Methods of biological dosimetry employing chromosome-specific staining

• US 6,132,961 A
• Filed: 06/07/1995
• Issued: 10/17/2000
• Est. Priority Date: 01/16/1986
• Status: Expired due to Term

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Pinkel, Daniel, Gray, Joe W.
• Primary Examiner(s): Marschel, Ardin H.
• Application Number:
  US08/479,526
• Time in Patent Office: 1,959 Days
• Field of Search: 435/6, 435/5, 435/91.2, 536/24.3-24.33, 422/50, 422/68.1
• US Class Current: 435/6.14
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6841 In situ hybridisation
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/6879 for sex determination
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2525/151 repeat or repeated sequences, e.g. VNTR, microsatellite, concatemer
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

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 are provided to disable the hybridization capacity of shared, high copy repetitive sequences and/or remove such sequences to provide for useful contrast. Still further methods are provided to produce chromosome-specific staining reagents which are made specific to the targeted chromosomal material, which can be one or more whole chromosomes, one or more regions on one or more chromosomes, subsets of chromosomes and/or the entire genome. Probes and test kits are provided for use in tumor cytogenetics, in the detection of disease related loci, in analysis of structural abnormalities, such as translocations, and for biological dosimetry. Further, methods and prenatal test kits are provided to stain targeted chromosomal material of fetal cells, including fetal cells obtained from maternal blood. Still further, the invention provides for automated means to detect and analyse chromosomal abnormalities.

Claims

1. A method of biological dosimetry comprising measuring the frequency of structurally aberrant chromosomes to determine the level of genetic damage suffered by a subject, wherein the frequency of structurally aberrant chromosomes is measured by staining target interphase chromosomal DNA according to a method which comprises:

(a) providing

     1) labeled nucleic acid that comprises fragments which are substantially complementary to nucleic acid segments within the chromosomal DNA for which detection is desired, and

     2) 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, blocking nucleic acid, and chromosomal DNA in in situ hybridization so that labeled repetitive segments are substantially blocked from binding to the chromosomal DNA, while hybridization of unique segments within the labeled nucleic acid 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;

wherein the detection of unique segments in the chromosomal DNA indicates structurally aberrant chromosomes and the frequency of structurally aberrant chromosomes is measured to determine levels of generic damage suffered by a subject.

2. The method of claim 1, wherein the structurally aberrant chromosome is a dicentric chromosome or a translocation.

3. The method according to claim 1, wherein the hybridized labeled nucleic acid containing unique segments are detected visually in a microscope or in an automated manner using computer assisted microscopy.

4. The method according to claim 1, wherein a translocation is discriminated from a dicentric by adding to the probe at least one sequence found at all of the chromosome centromeres.

5. The method according to claim 4, wherein the sequence found at the chromosome centromeres is differently labeled from that of the labeled nucleic acid which hybridizes to the unique segments of the chromosomal DNA.

6. The method according to claim 5, wherein the centromere sequence and the labeled nucleic acid are differently labeled by color.

7. The method according to claim 1, wherein one or more chromosomes are stained and wherein the labeled nucleic acid comprises labels of one or more colors to stain said chromosomes.

8. The method according to claim 1, wherein said fragments of labeled nucleic acid are labeled using more than one color.

9. The method according to claim 1, wherein the labeled nucleic acid is a high complexity probe comprising greater than or equal to 50 kb of nucleic acid sequences.

10. A method for biological dosimetry comprising measuring the frequency of structurally aberrant chromosomes by staining target interphase chromosomal DNA with a nucleic acid probe comprising:

providing labeled nucleic acid that comprises fragments which are substantially complementary to nucleic acid segments within the chromosomal DNA for which detection is desired, from which high copy repetitive nucleic acid sequences or genomic DNA that comprises fragments which are substantially complementary to repetitive segments have been removed; and

employing said labeled nucleic acid and chromosomal DNA in in situ hybridization so that hybridization of unique segments within the labeled nucleic acid to the chromosomal DNA is allowed, wherein removal of the repetitive segments from the labeled nucleic acid 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;

wherein the detection of unique segments in the chromosomal DNA indicates structurally aberrant chromosomes and the frequency of structurally aberrant chromosomes is measured to determine level of genetic damage suffered by a subject, wherein a translocation is discriminated from a dicentric by adding to the probe at least one sequence found at all of the chromosome centromeres.

11. The method according to claim 10, wherein the sequence found at the chromosome centromeres is differently labeled from that of the labeled nucleic acid which hybridizes to the unique segments of the chromosomal DNA.

12. The method according to claim 11, wherein the centromere sequence and the labeled nucleic acid are differently labeled by color.

13. The method according to claim 10, wherein said fragments of labeled nucleic acid are labeled using more than one color.