Comparative genomic hyridization
First Claim
1. A method of comparing copy numbers of different DNA sequences in a subject cell or cell population comprising the steps of:
- a) extracting the DNA from the subject cell or from a number of cells of the subject cell population;
b) amplifying said extracted subject DNA, if necessary;
c) labeling the subject DNA;
d) hybnidizing said labeled subject DNA in situ to reference metaphase chromosomes after substantially removing from the labeled DNA those repetitive sequences that could bind to multiple loci in the reference metaphase chromosomes, and/or after blocldng the binding sites for those repetitive sequences in the reference metaphase chromosomes by prehybridization with appropriate blocking nucleic acids, and/or blocking those repetitive sequences in the labeled DNA by prehybridization with appropriate blocking nucleic acid sequences, and/or including such blocking nucleic acid sequences for said repetitive sequences during said hybridization, wherein the DNA sequences in the labeled subject DNA that bind to single copy sequences in the reference metaphase chromosomes are substantially retained, and those single copy DNA sequences as well as their binding sites in the reference metaphase chromosomes remain substantially unblocked both before and during the hybridization;
e) rendering the bound, labeled DNA sequences visualizable, if necessary;
f) observing and/or measuring the intensity of the signal from the labeled subject DNA sequences as a function of position on the reference metaphase chromosomes; and
g) conmparing the copy numbers of different DNA sequences of the subject DNA by comparing the signal intensities at different positions on the reference metaphase chromosomes, wherein the greater the signal intensity at a given position, the greater the copy number of the sequences in the subject DNA that bind at that position.
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Abstract
Disclosed are new methods comprising the use of in situ hybridization to detect abnormal nucleic macid sequence copy numbers in one or more genomes wherein repetitive sequences that bind to multiple loci in a reference chromosome spread are either substantially removed and/or their hybridization signals suppressed. The invention termed Comparative Genomic Hybridization (CGH) provides for methods of determining the relative number of copies of nucleic acid sequences in one or more subject genomes or portions thereof (for example, a tumor cell) as a function of the location of those sequences in a reference genome (for example, a normal human genome). The intensity(ies) of the signals from each labeled subject nucleic acid and/or the differences in the ratios between different signals from the labeled subject nucleic acid sequences are compared to determine the relative copy numbers of the nucleic acid sequences in the one or more subject genomes as a function of position along the reference chromosome spread. Amplifications, duplications and/or deletions in the subject genome(s) can be detected. Also provided is a method of determining the absolute copy numbers of substantially all RNA or DNA sequences in subject cell(s) or cell population(s).
53 Citations
44 Claims
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1. A method of comparing copy numbers of different DNA sequences in a subject cell or cell population comprising the steps of:
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a) extracting the DNA from the subject cell or from a number of cells of the subject cell population;
b) amplifying said extracted subject DNA, if necessary;
c) labeling the subject DNA;
d) hybnidizing said labeled subject DNA in situ to reference metaphase chromosomes after substantially removing from the labeled DNA those repetitive sequences that could bind to multiple loci in the reference metaphase chromosomes, and/or after blocldng the binding sites for those repetitive sequences in the reference metaphase chromosomes by prehybridization with appropriate blocking nucleic acids, and/or blocking those repetitive sequences in the labeled DNA by prehybridization with appropriate blocking nucleic acid sequences, and/or including such blocking nucleic acid sequences for said repetitive sequences during said hybridization, wherein the DNA sequences in the labeled subject DNA that bind to single copy sequences in the reference metaphase chromosomes are substantially retained, and those single copy DNA sequences as well as their binding sites in the reference metaphase chromosomes remain substantially unblocked both before and during the hybridization;
e) rendering the bound, labeled DNA sequences visualizable, if necessary;
f) observing and/or measuring the intensity of the signal from the labeled subject DNA sequences as a function of position on the reference metaphase chromosomes; and
g) conmparing the copy numbers of different DNA sequences of the subject DNA by comparing the signal intensities at different positions on the reference metaphase chromosomes, wherein the greater the signal intensity at a given position, the greater the copy number of the sequences in the subject DNA that bind at that position. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 37, 41, 42, 43)
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19. A method of comparing copy numbers of different RNA sequences in a subject cell or cell population comprising the steps of:
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a) extracting the RNA from the subject cell or from a number of cells of the subject cell population;
b) amplifying said extracted subject RNA, if necessary;
c) labeling the subject RNA;
d) hybridizing said labeled subject RNA in situ to reference metaphase chromosomes after substantially removing from the labeled RNA those repetitive sequences that could bind to multiple loci in the reference metaphase chromosomes, and/or after blocking the binding sites for those repetitive sequences in the reference metaphase chromosomes by prehybridization with appropriate blocking nucleic acids, and/or blocking those repetitive sequences in the labeled RNA by prehybridization with appropriate blocking nucleic acid sequences, and/or including such blocking nucleic acid sequences for said repetitive sequences during said hybridization, wherein the RNA sequences in the labeled subject RNA that bind to single copy sequences in the reference metaphase chromosomes are substantially retained, and those RNA sequences as well as their binding sites in the reference metaphase chromosomes remain substantially unblocked both before and during the hybridization;
e) rendering the bound, labeled RNA sequences visualizable, if necessary;
f) observing and/or measuring the intensities of the signals from the labeled subject RNA sequences as a function of position on the reference metaphase chromosomes; and
g) comparing the copy numbers of different RNA sequences of the subject RNA by comparing the signal intensities at different positions on the reference metaphase chromosomes, wherein the greater the signal intensity at a given position, the greater the copy number of the sequences in the subject RNA that bind at that position.
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20. A method of comparing copy numbers of different DNA sequences in one subject cell or cell population relative to copy numbers of substantially identical sequences in another cell or cell population, said method comprising the steps of:
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a) extracting the DNA from both of the subject cells or cell populations;
b) amplifying said extracted subject DNAS, if necessary;
c) differentially labeling the subject DNAS;
d) hybridizing said differentially labeled subject DNAs in situ to reference metaphase chromosomes after substantially removing from the labeled DNAs those repetitive sequences that could bind to multiple loci in the reference metaphase chromosomes, and/or after blocking the binding sites for those repetitive sequences in the reference metaphase chromosomes by prehybridization with appropriate blocking nucleic acids, and/or blockidng those repetitive sequences in the labeled DNA by prehybridization with appropriate blocking nucleic acid sequences, and/or including such blocking nucleic acid sequences for said repetitive sequences during said hybridization;
e) rendering the bound, differentially labeled DNA sequences visualizable, if necessary;
f) observing and/or measuring the intensities of the signals from each subject DNA, and the relative intensities, as a function of position along the reference metaphase chromosomes; and
g) comparing the relative intensities among different locations along the reference metaphase chromosomes wherein the greater the intensity of the signal at a location due to one subject DNA relative to the intensity of the signal due to the other subject DNA at that location, the greater the copy number of the sequence that binds at that location in the first subject cell or cell population relative to the copy number of the substantially identical sequence in the second subject cell or cell population that binds at that location. - View Dependent Claims (21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 38, 39, 44)
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40. A method of comparing copy numbers of different RNA sequences in one subject cell or cell population relative to copy numbers of substantially identical sequences in another cell or cell population, said method comprising the steps of:
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a) extracting the RNA from both of the subject cells or cell populations;
b) amplifying said extracted subject RNAS, if necessary;
c) differentially labeling the subject RNAS;
d) hybridizing said differentially labeled subject RNAs in situ to reference metaphase chromosomes after substantially removing from the labeled RNAs those repetitive sequences that could bind to multiple loci in the reference metaphase chromosomes, and/or after blocking the binding sites for those repetitive sequences in the reference metaphase chromosomes by prehybridization with appropriate blocking nucleic acids, and/or blocking those repetitive sequences in the labeled RNA by prehybridization with appropriate blocking nucleic acid sequences, and/or including such blocking nucleic acid sequences for said repetitive sequences during said hybridization;
e) rendering the bound, differentially labeled RNA sequences visualizable, if necessary;
f) observing and/or measuring the intensities of the signals from each subject RNA, and the relative intensities as a function of position along the reference metaphase chromosomes; and
g) comparing the relative intensities among different locations along the reference metaphase chromosomes wherein the greater the intensity of the signal at a location due to one subject RNA relative to the intensity of the signal due to the other subject RNA at that location, the greater the copy number of the sequence that binds at that location in the first subject cell a cell population relative to the copy number of the substantially identical sequence in the second subject cell or cell population.
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Specification