Inactivatable target capture oligomers for use in the selective hybridization and capture of target nucleic acid sequences
View Patent ↗The present invention provides compositions, kits and methods for the selective hybridization and capture of a specific target nucleic acid. The specific target nucleic acid may be present in a heterogeneous mixture of nucleic acids. Selective hybridization and capture provides a target nucleic acid that is substantially free of non-target and/or contaminating nucleic acids. Target nucleic acids that have been selectively hybridized and captured using the current invention are then used in subsequent analysis, wherein the presence of non-target and/or contaminating nucleic acids that interfere with said subsequent analysis have been substantially reduced or eliminated, thereby providing improved analysis results. The invention offers the further advantage of requiring less stringent purification and/or sterility efforts than conventionally needed in order to ensure that enzymes and other reagents used in subsequent analysis, or present in the environment in which an assay is performed, are free of bacterial or other contaminating nucleic acids.
1. A preannealing reaction mixture consisting essentially of a target nucleic acid, an inactivatable target capture oligomer comprising at least a binding pair member, a target hybridizing region comprising a nucleotide sequence of 15 to 30 nucleobases in length complementary to the target nucleic acid and a tag-closing region comprising a nucleotide sequence of 3 to 20 nucleobases in length, wherein said binding pair member, said target hybridizing region and said tag-closing region are joined as a single molecule with said target hybridizing region joined at its 3′ end to the 5′ end of the tag-closing region and the tag-closing region is joined at its 3′ end to the binding pair member, wherein said binding pair member comprises a homopolymeric region that allows capture of the target capture oligomer by hybridizing to a complementary sequence of the other member of the binding pair linked to a support, and wherein at least 80% of nucleotides of said tag-closing region nucleotide sequence are complementary to corresponding nucleotides of a sequence contained within said target hybridizing region.
2. The reaction mixture of claim 1 , wherein said tag-closing region comprises a nucleotide sequence that contains one or more nucleotide residues selected from the group consisting of: an abasic nucleotide residue, a wobble nucleotide residue, a mismatched nucleotide residue relative to said residue's corresponding position in said sequence contained within said target hybridizing region, and combinations thereof.
3. The reaction mixture of claim 1 , wherein said tag-closing region comprises a nucleotide sequence of at least 6 to 14 nucleobases in length.
4. The reaction mixture of claim 1 , wherein said tag-closing region comprises a nucleotide sequence of at least 6 to 9 nucleobases in length.
5. The reaction mixture of claim 1 , wherein said tag-closing region comprises a nucleotide sequence of 7 nucleobases in length.
6. The reaction mixture of claim 1 , wherein said tag-closing region comprises a nucleotide sequence of at least 6 to 9 nucleobases in length and said target hybridizing regions comprises a nucleotide sequence of 17 nucleobases in length.
7. The reaction mixture of claim 1 , wherein said target hybridizing region is joined to said end of said tag-closing region using a nucleotide linker thereby forming a contiguous nucleotide sequence consisting essentially of said target hybridizing region and said tag-closing region.
8. The reaction mixture of claim 7 , wherein said binding pair member is joined to said end of said tag-closing region using a nucleotide linker thereby forming a contiguous nucleotide sequence consisting essentially of said binding pair member, said tag-closing region and said target hybridizing region.
9. The reaction mixture of claim 1 , further comprising at least one amplification oligomer.
10. The reaction mixture of claim 9 , wherein said at least one amplification oligomer is a heterologous amplification oligomer.
11. The reaction mixture of claim 1 , under conditions biasing said target hybridizing region towards hybridizing with the target nucleic acid and not towards hybridizing with said tag-closing region.
12. The reaction mixture of claim 1 , under conditions biasing said target hybridizing region towards hybridizing with said tag-closing region and not towards hybridizing with a non-target nucleic acid.
13. The reaction mixture of claim 1 , wherein the target capture oligomer lacks a detectable label.
14. The reaction mixture of claim 9 further comprising a terminating oligonucleotide to terminate extension from the amplification oligomer.