Nucleic acid analysis using non-templated nucleotide addition

Abstract

One embodiment of the invention is a method of producing an oligonucleotide extended by a single nucleotide base. An oligonucleotide and an extension terminating nucleotide are mixed with an enzyme having terminal transferase activity. The reaction produces an oligonucleotide extended by a single base. The extended oligonucleotide may be used as a size standard for single base extension reactions. Another embodiment of the invention is a method of producing a mixture of oligonucleotides extended by different single bases. An oligonucleotide, a first extension terminating nucleotide, and a second extension terminating nucleotide are mixed with an enzyme having terminal transferase activity. The first and second extension terminating nucleotides comprise different nucleotide bases and are labeled with different labels. The identity of the different extension terminating nucleotides (and hence the extended oligonucleotides) may be ascertained by reference to the specific label incorporated.

1 . A method of producing an oligonucleotide extended by a single nucleotide base, said method comprising, 
 mixing an oligonucleotide with an extension terminating nucleotide and a terminal transferase.    
     
     
         2 . The method of  claim 1 , wherein a label is attached to the extension terminating nucleotide.  
     
     
         3 . The method of  2 , wherein the label is a fluorescent dye.  
     
     
         4 . The method of  claim 1 , wherein a label is attached to the oligonucleotide.  
     
     
         5 . The method of  claim 4 , wherein the label is a fluorescent dye.  
     
     
         6 . A method of producing a mixture of oligonucleotides extended by a single nucleotide base, wherein the mixture comprises oligonucleotides extended by different single bases, said method comprising, 
 mixing an oligonucleotide with a first extension terminating nucleotide, a second extension terminating nucleotide, and a terminal transferase, wherein the first and second extension terminating nucleotides comprise different labels and different nucleotide bases.    
     
     
         7 . The method according to  claim 6 , wherein the labels are fluorescent dyes.  
     
     
         8 . A kit for producing oligonucleotides extended by a single nucleotide base, said kit comprising, 
 an extension terminating nucleotide, and an enzyme having terminal transferase activity.    
     
     
         9 . The kit according to  claim 8 , wherein the extension terminating nucleotides is labeled.  
     
     
         10 . The kit according to  claim 9 , wherein the label is a fluorescent label.  
     
     
         11 . The kit according to  claim 8 , said kit comprising a first extension terminating nucleotide and a second extension terminating nucleotide, wherein the first and second extension terminating nucleotides comprise different labels and different nucleotide bases.  
     
     
         12 . The kit according to  claim 11 , said kit comprising a four different extension terminating nucleotides, wherein each of the four extension terminating nucleotides has a different nucleotide base and a different label.  
     
     
         13 . The method of  claim 12 , wherein the labels are fluorescent dyes.  
     
     
         14 . A method of identifying the reaction product of a single nucleotide base extension reaction on a detection instrument, said method comprising, 
 forming a single base oligonucleotide extension product standard by mixing an oligonucleotide with an extension terminating nucleotide and a terminal transferase, and    resolving the single base oligonucleotide extension product standard on a detection instrument, whereby a signal indicative of the single base oligonucleotide extension product standard is created.    
     
     
         15 . The method of  claim 14 , comprising, 
 forming a single base extension reaction product standard by mixing a second oligonucleotide with an extension terminating nucleotide and an enzyme having DNA polymerase activity,    resolving the single base extension product on the detection instrument, whereby a signal indicative of the single base extension product is created,    comparing the signal indicative of the single base oligonucleotide extension product with the signal indicative of the single base extension product.    
     
     
         16 . A method of producing an oligonucleotide extended by a single nucleotide base, said method comprising, 
 mixing an oligonucleotide with an extension terminating nucleotide and an enzyme having terminal transferase activity, wherein the mixture does not contain a template that permits the oligonucleotide to function as a primer.    
     
     
         17 . A method of producing a mixture of oligonucleotides extended by a single nucleotide base, wherein the mixture comprises oligonucleotides extended by different single bases, said method comprising, 
 mixing an oligonucleotide with a first extension terminating nucleotide, a second extension terminating nucleotide, and an enzyme having terminal transferase activity and an enzyme having terminal transferase activity, wherein the first and second extension terminating nucleotides comprise different labels and different nucleotide bases, wherein the mixture does not contain a template that permits the oligonucleotide to function as a primer.    
     
     
         18 . A method of identifying the reaction product of a single nucleotide base extension reaction on a detection instrument, said method comprising, 
 forming a single base oligonucleotide extension product standard by mixing an oligonucleotide with an extension terminating nucleotide and an enzyme having terminal transferase activity, wherein the mixture does not contain a template that permits the oligonucleotide to function as a primer, and    resolving the single base oligonucleotide extension product standard on a detection instrument, whereby a signal indicative of the single base oligonucleotide extension product standard is created.