Connexin allele detection assays
View Patent ↗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. A kit for characterizing a target sequence for the presence or absence of Connexin 26 allele 30ΔG, wherein said kit comprises first and second oligonucleotides configured to form an invasive cleavage structure when in combination with said target sequence, wherein said first oligonucleotide comprises a 5′ portion and a 3′ portion, wherein said 3′ portion of said first oligonucleotide comprises the sequence 5′ CCCAGGATCGTCTG 3′ (SEQ ID NO:15) or 5′ CCCCAGGATCGTCT 3′ (SEQ ID NO:17) configured to hybridize to said target sequence, and wherein said 5′ portion of said first oligonucleotide is configured to not hybridize to said target sequence, and wherein said second oligonucleotide comprises a 5′ portion and a 3′ portion, wherein said 5′ portion of said second oligonucleotide comprises the sequence 5′ TGCTGGTGGAGTGTTTGTTCACACC 3′ (SEQ ID NO:16) configured to hybridize to said target sequence, and wherein said 3′ portion of said second oligonucleotide is configured to not hybridize to said target sequence.
2. The kit of claim 1 , wherein said kit comprises a plurality of first oligonucleotides, where said plurality of first oligonucleotides comprises a first first oligonucleotide comprising a 5′ portion and a 3′ portion, wherein said 3′ portion of said first first oligonucleotide comprises the sequence 5′ CCCAGGATCGTCT G 3′ (SEQ ID NO:15) configured to hybridize to a target sequence comprising a Connexin 26 allele , and wherein said 5′ portion of said first first oligonucleotide is configured to not hybridize to said target sequence, and a second first oligonucleotide comprising a 5′ portion and a 3′ portion, wherein said 3′ portion of said second first oligonucleotide comprises the sequence 5′ CCCCAGGATCGTCT 3′ (SEQ ID NO:17) configured to hybridize to a target sequence comprising a Connexin 26 allele, and wherein said 5′ portion of said second first oligonucleotide is configured to not hybridize to said target sequence.
3. The kit of claim 1 , further comprising a cleavage agent, wherein said cleavage agent is a 5′ nuclease.
4. The kit of claim 3 , wherein said 5′ nuclease comprises a FEN-1 endonuclease.
5. The kit of claim 3 , wherein said 5′ nuclease comprises a polymerase.
6. The kit of claim 1 , further comprising instructions for using said kit for said detecting the presence or absence of Connexin 26 allele 30ΔG.
7. The kit of claim 6 , wherein said instructions comprise instructions required by the United States Food and Drug Administration for labeling of analyte specific reagents.
8. The kit of claim 6 , wherein said instructions comprise instructions required by the United States Food and Drug Administration for labeling of in vitro diagnostics.
9. The kit of claim 1 , further comprising a FRET probe comprising a portion that is complementary to said 5′ portion of said first oligonucleotide.
10. The kit of claim 2 , further comprising a plurality of FRET probes, wherein said plurality of FRET probes comprises a first FRET probe comprising a portion that is complementary to said 5′ portion of said first first oligonucleotide, and a second FRET probe comprising a portion that is complementary to said 5′ portion of said second first oligonucleotide.
11. The kit of claim 10 , wherein said first FRET probe and said second FRET probe have different labels from each other.