to suppress cross-hybridization between said repetitive sequeneces in said probes amd in said sample and allow hybridization of said probes to specific target chromosomal sequeneces present in said sample, and
- c) detecting multiple hybridization signals simultaneously, wherein each detected signals is distinguishable and identifies a specific target chromosomal sequenece present in said sample.
4 more elements
A method of DNA hybridization for detecting target chromosomal DNA in situ in interphase cells of nuclei thereof, comprising:
- a) providing labeled DNA probes having sequences which specifically hybridize to target chromosomal DNA, but have repetitive sequences which cross-hybridize to non-target chromosomal DNA, and competitor DNA fragments which contain the repetitive sequences, wherein the labeled DNA probes and the competitor DNA fragments are DNA fragments which range from 150-250 nucleotides in length:
- b) combining
- i) the labeled DNA probes;
- ii) competitor DNA fragments;
- and
- iii) a sample of interphase cells, or nuclei thereof, treated to render chromosomal DNA therein available for hybridization with the labeled DNA probes,
- under hybridization conditions wherein cross-hybridization between repetitive sequences in th DNA probes and the sample are sufficiently suppressed to allow the DNA probes to hybridize specifically to the target chromosomal DNA;
- and
- c) detecting the labeled DNA probes in order to detect the target chromosomal DNA in situ in interphase cells or nuclei thereof.
6 more elements
A method of DNA hybridization for detecting target chromosomal DNA is situ, comprising:
- a) providing labeled DNA probes having sequences which specifically hybridize to target chromosomal DNA, but having repetitive sequences which cross-hybridize to non-target chromosomal DNA, and competitor DNA fragments containing the repetitive sequences;
- b) combining
- i) the label DNA probes at a concentration equivalent to 5 to 30 micrograms per milliliter of a genomic DNA library of and individual human chromosome;
- ii) the competitor DNA fragments at a concentration equivalent to about 100 to 200 micrograms per milliliter of total human genomic DNA;
- and
- iii) a sample of cells treated to render chromosomal DNA therein available for hybridization with the labeled DNA probes,
- under hybridization conditions wherein cross-hybridization between reptitive sequences in the DNA probes and the sample is sufficiently suppressed to allow the DNA probes to hybridize soecifically to the target chromosomal DNA;
- and
- c) detecting the labeled DNA probes in order to detect the target chromosomal chromosomal DNA in situ.
6 more elements
A method of DNA hybridization for detecting target chromosomal DNA in situ, comprising:
- a) providing labeled DNA probes having sequences which specifically hybridize to target chromosomal DNA, but having repetitive sequences which cross-hybridize to non-target chromosomal DNA, and competitor DNA fragments which contain the repetitive sequences;
- b) combining
- i) the labeled DNA probes;
- ii) the competitor DNA fragments;
- and
- iii) a sample of cells treated to render chromosomal DNA therein available for hybridization with the labeled DNA probes,
- under hybridization conditions wherein cross-hybridization between repetitive sequences in the DNA probes and the sample is sufficiently suppressed to allow the DNA probes to hybridize specifically to the target chromosomal DNA, wherein the concentrations of the labeled DNA probes and the competitor DNA fragments are selected such that the signal-to-noise confidence ratio interval (99%) for hybridization is at least 7.18±0.37 calculated using Fieller's theorem;
- and
- c) detecting the labeled DNA probes in order to detect the target chromosomal DNA in situ.
6 more elements
A method of in situ hybridization for distinguishably labeling individual human chromosomes, or regions thereof, in interphase cells of nuclei thereof comprising:
- (a) treating a sample of interphase cells or nuclei thereof to render chromosomal DNA present therein available for hybridization to complementary sequences,
- (b) contacting said sample with a pool of DNA, wherein said pool comprises:
- i) different labeled DNA probes, each DNA probe comprising either DNA fragments labeled with a single fluorophore or a mixture of DNA fragments separately labeled with different fluorophores, such that each DNA probe generates an optically distinguishable signal, and each probe being specific for one of said human chromosomes, of region thereof, suspected to be present in said sample;
- and
- ii) competitor DNA fragments, wherein said fragments comprise repetitive sequences complementary to sequences present in said sample and to sequences present in said probes,
- under conditions that allow hybridization of said competitor DNA fragments to said complementary repetitive sequences present in said sample and present in said probes, sufficient to suppress cross-hybridization between said repetitive sequences in said probes and in said sample, allowing hybridization of said probes to specific target chromosomal sequences present in said sample, to thereby distinguishable label individual human chromosomes, or regions thereof, in interphase cells of nuclei thereof.
3 more elements
A method of in situ hybridization for simultaneously visualizing individual human chromosomes, or regions thereof, in interphase cells or nuclei thereof comprising:
- (a) treating a sample of interphase cells or nuclei thereof to render chromosomal DNA present therein available for hybridization to complementary sequences,
- (b) contacting said sample with a pool of DNA, wherein said pool comprises:
- i) different labeled DNA probes, each DNA probe comprising either DNA fragments labeled with a single fluorophore or a mixture of DNA fragments separately labeled with different fluorophores, such that each DNA probe generates an optically distinguishable signal, and each probe being specific for one of said human chromosomes, or region thereof, suspected to be present in said sample;
- and
- ii) competitor DNA fragments, wherein said fragments comprise repetitive sequences complementary to sequences present in said sample and to sequences present in said probes,
- under conditions that allow hybridization of said competitor DNA fragments to said complementary repetitive sequences present in said sample and present in said probes, sufficient to suppress cross-hybridization between said repetitive sequences in said probes and in said sample and allow hybridization of said probes to specific target chromosomal sequences present in said sample;
- and
- c) detecting the optically distinguishable signals generated by DNA probes to simultaneously visualize each human chromosome, or regions thereof, in interphase cells or nuclei thereof.
5 more elements
A method of in situ hybridization to simultaneously detect multiple target sequences of interest in chromosomal DNA from interphase cells or nuclei thereof, the method comprising:
- (a) treating a sample of interphase cells or nuclei thereof to render chromosomal DMA present therein available for hybridization to sequences complementary thereto,
- (b) contacting said sample with a pool of DNA, wherein said pool comprises:
- i) different labeled DNA probes, each DNA probe comprising either DNA fragments labeled with a single fluorophore or a mixture of DNA fragments separately labeled with different fluorophores, such that each DNA probe generates an optically distinguishable signal, and each probe being specific for one of said human chromosomes, or region thereof, suspected to be present in said sample;
- and
- ii) competitor DNA fragments, wherein said fragments comprise repetitive sequences complementary to sequences present in said sample and to sequences present in said probes,
- wherein the labeled DNA probes and the competitor DNA fragments are DNA fragments ranging from 150-250 nucleotides, under conditions that allow hybridization of said competitor DNA fragments to said complementary repetitive sequences present in said sample and present in said probes, sufficient to suppress cross-hybridization between said repetitive sequences in said sample and allow hybridization of said probes to specific target chromosomal sequences present in said sample;
- and
- c) detecting multiple hybridization signals simultaneously, wherein each detected signal is distinguishable and identifies a specific target chromosomal sequence present in said sample.
5 more elements
Dependencies are inferred from the claim text.