4,7-dichlorofluorescein dyes as molecular probes

Abstract

Long wavelength, narrow emission bandwidth fluorecein dyes are provided for detecting spacially overlapping target substances. The dyes comprise 4,7-dichlorofluoresceins, and particularly 2′,4′,5′,7′-tetrachloro-4,7-dichloro-5- (and 6-)carboxyfluoresceins. Methods and kits for using the dyes in DNA analysis are provided.

1 . In a method of detecting a plurality of electrophoretically separated classes of DNA fragments, and improvement comprising labelling DNA fragments of at least one class with a 4,7-dichlorofluorescein dye.  
     
     
         2 . The method of  claim 1  wherein said 4,7-dichlorofluorescein dye is defined by the formula:  
       
         
           
           
               
               
           
         
       
       wherein: 
 A′ hydrogen, fluoro, chloro, a linking functionality, or a group that may be converted to a linking functionality;  
 B′ is fluoro, chloro, or an acidic anionic group;  
 X′ is hydrogen, fluoro, or chloro;  
 Z 1  is hydrogen or,m when taken with Z 2 , benzo;  
 Z 2  when taken alone, is hydrogen, halo, lower alkyl, lower alkyloxy, a linking functionality, or a group that may be converted to a linking functionality, or when taken with Z 1 , benzo;  
 Z 3  and Z 4  are separately hydrogen, halo, lower alkyl, lower alkyloxy, a linking functionality, or a group that may be converted to a linking functionality;  
 Z 5 , when taken alone, is hydrogen, halo, lower alkyl, lower alkyloxy, a linking functionality, or a group that may be converted to a linking functionality, or when taken with Z 6 , benzo;  
 Z 6  is hydrogen or, when taken with Z 5 , benzo; and wherein at least one of A, Z 2 , Z 3 , Z 4 , and Z 5  is a linking functionality or a group that may be converted to a linking functionality.  
 
     
     
         3 . The method of claim 2 wherein: 
 A′ is carboxyl, sulfonyl, isothiocyanate, succinimidyl carboxylate, phosphoramidite, or amino;    B′ is carboxyl or sulfonyl;    X′ is hydrogen or chloro;    Z 2 , when taken alone, is hydrogen, methyl, ethyl, methoxy, ethoxy, or chloro;    Z 3  , and Z 4  are separately hydrogen, methyl, ethyl, methoxy, ethoxy, chloro, carboxyl, sulfonyl, isothiocyanate, succinimidyl carboxylate, phosphoramidite, or methylamino;    Z 5 , when taken alone, is hydrogen, methyl, ethyl, methoxy, ethoxy, or chloro; and    wherein only one of A′, Z 3 , and Z 4  is carboxyl, sulfonyl, methylamino, isothiocyanate, succinimidyl carboxylate, phosphoramidite, or amino.    
     
     
         4 . The method of  claim 3  wherein Z 3 , and Z 4  are separately hydrogen, methyl, ethyl, methoxy, ethoxy, or chloro.  
     
     
         5 . The method of  claim 4  wherein Z 2 , when taken alone, is hydrogen, methoxy, ethoxy, or chloro; 
 Z 3 , and Z 4  are separately hydrogen, methoxy, ethoxy, chloro; and    Z 5 , when taken alone, is hydrogen, methoxy, ethoxy, or chloro.    
     
     
         6 . The method of  claim 5  wherein B′ is carboxy and A′ is carboxy, succinimidyl carboxylate, or phosphoramidite.  
     
     
         7 . A compound having the formula:  
       
         
           
           
               
               
           
         
       
       wherein: 
 A′ is hydrogen, fluoro, chloro, a linking functionality, or a group that may be converted to a linking functionality;  
 B′ is fluoro, chloro, or an acidic anionic group;  
 X′ is hydrogen, fluoro, or chloro;  
 Z 3  and Z 4  are separately hydrogen, halo, a linking functionality, or a group that may be converted to a linking functionality; and  
 wherein at least one of A′, Z 3 , and Z 4  is a linking functionality or a group that may be converted to a linking functionality.  
 
     
     
         8 . The compound of  claim 7  wherein A′ is carboxyl, sulfonyl, isothioncyanate, succinimidyl carboxylate, phosphoramidite, or amino; B′ is carboxyl or sulfonyl; X′ is hydrogen; Z 3  and Z 4  are separately hydrogen, halo, carboxyl, sulfonyl, or methylamino.  
     
     
         9 . The compound of  claim 8  wherein only one of A′, Z 3 , and Z 4  is carboxyl, sulfonyl, methylamino, or amino.  
     
     
         10 . The compound of  claim 9  wherein A′ and B′ are carboxyl, Z 3  is hydrogen or chloro, and Z 4  is hydrogen or chloro.  
     
     
         11 . A kit for detecting a plurality of electrophoretically separated classes of DNA fragments comprising an oligonucleotide labelled with a 4,7-dichlorofluorescein dye.  
     
     
         12 . The kit of  claim 11  further comprising: 
 an enzyme selected from the group consisting of nucleic acid polymerase and nucleic acid ligase; and    a reaction buffer.    
     
     
         13 . The kit of  claim 12  wherein said enzyme is a nucleic acid polymerase and wherein said kit further includes a nucleoside triphosphate mix.  
     
     
         14 . The kit of  claim 12  wherein said enzyme is a nucleic acid ligase.  
     
     
         15 . A kit sequencing DNA comprising: 
 an oligonucleotide labelled with a 4,7-dichlorofluorescein dye;    a nucleic acid polymerase;    a reaction buffer; and    a nucleoside triphosphate mix.    
     
     
         16 . The kit of  claim 15  wherein said 4,7-dichlorofluorescein dye is defined by the formula:  
       
         
           
           
               
               
           
         
       
       wherein: 
 A′ is hydrogen, fluoro, chloro, or a group that may be converted to a linking functionality;  
 B′ is fluoro, chloro, or an acidic anionic group;  
 X′ is hydrogen, fluoro, or chloro;  
 Z 1  is hydrogen or, when taken with Z 2 , benzo;  
 Z 2 , when taken alone, is hydrogen, halo, lower alkyl, lower alkyloxy, or a group that may be converted to a linking functionality, or when taken with Z 1 , benzo;  
 Z 3  and Z 4  are separately hydrogen, halo, lower alkyl, lower alkyloxy, or a group that may be converted to a linking functionality;  
 Z 5 , when taken alone, is hydrogen, halo, lower alkyl, lower alkyloxy, or a group that may be converted to a linking functionality, or when taken with Z 6 , benzo;  
 Z 6  is hydrogen or, when taken with Z 5 , benzo; and wherein at least one of A′, Z 2 , Z 3 , Z 4 , and Z 5  is a group that may be converted to a linking functionality.  
 
     
     
         17 . The kit of  claim 16  wherein: 
 A′ and B′ are carboxyl;    X′ is hydrogen or chloro;    Z 2 , when taken alone, is hydrogen, methyl, ethyl, methoxy, ethoxy, or chloro;    Z 3 , and Z 4  are separately hydrogen, methyl, ethyl, methoxy, ethoxy, chloro,; and    Z 5 , when taken alone, is hydrogen, methyl, ethyl, methoxy, ethoxy, or chloro.    
     
     
         18 . A kit for sequencing DNA comprising: 
 a dye-terminator mix wherein at least one dye-terminator is labelled with a 4,7-dichlorofluorescein dye;    a nucleic acid polymerase;    a nucleoside triphosphate mix; and    a reaction buffer.    
     
     
         19 . The kit of  claim 18  wherein said dye-terminator mix comprises dideoxynucleoside triphosphates selected from the group consisting of dideoxyadenosine triphosphate, dideoxycytidine triphosphate, dideoxyguanosine triphosphate, and dideoxythymidine triphosphate wherein each of said dideoxynucleoside triphosphate is separately labelled with a dye selected from the group consisting of 5- and 6-carboxyfluorescein, 5- and 6-carboxy-4,7-dichlorofluorescein, 2′,7′-dimethoxy-5- and 6-carboxy-4,7-dichlorofluorescein, 2′,7′-dimethoxy-4′,5′-dichloro-5- and 6-carboxyfluorescein, 2′,7′-dimethoxy-4′,5′-dichloro-5- and 6-carboxy-4,7-dichlorofluorescein, 1′,2′,7′,8′-dibenzo-5- and 6carboxy-4,7-dichlorofluorescein, 1′,2′,7′,8′-dibenzo-4′,5′-dichloro-5- and 6-carboxy-4,7-dichlorofluorescein, 2′,7′-dichloro-5- and 6-carboxy-4,7-dichlorofluorescein, and 2′,4′,5′,7′-tetrachloro-5- and 6-carboxy-4,7-dichlorofluorescein.  
     
     
         20 . The kit of  claim 19  wherein said dideoxythymidine triphosphate is labelled with 6-carboxyfluorescein, said dideoxycytidine triphosphate is labelled with 2′,4′,5′,7′-tetrachloro-5-carboxyfluorescein, said dideoxyadenosine triphosphate is labelled with 2′,4′,5′,7′-tetrachloro-4,7-dichloro-5-carboxyfluorescein, and said dideoxyguanosine triphosphate is labelled with 1′,2′,7′,8′-dibenzo-4,7-dichloro-5-carboxyfluorescein.  
     
     
         21 . The kit of  claim 20  wherein said nucleic acid polymerase is Sequenase™.