Ligation assays in liquid phase
Ligation assays in liquid phase for detecting nucleic acid sequences.
1. A method for detecting target nucleic acid sequences in a sample, wherein a target sequence has a downstream region (DR) and an upstream region (UR), comprising
(a) contacting the sample with a pair of detector oligos, which pair comprises
a downstream detector oligo (DD) having a complementary downstream region (DR′) and
a separate upstream detector oligo (UD) having a complementary upstream region (UR′),
wherein at least one of the DD or UD contains DNA and RNA,
thereby allowing the pair of detectors to hybridize specifically to the target nucleic acids;
(b1) ligating the DR′ and UR′ if both are specifically hybridized to the DR and UR of a target sequence, wherein the ligase efficiently joins DNA to a ribonucleotide or RNA while both are hybridized to an RNA template;
(b2) exposing hybridization complexes to at least one nuclease; and
whereby the ligation product indicates the presence of the target sequence in the sample.
2. The method of claim 1 , downstream region (DR) and an upstream region (UR), comprising
wherein at least one of the DD or UD has a second complementary region (DR2′ or UR2′) separated from the DR′ or UR′ by a noncomplementary region (CP1) that does not hybridize to the target nucleic acid, whereby the DR2′ or UR2′ can specifically hybridize to a DR2 or UR2 of the target nucleic acid.
3. The method of claim 1 , wherein step (a), (b1), or both are performed in liquid phase.
4. The method of claim 1 , wherein step (a), (b1) or both are performed while the target nucleic acid is attached to a solid surface.
5. The method of claim 1 , wherein sets of DDs and UDs, having different DRs or URs, provide measurement of a portion of the target sequence that potentially harbors multiple mutations.
6. The method of claim 1 , wherein the target nucleic acid is an RNA selected from the group comprising mRNA, siRNA, antisense RNA, long noncoding RNA, circular RNA and microRNA, or portion thereof.
7. The method of claim 6 , wherein the microRNA or portion thereof is 3′-polyadenylated prior to step (a) and the DD has a poly-T region adjacent to the DR′.
8. The method of claim 1 , wherein the sample is a tissue sample, mounted on a slide, fixed or a formalin-fixed, paraffin-embedded (FFPE) sample.
9. The method of claim 1 , further comprising the step of permeabilizing the cell walls, cell membranes, or subcellular structures.
10. The method of claim 1 , wherein the target nucleic acid is in situ.
11. The method of claim 10 , wherein the ligation product is cross-linked in situ.
12. The method of claim 11 , wherein the cross-linking is reversible.
13. The method of claim 12 , wherein the reversible cross-linking is photocleavable.
14. The method of claim 1 , wherein the DD has a first amplification region (P1) downstream of the DR′, and the UD has a second amplification region (P2′) upstream of the UR′.
15. The method of claim 1 , wherein a detector has a photocleavable site.
16. The method of claim 1 , wherein the method further comprises any of
(a1) imaging or detecting another analyte at or near the location of the target nucleic acid in situ;
(a2) before the ligation step, performing a wash step;
(b3) performing a photocleavage step;
(c1) after the ligation step, adding an EDTA solution;
(c2) after the ligation step, performing a wash step;
(d) amplifying the ligation products;
where the DR and UR are separated by at least one nucleotide,
(b0) extending the DR′ using the sample as template; or
(e) detecting the ligation or amplification product.
17. The method of claim 1 , further comprising the step of eluting the ligation product.
18. The method of claim 17 , wherein the elution is effected by addition of a base.
19. The method of claim 17 , wherein the elution is effected by low salt concentration.
20. The method of claim 1 , wherein the sample is from a tissue culture.
21. The method of claim 1 , wherein the 3′ end of the DD has one or more ribonucleotides.
22. The method of claim 1 , wherein a UD is phosphorylated at the 5′ end.
23. The method of claim 1 , further comprising the step of
(b3) releasing ligation products from RNA targets.
24. The method of claim 21 , wherein a UD is phosphorylated at the 5′ end.
25. The method of claim 21 , further comprising the step of (b3) releasing ligation products from RNA targets.
26. The method of claim 22 , further comprising the step of (b3) releasing ligation products from RNA targets.
27. The method of claim 26 , wherein the 3′ end of the DD has one or more ribonucleotides.