Method of staining target interphase chromosomal DNA

• US 6,872,817 B1
• Filed: 06/05/1995
• Issued: 03/29/2005
• 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:
  US08/463,830
• Time in Patent Office: 3,585 Days
• Field of Search: 536/23.1, 536/24.1, 536 243- 2433, 536/25.3, 536/26.3, 435/4, 435/6, 435/810, 436/501, 935/77, 935/78
• US Class Current: 536/25.3
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• 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 chromosome-specific staining are provided. Compositions comprise heterogeneous 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 by in situ hybridization a target chromosome, over the entire length of the body of the target chromosome, the method comprising:

providing a heterogeneous mixture comprising labeled unique sequence nucleic acid fragments which are substantially complementary to nucleic acid segments substantially uniformly distributed over the entire length of the body of the target chromosome, and which have been prepared from chromosome-specific DNA which has been isolated from a pre-selected chromosome, wherein the pre-selected chromosome has first been isolated away from other chromosomes; and

employing the heterogeneous mixture and the target chromosome in in situ hybridization so that hybridization of unique segments within the labeled nucleic acid to the target chromosome is allowed and the presence of fluorescently labeled unique sequence nucleic acid fragments over substantially the entire length of the body of the target chromosome is detected, wherein the target chromosome is present as a morphologically identifiable chromosome during the in situ hybridization, and wherein substantially all of the repetitive sequences within the target chromosome are not stained by in situ hybridization.

2. The method of claim 1, wherein the heterogeneous mixture comprises repetitive sequences, and wherein the step of employing the hetorogenous mixture and the target chromosome in in situ hybridization further comprises employing blocking nucleic acid that comprise unlabeled fragments which are substantially complementary to the repetitive sequences in the heterogeneous mixture, whereby substantially all of the repetitive sequences within the target chromsome arc not stained.

3. The method of claim 2, wherein the heterogeneous mixture and the blocking nucleic acid are employed simultaneously.

4. The method of claim 1, wherein the heterogeneous mixture is substantially free of repetitive sequences, whereby substantially all of the repetitive sequences within the target chromosome are not stained.

5. The method of claim 1, wherein the heterogeneous mixture comprises fragments which arc designed to allow detection of extra or missing chromosomes.

6. The method of claim 1, wherein the heterogeneous mixture comprises fragments which are designed to allow detection of extra or missing portions of a chromosome.

7. The method of claim 1, wherein the heterogeneous mixture comprises fragments which are designed to allow detection of chromosomal arrangements.

8. The method of claim 1, wherein the labeled unique sequence nucleic acid fragments are labeled by direct attachment of a fluorescent label.

9. The method of claim 1, wherein the labeled unique sequence nucleic acid fragments are detected by detecting the presence of fluorescein.

10. The method of claim 1, wherein the labeled unique sequence nucleic acid fragments are labeled by modifying the nucleic acid to render it detectable by an affinity reaction.

11. The method of claim 10, wherein the labeled unique sequence nucleic acid fragments are modified by direct attachment of biotin.

12. The method of claim 1, wherein the target chromosomes are human chromosomes.

13. The method of claim 12, wherein the heterogeneous mixture comprises fragments which are designed to allow detection of extra or missing chromosomes.

14. The method of claim 12, wherein the heterogeneous mixture comprises fragments which are designed to allow detection of extra or missing portions of a chromosome.

15. The method of claim 12, wherein the heterogeneous mixture comprises fragments which are designed to allow detection of chromosomal rearrangements.