Methods of capturing a target nucleic acid for amplification and detection using an inactivatable target capture oligomer
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 method for the specific hybridization and capture of a target nucleic acid strand, said method comprising the steps of:
a. treating a nucleic acid sample with an inactivatable target capture oligomer,
wherein said inactivatable target capture oligomer comprises a target hybridizing region, a binding pair member and a tag-closing region,
wherein said target hybridizing region stably hybridizes to said target nucleic acid strand under a first set of conditions,
wherein said tag-closing region is from 3 contiguous nucleobases in length to 20 contiguous nucleobases in length, does not stably hybridize to said target hybridizing region under said first set of conditions, is substantially complementary to a portion of said target hybridizing region such that the tag-closing region and target hybridizing region will stably hybridize under a second set of conditions less stringent than the first set of conditions, and does not stably hybridize to said target nucleic acid strand under the first or second set of conditions, and
wherein said target hybridizing region, said binding pair member and said tag-closing region are joined as a single molecule;
b. providing said first set of conditions, whereby said target hybridizing region of molecules of said inactivatable target capture oligomer stably hybridizes with said target nucleic acid present in said nucleic acid sample and does not stably hybridize with said tag-closing region;
c. providing said second set of less stringent conditions, thereby allowing molecules of said inactivatable target capture oligomer that are not stably hybridized with said target nucleic acid to form an inactive configuration by stably hybridizing said tag-closing region with said target hybridizing region;
d. performing a capture step wherein a complex comprising said molecules of said inactivatable capture oligomer stably hybridized with said target nucleic acid strand in step b is captured;
e. determining the specific hybridization and capture of said target nucleic acid strand in said nucleic acid sample by performing a reaction following step d, wherein said reaction comprises an amplification reaction and a detection reaction;
wherein stable hybridization of two nucleic acid strands in a reaction mixture means the temperature of a reaction mixture including the strands is at least 2° C. below the melting temperature of the duplex formed by the strands.
2. The method of claim 1 , wherein step d. comprises providing a solid support material consisting essentially of: (a) a solid support material and (b) a complementary binding pair member; and providing conditions that allow for binding of said binding pair member of said inactivatable capture probe with said complementary binding member.
3. The method of claim 2 , wherein said solid support material is a paramagnetic bead and wherein said complementary binding pair member is covalently attached to said paramagnetic bead.
4. The method of claim 3 , wherein said complementary binding pair member is an immobilized probe and said binding pair member of said inactivatable capture probe is a polynucleotide region with a nucleotide sequence that is substantially complementary to said immobilized probe.
5. The method of claim 2 , further comprising a washing step wherein said captured target nucleic acid strand from step d. is retained while one or more of non-captured nucleic acids, non-target nucleic acids, contaminating nucleic acids, inactivated inactivatable target capture oligomer, reagents and sample debris are separated from said captured target nucleic acid.
6. The method of claim 1 , wherein said binding pair member of said inactivatable target capture oligomer is a polynucleotide region consisting essentially of a nucleotide sequence.
7. The method 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.