Methods and compositions for chromosome-specific staining

• US 6,596,479 B1
• Filed: 06/05/1995
• Issued: 07/22/2003
• Est. Priority Date: 01/16/1986
• Status: Expired due to Fees

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Gray, Joe W., Pinkel, Daniel
• Primary Examiner(s): Marschel, Ardin H.
• Application Number:
  US08/465,221
• Time in Patent Office: 2,969 Days
• Field of Search: 435/6, 435/810, 536/22.1, 536/23.1, 536/24.1, 536/24-- 2, 536/25.3, 536/24.5, 436/501, 935/77, 935/78
• 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 interphase chromosomal DNA to detect an extra or missing portion or portions of a chromosome, or a translocation or an inversion of a portion or portions of a chromosome, the method comprising:

(a) providing a heterogeneous mixture of labeled unique sequence nucleic acid fragments which are substantially complementary to nucleic acid segments within the interphase chromosomal DNA for which detection is desired and are designed to allow detection of an extra or missing portion or portions of a chromosome, or a translocation or an inversion of a portion or portions of a chromosome;

(b) employing the heterogeneous mixture and interphase chromosomal DNA in in situ hybridization to permit detection of labeled nucleic acid fragments which are hybridized to interphase chromosomal DNA, wherein the chromosomal DNA is present in a morphologically identifiable cell nucleus during the in situ hybridization; and

(c) detecting the labeled nucleic acid fragments which are hybridized to the interphase chromosomal DNA to determine whether an extra or missing portion or portions of a chromosome, or a translocation or an inversion of a portion or portions of a chromosome is present in the target interphase chromosomal DNA.

2. The method of claim 1 wherein the labeled nucleic acid comprises fragments complementary to a portion of each chromosome in the total genomic complement of chromosomes.

3. The method of claim 1 wherein the labeled nucleic acid comprises fragments complementary to a single chromosome.

4. The method of claim 1 wherein the labeled nucleic acid comprises fragments complementary to a subset of chromosomes.

5. The method of claim 1 wherein the labeled nucleic acid comprises fragments complementary to a subregion of a single chromosome.

6. The method of claim 3 wherein the fragments are selected from the nucleic acid of normal human chromosomes 1 through 22, X, and Y.

7. The method of claim 4 wherein the fragments are selected from the nucleic acid of normal human chromosomes 1 through 22, X, and Y.

8. The method of claim 5 wherein the fragments are selected from the nucleic acid of normal human chromosomes 1 through 22, X, and Y.

9. The method of claim 1 wherein the labeled unique sequence fragments comprise fluorescent labels.

10. The method of claim 1 wherein the labeled unique sequence fragments comprise immunochemical labels.

11. The method of claim 4 wherein the labeled unique sequence fragments comprise biotin.

12. The method of claim 1, wherein the heterogeneous mixture further comprises repetitive sequences.

13. The method of claim 12, further comprising the step of disabling the hybridization capacity of repetitive sequences in the heterogenous mixture.

14. The method of claim 13, wherein the step of disabling the hybridization capacity of the repetitive sequence is carried out by contacting the chromosomal DNA with unlabeled blocking nucleic acids comprising repetitive sequences.

15. The method of claim 14, wherein the unlabeled blocking nucleic acids are C_ot-1 DNA.

16. The method of claim 1, wherein hybridization is detected on more than one chromosome in the morphologically identifiable cell nucleus.

17. The method of claim 13, wherein the step of disabling the hybridization capacity of the repetitive sequence is carried out by contacting the labeled nucleic acid fragments with unlabeled blocking nucleic acids comprising repetitive sequences.

18. The method of claim 1 wherein the labeled nucleic acid comprises fragments which are designed to allow detection of an extra portion or portions of a chromosome.

19. The method of claim 18 wherein the extra portion of a chromosome is from chromosome 21.

20. The method of claim 1 wherein the labeled nucleic acid comprises fragments which are designed to allow detection of a missing portion or portions of a chromosome.

21. The method of claim 1 wherein the labeled nucleic acid comprises fragments which are designed to allow detection of an inversion or a translocation of a portion or portions of a chromosome.

22. The method of claim 1 wherein the labeled nucleic acid is designed allow detection of a translocation consistent with chronic myelogenous leukemia.