Detection of immobilized nucleic acid

Abstract

The present invention provides methods for determining the presence of immobilized nucleic acid employing unsymmetrical cyanine dyes that are derivatives of thiazole orange, a staining solution and select fluorogenic compounds that are characterized as being essentially non-genotoxic. The methods comprise immobilizing nucleic acid, single or double stranded DNA, RNA or a combination thereof, on a solid or semi solid support, contacting the immobilized nucleic acid with an unsymmetrical cyanine dye compound and then illuminating the immobilized nucleic acid with an appropriate wavelength whereby the presence of the nucleic acid is determined. The cyanine dye compounds are typically present in an aqueous staining solution comprising the dye compound and a tris acetate or tris borate buffer wherein the solution facilitates the contact of the dye compound and the immobilized nucleic acid.

1 . A compound for the detection of nucleic acid immobilized on a solid or semi solid support, wherein the compound is an unsymmetrical cyanine dye compound that is characterized as being essentially non-genotoxic in eukaryotic cells. 
     
     
         2 . The compound according to  claim 1 , wherein the compound is in an aqueous solution. 
     
     
         3 . The compound according to  claim 1 , wherein the compound is immobilized on or in a solid or semi-solid matrix. 
     
     
         4 . The compound according to  claim 3 , wherein the solid or semi-solid matrix comprises agarose. 
     
     
         5 . The compound according to  3 , wherein the solid or semi-solid matrix is a polymeric membrane. 
     
     
         6 . The compound according to  claim 1 , wherein the cyanine compound has the formula 
       
         
           
           
               
               
           
         
       
     
     
         7 . A kit for the detection of immobilized nucleic acid, the kit comprising:
 a. a compound having the formula   
       
         
           
           
               
               
           
         
         
           wherein X is O, S or C(CH 3 ) 2 ; 
           R 1  is a fused benzene, methoxy, a C 1 -C 6  alkyl; 
           R 2  and R 3  are independently a C 1 -C 6  alkyl; 
           R 4  is a C 1 -C 6  alkyl or a methoxy; 
           t is independently 0, 1, 2, 3, or 4; 
           s is independently 0, 1, 2, 3 or 4 and 
           n is 0, 1, 2 or 3; 
         
       
       with the proviso that the cyanine dye compound is not thiazole orange. 
     
     
         8 . The kit according to  claim 7 , wherein the compound is in an aqueous solution. 
     
     
         9 . The kit according to  claim 8 , wherein the aqueous solution comprises tris borate or tris acetate. 
     
     
         10 . The kit according to  claim 7 , wherein the compound is dissolved in an alcohol or DMSO. 
     
     
         11 . The kit according to  claim 7 , wherein the compound is immobilized on or in a solid or semi-solid matrix. 
     
     
         12 . The kit according to  claim 11 , wherein the matrix comprises agarose. 
     
     
         13 . The lit according to  claim 11 , wherein the matrix is a polymeric membrane. 
     
     
         14 . The kit according to  claim 7 , wherein the compound is 
       
         
           
           
               
               
           
         
       
       and is characterized as being essentially non-genotoxic.