DDAO compounds as fluorescent reference standards

Patent metadata

Field	Value
Publication number	US-10988798-B2
Application number	US-201816164348-A
Country	US
Kind code	B2
Filing date	Oct 18, 2018
Priority date	Dec 29, 2010
Publication date	Apr 27, 2021
Grant date	Apr 27, 2021

Abstract

According to the present teachings, methods and compositions are provided that utilize at least one reference dye of formula (I):

In some embodiments, a method comprises measuring a detection signal of a reporter dye and at least one reference dye of formula (I). In some embodiments, a composition comprises a reference dye of formula (1), a buffer, a selection of nucleotides and a protein.

First claim

What is claimed is:

1. A multiplex assay technique comprising
(a) combining in a mixture:
at least two different targets,
at least two probes each comprising a target-specific moiety, a reporter dye, and a quencher dye, wherein each target-specific moiety is an oligonucleotide and is specific for a different target and each reporter dye is different from the other reporter dyes and is different from the reference dye, and
a reference dye having a structure according to formula (I):

wherein:
each of R 1 to R 3 and R 6 to R 8 is independently —H, halogen, —CO 2 H, —CO 2 R, —SO 3 H, —SO 3 R, —CH 2 CO 2 H, —CH 2 CO 2 R, —CH 2 SO 3 H, —CH 2 SO 3 R, —CH 2 NH 2 , —CH 2 NHR, —NO 2 , C 1 -C 6 alkyl, substituted C 1 -C 6 alkyl, C 1 -C 6 alkoxy, and substituted C 1 -C 6 alkoxy, wherein R is C 1 -C 6 alkyl, substituted C 1 -C 6 alkyl, C 1 -C 6 alkoxy, and substituted C 1 -C 6 alkoxy;
R 4 and R 5 taken separately are selected from C 1 -C 6 alkyl, and C 1 -C 6 substituted alkyl, or R 4 and R 5 taken together are selected from C 3 -C 7 cycloalkyl, C 4 -C 7 unsaturated cycloalkyl, C 3 -C 7 substituted cycloalkyl, or C 4 -C 7 substituted unsaturated cycloalkyl;
(b) amplifying the mixture of (a) to form an amplified mixture;
(c) irradiating the mixture with a first excitation wave length; and
(d) detecting the radiation emitted by the at least two reporter dyes, which can each be measured in the presence of the other reporter dyes and the reference dye;
wherein the assay indicates the presence, absence, amount, and/or identity of the at least two different targets in the mixture.

2. The multiplex assay technique of claim 1 , wherein at least one reporter dye is a fluorescence resonance energy transfer (FRET) dye.

3. The multiplex assay technique of claim 1 , wherein in step f) the amplified mixture is irradiated with a second excitation wave length.

4. The multiplex assay technique of claim 1 , wherein the mixture comprises at least three different targets and at least three different probes, and wherein the assay indicates the presence, absence, amount, and/or identity of the at least three different targets in the mixture.

5. The multiplex assay technique of claim 4 , wherein the mixture comprises at least four different targets and at least four different probes, and wherein the assay indicates the presence, absence, amount, and/or identity of the at least four different targets in the mixture.

6. The multiplex assay technique of claim 5 , wherein the mixture comprises at least five different targets and at least five different probes, and wherein the assay indicates the presence, absence, amount, and/or identity of the at least five different targets in the mixture.

7. The multiplex assay technique of claim 6 , wherein the mixture comprises at least six different targets and at least six different probes, and wherein the assay indicates the presence, absence, amount, and/or identity of the at least six different targets in the mixture.

8. The multiplex assay technique of claim 1 , wherein R 1 is selected from hydrogen, halogen, methyl, and ethyl; R 2 and R 3 are each independently a halogen; R 4 and R 5 are each independently selected from methyl and ethyl; R 6 is selected from hydrogen, halogen, methyl, and ethyl; R 7 is selected from hydrogen, halogen, methyl, ethyl, and SO 3 H; and R 8 is selected from hydrogen, halogen, methyl, and ethyl.

9. The multiplex assay technique of claim 8 , wherein R 2 and R 3 are each chlorine and R 7 is hydrogen or SO 3 H.

10. The multiplex assay technique of claim 1 , wherein the reference dye is selected from:

Assignees

• Life Technologies Corp

Inventors

• Benson Scott C
• Monighetti Cinna
• Koepf Sandy M

Classifications

• C09B15/00 Primary

  Acridine dyes · CPC title

• C12Q1/6818 Primary

  involving interaction of two or more labels, e.g. resonant energy transfer · CPC title

• C12Q2563/107

  fluorescence · CPC title

• C12Q1/6844

  Nucleic acid amplification reactions · CPC title

• C12Q1/6851

  Quantitative amplification · CPC title