Ddao compounds as fluorescent reference standards

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.

1 . A method comprising:
 (a) forming a mixture comprising at least one probe and a reference dye, wherein each of the at least one probes comprises a target-specific moiety and a reporter dye, wherein each target-specific moiety is specific for a different target and each reporter dye is different from the others, and wherein the reference dye has 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; and 
         (b) measuring fluorescence emitted by the reference dye; and 
         (c) measuring fluorescence emitted by at least one reporter dye. 
       
     
     
         2 . The method of  claim 1 , further comprising adjusting the measured fluorescence emitted by the at least one reporter dye based on the measured fluorescence emitted by the reference dye, to form an adjusted measurement. 
     
     
         3 . The method of  claim 1 , wherein the target-specific moiety is an oligonucleotide. 
     
     
         4 . The method of  claim 1 , wherein the probe comprises an oligonucleotide, a reporter dye, and a quencher dye. 
     
     
         5 . The method of  claim 1 , wherein the method comprises: irradiating the mixture with a first excitation wavelength range;
 detecting radiation emitted by at least a first reporter dye; and   detecting radiation emitted by at least a second reporter dye;   wherein the radiation emitted by each reporter dye can be measured in the presence of the other reporter dyes and the reference dye in the mixture.   
     
     
         6 . The method 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.   
     
     
         7 . The method of  claim 7 , wherein R 2  and R 3  are each chlorine and R 7  is hydrogen or SO 3 H. 
     
     
         8 . The method of  claim 1 , wherein the reference dye is selected from: 
       
         
           
           
               
               
           
         
       
     
     
         9 . A composition comprising:
 a reference dye has 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; and 
 
         a buffer, a selection of nucleotides, and a protein. 
       
     
     
         10 . The composition of  claim 9 , wherein the buffer is selected from tris, tricene, bicene, HEPES, and MOPS 
     
     
         11 . The composition of  claim 10 , wherein the buffer further comprises a chloride salt selected from potassium, lithium, magnesium and manganese, and a surfactant. 
     
     
         12 . The composition of  claim 12 , wherein the surfactant is selected from a polysorbate surfactant, a polyoxyethylene surfactant, a polyethoxylated phenol surfactant, and a zwitterionic surfactant. 
     
     
         13 . The composition of  claim 9 , wherein the selection of nucleotides is from deoxynucloetides (dNPTs). 
     
     
         14 . The composition of  claim 13 , wherein the selection of nucleotides is further from dideoxynucleotides (didNTPs), deaza-GTP, deaza-dGTP, and 2′-deoxyinosine 5′-triphosphate (dITP). 
     
     
         15 . The composition of  claim 9 , 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.