Novel quencher and reporter dye combinations

Abstract

Disclosed is a probe for use in biological assays. The probe includes a fluorescent dye bound to a quencher compound through an oligonucleotide linker. Also disclosed are methods of using the probe, such as for a polymerase chain reaction (PCR), such as in a quantitative PCR reaction (qPCR), as well as kits including the probe.

We claim: 
     
         1 . A probe comprising a product of conjugation of:
 a) a dye having a general Formula Ia, Formula Ib, Formula Ic, or Formula Id   
       
         
           
           
               
               
           
         
         wherein 
         each of R 4 , R 13 , and R 14 , and R 3  when present, is the same or different and is selected from the group consisting of H, an aliphatic, a heteroaliphatic, a sulfoalkyl, a heteroaliphatic with terminal SO 3 , a benzyl, and a substituted benzyl, where the substituted benzyl comprises at least one carboxy group, at least one sulfonate group, —F, —Cl, —Br, or a combination thereof; 
         each of R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , and R 22  is the same or different and is selected from the group consisting of H and SO 3 ; 
         each of R 2  and R 12  is the same or different and is selected from the group consisting of H 
         X is selected from the group consisting of —OH, —SH, —NH 2 , —NH—NH 2 , —F, —Cl, —Br, I, NHS (hydroxysuccinimidyl/sulfosuccinimidyl), —O-TFP (2,3,5,6-tetrafluorophenoxy), —O-STP (4-sulfo-2,3,5,6-tetrafluorophenoxy), —O-benzotriazole, -benzotriazole, —NR-L-OH, —NR-L-O-phosphoramidite, —NR-L-SH, —NR-L-NH 2 , —NR-L-NH—NH 2 , —NR-L-CO2H, —NR-L-CO—NHS, —NR-L-CO-STP, —NR-L-CO-TFP, —NR-L-CO-benzotriazole, —NR-L-CHO, —NR-L-maleimide, —NH(CH 2 CH 20 ) z CH 2 CH 2 N 3 , —NR-L-NH—CO—CH 2 —I, and an azide (N 3 )-containing group, where R is —H or an aliphatic or heteroaliphatic group, z is an integer from 1 to 5 inclusive, and L is selected from the group consisting of a divalent linear, crossed, or cyclic alkyl group optionally substituted by at least one oxygen atom and/or sulfur atom; 
         Kat is a number of Na + , K + , Ca 2+ , ammonia, or other cation(s) needed to compensate the negative charge brought by the cyanine; 
         m is an integer from 0 to 5 inclusive; n is an integer from 1 to 3 inclusive; o is an integer from 0 to 12 inclusive; and p is an integer from 0 to 5 inclusive; 
         b) a quencher having general Formula II 
       
       
         
           
           
               
               
           
         
       
       wherein
 each of R 23 , R 24 , R 25 , R 26 , R 27 , R 28 , R 29 , and R 30  is the same or different and is independently selected from either H or SO 3 ; 
 Z is OR, where R is H or alkyl, or Z is NH-L, where L is 
 
       
         
           
           
               
               
           
         
       
       and Y is either H or a linkage to a solid support; and
 c) an oligonucleotide linker joining the dye and the quencher.