Detection of trinucleotide repeats by in situ hybridization

• US 5,962,332 A
• Filed: 12/11/1995
• Issued: 10/05/1999
• Est. Priority Date: 03/17/1994
• Status: Expired due to Fees

• Current Assignee: University Of Massachusetts
• Original Assignee: University Of Massachusetts
• Inventors: Taneja, Krishan L., Singer, Robert H.
• Primary Examiner(s): Sisson, Bradley L.
• Application Number:
  US08/570,155
• Time in Patent Office: 1,394 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/183, 536/23.1, 536/23.5, 536/24.3, 536/24.31, 536/24.32, 536/25.32, 536/25.6, 935/77, 935/78, 436/94, 436/164, 436/172, 436/806, 436/805, 436/808
• US Class Current: 436/94
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6841 In situ hybridisation
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine
• CPC Class Codes: Y10S 436/805 Optical property
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

Disclosed is a method of detecting a trinucleotide repeat expansion by in situ hybridization. The disclosed method uses a sample of nucleated cells, a labeled trinucleotide repeat-specific probe and detection of the hybridized probe by a means whose sensitivity distinguishes between the signal from probes hybridized to an expanded repeat and the signal from probes hybridized to a non-expanded repeat.

Claims

1. An in situ hybridization method for detecting a trinucleotide repeat expansion, wherein said method comprises the steps of:

(a) providing a sample of nucleated cells;

(b) contacting said sample with a trinucleotide repeat-specific oligonucleotide probe, which probe comprises a covalently attached detectable label, under conditions that allow said probe to hybridize with said trinucleotide repeat expansion in said sample; and

(c) detecting the hybridized probe by means of a detectable label, using a labeled detection threshold that is below signal strength of a signal from probes hybridized to an expanded trinucleotide repeat and above signal strength of a signal from probes hybridized to a non-expanded trinucleotide repeat.

2. The method of claim 1, wherein said detectable label is a fluorescent moiety.

3. The method of claim 2, wherein said fluorescent moiety is selected from the group consisting of fluoroscein isothiocyanate , CY3, and Texas red.

4. The method of claim 1, wherein said hybridized probe is detected by fluorescence microscopy.

5. The method of claim 1, wherein said hybridized probe is detected by image cytometry.

6. The method of claim 1, wherein said oligonucleotide probe is an antisense probe.

7. The method of claim 6, wherein said antisense probe comprises a trinucleotide repeat selected from the group consisting of CAG, CCG, and CTG.

8. The method of claim 1 wherein said oligonucleotide probe is a sense probe.

9. The method of claim 8, wherein said sense probe comprises a trinucleotide repeat selected from the group consisting of, CTG, and CAG.

10. The method of claim 8, further comprising a DNA denaturation step prior to contacting said sample with said trinucleotide repeat-specific oligonucleotide probe.

11. The method of claim 1, wherein said oligonucleotide probe comprises from 5 to 15 trinucleotide repeats.

12. The method of claim 11, wherein said oligonucleotide probe comprises from 8 to 10 trinucleotide repeats.

13. The method of claim 1, wherein said nucleated cells are from an asymptomatic patient.

14. The method of claim 1, wherein said nucleated cells are selected from the group consisting of white blood cells, epithelial cells, myocytes, and fibroblasts.

15. The method of claim 1, wherein said nucleated cells are fetal cells.

16. The method of claim 15, wherein said fetal cells are from a sample of amniotic fluid or chorionic villus.

17. The method of claim 1, wherein said trinucleotide repeat expansion is in a transcript from an Mt-PK gene.

18. The method of claim 17, wherein said transcript is in a myocyte.

19. The method of claim 1, wherein said trinucleotide repeat expansion is in a transcript from an FMR-1 gene.

20. The method of claim 19, wherein said transcript is in a lymphocyte.

21. An in situ hybridization method for differentially detecting sense and antisense sequences of a trinucleotide repeat expansion, said method comprising the steps of:

(a) providing a sample of nucleated cells;

(b) denaturing DNA in said cells;

(c) hybridizing to said antisense sequence a trinucleotide repeat-specific sense probe, said probe being labeled with a first detectable label; and

(d) hybridizing to said sense sequence a trinucleotide repeat-specific antisense probe, said probe being labeled with a second detectable label;

(e) differentially detecting said first detectable label and said second detectable label.

22. The method of claim 21, wherein said sense sequence is present in an mRNA molecule.