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 method of capturing a target nucleic acid, 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, which first and second hairpin stem segments are polyA and polyT segments respectively or vice versa differing in length by at least five nucleobase units flanking a target-binding segment complementary to target nucleic acid, the target capture oligomer being in the form of or formable into 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 an 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 separating or keeping separate 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 thereby capturing the target nucleic acid; and if the sample does not contain the target nucleic acid, the first and second segments are intramolecularly hybridized as a stem.
2. The method of claim 1 , wherein the target nucleic acid is present in the sample.
3. The method of claim 1 , further comprising separating the support-bound capture hybrid from the sample.
4. The method of claim 3 , wherein the method further comprises following the contacting step, incubating the sample at a first temperature followed by a second temperature lower than the first temperature, wherein the first temperature is between the melting temperature of a duplex formed between the first and second stem segments and the melting temperature of a duplex formed between the target binding segment and the target nucleic acid and the second temperature is below both the melting temperature of the duplex formed between the first and second stem segments and the melting temperature of a duplex formed between the second stem segment and the immobilized probe.
5. The method of claim 4 , wherein the separating step is performed at the second temperature.
6. The method of claim 1 , further comprising releasing the target nucleic acid from the capture hybrid.
7. The method of claim 1 , further comprising detecting the target nucleic acid.
8. The method of claim 7 , further comprising sequencing the target nucleic acid.
9. The method of claim 1 , wherein the target capture oligomer is one of a plurality of target capture oligomers having different target binding segments complementary to different targets.
10. The method of claim 1 , wherein the first hairpin stem segment is a homopolymer of 10-40 adenine residues and the second hairpin stem segment is a homopolymer of 10-30 thymine residues or vice versa.