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. Typically the solid or semi-solid support is selected from the group consisting of a polymeric gel, a membrane, an array, a glass bead, a glass slide, and a polymeric microparticle. Preferably, the polymeric gel is agarose or polyacrylamide. The methods employing the non-genotoxic compounds represent an improvement over commonly used methods employing ethidium bromide wherein the present methods retain the advantages of ethidium bromide, ease of use and low cost, but without the disadvantageous, known mutagen requiring special handling and waste procedures.

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.