Claims for Patent: 5,955,272

Summary for Patent: 5,955,272

Title:	Detection of individual gene transcription and splicing
Abstract:	Disclosed are in situ hybridization methods for differentially detecting an RNA and the gene from which it was transcribed, while preserving the spatial relationship of the RNA transcript and the gene. Also disclosed are in situ hybridization methods for simultaneously detecting two alleles of the same gene in a single cell, while differentially detecting RNA transcribed each of the two alleles. Also disclosed are in situ hybridization methods for detecting normal and defective RNA splicing.
Inventor(s):	Lawrence; Jeanne Bentley (Mapleville, RI), Johnson; Carol Villnave (Stowe, MA), Xing; Yigong (Northborough, MA)
Assignee:	University of Massachusetts (Boston, MA)
Application Number:	08/682,924
Patent Claims:	1. An in situ hybridization method for detecting normal splicing, if present, of RNA transcribed from a gene of interest in a cell, said method comprising the steps of: (a) fixing said cell with a fixative such that the nucleus remains penetrable by labeled probes, and the nucleic acids are preserved in place; (b) hybridizing said fixed cell with an intron-specific nucleic acid probe able to hybridize to an intron of interest encoded by a gene of interest, said intron-specific probe bearing a first label; (c) hybridizing said fixed cell with an exon-specific nucleic acid probe able to hybridize to at least one exon in said gene of interest, said exon-specific probe bearing a second label, said second label being distinguishable from said first label; (d) detecting said second label as a large focus or track; and (e) detecting said first label as a small focus or track within said large focus or track, said small focus or track having a size and shape such that it does not substantially totally overlap said large focus or track, as an indication of normal splicing of RNA transcribed from said gene of interest in said cell. 2. The method of claim 1, further comprising, as an initial step, permeabilizing the nucleus of said cell while minimizing mRNA degradation. 3. The method of claim 1, wherein said first label and said second label are different fluorochromes whose emission wavelengths are clearly distinguishable. 4. An in situ hybridization method for detecting defective splicing, if present, of RNA transcribed from a gene of interest in a cell, said method comprising the steps of: (a) fixing said cell with a fixative such that the nucleus remains penetrable by labeled probes, and the nucleic acids are preserved in place; (b) hybridizing said fixed cell with an intron-specific nucleic acid probe able to hybridize to an intron of interest encoded by a gene of interest, said intron-specific probe bearing a detectable label; (c) detecting said label as a large focus, as an indication of defective splicing of RNA transcribed from said gene of interest in said cell. 5. The method of claim 4, further comprising, as an initial step, permeabilizing the nucleus of said cell while minimizing mRNA degradation. 6. The method of claim 4, wherein said label is a fluorochrome. 7. An in situ hybridization method for detecting defective splicing, if present, of RNA transcribed from a gene of interest in a cell, said method comprising the steps of: (a) fixing said cell with a fixative such that the nucleus remains penetrable by labeled probes, and the nucleic acids are preserved in place; (b) hybridizing said fixed cell with an intron-specific nucleic acid probe able to hybridize to an intron of interest encoded by a gene of interest, said intron-specific probe bearing a first label; (c) hybridizing said fixed cell with an exon-specific nucleic acid probe able to hybridize to at least one exon in said gene of interest, said exon-specific probe bearing a second label, said second label being distinguishable from said first label; (d) detecting said second label as a signal focus that substantially completely overlaps a signal focus from said first label, as an indication of defective splicing of RNA transcribed from said gene of interest in said cell. 8. The method of claim 7, further comprising, as an initial step, permeabilizing the nucleus of said cell while minimizing mRNA degradation. 9. The method of claim 7, wherein said first label and said second label are different fluorochromes whose emission wavelengths are clearly distinguishable. 10. The method of claim 7, wherein steps (b) and (c) are performed simultaneously by having said intron-specific probe and said exon-specific probe present in a single hybridization reaction. 11. An in situ hybridization method for detecting defective splicing, if present, of RNA transcribed from a gene of interest in a cell, said method comprising the steps of: (a) fixing said cell with a fixative such that the nucleus remains penetrable by labeled probes, and the nucleic acids are preserved in place; (b) subjecting said fixed cell to an in situ hybridization reaction comprising: (1) a labeled, full-length genomic probe comprising the antisense sequence of an entire gene of interest, said full-length genomic probe bearing a label, and said full-length genomic probe being present in said hybridization reaction at a concentration between 0.5 .mu.g/ml and 20 .mu.g/ml; and (2) an unlabeled full-length cDNA probe comprising the complete coding region of said gene of interest, said cDNA probe being present in said hybridization reaction at a concentration between 50-fold greater and 500-fold greater than the concentration of said labeled genomic probe; and (c) detecting said label as a large focus in the nucleus of said cell, as an indication of defective splicing in RNA transcribed from said gene of interest in said cell. 12. The method of claim 11, further comprising, as an initial step, permeabilizing the nucleus of said cell while minimizing mRNA degradation. 13. The method of claim 11, wherein said label is a fluorochrome. 14. An in situ hybridization method for detecting defective splicing, if present, of RNA transcribed from a gene of interest in a cell, said method comprising the steps of: (a) fixing said cell with a fixative such that the nucleus remains penetrable by labeled probes, and the nucleic acids are preserved in place; (b) hybridizing said fixed cell with a cDNA bearing a first label; (c) fixing said cell to preserve in place the hybridized cDNA of step (b); (d) hybridizing said fixed cell with a full-length genomic probe comprising the antisense sequence of an entire gene of interest, said full-length genomic probe bearing a second label, said second label being distinguishable from said first label; (e) detecting said second label as a focus that substantially completely overlaps a signal focus from said first label, as an indication of a defective splicing of RNA transcribed from said gene of interest in said cell. 15. The method of claim 14, further comprising, as an initial step, permeabilizing the nucleus of said cell while minimizing mRNA degradation. 16. The method of claim 14, wherein said first label and said second label are different fluorochromes whose emission wavelengths are clearly distinguishable.

Details for Patent 5,955,272

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132	06/04/1986		2013-02-26
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132			2013-02-26
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	Injection	103132			2013-02-26