Materials and methods for detection of nucleic acids

Abstract

Assays using non-natural bases are described. In one embodiment, the method involves contacting a sample suspected of containing the target nucleic acid with a polymerase and first and second primers; amplifying the target nucleic acid by PCR using the first and second primers to generate an amplification product having a double-stranded region and a single-stranded region that comprises the non-natural base; contacting the sample with a reporter comprising a label and a non-natural base that is complementary to the non-natural base of the single-stranded region; annealing at least a portion of the reporter to the single-stranded region of the amplification product; and correlating a signal of the label with the presence of the target nucleic acid in the sample. The invention also provides corresponding kits for use in detecting target nucleic acids in a sample.

1 . A kit for detecting a target nucleic acid comprising:
 a) a first oligonucleotide primer comprising a sequence complementary to a first portion of the target nucleic acid;   b) a second oligonucleotide primer comprising a first region and a second region, the first region comprising a sequence complementary to a second portion of the target nucleic acid and the second region comprising a non-natural base; and   c) a reporter comprising a label and a non-natural base that is complementary to the non-natural base of the second region.   
     
     
         2 . The kit of  claim 1 , wherein the reporter comprises an oligonucleotide comprising the non-natural base. 
     
     
         3 . The kit of  claim 1 , wherein the reporter does not include any base other than the non-natural base. 
     
     
         4 . The kit of  claim 1 , wherein the second region of the second oligonucleotide primer further comprises a label and the labels of the reporter and the second region of the second oligonucleotide primer comprise a pair of fluorophores where the emission of one of the fluorophores stimulates the emission of the other fluorophore. 
     
     
         5 . The kit of  claim 1 , wherein the second region of the second oligonucleotide primer further comprises a label and the labels of the reporter and the second region of the second oligonucleotide primer comprise a signal generating element and a signal quenching element. 
     
     
         6 . A kit for amplifying a target nucleic acid comprising:
 a) a thermostable polymerase;   b) a pair of primers, wherein at least one primer of the pair comprises at least one non-natural base; and   c) a reporter comprising at least one nucleotide and at least one non-natural base that base-pairs with the non-natural base of the at least one primer.   
     
     
         7 . The kit of  claim 6 , wherein the at least one primer further comprises a label. 
     
     
         8 . The kit of  claim 6 , wherein the reporter further comprises a label. 
     
     
         9 . A method for detecting a target nucleic acid in a sample comprising:
 a) amplifying the target nucleic acid using a reaction mixture comprising:
 i) a pair of primers, wherein at least one primer of the pair comprises at least one non-natural base; and 
 ii) a nucleotide or oligonucleotide that comprises a non-natural base that base-pairs with the non-natural base of the at least one primer; and 
   b) detecting the target nucleic acid.   
     
     
         10 . The method of  claim 9 , wherein the target nucleic acid is detected during amplification. 
     
     
         11 . The method of  claim 9 , wherein the non-natural base is selected from the group consisting of iso-cytosine and iso-guanine. 
     
     
         12 . The method of  claim 9 , wherein the at least one primer further comprises a label. 
     
     
         13 . The method of  claim 12 , wherein the label is a fluorophore. 
     
     
         14 . The method of  claim 9 , wherein the nucleotide or oligonucleotide further comprises a label. 
     
     
         15 . The method of  claim 14 , wherein the label is a fluorophore. 
     
     
         16 . The method of  claim 14 , wherein the label is a quencher. 
     
     
         17 . The method of  claim 9 , wherein the at least one primer further comprises a first fluorophore and the nucleotide or oligonucleotide further comprises a second fluorophore, wherein the first fluorophore and second fluorophore exhibit fluorescence resonance energy transfer. 
     
     
         18 . The method of  claim 9 , wherein the at least one primer further comprises a fluorophore and the nucleotide or oligonucleotide further comprises a quencher that quenches the fluorophore. 
     
     
         19 . The method of  claim 9 , wherein the target nucleic acid is detected quantitatively. 
     
     
         20 . The method of  claim 9 , wherein the target nucleic acid comprises RNA and the method further comprises reverse transcribing the RNA.