Asymmetric hairpin target capture oligomers
AssigneeGen-Probe Incorporated
InventorsJames Carlson, Reinhold Pollner, Steven T. Brentano
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Abstract
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).
Claims32
A method of capturing a target nucleic acid in, the method comprising:
- contacting a sample suspected of containing the target nucleic acid with a target capture oligomer and an immobilized probe;
- the target capture oligomer comprising first and second hairpin stem segments differing in length by at least two nucleobase units flanking a target-binding segment complementary to target nucleic acid, the target capture oligomer being in the form of a hairpin stem-loop, the stem being formed by intramolecular hybridization of the first and second hairpin stem segments and the target-binding segment constituting the loop;
- the immobilized probe comprising a support bearing a probe comprising a segment complementary to the first or second hairpin stem segment;
- wherein if the sample contains the target nucleic acid, the target nucleic acid hybridizes to the target-binding segment disrupting the intramolecular hybridization of the first and second hairpin stem segments, as a result of which the first or second hairpin segment hybridizes to the complementary segment on the immobilized probe forming a support-bound capture hybrid;
- and if the sample does not contain the target nucleic acid the first and second segments remain intramolecularly hybridized as a stem.
A target capture oligomer comprising first and second stem segments differing in length by at least two 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, intramolecular hybridization of the first and second stem segments forming the stem, and the target-binding segment forming the loop;
- and
- in the presence of the target nucleic acid, the target-binding segment hybridizes to the target nucleic acid disrupting the intramolecular hybridization of the first and second stem segments resulting in the first stem segment being accessible to hybridize to a complementary immobilized probe.
A kit comprising a target capture oligomer as defined in any preceding claim and an immobilized probe immobilized probe comprising a support bearing a probe comprising a segment complementary to the first or second hairpin stem segment.
A reaction mixture comprising a target capture oligomer as defined in any preceding claim, an immobilized probe immobilized probe comprising a support bearing a probe comprising a segment complementary to the first or second hairpin stem segment and a target nucleic acid that hybridizes to the target-binding segment of the target capture oligomer.
Dependencies are inferred from the claim text.