Methods and compositions for chromosome-specific staining

• US 20030108943A1
• Filed: 12/30/2002
• Published: 06/12/2003
• Est. Priority Date: 01/16/1986
• Status: Abandoned Application

• Current Assignee: Daniel Pinkel, Joe W. Gray
• Original Assignee: Daniel Pinkel, Joe W. Gray
• Inventors: Pinkel, Daniel, Gray, Joe W.
• Application Number:
  US10/330,089
• Publication Number:
  US 20030108943A1
• Time in Patent Office: Days
• US Class Current: 435/6
• 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 chromosome-specific staining reagent comprising a heterogeneous mixture of labeled nucleic acid fragments, the labeled nucleic acid fragments being derived from the same chromosome type, and being substantially free of repetitive nucleic acid sequences having hybridization capacity.

2. The chromosome-specific staining reagent according to claim 1 wherein said chromosome type is an abnormal human chromosome type.

3. The chromosome-specific staining-reagent according to claim 1 wherein said chromosome type is selected from the group consisting of normal human chromosomes 1 through 22, X, and Y.

4. The chromosome-specific staining reagent of claim 3 wherein said heterogeneous mixture comprises labeled nucleic acid fragments derived from substantially equal amounts of between about 10-1000 distinct cloned inserts.

5. The chromosome-specific staining reagent of claim 4 wherein said labeled nucleic acid fragments are derived from substantially equal amounts of between about 10-1000 distinct cloned inserts each having a length within the range of between about 20-45 kilobases.

6. The chromosome-specific staining reagent of claim 5 wherein said distinct cloned inserts form said labeled nucleic acid fragments, each fragment having a length within the range of between about 50-500 bases.

7. The chromosome-specific staining reagent of claim 6 wherein said labeled nucleic acid fragments are single stranded.

8. The chromosome-specific staining reagent of claim 7 wherein said labeled nucleic acid fragments are biotinylated or modified with N-acetoxy-N-2-acetylamino-fluorene.

9. The chromosome-specific staining reagent of claim 4 wherein said labeled nucleic acid fragments are derived from substantially equal amounts of between about 100-400 distinct cloned unique sequence inserts.

10. The chromosome-specific staining reagent of claim 9 wherein said distinct cloned inserts form said labeled nucleic acid fragments, each fragment having a length within the range of between about 50-5000 bases.

11. The chromosome-specific staining reagent of claim 10 wherein said distinct cloned inserts form said labeled nucleic acid fragments, each fragment having a length within the range of between about 50-500 bases.

12. The chromosome-specific staining reagent of claim 11 wherein said labeled nucleic acid fragments are single stranded.

13. The chromosome-specific staining reagent of claim 12 wherein said labeled nucleic acid fragments are biotinylated or modified with N-acetoxy-N-2-acetylamino-fluorene.

14. A chromosome-specific staining reagent produced by the process of:

isolating chromosome-specific DNA;

cloning pieces of the isolated chromosome-specific DNA;

disabling the hybridization capacity of repeated sequences contained in the cloned pieces of the isolated chromosome-specific DNA to form a collection of nucleic acid fragments which hybridize to unique chromosomal DNA sequences;

labeling the nucleic acid fragments of the collection to form a heterogeneous mixture of nucleic acid fragments.

15. The chromosome-specific staining reagent of claim 14, wherein said step of disabling said hybridization capacity includes selecting cloned pieces of said isolated chromosome-specific DNA which have substantially unique base sequences.

16. The chromosome-specific staining reagent of claim 14 wherein said step of disabling said hybridization capacity includes prereassociating said cloned pieces of said isolated chromosome-specific DNA with unlabeled repetitive sequence nucleic acid fragments.

17. A method of staining chromosomal DNA of a particular chromosome type or portion thereof, or a particular group of chromosome types, the method comprising the steps of:

providing a heterogeneous mixture of labeled nucleic acid fragments, substantial portions of each labeled nucleic acid fragment in the heterogeneous mixture having base sequences substantially complementary to base sequences of the chromosomal DNA; and

reacting the heterogeneous mixture with the chromosomal DNA by in situ hybridization.