Method of nucleic acid sequence detection
Barcoded ligation assay products from individual samples.
1. A method for detecting target nucleic acid sequences in samples, wherein a target sequence has a downstream region (DR) and an upstream region (UR), comprising
(a) contacting the samples with a pair of detector oligos (DOs), 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 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′ specifically hybridizes to a DR2 or UR2 of the target nucleic acid,
thereby allowing the pair of detectors to hybridize specifically to target nucleic acids;
(b) ligating the DR′ and UR′ if both are specifically hybridized to the DR and UR of a target sequence;
(c) labeling the ligated detectors of step (b) with a barcode sequence in a plurality of samples; and
(d) labeling the product of step (c) with a barcode sequence in a different plurality of samples;
whereby the barcoded ligation product indicates the presence of the target sequence and identifies the sample.
2. The method of claim 1 , further comprising the step of exposing hybridization complexes to at least one nuclease that degrades single strands but does not significantly degrade double strands.
3. The method of claim 1 , wherein step (c) or (d) is performed by attaching an oligo having a barcode sequence.
4. The method of claim 1 , further comprising the step of mixing the labeled products of step (c) or (d), and dividing the mixed labeled products into sets of different pluralities of samples.
5. The method of claim 1 , further comprising repeating step (d) with a different plurality of samples.
6. The method of claim 1 , wherein
step (c) is performed on one end of the ligated product; and
step (d) is performed on the other end of the ligated product.
7. The method of claim 1 , 2 , 3 , 4 , 5 , or 6 , wherein step (d) is performed by attaching oligos having an amplification sequence.
8. The method of claim 1 , 2 , 3 , 4 , 5 , or 6 , further comprising the step of
permeabilizing cell walls, cell membranes, or subcellular structures;
dissociating individual cells;
cross-linking detectors to target sequence; or
eluting the ligation product.
9. The method of claim 1 , 2 , 3 , 4 , 5 , or 6 , further comprising the step of capturing the target nucleic acids, the ligation products, or amplification products thereof with a capture oligo.