Method of analyzing chromosomal inversions
First Claim
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1. A chromogenic in situ hybridization method for determining whether a sample comprises a chromosomal rearrangement, the chromosomal rearrangement occurring as a result of a breakpoint within a gene, comprising:
- contacting the sample witha first nucleic acid probe comprising a first sequence configured to hybridize to a first chromosomal DNA target located 5′
to the breakpoint,a second nucleic acid probe comprising a second sequence configured to hybridize to a second chromosomal DNA target located 3′
to the breakpoint, anda third nucleic acid probe comprising a third sequence having a 5′
portion and a 3′
portion, the 5′
portion configured to hybridize to a portion of a third chromosomal DNA target that is 5′ and
adjacent to the breakpoint, and the 3′
portion configured to hybridize to a portion of the third chromosomal DNA target that is 3′ and
adjacent to the breakpoint, such that in an absence of a rearrangement the third nucleic acid probe hybridizes to a region of the third chromosomal DNA target spanning the breakpoint;
establishing conditions suitable for the first, second, and third probes to hybridize to the respective chromosomal DNA targets in the sample;
contacting the sample with first, second, and third detection reagents, the first detection reagent comprising components to label the first chromosomal DNA target with a first chromogen, the second detection reagent comprising components to label the second chromosomal DNA target with a second chromogen, and the third detection reagent comprising components to label the third chromosomal DNA target with a third chromogen, where each of the first, second, and third chromogens provide different detectable signals;
detecting a colocalization of a third signal from the third labeled chromosomal DNA target with a first signal from the first labeled chromosomal DNA target;
detecting a colocalization of the third signal from the third labeled chromosomal DNA target with a second signal from the second labeled chromosomal DNA target; and
identifying the chromosomal rearrangement based on the detected colocalizations.
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Abstract
The present disclosure relates to systems and methods for analyzing chromosomal translocations, and in particular to analysis of chromosomal translocation by in situ hybridization.
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Citations
13 Claims
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1. A chromogenic in situ hybridization method for determining whether a sample comprises a chromosomal rearrangement, the chromosomal rearrangement occurring as a result of a breakpoint within a gene, comprising:
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contacting the sample with a first nucleic acid probe comprising a first sequence configured to hybridize to a first chromosomal DNA target located 5′
to the breakpoint,a second nucleic acid probe comprising a second sequence configured to hybridize to a second chromosomal DNA target located 3′
to the breakpoint, anda third nucleic acid probe comprising a third sequence having a 5′
portion and a 3′
portion, the 5′
portion configured to hybridize to a portion of a third chromosomal DNA target that is 5′ and
adjacent to the breakpoint, and the 3′
portion configured to hybridize to a portion of the third chromosomal DNA target that is 3′ and
adjacent to the breakpoint, such that in an absence of a rearrangement the third nucleic acid probe hybridizes to a region of the third chromosomal DNA target spanning the breakpoint;establishing conditions suitable for the first, second, and third probes to hybridize to the respective chromosomal DNA targets in the sample; contacting the sample with first, second, and third detection reagents, the first detection reagent comprising components to label the first chromosomal DNA target with a first chromogen, the second detection reagent comprising components to label the second chromosomal DNA target with a second chromogen, and the third detection reagent comprising components to label the third chromosomal DNA target with a third chromogen, where each of the first, second, and third chromogens provide different detectable signals; detecting a colocalization of a third signal from the third labeled chromosomal DNA target with a first signal from the first labeled chromosomal DNA target; detecting a colocalization of the third signal from the third labeled chromosomal DNA target with a second signal from the second labeled chromosomal DNA target; and identifying the chromosomal rearrangement based on the detected colocalizations. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12)
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13. A chromogenic in situ hybridization method for determining whether a sample comprises a chromosomal rearrangement associated with cancer, the chromosomal rearrangement occurring as a result of a breakpoint within a gene, comprising:
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contacting the sample with a first nucleic acid probe comprising a first sequence configured to hybridize to a first chromosomal DNA target located 5′
to the breakpoint, the first nucleic acid probe conjugated to a first hapten;a second nucleic acid probe comprising a second sequence configured to hybridize to a second chromosomal DNA target located 3′
to the breakpoint, the second nucleic acid probe conjugated to a second hapten;a third nucleic acid probe comprising a third sequence having a 5′
portion and a 3′
portion, the 5′
portion configured to hybridize to a portion of a third genomic DNA target that is 5′ and
adjacent to the breakpoint, and the 3′
portion configured to hybridize to a portion of the third chromosomal DNA target that is 3′ and
adjacent to the breakpoint, such that in an absence of a rearrangement the third nucleic acid probe hybridizes to a region of the third chromosomal DNA target spanning the breakpoint, the third nucleic acid probe conjugated to a third hapten;establishing conditions suitable for the first, second, and third probes to hybridize to the respective chromosomal DNA targets in the sample; contacting the sample with first, second, and third antibodies that are specific to the first, second, and third haptens, respectively, and wherein the first, second, and third antibodies are each conjugated to an enzyme; contacting the sample with first, second, and third chromogenic substrates, to provide first, second, and third labeled chromosomal DNA targets, wherein each of the chromogenic substrates provide different signals; detecting a colocalization of a third signal from the third labeled chromosomal DNA target with a first signal from the first labeled chromosomal DNA target; detecting a colocalization of the third signal from the third labeled chromosomal DNA target with a second signal from the second labeled chromosomal DNA target; and identifying the chromosomal rearangement based on the detected colocalizations.
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Specification