Claims for Patent: 5,358,649

Summary for Patent: 5,358,649

Title:	Diagnosis for porcine malignant hyperthermia
Abstract:	A purified DNA molecule comprises a DNA sequence of approximately 15.1 kb coding for normal or mutant RYR1 protein having a molecular weight of approximately 564,740 daltons. The DNA molecule has an endonuclease restriction map of FIG. 1 and a sequence of FIG. 2.
Inventor(s):	MacLennan; David H. (Toronto, CA), O\'Brien; Peter J. (Guelph, CA)
Assignee:	University of Guelph (Ontario, CA) The University of Toronto Innovations Foundation (Ontario, CA)
Application Number:	08/030,159
Patent Claims:	1. A method for screening a pig to determine if said pig is susceptible to malignant hyperthermia comprising: i) providing a biological sample which was removed from said pig to be screened, and ii) conducting a biological assay to determine presence in said biological sample of mutant RYR1 gene having a nucleotide mutation of C1843 to T1843 in the DNA sequence of FIG. 2. 2. A method of claim 1 wherein said biological sample includes at least part of a genome of said pig, said part including at least a portion of a nucleic acid sequence of the normal or mutant RYR1 gene represented by said DNA sequence of FIG. 2 or FIG. 6, said portion of said nucleic acid sequence including either the C1843 or T1843 nucleotide of the DNA sequence of FIG. 2. 3. A method of claim 2 wherein said assay comprises a DNA hybridization assay in which a labelled DNA probe is used, said probe having a sequence of at least 12 consecutive nucleotides of said DNA sequence of FIG. 2 and containing either the C1843 or T1843 nucleotide. 4. A method of claim 2 wherein said assay comprises a one step, allele specific, PCR-based diagnosis of the DNA sequence in said biological sample, said assay comprising a PCR amplification of DNA in said biological sample by use of forward and reverse primer oligonucleotide sequences, said forward primer sequence having at its 3' end a C1843 base or its complement for specific detection of a normal RYR1 gene or said forward primer having at its 3' end a T1843 base or its complement for specific detection of a mutant RYR1 gene and wherein the remaining sequence of said selected forward primers and all of said reverse primers comprise a DNA sequence of consecutive nucleotides of a portion of said DNA sequence to either side of nucleotide 1843 of FIG. 2. 5. A method of claim 2 wherein said assay comprises restriction endonuclease digestion of said nucleic acid sequence fragments of said genome part in said biological sample, said fragments having been amplified to facilitate detection, said digestion comprising use of an enzyme selected from the group of enzymes for digesting said fragments at enzyme digestion sites which are cleaved by enzymes HinP1 and HgiA1, detecting either said HinP1 or HgiA1 site in said amplified DNA fragment cleaved by selected enzyme cleavage at said HinP1 site being indicative of normal RYR1 gene and cleavage at said HgiA1 site being indicative of mutant RYR1 gene. 6. A method of claim 3 wherein said DNA probes are selected from the group consisting of (17-c) 5'-TGGCCGTGCGCTCCAAC-3' (nucleotide positions 1835 to 1851 of SEQ ID No. 1) and (15-t) 5'-GGCCGTGTGCTCCAA-3' (nucleotide positions 1836 to 1850 of SEQ ID No. 1). 7. A method of claim 2 wherein said assay comprises PCR amplification of DNA in said biological sample by use of forward and reverse primer oligonucleotide sequences, each of said selected forward or reverse primer sequence comprises a consecutive nucleotide sequence of a portion of said DNA sequence to either side of nucleotide 1843 of FIG. 2 or FIG. 6, said forward and reverse primers being on opposite sides of said nucleotide 1843 to permit amplification of said biological sample DNA containing said nucleotide at position 1843 of FIG. 2. 8. A method of claim 7 wherein said forward and reverse primers are selected from the group of sets of primer sequences consisting of: a) i) a forward primer sequence having nucleotide positions 1771 to 1794 of FIG. 2 (SEQ ID No. 1); ii) a reverse primer sequence having the reverse complement of nucleotide positions 1929 to 1952 of FIG. 2 (SEQ ID No. 1); b) i) a forward primer sequence having the intron sequence of FIG. 6: 5'-TCCAGTTTGCCACAGGTCCTACCA-3' (SEQ ID No. 3--positions 520 to 523); ii) a reverse primer sequence having the reverse complement of the intron sequence of FIG. 6: 5'-ACTCAGAGACTCCACTCCGGTGAA-3' (SEQ ID No. 3--positions 1134 to 1157); c) i) a forward primer sequence having nucleotide positions 1811 to 1834 of FIG. 2 (SEQ ID No. 1); ii) a reverse primer sequence having the reverse complement of nucleotide positions 1861 to 1884 of FIG. 2 (SEQ ID No. 1). 9. A method of claim 5 wherein said DNA fragment is amplified by PRC and forward and reverse primers selected for such amplification of DNA are selected from the group consisting of: a) i) a forward primer sequence having nucleotide positions 1771 to 1794 of FIG. 2 (SEQ ID No. 1); ii) a reverse primer sequence having the reverse complement of nucleotide positions 1929 to 1952 of FIG. 2 (SEQ ID No. 1); b) i) a forward primer sequence having the sequence: 5'-TCCAGTTTGCCACAGGTCCTACCA-3' (SEQ ID No. 3--positions 520 to 523); ii) a reverse primer sequence having the reverse complement of the sequence: 5'-ACTCAGAGACTCCACTCCGGTGAA-3' (SEQ ID No. 3--positions 1134 to 1157); c) i) a forward primer sequence having nucleotide positions 1811 to 1834 of FIG. 2 (SEQ ID No. 1); ii) a reverse primer sequence having the reverse complement of nucleotide positions 1861 to 1884 of FIG. 2 (SEQ ID No. 1).

Details for Patent 5,358,649

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		2011-10-25
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132			2011-10-25
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	Injection	103132			2011-10-25