Methods for 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 (DDO) having a complementary downstream region (DR′) and
a separate upstream detector oligo (UDO) having a complementary upstream region (UR′),
wherein at least one of the DDO or UDO 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,
and where at least one of the DDO or UDO has been labeled with a barcode sequence,
thereby allowing the pair of detectors to hybridize specifically to target nucleic acids;
(b) whereby the DR′ and UR′ are ligated if both are specifically hybridized to the DR and UR of a target sequence; and
(c) optionally labeling the ligated detectors with a barcode sequence in a 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 , wherein step (c) is performed by attaching an oligo having a barcode sequence.
3 . The method of claim 1 , further comprising repeating step (c) with a different plurality of samples.
4 . The method of claim 1 , further comprising the steps of mixing the labeled products of step (b), and dividing the mixed labeled products into sets of different pluralities of samples to perform step (c).
5 . The method of claim 1 , further comprising the step of extending a strand.
6 . The method of claim 1 , wherein
step (c) is performed on one end of the ligated product; and
step (c) is repeated on the other end of the ligated product.
7 . The method of claim 1 , further comprising providing a terminal set of barcoded oligos having an amplification sequence.
8 . The method of claim 1 , wherein the sample is a tissue sample.
9 . The method of claim 1 , wherein the samples are dissociated individual cells.
10 . The method of claim 1 , wherein a sample is a single cell.
11 . The method of claim 1 , wherein the number of different barcode sequences is at least 96.
12 . The method of claim 1 , further comprising the step of
permeabilizing the cell walls, cell membranes, or subcellular structures;
dissociating individual cells;
cross-linking detectors to target sequence; or
eluting the ligation product.
13 . 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.
14 . The method of claim 1 , wherein step (c) is performed by attaching oligos having an amplification sequence or its complement.
15 . 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 (DDO) having a complementary downstream region (DR′) and
a separate upstream detector oligo (UDO) having a complementary upstream region (UR′),
wherein at least one of the DDO or UDO 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,
and where at least one of the DDO or UDO contains a barcode sequence,
thereby allowing the pair of detectors to hybridize specifically to target nucleic acids;
(b) whereby the DR′ and UR′ are ligated if both are specifically hybridized to the DR and UR of a target sequence; and
(c) optionally labeling the ligated detectors with a barcode sequence in a plurality of samples;
whereby the barcoded ligation product indicates the presence of the target sequence and identifies the sample.
16 . The method of claim 15 , wherein step (c) is performed by attaching an oligo having a barcode sequence.
17 . The method of claim 15 , further comprising repeating step (c) with a different plurality of samples.
18 . The method of claim 15 , further comprising the steps of mixing the labeled products of step (b), and dividing the mixed labeled products into sets of different pluralities of samples to perform step (c).
19 . The method of claim 15 , further comprising the step of extending a strand.
20 . The method of claim 15 , wherein
step (c) is performed on one end of the ligated product; and
step (c) is repeated on the other end of the ligated product.
21 . The method of claim 15 , further comprising providing a terminal set of barcoded oligos having an amplification sequence.
22 . The method of claim 15 , wherein the sample is a tissue sample.
23 . The method of claim 15 , wherein the samples are dissociated individual cells.
24 . The method of claim 15 , wherein a sample is a single cell.
25 . The method of claim 15 , wherein the number of different barcode sequences is at least 96.
26 . The method of claim 15 , further comprising the step of
permeabilizing the cell walls, cell membranes, or subcellular structures;
dissociating individual cells;
cross-linking detectors to target sequence; or eluting the ligation product.
27 . The method of claim 15 , further comprising the step of exposing hybridization complexes to at least one nuclease that degrades single strands but does not significantly degrade double strands.
28 . The method of claim 15 , wherein step (c) is performed by attaching oligos having an amplification sequence or its complement.