Claims for Patent: 8,092,996

Summary for Patent: 8,092,996

Title:	Method for detecting cystic fibrosis
Abstract:	The present invention relates to methods for amplifying various regions of the cystic fibrosis transmembrane regulator (CFTR) gene. Methods are provided for amplifying one or all 27 exons of the CFTR gene and a portion of the CFTR promoter region in a single tube. The method can identify the presence or absence of CF deletions or insertions in a sample and assist in the diagnosis of a genetic predisposition to cystic fibrosis.
Inventor(s):	Hantash; Feras (Dana Point, CA)
Assignee:	Quest Diagnostics Investments Incorporated (Wilmington, DE)
Application Number:	10/942,757
Patent Claims:	1. A method for detecting deletions in the human cystic fibrosis transmembrane conductance regulator gene (CFTR) in a sample comprising nucleic acids, said method comprising: (a) amplifying at least seven different target segments of the human CFTR gene in a single tube using an oligonucleotide primer pair specific to each of said target segments, wherein the at least seven target segments comprise at least one target segment from each of the promoter, exon 1, exon 2, exon 22, exon 23, and exon 24; and (b) identifying the amplified target segments; and (c) determining the amount of each target segment amplified versus that for a wildlife human CFTR gene to determine the presence or absence of a deletion selected from the group consisting of: (i) a deletion in a segment of the CFTR promoter region including the adjoining CFTR exon 1 comprising at least about 1,800 nucleotides in length; (ii) a deletion in a segment of the CFTR promoter region including the adjoining CFTR exons 1 and 2 comprising at least about 28,000 nucleotides in length; and, (iii) a deletion of exons 22,23, and 24 but no other CFTR exons, wherein a substantial decrease in the amount of amplified target segments observed in the sample versus that for a human wildtype CFTR gene indicates a deletion in the CFTR segment in the sample. 2. The method according to claim 1 wherein identifying involves separating the amplified target segments by size. 3. The method according to claim 2 wherein size separation is performed by gel electrophoresis. 4. The method according to claim 1 wherein at least one primer of each said primer pair is detectably labeled. 5. The method according to claim 4 wherein identifying involves separating the amplified target segments by size and by detectable moiety. 6. The method according to claim 5 wherein said primer is labeled with a fluorescent dye. 7. The method according to claim 1 wherein at least one primer pair for each target segment is labeled with a fluorescent dye; wherein at least two different fluorescent dyes are used. 8. The method according to claim 1 wherein said at least 7 target segments together comprise sequence from all or a portion of at least 5 different exons of the CFTR gene. 9. The method according to claim 1 wherein at least 17 target segments of the CFTR gene are amplified. 10. The method according to claim 9 wherein said at least 17 target segments together comprise sequence from all or a portion of at least 15 different exons of the CFTR gene. 11. The method according to claim 1 wherein at least 28 target segments of the CFTR gene are amplified. 12. The method according to claim 11 wherein at least 28 target segments together comprise sequence from all or a portion of at least 20 different exons of the CFTR gene. 13. The method according to claim 1 further comprising at least one primer pair for amplifying at least one internal control target segment that does not correspond to the CFTR gene. 14. The method according to claim 13 wherein at least 3 internal control target segments are amplified. 15. The method according to claim 1 wherein a total of at least 32 target segments are amplified, said segments representing 27 exons of the CFTR gene, a portion of a CFTR intron, and three internal control segments. 16. The method according to claim 1 wherein at least one of said oligonucleotide primer pairs includes SEQ ID NO: 1-2. 17. The method according to claim 1 wherein said CFTR gene having a mutation comprising a deletion in a segment of the CFTR promoter region including the adjoining CFTR exon 1 comprising at least about 1,800 nucleotides in length has no other exon deletions. 18. the method according to claim 1 wherein said CFTR gene having a mutation comprising a deletion in a segment of the CFTR promoter region including the adjoining CFTR exons 1 and 2 comprising at least about 28,000 nucleotides in length has no other exon deletions. 19. A method for determining the cystic fibrosis status of an individual human comprising: (a) determining the presence or absence of a deletion in both alleles of the human cystic fibrosis transmembrane conductance regulator (CFTR) gene, said deletion selected from the group consisting of (i) a deletion in a segment of the CFTR promoter region including the adjoining CFTR exon 1 comprising at least about 1,800 nucleotides in length, (ii) a deletion in a segment of the CFTR promoter region including the adjoining CFTR exons 1 and 2 comprising at least about 28,000 nucleotides in length, and (iii) a deletions of exons 22,23, and 24 but no other CFTR exons, wherein determining involves (A) amplifying at least seven different target segments of the CFTR gene in a single tube using an oligonucleotide primer pair specific to each of said target segments, wherein the at least seven target segments comprise at least one target segment from each of the promoter, exon 1, exon 2, exon 22, exon 23,and exon 24, (B) identifying the amplified target segments, and (C) comparing the amount of each amplified target segment to the amount of a similarly amplified human wildtype target segment, wherein a decrease of at least 30-50% in the amount of at least one amplified segment is observed in the sample compared with the amount of amplified wildtype target segment; and (b) identifying the individual (i) as having cystic fibrosis when the individual is homozygous for at least one of said deletions in the CFTR gene, (ii) as being a cystic fibrosis carrier when the individual is heterozygous for at least one of said deletions in the CFTR gene, or (iii) as having no cystic fibrosis or carrier status caused by said deletions in the CFTR gene when each of said deletions is absent from both alleles of the CFTR gene. 20. The method according to claim 19 wherein identifying involves separating the amplified target segments by size. 21. The method according to claim 20 wherein size separation is performed by gel electrophoresis. 22. The method according to claim 19 wherein at least one primer of each said primer pairs is detectably labeled. 23. The method according to claim 22 wherein identifying involves separating the amplified target segments by size and by detectable moiety. 24. The method according to claim 22 wherein said primer is labeled with a fluorescent dye. 25. The method according to claim 19 wherein at least one primer pair for each target segment is labeled with a fluorescent dye; wherein at least two different fluorescent dyes are used. 26. The method according to claim 19 wherein said at least 7 target segments together comprise sequence from all or a portion of the at least 5 different exons of the CFTR gene. 27. The method according to claim 19 wherein at least 17 target segments of the CFTR gene are amplified. 28. The method according to claim 27 wherein said at least 17 target segments together comprise sequence from all or a portion of the least 15 different exons of the CFTR gene. 29. The method according to claim 19 wherein at least 28 target segments of the CFTR gene are amplified. 30. The method according to claim 29 wherein at least 28 target segments together comprise sequence from all or a portion of the at least 20 different exons of the CFTR gene. 31. The method according to claim 19 further comprising at least one primer pair for amplifying at least one internal control target segment that does not correspond to the CFTR gene. 32. The method according to claim 31 wherein at least 3 internal control target segments are amplified. 33. The method according to claim 19 wherein a total of at least 32 target segments are amplified, said target segments representing 27 exons of the CFTR gene, a portion of a CFTR intron, and three internal control segments. 34. The method according to claim 19 wherein at least one of said target segments is amplified by an oligonucleotide primer pair comprising the sequences of SEQ ID NO: 1-2. 35. The method according to claim 19 wherein said CFTR gene having a mutation comprising a deletion in a segment of the CFTR promoter region including the adjoining CFTR exon 1 comprising at least about 1,800 nucleotides in length has no other exon deletions. 36. The method according to claim 19 wherein said CFTR gene having a mutation comprising a deletion in a segment of the CFTR promoter region including the adjoining CFTR exons 1 and 2 comprising at least about 28,000 nucleotides in length has no other exon deletions.

Details for Patent 8,092,996

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		2039-02-26
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132			2039-02-26
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	Injection	103132			2039-02-26