Method of detecting genetic translocations identified with chromosomal abnormalities
First Claim
1. A method of staining target chromosomal DNA to detect in an interphase cell one or more genetic translocations identified with chromosomal abnormalities, said method comprising:
- (a) providing a heterogeneous mixture of two or more nucleic acid probes having a combined complexity of at least 40 kb, which probes contain nucleic acid segments which are substantially complementary to nucleic acid segments that flank and/or extend partially or fully across breakpoint regions known to be associated with genetic translocations, wherein each probe comprises a distinct fluorescent label;
(b) reacting the heterogeneous mixture with the targeted chromosomal DNA by in situ hybridization; and
(c) observing the proximity or overlap of the regions stained by each probe, to determine whether said translocation is present in the interphase cell.
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Abstract
Methods and compositions for staining based upon nucleic acid sequence that employ nucleic acid probes are provided. Said methods produce staining patterns that can be tailored for specific cytogenetic analyses. Said probes are appropriate for in situ hybridization and stain both interphase and metaphase chromosomal material with reliable signals. The nucleic acid probes are typically of a complexity greater than 50 kb, the complexity depending upon the cytogenetic application. Methods and reagents are provided for the detection of genetic rearrangements. Probes and test kits are provided for use in detecting genetic rearrangements, particularly for use in tumor cytogenetics, in the detection of disease related loci, specifically cancer, such as chronic myelogenous leukemia (CML) and for biological dosimetry. Methods and reagents are described for cytogenetic research, for the differentiation of cytogenetically similar but genetically different diseases, and for many prognostic and diagnostic applications.
78 Citations
31 Claims
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1. A method of staining target chromosomal DNA to detect in an interphase cell one or more genetic translocations identified with chromosomal abnormalities, said method comprising:
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(a) providing a heterogeneous mixture of two or more nucleic acid probes having a combined complexity of at least 40 kb, which probes contain nucleic acid segments which are substantially complementary to nucleic acid segments that flank and/or extend partially or fully across breakpoint regions known to be associated with genetic translocations, wherein each probe comprises a distinct fluorescent label;
(b) reacting the heterogeneous mixture with the targeted chromosomal DNA by in situ hybridization; and
(c) observing the proximity or overlap of the regions stained by each probe, to determine whether said translocation is present in the interphase cell. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 23, 28)
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16. A method of distinguishing normal and malignant cells comprising staining target chromosomal DNA to detect in an interphase cell one or more genetic translocations identified with chromosomal abnormalities of malignant cells, said method comprising:
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(a) providing a heterogeneous mixture of two or more nucleic acid probes having a combined complexity of at least 40 kb, which probes contain nucleic acid segments which are substantially complementary to nucleic acid segments that flank and/or extend partially or fully across breakpoint regions known to be associated with genetic translocations, wherein each probe comprises a distinct fluorescent label;
(b) reacting the heterogeneous mixture with the targeted chromosomal DNA by in situ hybridization;
(c) observing the proximity or overlap of the regions stained by each probe to determine whether said translocation is present in the interphase cell, wherein said translocation is indicative of a malignant cell.
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17. A method of determining prognosis for a patient and/or determining the effectiveness of a therapy comprising staining target chromosomal DNA to detect in an interphase cell one or more genetic translocations identified with chromosomal abnormalities of malignant cells, said method comprising:
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(a) providing a heterogeneous mixture of two or more nucleic acid probes having a combined complexity of at least 40 kb, which probes contain nucleic acid segments which are substantially complementary to nucleic acid segments that flank and/or extend partially or fully across breakpoint regions known to be associated with genetic translocations, wherein each probe comprises a distinct fluorescent label;
(b) reacting the heterogeneous mixture with the targeted chromosomal DNA by in situ hybridization;
(c) observing the proximity or overlap of the regions stained by each probe to determine whether said translocation is present in the interphase cell, wherein the occurrence of a translocation is indicative of the prognosis of the patient and/or the effectiveness of therapy. - View Dependent Claims (18)
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19. A method of staining target chromosomal DNA to detect in an interphase cell one or more genetic translocations identified with chromosomal abnormalities, said method comprising:
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(a) providing a heterogeneous mixture of two or more nucleic acid probes having a combined complexity of at least 40 kb, which probes contain nucleic acid segments which are substantially complementary to nucleic acid segments that flank and/or extend partially or fully across breakpoint regions known to be associated with genetic translocations;
(b) reacting the heterogeneous mixture with the targeted chromosomal DNA by in situ hybridization;
(c) adding a distinct fluorescent label to each of said nucleic acid probes; and
(d) observing the proximity or overlap of the regions stained by each probe, to determine whether said translocation is present in the interphase cell. - View Dependent Claims (20, 21, 22, 24, 25, 26)
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27. A method of staining target chromosomal DNA to detect in an interphase cell one or more genetic translocations identified with chromosomal abnormalities, said method comprising:
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(a) providing a heterogeneous mixture of two or more nucleic acid probes having a combined complexity of about 50 kb, which probes contain nucleic acid segments which are substantially complementary to nucleic acid segments that flank and/or extend partially or fully across breakpoint regions known to be associated with genetic translocations, wherein each probe comprises a distinct fluorescent label;
(b) reacting the heterogeneous mixture with the targeted chromosomal DNA by in situ hybridization; and
(c) observing the proximity or overlap of the regions stained by each probe, to determine whether said translocation is present in the interphase cell.
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29. A method of distinguishing normal and malignant cells comprising staining target chromosomal DNA to detect in an interphase cell one or more genetic translocations identified with chromosomal abnormalities of malignant cells, said method comprising:
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(a) providing a heterogeneous mixture of two or more nucleic acid probes having a combined complexity of about 50 kb, which probes contain nucleic acid segments which are substantially complementary to nucleic acid segments that flank and/or extend partially or fully across breakpoint regions known to be associated with genetic translocations, wherein each probe comprises a distinct fluorescent label;
(b) reacting the heterogeneous mixture with the targeted chromosomal DNA by in situ hybridization;
(c) observing the proximity or overlap of the regions stained by each probe to determine whether said translocation is present in the interphase cell, wherein said translocation is indicative of a malignant cell.
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30. A method of determining prognosis for a patient and/or determining the effectiveness of a therapy comprising staining target chromosomal DNA to detect in an interphase cell one or more genetic translocations identified with chromosomal abnormalities of malignant cells, said method comprising:
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(a) providing a heterogeneous mixture of two or more nucleic acid probes having a combined complexity of about 50 kb, which probes contain nucleic acid segments which are substantially complementary to nucleic acid segments that flank and/or extend partially or fully across breakpoint regions known to be associated with genetic translocations, wherein each probe comprises a distinct fluorescent label;
(b) reacting the heterogeneous mixture with the targeted chromosomal DNA by in situ hybridization;
(c) observing the proximity or overlap of the regions stained by each probe to determine whether said translocation is present in the interphase cell, wherein the occurrence of a translocation is indicative of the prognosis of the patient and/or the effectiveness of therapy.
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31. A method of staining target chromosomal DNA to detect in an interphase cell one or more genetic translocations identified with chromosomal abnormalities, said method comprising:
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(a) providing a heterogeneous mixture of two or more nucleic acid probes having a combined complexity of about 50 kb, which probes contain nucleic acid segments which are substantially complementary to nucleic acid segments that flank and/or extend partially or fully across breakpoint regions known to be associated with genetic translocations;
(b) reacting the heterogeneous mixture with the targeted chromosomal DNA by in situ hybridization;
(c) adding a distinct fluorescent label to each of said nucleic acid probes; and
(d) observing the proximity or overlap of the regions stained by each probe, to determine whether said translocation is present in the interphase cell.
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Specification