Methods for chromosome-specific staining

• US 5,447,841 A
• Filed: 12/14/1990
• Issued: 09/05/1995
• 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): Parr, Margaret
• Assistant Examiner(s): Marschel, Ardin H.
• Application Number:
  US07/627,707
• Time in Patent Office: 1,726 Days
• Field of Search: 435/6, 435/91, 435/91.1, 435/91.2, 435/5, 435/243, 435/805, 435/820, 436/501, 436/504, 436/63, 436/64, 536/27, 536/22.1, 536/23.1, 536/24.3-24.33, 536/25.42, 935/23, 935/78, 935/77
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Q 1/6832 Enhancement of hybridisation reaction
• 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 2525/186 incorporating a non-extendable or blocking moiety
• 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
• CPC Class Codes: Y10S 435/805 Test papers
• CPC Class Codes: Y10S 435/82 Subcellular parts of microorganisms

Abstract

Methods and compositions for chromosome-specific staining are provided. Compositions comprise heterogenous mixtures of labeled nucleic acid fragments having substantially complementary base sequences to unique sequence regions of the chromosomal DNA for which their associated staining reagent is specific. Methods include methods for making the chromosome-specific staining compositions of the invention, and methods for applying the staining compositions to chromosomes.

Claims

1. A method of staining target chromosomal DNA comprising:

(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.

2. The method of claim 1 wherein the blocking nucleic acid is hybridized with the labeled nucleic acid prior to hybridization of the labeled nucleic acid with the chromosomal DNA.

3. The method of claim 1 wherein the blocking nucleic acid is hybridized with the chromosomal DNA prior to hybridization of the labeled nucleic acid with the chromosomal DNA.

4. The method of claim 1 wherein the blocking nucleic acid is hybridized with the labeled nucleic acid and separately with the chromosomal DNA prior to hybridization of the labeled nucleic acid with the chromosomal DNA.

5. The method of claim 1 wherein the labeled nucleic acid, the blocking nucleic acid, and the chromosomal DNA are hybridized together during in situ hybridization step.

6. The method of claim 1 wherein the labeled nucleic acid comprises fragments which are designed to allow detection of extra or missing chromosomes, extra or missing portions of a chromosome, or chromosomal rearrangements.

7. The method of claim 6 wherein the chromosomal rearrangement is a translocation or an inversion.

8. The method of claim 6 wherein the labeled nucleic acid is designed to allow detection of a chromosomal rearrangement consistent with chronic myelogenous leukemia.

9. The method of claim 6 wherein the labeled nucleic acid is designed to allow detection of aneuploidy.

10. The method of claim 6 wherein the extra chromosome is chromosome 21.

11. The method of claim 1 wherein the labeled nucleic acid comprises fragments complementary to the total genomic complement of chromosomes, fragments complementary to a single chromosome, fragments complementary to a subset of chromosomes, or fragments complementary to a subregion of a single chromosome.

12. The method of claim 11 wherein the fragments are selected from the nucleic acid of normal human chromosomes 1 through 22, X, and Y.

13. The method of claim 1 wherein the chromosomal DNA is metaphase or interphase chromosomal DNA.

14. The method of claim 1 wherein the repetitive segments comprise segments which are substantially complementary to highly repetitive segments and middle-repetitive segments.

15. The method of claim 14 wherein the repetitive segments are substantially complementary to satellite repetitive segments.

16. The method of claim 14 wherein the repetitive segments are substantially complementary to tandem repetitive segments or repetitive segments located in regions of centromeric heterochromatin.

17. The method of claim 14 wherein the repetitive segments are substantially complementary to interspersed repetitive segments.