Connexin allele detection assays

Abstract

The present invention provides compositions and methods for the detection and characterization of mutations associated with non-syndromic hearing impairment. More particularly, the present invention provides compositions, methods and kits for using invasive cleavage structure assays (e.g. the INVADER assay) to screen nucleic acid samples, e.g., from patients, for the presence of any one of a collection of mutations in the Connexin 26, or gap junction beta 2, gene associated with non-syndromic hearing loss.

1 - 29 . (canceled) 
     
     
         30 . A kit for characterizing a target sequence for the presence or absence of Connexin 26 allele 167ΔT, wherein said kit comprises first and second oligonucleotides configured to form an invasive cleavage structure in combination with said target sequence. 
     
     
         31 . The kit of  claim 30 , wherein said first oligonucleotide comprises a 5′ portion and a 3′ portion, wherein said 3′ portion of said first oligonucleotide is configured to hybridize to said target sequence, and wherein said 5′ portion of said oligonucleotide is configured not to hybridize to said target sequence. 
     
     
         32 . The kit of  claim 31 , wherein said second oligonucleotide comprises a 5′ portion and a 3′ portion, wherein said 5′ portion of said second oligonucleotide is configured to hybridize to said target sequence, and wherein said 3′ portion of said second oligonucleotide is configured not to hybridize to said target sequence. 
     
     
         33 . The kit of  claim 30 , further comprising a cleavage agent. 
     
     
         34 . The kit of  claim 33 , wherein said cleavage agent comprises a structure-specific nuclease. 
     
     
         35 . The kit of  claim 33 , wherein said cleavage agent comprises a 5′ nuclease. 
     
     
         36 . The kit of  claim 35 , wherein said 5′ nuclease comprises a FEN-1 endonuclease. 
     
     
         37 . The kit of  claim 30 , further comprising a FRET probe comprising a portion that is complementary to said 5′ portion of said first oligonucleotide. 
     
     
         38 . The kit of  claim 30 , wherein said first oligonucleotide consists of SEQ ID NO:10 or SEQ ID NO:11. 
     
     
         39 . The kit of  claim 30 , wherein said second oligonucleotide consists of SEQ ID NO:12. 
     
     
         40 . A kit for characterizing a target sequence for the presence or absence of the 167ΔT mutation on the Connexin 26 allele, wherein said kit comprises a first and second oligonucleotide, wherein said first oligonucleotide comprises a 5′ portion and a 3′ portion, and wherein said second oligonucleotide comprise a 5′ portion and a 3′ portion, wherein said 3′ portion of said first oligonucleotide and said 3′ portion of said second oligonucleotide are configured to both hybridize with the wild type and 167ΔT mutant Connexin 26 allele and form an invasive cleavage structure in combination with said target sequence. 
     
     
         41 . The kit of  claim 40 , wherein said 5′ portion of said first oligonucleotide and said 5′ portion of said second oligonucleotide are configured not to hybridize to said target sequence. 
     
     
         42 . The kit of  claim 40 , further comprising a cleavage agent. 
     
     
         43 . The kit of  claim 42 , wherein said cleavage agent comprises a structure-specific nuclease. 
     
     
         44 . The kit of  claim 42 , wherein said cleavage agent comprises a 5′ nuclease. 
     
     
         45 . The kit of  claim 44 , wherein said 5′ nuclease comprises a FEN-1 endonuclease. 
     
     
         46 . The kit of  claim 40 , further comprising a plurality of FRET probes, wherein said plurality of FRET probes comprising a first FRET probe comprising a portion that is complementary to said 5′ portion of said first oligonucletoide, and a second FRET probe comprising a portion that is complementary to said 5′ portion of said second oligonucleotide. 
     
     
         47 . The kit of  claim 46 , wherein said first FRET probe and said second FRET probe have different labels from each other.