Sets of oligonucleotide-binding e-tag probes
View Patent ↗Probe sets for the multiplexed detection of known, selected nucleotide target sequences are provided. Detection involves the release of identifying tags as a consequence of target recognition. The probe sets include electrophoretic tag probes or “e-tag probes”, comprising a detection region and a mobility-defining region called the mobility modifier, both linked to a target-binding moiety. The target-binding moiety of the e-tag probes hybridizes to complementary target sequences followed by nuclease cleavage of the e-tag probes and release of detectable e-tags or e-tag reporters. The mixture is exposed to a capture agent which binds uncleaved and/or partially cleaved e-tag probes, followed by electrophoretic separation. In a multiplexed assay, different released e-tag reporters may be separated and detected providing for target identification.
1. A set of electrophoretic probes for detecting the presence or absence of one or more of a plurality nucleotide sequences in a sample, the set comprising a plurality of electrophoretic probes selected from the group defined by the formula:
(D, M)-N-T
wherein:
(D, M)-N is an e-tag reporter released from an electrophoretic probe of the set upon digestion of the electrophoretic probe by a nuclease, the e-tag reporter having a charge;
D is a detection moiety;
M is a mobility modifier consisting of from 1 to 500 atoms selected from the group consisting of carbon, hydrogen, oxygen, phosphorus, nitrogen, sulfur, and boron;
N is a nucleotide; and
T is an oligonucleotide specific for at least one of the plurality of nucleotide sequences, each T having a length in the range of from 12 to 60 nucleotides such that at least one nucleotide of T has a capture ligand attached;
and wherein each e-tag reporter of the plurality of electrophoretic probes has a distinct charge/mass ratio so that e-tag reporters of different electrophoretic probes form distinct peaks in an electropherogram upon electrophoretic separation;
and wherein the capture ligand specifically binds to a capture agent that has a charge opposite to that of e-tag reporters so that undigested electrophoretic probes are excluded from the electropherogram.
2. The set of claim 1 wherein said plurality is in the range of from 5 to 100 and wherein M is a mobility modifier consisting of from 1 to 300 atoms selected from the group consisting of carbon, hydrogen, oxygen, phosphorus, nitrogen, sulfur, and boron.
3. The set of claim 2 wherein D is a fluorophore, chromophore, or an electrochemical label.
4. The set according to claim 1 , 2 , or 3 wherein said formula is D-M-N-T.
5. The set of claim 4 wherein said capture ligand is biotin and wherein said capture agent is avidin.
6. The set of claim 4 wherein said oligonucleotide has at least one nuclease-resistant linkage.
7. The set of claim 4 wherein said fluorophore is a fluorescern.
8. The set of claim 4 wherein M consists of from 2 to 100 atoms selected from the group consisting of carbon, hydrogen, oxygen, phosphorus, nitrogen, sulfur, and boron.
9. A set of electrophoretic probes for detecting the presence or absence of one or more of a plurality nucleotide sequences in a sample, the set comprising a plurality of electrophoretic probes selected from the group defined by the formula:
(D, M)-N-T
wherein:
(D, M)-N is an e-tag reporter released from an electrophoretic probe of the set upon digestion of the electrophoretic probe by a nuclease, the e-tag reporter having a charge;
D is a detection moiety;
M is a mobility modifier having a molecular weight of between 35 and 1500 daltons;
N is a nucleotide; and
T is an oligonucleotide specific for at least one of the plurality of nucleotide sequences, each T having a length in the range of from 12 to 60 nucleotides such that at least one nucleotide of T has a capture ligand attached;
and wherein each e-tag reporter of the plurality of electrophoretic probes has a distinct charge/mass ratio so that e-tag reporters of different electrophoretic probes form distinct peaks in an electropheroeram upon electrophoretic separation;
and wherein the capture ligand specifically binds to a capture agent that has a charge opposite to that of e-tag reporters so that undigested electrophoretic probes are excluded from the electropherogram.
10. The set of claim 9 wherein D is a fluorophore, chromophore, or an electrochemical label.
11. The set according to claim 10 wherein said formula is D-M-N-T.
12. The set of claim 11 wherein said capture ligand is biotin and wherein said capture agent is avidin.
13. The set of claim 11 wherein said e-tag reporter is selected from the group consisting of the following compounds: