DDAO compounds as fluorescent reference standards

Patent metadata

Field	Value
Publication number	US-9790544-B2
Application number	US-201615066248-A
Country	US
Kind code	B2
Filing date	Mar 10, 2016
Priority date	Dec 29, 2010
Publication date	Oct 17, 2017
Grant date	Oct 17, 2017

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. An amplification master mix composition comprising: a buffer, a selection of nucleotides, at least one protein moiety, and a reference dye, wherein the reference dye has 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.

2. The composition of claim 1 , wherein the buffer is selected from tris, tricene, bicene, HEPES, and MOPS.

3. The composition of claim 2 , wherein the buffer further comprises a chloride salt selected from potassium, lithium, magnesium and manganese, and a surfactant.

4. The composition of claim 3 , wherein the surfactant is selected from a polysorbate surfactant, a polyoxyethylene surfactant, a polyethoxylated phenol surfactant, and a zwitterionic surfactant.

5. The composition of claim 1 , wherein the selection of nucleotides is from deoxynucloetides (dNPTs).

6. The composition of claim 5 , wherein the selection of nucleotides is further from dideoxynucleotides (didNTPs), deaza-GTP, deaza-dGTP, and 2′-deoxyinosine 5′-triphosphate (dITP).

7. The composition of claim 1 , wherein the protein is selected from a DNA polymerase, a ligase, a reverse transcriptase, a ribonuclease, a glycosylase, a single strand binding protein, a pyrophosphatase, an albumin, and a gelatin.

8. The composition of claim 1 , wherein the composition further comprises primers.

9. The composition of claim 1 , wherein the composition is lyophilized.

10. A multiwell plate comprising an amplification master mix composition, wherein the composition comprises: a buffer, a selection of nucleotides, at least one protein moiety, and a reference dye, wherein the reference dye has 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.

11. The multiwell plate of claim 10 , wherein the buffer selected from tris, tricene, bicene, HEPES, and MOPS.

12. The multiwell plate of claim 10 , wherein the buffer further comprises a chloride salt selected from potassium, lithium, magnesium and manganese, and a surfactant.

13. The multiwell plate of claim 12 , wherein the surfactant is selected from a polysorbate surfactant, a polyoxyethylene surfactant, a polyethoxylated phenol surfactant, and a zwitterionic surfactant.

14. The multiwell plate of claim 10 , wherein the selection of nucleotides is from deoxynucloetides (dNPTs).

15. The multiwell plate of claim 14 , wherein the selection of nucleotides is further from dideoxynucleotides (didNTPs), deaza-GTP, deaza-dGTP, and 2′-deoxyinosine 5′-triphosphate (dITP).

16. The multiwell plate of claim 10 , wherein the protein is selected from a DNA polymerase, a ligase, a reverse transcriptase, a ribonuclease, a glycosylase, a single strand binding protein, a pyrophosphatase, an albumin, and a gelatin.

17. The multiwell plate of claim 10 , wherein the composition further comprises primers.

18. The multiwell plate of claim 10 , wherein composition lyophilized.

Assignees

• Life Technologies Corp

Inventors

• Benson Scott C
• Monighetti Cinna
• Koepf Sandy M

Classifications

• C09B15/00 Primary

  Acridine dyes · CPC title

• C12Q1/6851

  Quantitative amplification · CPC title

• C12Q1/6818 Primary

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

• C12Q2561/113

  Real time assay · CPC title

• C12Q2527/125

  Specific component of sample, medium or buffer · CPC title