Methods for detection by in situ hybridization of multiple chromosomes or regions thereof

• US 5,776,688 A
• Filed: 01/10/1997
• Issued: 07/07/1998
• Est. Priority Date: 09/20/1990
• Status: Expired due to Term

• Current Assignee: Vysis, Inc. (Abbott Laboratories)
• Original Assignee: Vysis, Inc. (Abbott Laboratories)
• Inventors: Morrison, Larry E., Bittner, Michael L., Legator, Mona S.
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): WHISENANT, ETHAN C
• Application Number:
  US08/781,682
• Time in Patent Office: 543 Days
• Field of Search: 435/6, 536/23.1, 536/24.3, 935/76, 935/77, 935/78
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6841 In situ hybridisation
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2565/102 Multiple non-interacting labels
• CPC Class Codes: Y10S 435/81 Packaged device or kit

Abstract

Direct label probe compositions which stain DNA of a preselected single chromosome or region of a chromosome of a multi-chromosomal genome are provided that comprise mixed DNA segments which are covalently bound to fluorophore groups through linking groups. The mixed DNA segments are derived from the DNA present in the preselected chromosome or chromosome region. These probe compositions can be used concurrently or sequentially with other probe compositions.

Claims

1. A method for the detection by in situ hybridization of a plurality of target chromosomes or a plurality of target regions of chromosomes comprising:

(a) contacting under hybridizing conditions a plurality of target chromosomes or a plurality of target regions of chromosomes, which are contained within a cytological or histological preparation or specimen, with a multiple probe composition comprising a plurality of direct label DNA probe compositions, wherein;

(i) each of the direct label DNA probe compositions is detectable by use of a different fluorescent label, each label being detectable in the presence of the other fluorescent label or labels, and

(ii) each of the direct label DNA probe compositions comprises direct label DNA segments which;

(1) are complementary to different target chromosomes or target regions of a chromosome to be detected,

(2) have multiple fluorescent labels covalently attached thereto, and

(3) have 0.3 to 6 mole percent of total nucleotides in each direct label DNA segment fluorescently labeled, and

(b) detecting the presence or absence of hybridization of each of the direct label DNA probe compositions to the chromosomes or regions of a chromosome by fluorescent techniques.

2. The method of claim 1 which further comprises adding an excess of blocking DNA segments to the multiple probe composition to bind with nonspecific DNA in the multiple probe composition during the contacting step (a).

3. The method of claim 1 wherein the direct label DNA segments comprise fluorescent labels covalently linked to the direct label DNA segments via transaminated cytosine sites.

4. The method of claim 1 wherein the multiple probe composition comprises at least three direct label DNA probe compositions.

5. The method of claim 1 wherein the target chromosomes or target regions of a chromosome are contained in amniotic cells.

6. The method of claim 1 wherein the target chromosomes or target regions of a chromosome are contained in cells grown in culture.

7. The method of claim 1 wherein the target chromosomes or target regions of a chromosome are contained in at least one interphase cell.

8. The method of claim 1 wherein the target chromosomes or target regions of a chromosome are extracted from tissue.

9. The method of claim 1 wherein the target chromosomes or target regions of a chromosome are contained in animal cells.

10. The method of claim 1 wherein said fluorescent labels are each derivatives of a compound selected from the group consisting of 7-amino-4-methylcoumarin-3-acetic acid, succinimidyl ester;

Texas Red sulfonyl chloride;

5-(and 6-)-carboxy-X-rhodamine, succinimidyl ester;

Lissamine rhodamine B sulfonyl chloride;

5-(and 6-)-carboxyfluorescein, succinimidyl ester;

fluorescein-5-isothiocyanate;

7-diethylaminocoumarin-3-carboxylic acid, succinimidyl ester;

tetramethylrhodamine 5-(and 6-)isothiocyanate;

5-(and 6-)-carboxytetramethylrhodamine, succinimidyl ester;

7-hydroxycoumarin-3-carboxylic acid, succinimidyl ester; and

6- fluorescein-5-(and -6)-carboxamido!hexanoic acid, succinimidyl ester.