Direct label transaminated DNA probe compositions for chromosome identification and methods for their manufacture

• US 5,491,224 A
• Filed: 04/04/1994
• Issued: 02/13/1996
• Est. Priority Date: 09/20/1990
• Status: Expired due to Term

• Current Assignee: Vysis, Inc. (Abbott Laboratories Incorporated)
• Original Assignee: Larry E. Morrison, Michael L. Bittner, Mona S. Legator
• Inventors: Morrison, Larry E., Bittner, Michael L., Legator, Mona S.
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): Marschel, Ardin H.
• Application Number:
  US08/222,167
• Time in Patent Office: 680 Days
• Field of Search: 435/6, 435/810, 436/501, 536/22.1, 536/23.1, 536/24.1, 536/25.3, 536/25.4, 935/77, 935/78, 935/88
• US Class Current: 536/22.1
• 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 direct label probe composition for staining the DNA present in a chromosome or region of a chromosome comprising multiple DNA segments complementary to different portions of said chromosome or chromosome region to be detected wherein (a) said DNA segments include multiple fluorescent labels covalently linked to the DNA segments via transaminated cytosine sites and (b) 0.3 to 6 mole percent of bases in the DNA segments are fluorescently labeled.

2. The probe composition according to claim 1 wherein the DNA sequences have an average length of about 150 to 600 bp.

3. The probe composition according to claim 1 wherein 1 to 30 mole percent of cytosine sites in the DNA segments are aminated by bisulfite catalyzed transamination with ethylenediamine.

4. The direct label probe composition of claim 1 wherein said fluorescent labels are 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.

5. A direct label probe composition for detecting the presence by hybridization of DNA comprising a single preselected chromosomal entity of a multi-chromosome genome, said chromosomal entity being selected from the group consisting of (a) a whole chromosome, and (b) a region of a chromosome, said probe composition comprising:

(a) a mixture of different DNA segments that are derived from the DNA sequences comprising said preselected chromosomal entity and that are complementary to DNA segmental portions occurring throughout said DNA sequences, said DNA segments having an average size in the range of 150 to 600 base pairs, and

(b) said DNA segments being covalently substituted on 1 to 30 mole percent of the total deoxycytidine nucleotides thereof with a linking group, said linking group being additionally covalently bonded to a fluorophore group, wherein 0.3 to 6 mole percent of bases in the DNA segments are labeled with said fluorophore group, so that individual ones of such fluorophore-group containing DNA segments are hybridizable to said complementary DNA segmental portions for achieving substantially complete staining of said DNA sequences.

6. The probe composition of claim 5 wherein said DNA segments are reasonably representative of a single preselected chromosome and comprise fragmented DNA that is obtained from one member of the group consisting of (a) a single preselected chromosome of a multi-chromosomal genome, (b) a chromosome library of a single preselected chromosome of a multi-chromosome genome, and (c) an inter species hybrid which incorporates the DNA of a single preselected chromosome of a multi-chromosomal genome.

7. The probe composition of claim 5 wherein said DNA segments are reasonably representative of a single predetermined region of a single chromosome and comprise fragmented DNA that is obtained from one member of the group consisting of (a) a single preselected region of a single chromosome, and (b) cloned DNA derived from DNA that occurs in said chromosomal region.

8. The probe composition of claim 7 wherein said cloned DNA contains DNA repeated segments.

9. The probe composition of claim 5 wherein said preselected chromosome is selected from the human genome.

10. The probe composition of claim 5 which is in admixture with at least one other probe composition.

11. The probe composition of claim 5 that is in admixture with a blocking DNA composition which comprises a mixture of different blocking DNA segments that are derived from total genomic DNA of said genome, said blocking DNA segments having an average size in the range of 150 to 600 base pairs.

12. The probe composition of claim 5 wherein said difunctional linking group is characterized by the formula:

##STR8## wherein X is selected from the group of moieties consisting of ##STR9## R₁ and R₂ are each independently selected from the group consisting of hydrogen and lower alkyl;

m and n are each an independently selected integer of 1 through 6 inclusive; and

p is the integer 0 or 1; and

wherein each moiety ##STR10## is joined to a different said deoxycytidine nucleotide and each moiety --X-- is covalently bonded to fluorophore group containing a radical that has reacted with --X-- resulting in covalent bond formation.

13. The probe composition of claim 5 wherein said fluorophore group is a derivative 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;

6-[fluorescein-5-(and-6-) carboxamido]hexanoic acid, succinimidyl ester;

5,7-dimethyl BODIPY™

propionic acid;

succinimidyl ester "activated fluorescein derivative" FAP;

eosin-5-isothiocyanate, cascade blue acetylazide, and erythrosin-5-isothiocyanate.

14. The probe composition of claim 5 and wherein each said fluorophore group is bonded to said linking group through a reactive group that is selected from the group consisting of succinimidyl esters, sulfonic acid chlorides, acetyl azides, and isothiocyanates, wherein each said fluorophore group contains one fluorophore radical, and wherein each said fluorophore group has an extinction coefficient of at least 6 000 M^-1 cm^-1 at the wavelength region of the excitation light incident upon the specimen and also a quantum yield of at least 0.02.

15. A method for making a reagent for in situ detection of a chromosome comprising:

(a) disrupting DNA complementary to the chromosome or region of the chromosome to be detected into fragments,

(b) transaminating cytosine bases in said DNA fragments, and

(c) covalently linking a fluorescent dye to said transaminated DNA fragments, wherein 0.3 to 6 mole percent of bases in the DNA fragments are fluorescently labeled.

16. A process for making a probe composition which stains the DNA present in a single preselected chromosomal entity selected from the group consisting of (1) a whole chromosome, and (2) a region of a chromosome, said process comprising the steps of:

(a) fragmenting into a mixture of segments a plurality of starting DNA sequences which are derived from the DNA that is present in said preselected chromosomal entity, said DNA segments having average sizes in the range of 150 to 600 base pairs;

(b) transaminating said segments with a difunctional linking compound one of whose two functional substituents is reactive with deoxycytidine nucleotides, said transaminating being performed in an aqueous medium which has dissolved therein an alkali metal bisulfite catalyst and which has a pH in the range of 4.5 to 7.5, said aqueous medium being maintained at a temperature in the range of 20 to 60 degrees C. until 1 to 30 mole percent of the total deoxycytidine nucleotides existing in said segments have been substituted by one of said functional substituents, thereby producing transaminated segments; and

(c) covalently bonding to the second remaining functional substituent of at least some of said so transaminated linking compounds a fluorescent compound which incorporates both at least one fluorophore substituent and also a reactive substituent which is reactive with said second functional substituent, said covalent bonding being carried out by contacting under aqueous liquid phase conditions said so transaminated segments with a substantial molar excess of said fluorescent compound while maintaining a temperature in the range of 4 to 50 degrees C., to produce a probe composition having 0.3 to 6 mole percent of bases in the DNA segments fluorescently labeled.