Methods of staining target chromosomal DNA employing high complexity nucleic acid probes
First Claim
1. A method of staining target chromosomal material comprising:
- (a) providing at least one labeled nucleic acid probe having a complexity greater than about 40 kb, which labeled nucleic acid probe comprises fragments which are substantially complementary to unique nucleic acid segments within the chromosomal material for which detection is desired, and providing blocking nucleic acid that comprises fragments which are substantially complementary to repetitive segments in the labeled nucleic acid; and
(b) employing said labeled nucleic acid probe, blocking nucleic acid, and chromosomal DNA in in situ hybridization so that labeled repetitive segments, if present, are substantially blocked from binding to the chromosomal DNA, while hybridization of unique segments within the labeled nucleic acid probe to the chromosomal DNA is allowed, wherein blocking of the labeled repetitive segments is sufficient to permit detection of hybridized labeled nucleic acid containing unique segments, and wherein the chromosomal DNA is present in a morphologically identifiable chromosome or cell nucleus during the in situ hybridization.
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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), retinoblastoma, ovarian and uterine cancers, 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.
42 Citations
51 Claims
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1. A method of staining target chromosomal material comprising:
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(a) providing at least one labeled nucleic acid probe having a complexity greater than about 40 kb, which labeled nucleic acid probe comprises fragments which are substantially complementary to unique nucleic acid segments within the chromosomal material for which detection is desired, and providing blocking nucleic acid that comprises fragments which are substantially complementary to repetitive segments in the labeled nucleic acid; and
(b) employing said labeled nucleic acid probe, blocking nucleic acid, and chromosomal DNA in in situ hybridization so that labeled repetitive segments, if present, are substantially blocked from binding to the chromosomal DNA, while hybridization of unique segments within the labeled nucleic acid probe to the chromosomal DNA is allowed, wherein blocking of the labeled repetitive segments is sufficient to permit detection of hybridized labeled nucleic acid containing unique segments, and wherein the chromosomal DNA is present in a morphologically identifiable chromosome or cell nucleus during the in situ hybridization. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22)
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23. A method of staining target interphase chromosomal DNA comprising:
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(a) providing at least one labeled nucleic acid probe having a complexity greater than about 40 kb which labeled nucleic acid probe comprises fragments which are substantially complementary to unique nucleic acid segments within the chromosomal DNA for which detection is desired, wherein the nucleic acid probe is substantially free of repetitive segments which are complementary to repetitive segments in the target interphase chromosomal material; and
(b) employing said labeled nucleic acid probe and chromosomal DNA in in situ hybridization so that hybridization of unique segments within the labeled nucleic acid probe to the chromosomal DNA is allowed, and hybridized labeled nucleic acid containing unique segments are detected, and wherein the interphase chromosomal DNA is present in a morphologically identifiable cell nucleus during the in situ hybridization. - View Dependent Claims (25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44)
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24. A method of staining target interphase chromosomal DNA comprising:
providing at least one labeled nucleic acid probe having a complexity greater than about 40 kb which labeled nucleic acid probe comprises fragments which are substantially complementary to unique nucleic acid segments within the chromosomal DNA for which detection is desired, wherein the nucleic acid probe is substantially free of repetitive segments which are complementary to repetitive segments in the target interphase chromosomal material;
further comprisingproviding blocking nucleic acid that comprises fragments which are substantially complementary to repetitive segments in the labeled nucleic acid probe and employing said labeled nucleic acid probe, blocking nucleic acid, and chromosomal DNA in in situ hybridization so that hybridization of unique segments within the labeled nucleic acid probe to the chromosomal DNA is allowed, labeled repetitive segments, if present, are substantially blocked from binding to the chromosomal DNA, and hybridized labeled nucleic acid containing unique segments are detected, and wherein the interphase chromosomal DNA is present in a morphologically identifiable cell nucleus during the in situ hybridization.
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45. A method of staining target chromosomal material comprising:
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(a) providing at least one labeled nucleic acid probe having a complexity greater than about 50 kb, which labeled nucleic acid probe comprises fragments which are substantially complementary to unique nucleic acid segments within the chromosomal material for which detection is desired, and providing blocking nucleic acid that comprises fragments which are substantially complementary to repetitive segments in the labeled nucleic acid; and
(b) employing said labeled nucleic acid probe, blocking nucleic acid, and chromosomal DNA in in situ hybridization so that labeled repetitive segments, if present, are substantially blocked from binding to the chromosomal DNA, while hybridization of unique segments within the labeled nucleic acid probe to the chromosomal DNA is allowed, wherein blocking of the labeled repetitive segments is sufficient to permit detection of hybridized labeled nucleic acid containing unique segments, and wherein the chromosomal DNA is present in a morphologically identifiable chromosome or cell nucleus during the in situ hybridization. - View Dependent Claims (46, 47, 48)
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49. A method of staining target interphase chromosomal DNA comprising:
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(a) providing at least one labeled nucleic acid probe having a complexity greater than about 50 kb which labeled nucleic acid probe comprises fragments which are substantially complementary to unique nucleic acid segments within the chromosomal DNA for which detection is desired, wherein the nucleic acid probe is substantially free of repetitive segments which are complementary to repetitive segments in the target interphase chromosomal material; and
(b) employing said labeled nucleic acid probe and chromosomal DNA in in situ hybridization so that hybridization of unique segments within the labeled nucleic acid probe to the chromosomal DNA is allowed, and hybridized labeled nucleic acid containing unique segments are detected, and wherein the interphase chromosomal DNA is present in a morphologically identifiable cell nucleus during the in situ hybridization. - View Dependent Claims (50, 51)
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Specification