Attenuators
Methods for detecting nucleic acid sequences, where attenuator oligonucleotides are provided to reduce the number of detection products resulting from highly abundant sequences.
1 . A method for detecting a plurality of target nucleic acid sequences of interest in samples, wherein each target sequence has an upstream region (UR) and a downstream region (DR), the method comprising:
(a) contacting the sample with a pool of detector oligos comprising, for each target sequence,
(1) a downstream detector oligo (DDO) comprising a portion complementary to the downstream region of the target sequence (DR′), and
(2) an upstream detector oligo (UDO) comprising a portion complementary to the upstream region of the target sequence (UR′),
wherein, for a predetermined set of potentially high abundance target sequences (HATs) in the sample, also contacting the sample with
(3) an attenuator oligonucleotide that comprises a portion at least partially complementary to at least one of the downstream or upstream regions of a HAT, wherein the portion is of sufficient length to specifically bind to the downstream or upstream region of the HAT, and further comprises a nonextendable or nonligatable blocking group, or a nonamplifiable segment, and
(b) ligating a downstream detector oligo for a target sequence to a corresponding upstream detector oligo if both are hybridized to the downstream and upstream regions of a target sequence in the sample; and
(c) labeling the product of step (b) in a sample with a barcode sequence;
whereby the number of HAT products to be detected is attenuated and the barcoded ligation product indicates the presence of the target sequence and identifies the sample.
2 . The method of claim 1 , wherein at least one of the detector oligos is detectably labeled.
3 . The method of claim 1 , wherein at least one of the detector oligos has a barcode sequence.
4 . The method of claim 1 , further comprising repeating step (c) on multiple samples from step (b).
5 . The method of claim 1 , further comprising the step of amplifying the ligated oligos.
6 . The method of claim 5 , wherein the amplified products incorporate a barcode sequence that is specific to the sample.
7 . The method of claim 1 , wherein an attenuator further has a portion that is complementary to a region adjacent to the downstream or upstream region of a HAT.
8 . The method of claim 1 , wherein an attenuator has a portion that is complementary to at least a portion of the downstream region of a HAT and another portion that is complementary to at least a portion of the upstream region of the HAT.
9 . The method of claim 1 , wherein an attenuator is complementary to a UR, a DR, or both.
10 . The method of claim 1 , wherein the attenuator oligonucleotide comprises a nonextendable blocking group.
11 . The method of claim 1 , wherein the attenuator oligonucleotide comprises a nonligatable blocking group.
12 . The method of claim 1 , wherein the blocking group is a nonhybridizing portion.
13 . The method of claim 1 , wherein the attenuator oligonucleotide comprises a nonamplifiable segment.
14 . The method of claim 13 , wherein the nonamplifiable segment comprises a nonamplifiable tail.
15 . The method of claim 13 , wherein the attenuator oligonucleotide is not phosphorylated.
16 . The method of claim 1 , wherein the attenuator oligonucleotide is phosphorylated.
17 . The method of claim 16 , wherein the attenuator oligonucleotide is circularizing.
18 . The method of claim 1 , wherein the attenuator oligonucleotide specifically binds to a downstream region of an exon junction of a target sequence.
19 . The method of claim 1 , wherein the attenuator oligonucleotide specifically binds to an upstream region of an exon junction of a target sequence.