Asymmetric hairpin target capture oligomers
The invention provides an improved stem-loop target capture oligomer and methods of use. Such a target capture oligomer has a target-binding segment forming a loop flanked by stem segments forming a stem. The stem segments are of unequal length. Such probes show little or no binding to immobilized probes in the absence of a target nucleic acid but offer good target sensitivity. The probes are particularly useful in multiplex methods of detection in which multiple target capture oligomers are present for detecting of multiple target nucleic acids (for example, detecting multiple polymorphic forms of a target gene).
1. A target capture oligomer comprising a first stem segment and a second stem segment comprising complementary segments of polyA and polyT differing in length by at least five nucleobases flanking a target-binding segment complementary to a target nucleic acid, wherein under hybridizing conditions:
in the absence of the target nucleic acid, the target capture oligomer forms a stem-loop structure such that, in the stem-loop structure, intramolecular hybridization of the first stem segment and the second stem segment forms the stem, and the target-binding segment forms the loop; and
in the presence of the target nucleic acid, the target-binding segment hybridizes to the target nucleic acid separating or keeping separate the first stem segment and the second stem segment and resulting in the first stem segment being accessible to hybridize to a complementary immobilized probe.
2. The target capture oligomer of claim 1 , wherein the first stem segment and the second stem segment occupy the 5′ and 3′ ends of the target capture oligomer respectively and the first stem segment is complementary to the immobilized probe.
3. The target capture oligomer of claim 1 , wherein the melting temperature of a duplex formed between the target binding segment and the target nucleic acid is greater than the melting temperature of a duplex formed between the first stem segment and the second stem segment, which is greater than the melting temperature of a duplex formed between the first stem segment and the immobilized probe.
4. The target capture oligomer of claim 1 , wherein the target binding segment comprises at least one methyoxynucleobase.
5. The target capture oligomer of claim 1 , wherein the oligomer is formed by deoxyribonucleobases.
6. A kit comprising the target capture oligomer of claim 1 and an immobilized probe comprising a support bearing a probe comprising a segment complementary to the first stem segment or the second stem segment.
7. A reaction mixture comprising the target capture oligomer of claim 1 , an immobilized probe comprising a support bearing a probe comprising a segment complementary to the first stem segment or the second stem segment and a target nucleic acid that hybridizes to the target-binding segment of the target capture oligomer.