Methods and compositions to detect nucleic acids in a biological sample
View Patent ↗Methods of the invention separate a target nucleic acid from a sample by using at least one capture probe oligonucleotide that contains a target-complementary region and a member of a specific binding pair that attaches the target nucleic acid to an immobilized probe on a capture support, thus forming a capture hybrid that is separated from other sample components before the target nucleic acid is released from the capture support and hybridized to a detection probe to form a detection hybrid that produces a detectable signal that indicates the presence of the target nucleic acid in the sample. Compositions for practicing the methods of the invention include a capture probe oligonucleotide made up a target-complementary region sequence and a covalently linked capture region sequence that includes a member of a specific binding pair.
1. A method of detecting the presence of a target nucleic acid in a sample comprising the steps of:
a) providing a biological sample containing a target nucleic acid,
b) mixing said sample with a nucleic acid hairpin capture probe,
wherein said hairpin capture probe has a structure containing an internal target complementary sequence of 20 nucleobases that is flanked on one of its 5′ end or its 3′ end by a homopolymeric nucleic acid capture sequence consisting of polyA or polyT sequence and on its other end by a terminal nucleic acid sequence,
wherein under hybridizing conditions said capture sequence hybridizes to said terminal sequence forming an intramolecular double-stranded stem portion of said hairpin capture probe; and said target-complementary sequence forms a single-stranded loop portion of said hairpin capture probe;
wherein the stem portion of the hairpin structure is at least from 20 to 30 nucleobases in length and wherein the loop portion of the hairpin is equal to or shorter than the stem section, and wherein said terminal region and said capture region are configured to not hybridize to said target nucleic acid, and
c) specifically hybridizing said target-complementary sequence to a target sequence in said target nucleic acid,
d) binding said capture sequence to a complementary immobilized probe sequence, thereby forming a capture hybrid made up of said target nucleic acid, said capture probe, and said immobilized probe attached to said capture support,
e) separating said capture hybrid attached to said capture support from sample components,
f) releasing said target nucleic acid from said capture hybrid,
g) hybridizing a detection probe to said target nucleic acid to form a detection hybrid, and
h) detecting a signal produced in a homogeneous reaction from said detection hybrid to indicate the presence of said target nucleic acid.
2. The method of claim 1 , wherein the capture region is located near the 3′ end of the capture probe and the terminal region is located near the 5′ end of the capture probe.
3. The method of claim 1 , wherein the capture region is located near the 5′ end of the capture probe and the terminal region is located near the 3′ end of the capture probe.
4. The method of claim 1 , wherein binding the capture region to the immobilized probe hybridizes complementary sequences of the capture region and the immobilized probe.
5. The method of claim 1 , wherein binding the capture region to the immobilized probe binds together non-nucleic acid members of a specific binding pair.
6. The method of claim 1 , wherein releasing the target nucleic acid from the capture hybrid further releases the capture probe from the immobilized probe.
7. The method of claim 1 , wherein the detection probe hybridizes specifically to a target sequence that is the same target sequence that hybridizes to the target-complementary sequence of the capture probe.
8. The method of claim 1 , wherein the detection probe hybridizes specifically to a target sequence that differs from or overlaps the target sequence that hybridizes to the target-complementary sequence of the capture probe.
9. The method of claim 1 , wherein said target nucleic acid is an RNA made up of at least 10 or more bases linked by a backbone structure.
10. The method of claim 1 , wherein said target nucleic acid is a portion of a nucleic acid selected from: a bacterial gene, an RNA encoded by a bacterial gene, a human gene, an RNA encoded by a human gene, a human mitochondria) nucleic acid, a viral gene, or an RNA encoded by a viral gene.
11. The method of claim 1 , wherein said terminal nucleic acid sequence is fully complementary to said capture sequence.
12. The method of claim 1 , wherein said target-complementary sequence is synthesized with 2′MO-methyl linkages.
13. The method of claim 1 , wherein the stem portion of the hairpin structure is 30 nucleobases in length and the target region is 20 nucleobases.
14. The method of claim 1 , further comprising detecting the presence of said target nucleic acid or said amplified target nucleic acid.
15. The method of claim 14 , wherein said detecting step is a detection probe based detection step.
16. The method of claim 1 , wherein the stem portion of the hairpin structure is 20 nucleobases in length and the target region is 20 nucleobases.
17. A method of detecting the presence of a target nucleic acid in a sample comprising the steps of:
a) providing a biological sample containing a target nucleic acid,
b) mixing said sample with a nucleic acid hairpin capture probe,
wherein said hairpin capture probe has a structure containing an internal target complementary sequence of 15-25 nucleobases that is flanked on its 5′ end or its 3′ end by a polymeric nucleic acid capture sequence comprising polyA and polyT sequence and on its other end by a terminal nucleic acid sequence, wherein the polarity of the hairpin capture probe is uniform throughout,
wherein under hybridizing conditions said capture sequence hybridizes to said terminal sequence forming an intramolecular double-stranded stem portion of said hairpin capture probe; and said target-complementary sequence forms a single-stranded loop portion of said hairpin capture probe;
wherein the stem portion of the hairpin structure is from 20 to 30 nucleobases in length and wherein the loop portion of the hairpin is equal to or shorter than the stem section, and wherein said terminal region and said capture region are configured to not hybridize to said target nucleic acid, and
c) specifically hybridizing said target-complementary sequence to a target sequence in said target nucleic acid,
d) binding said capture sequence to a complementary immobilized probe sequence, thereby forming a capture hybrid made up of said target nucleic acid, said capture probe, and said immobilized probe attached to said capture support,
e) separating said capture hybrid attached to said capture support from sample components,
f) releasing said target nucleic acid from said capture hybrid,
g) hybridizing a detection probe to said target nucleic acid to form a detection hybrid, and
h) detecting a signal produced in a homogeneous reaction from said detection hybrid to indicate the presence of said target nucleic acid.
18. The method according to claim 17 , wherein said target complementary sequence is 23 nucleobases.
19. A method of detecting the presence of a target nucleic acid in a sample comprising the steps of:
a) providing a biological sample containing a target nucleic acid,
b) mixing said sample with a nucleic acid hairpin capture probe,
wherein said hairpin capture probe has a structure comprising, in a contiguous and uniform polarity, an internal target complementary sequence of 10-25 nucleobases that is flanked on one of its 5′ end or its 3′ end by a polymeric nucleic acid capture sequence comprising sections of polyA sequence, polyT sequence or both, and on its other end by a terminal nucleic acid sequence,
wherein under hybridizing conditions said capture sequence hybridizes to said terminal sequence forming an intramolecular double-stranded stem portion of said hairpin capture probe, wherein said double-stranded region has antiparallel pairing; and said target-complementary sequence forms at least a section of a single-stranded loop portion of said hairpin capture probe;
wherein the stem portion of the hairpin structure is at least from 20 to 30 nucleobases in length and wherein the loop portion of the hairpin is equal to or shorter than the stem section, and wherein said terminal region and said capture region are configured to not hybridize to said target nucleic acid, and
c) specifically hybridizing said target-complementary sequence to a target sequence in said target nucleic acid,
d) binding said capture sequence to a complementary immobilized probe sequence, thereby forming a capture hybrid made up of said target nucleic acid, said capture probe, and said immobilized probe attached to said capture support,
e) separating said capture hybrid attached to said capture support from sample components,
f) releasing said target nucleic acid from said capture hybrid,
g) hybridizing a detection probe to said target nucleic acid to form a detection hybrid, and
h) detecting a signal produced in a homogeneous reaction from said detection hybrid to indicate the presence of said target nucleic acid.
20. The method according to claim 19 , wherein said target complementary sequence is 23 nucleobases.