Nucleic acid probes and methods to detect and/or quantify nucleic acid analytes

Abstract

The invention comprises novel methods and strategies to detect and/or quantify nucleic acid analytes. The methods involve nucleic acid probes with covalently conjugated dyes, which are attached either at adjacent nucleotides or at the same nucleotide of the probe and novel linker molecules to attach the dyes to the probes. The nucleic acid probes generate a fluorescent signal upon hybridization to complementary nucleic acids based on the interaction of one of the attached dyes, which is either an intercalator or a DNA groove binder, with the formed double stranded DNA. The methods can be applied to a variety of applications including homogeneous assays, real-time PCR monitoring, transcription assays, expression analysis on nucleic acid microarrays and other microarray applications such as genotyping (SNP analysis). The methods further include pH-sensitive nucleic acid probes that provide switchable fluorescence signals that are triggered by a change in the pH of the medium.

1 . A nucleic acid probe, wherein said nucleic acid probe is comprised of a nucleic acid that is derivatized with two or more non-identical covalently attached dyes, wherein at least one dye is fluorescent, and wherein at least one dye has a high affinity to double stranded nucleic acids, wherein the dyes are attached at either the same or at adjacent nucleotides of the nucleic acid probe.  
     
     
         2 . The nucleic acid probe of  claim 1  wherein the dye that has a high affinity to double stranded nucleic acids is a fluorescent intercalator.  
     
     
         3 . The nucleic acid probe of  claim 1  wherein the dye that has a high affinity to double stranded nucleic acids is a fluorescent groove binder.  
     
     
         4 . The nucleic acid probe of  claim 1  wherein the nucleic acid probe comprises a fluorescent intercalator and a non-fluorescent quencher.  
     
     
         5 . The nucleic acid probe of  claim 1  wherein the nucleic acid probe comprises a fluorescent intercalator and a second fluorescent dye wherein the second fluorescent dye functions as the donor of a FRET system formed between the intercalator and the second dye.  
     
     
         6 . The nucleic acid probe of  claim 1  wherein the nucleic acid probe comprises a fluorescent intercalator and a second fluorescent dye wherein the second fluorescent dye functions as the acceptor of a FRET system formed between the intercalator and the second dye.  
     
     
         7 . The nucleic acid probe of  claim 1  wherein the nucleic acid probe comprises a fluorescent intercalator and two dyes that form an excimer pair.  
     
     
         8 . The nucleic acid probe of  claim 1  wherein the nucleic acid probe comprises a fluorescent intercalator and two dyes that form an exciplex pair.  
     
     
         9 . The nucleic acid probe of  claim 1  wherein the nucleic acid probe comprises a fluorescent groove bonder and a non-fluorescent quencher.  
     
     
         10 . The nucleic acid probe of  claim 1  wherein the nucleic acid probe comprises a fluorescent groove binder and a second fluorescent dye wherein the second fluorescent dye functions as the donor of a FRET system formed between the intercalator and the second dye.  
     
     
         11 . The nucleic acid probe of  claim 1  wherein the nucleic acid probe comprises a fluorescent groove binder and a second fluorescent dye wherein the second fluorescent dye functions as the acceptor of a FRET system formed between the intercalator and the second dye.  
     
     
         12 . The nucleic acid probe of  claim 1  wherein the nucleic acid probe comprises a fluorescent groove binder and two dyes that form an excimer pair.  
     
     
         13 . The nucleic acid probe of  claim 1  wherein the nucleic acid probe comprises a fluorescent groove binder and two dyes that form an exciplex pair.  
     
     
         14 . The nucleic acid probe of  claim 2  wherein the fluorescent intercalator is selected from thiazole orange or ethidium.  
     
     
         15 . The nucleic acid probe of  claim 3  wherein the fluorescent groove binder is selected from thiazole orange or ethidium.  
     
     
         16 . The nucleic acid probe of  claim 4  wherein the fluorescent intercalator is selected from thiazole orange or ethidium.  
     
     
         17 . The nucleic acid probe of  claim 5  wherein the fluorescent intercalator is selected from thiazole orange or ethidium.  
     
     
         18 . The nucleic acid probe of  claim 6  wherein the fluorescent intercalator is selected from thiazole orange or ethidium.  
     
     
         19 . The nucleic acid probe of  claim 7  wherein the fluorescent intercalator is selected from thiazole orange or ethidium.  
     
     
         20 . The nucleic acid probe of  claim 8  wherein the fluorescent intercalator is selected from thiazole orange or ethidium.