Fluorescent quenching detection reagents and methods

• US 6,653,473 B2
• Filed: 02/26/2002
• Issued: 11/25/2003
• Est. Priority Date: 12/08/1999
• Status: Expired due to Term

• Current Assignee: ELITechGroup Inc. (Bruker Corporation)
• Original Assignee: Epoch Biosciences Inc. (Ngen, Inc.)
• Inventors: Gall, Alexander A., Dempcy, Robert O., Lukhtanov, Eugeny Alexander, Vermeulen, Nicolaas M. J., Reed, Michael W.
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US10/084,818
• Publication Number:
  US 20030008304A1
• Time in Patent Office: 637 Days
• Field of Search: 536/25.3, 536/26.6, 544/10.2, 435/94
• US Class Current: 544/102
• CPC Class Codes: C07B 2200/11 Compounds covalently bound to a solid support
• CPC Class Codes: C07C 245/08 with the two nitrogen atoms of azo groups bound to carbon atoms of six-membered aromatic rings, e.g. azobenzene
• CPC Class Codes: C07D 519/00 Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00
• CPC Class Codes: C07F 9/2408 of hydroxyalkyl compounds
• CPC Class Codes: C07F 9/572 Five-membered rings
• CPC Class Codes: C07F 9/65522 condensed with carbocyclic rings or carbocyclic ring systems
• CPC Class Codes: C07F 9/6561 containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings
• 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: C09B 29/0813 substituted by OH, O-C(=X)-R, O-C(=X)-X-R, O-R (X being O,S,NR; R being hydrocarbonyl)
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 2521/319 Exonuclease
• CPC Class Codes: C12Q 2565/1025 labels being on the same oligonucleotide
• CPC Class Codes: C40B 40/00 Libraries per se, e.g. arrays, mixtures

Abstract

Oligonucleotide probes containing two labels are provided and are useful in hybridization assays. The probes can also contain a minor groove binding group.

Claims

1. A resorufin phosphoramidite reagent having the formula:

wherein,

Z is O or S;

each of R₂₁, R₂₂, R₂₃, R₂₄, R₂₅, R₂₆ and R₂₇ is independently selected from H, halogen, NO₂, SO₃R, SO₂N(R)₂, C(O)OR, C(O)N(R)₂, CN, CNS, OR, OC(O)R, SR, CF₃, NHC(O)R, N(R)₂, N[R]₃ and —

L—

X;

wherein each R is independently H, (C₁-C₈)alkyl, aryl or heteroaryl, or a blocking group compatible with oligonucleotide synthesis;

—

L—

X is a linker of from 1 to 20 atoms, that is acyclic, cyclic or a combination thereof, having main chain atoms selected from C, N, O, S, Si and P; and

X is a phosphoramidite;

with the proviso that at least one of R₂₁, R₂₂, R₂₃, R₂₄, R₂₅, R₂₆, and R₂₇ is —

L—

X.

2. A resorufin phosphoramidite reagent of claim 1 wherein Z is O.

3. A resorufin phosphoramidite reagent of claim 1, wherein Z is O and R₂₂, R₂₃, R₂₄, R₂₆ and R₂₇ are H.

4. A resorufin phosphoramidite reagent of claim 1, wherein Z is O;

R₂₁ is —

L—

X;

R₂₅ is OC(O)R; and

R₂₂, R₂₃, R₂₄, R₂₆ and R₂₇ are H.

5. A resorufin phosphoramidite reagent of claim 4, wherein X is O—

P(OCH₂CH₂CN)N(iPr)₂.

6. A resorufin phosphoramidite reagent of claim 5, wherein L is a linker having a combination of cyclic and acyclic components.

7. A resorufin phosphoramidite reagent of claim 6, wherein R is selected from (C₁-C₈)alkyl.

8. A resorufin phosphoramidite reagent of claim 7, wherein R is C(CH₃)₃.

9. A resorufin phosphoramidite reagent of claim 5, wherein L is a linker having the formula —

(CH₂)_h—

, wherein the subscript h is an integer of from 1 to 20.

10. A resorufin phosphoramidite reagent of claim 9, wherein the subscript h is 3.