Kits employing oligonucleotide-binding e-tag probes

• US 6,955,874 B2
• Filed: 04/02/2001
• Issued: 10/18/2005
• Est. Priority Date: 04/30/1999
• Status: Expired due to Fees

• Current Assignee: Monogram Biosciences, Inc. (Laboratory Corporation Of America Holdings)
• Original Assignee: Aclara Biosciences, Inc (Laboratory Corporation Of America Holdings)
• Inventors: Singh, Sharat, Chenna, Ahmed, Matray, Tracy
• Primary Examiner(s): Horlick, Kenneth R.
• Assistant Examiner(s): Tung, Joyce
• Application Number:
  US09/824,905
• Publication Number:
  US 20020009737A1
• Time in Patent Office: 1,660 Days
• Field of Search: 435/6, 435/810, 536/23.1, 536/24.3, 536/25.32, 204/450, 206/569
• US Class Current: 435/6.1
• CPC Class Codes: C07H 19/06 Pyrimidine radicals
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C40B 70/00 Tags or labels specially adapted for combinatorial chemistry or libraries, e.g. fluorescent tags or bar codes
• CPC Class Codes: G01N 33/561 Immunoelectrophoresis
• CPC Class Codes: Y10S 435/81 Packaged device or kit

Abstract

Kits 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 kits include sets of electrophoretic tag probes or e-tag probes, capture agent and optionally a nuclease. The e-tag probes comprise a detection region and a mobility-defining region called the mobility modifier, both linked to a target-binding moiety. In using the kits, 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.

Claims

1. A kit for detecting the presence or absence of one or more of a plurality nucleotide sequences in a sample, the kit comprising:

a capture agent; and

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 upon digestion of the electrophoretic probe by a nuclease, the e-tag reporter having a charge;

D is a detection group;

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 the e-tag reporters form distinct peaks in an electropherogram upon electrophoretic separation;

and wherein the capture ligand specifically binds to the 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 kit of claim 1 wherein said formula is D-M-N-T and wherein M is a non-oligomeric compound consisting of from 1 to 300 atoms selected from the group consisting of carbon, hydrogen, oxygen, phosphorus, nitrogen, sulfur, and boron.

3. The kit of claim 2 wherein D is a fluorophore, chromophore, or an electrochemical label.

4. The kit of claim 3 wherein said plurality is in the range of from 5 to 100.

5. The kit of claim 4 further including a nuclease.

6. The kit of claim 4 wherein said fluorescent label is a fluorescein.

7. The kit of claim 4 wherein said capture ligand is biotin wherein said capture agent is avidin or streptavidin.

8. The kit of claim 4 further including said capture agent attached to a solid support.

9. A kit for detecting the presence or absence of one or more of a plurality nucleotide sequences in a sample, the kit comprising:

a capture agent; and

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;

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 electropherogram upon electrophoretic separation;

and wherein the capture ligand specifically binds to the capture agent to exclude undigested electrophoretic probes from the electropherogram.

10. The kit of claim 9 wherein D is a fluorophore, chromophore, or an electrochemical label.

11. The kit according to claim 10 wherein said formula is D-M-N-T and wherein said plurality is in the range of from 5 to 100.

12. The kit of claim 11 wherein said capture ligand is biotin and wherein said capture agent is avidin or streptavidin.

13. The kit of claim 11 further including a nuclease.

14. The kit of claim 11 wherein said e-tag reporter is selected from the group consisting of the following compounds: