DNA Ligation on RNA Template

Abstract

Disclosed are methods and compositions for detection and amplification of nucleic acids, wherein two DNA strands hybridized to an RNA strand are ligated. In one aspect, the disclosed methods include removal of an energy source, such as ATP, upon charging a ligase to form an enzyme-AMP intermediate, and then adding substrate, which results in one complete round of RNA-templated DNA ligation. In another aspect, the ligation reaction is accomplished by use of a mixture of at least two different ligase enzymes. The disclosed methods and compositions for RNA-templated DNA ligation may be particularly useful for detection of RNA sequence variants, for example RNA splice variants, and for quantitative expression analysis.

1 - 20 . (canceled) 
     
     
         21 . A method for detecting a plurality of target nucleic acids from a nucleic acid sample, comprising:
 combining a first probe with an interrogation probe and a nucleic acid sample comprising at least one RNA target nucleic acid,   wherein the first probe and the interrogation probe hybridize to the target nucleic acid such that the 3′ end of the first probe and the 5′ end of the interrogation probe are directly adjacent to one another and ligatable by DNA ligase;   adding an effective amount of ATP to a solution of DNA ligase, thereby charging the DNA ligase to form a DNA ligase-AMP intermediate;   depleting ATP from the solution of DNA ligase-AMP intermediate;   ligating the 3′ end of the first probe to the 5′ end of the interrogation probe using the solution of DNA ligase-AMP intermediate; and   detecting the ligated product.   
     
     
         22 . The method of  claim 21  wherein ATP depletion is accomplished by adding an effective amount of apyrase to catalytically convert ATP in solution to ADP and/or AMP. 
     
     
         23 . The method of  claim 21  wherein ATP depletion is accomplished by adding an effective amount of hexokinase to catalytically convert ATP in solution to ADP and/or AMP. 
     
     
         24 . The method of  claim 21 , wherein the DNA ligase is T4 DNA ligase. 
     
     
         25 . The method of  claim 22 , wherein the effective amount of apyrase is about 10 mU apyrase per μl of ATP depletion mix. 
     
     
         26 . The method of  claim 23  wherein the effective amount of hexokinase is about 0.4 U hexokinase per μl of ATP depletion mix. 
     
     
         27 . The method of  claim 21  wherein the effective amount of ATP is between 200 and 500 mM in a ligase charging mix. 
     
     
         28 . The method of  claim 21  wherein the effective amount of ATP is between 100 and 1000 mM in a ligase charging mix. 
     
     
         29 . The method of  claim 21  wherein the effective amount of ATP is between 0.1 and 200 mM in a ligase charging mix. 
     
     
         30 . The method of  claim 21  wherein the effective amount of ATP is between 10 and 100 mM in a ligase charging mix. 
     
     
         31 . The method of  claim 21  wherein the interrogation probed includes a detectable label. 
     
     
         32 . The method of  claim 21  wherein the step of detecting the ligated product is carried out by staining. 
     
     
         33 . The method of  claim 21  wherein the step of detecting the ligated product is carried out by staining with SYBR green or ethidium bromide. 
     
     
         34 . A method for detecting a plurality of target nucleic acids from a nucleic acid sample, comprising:
 contacting an open circle DNA probe with an RNA nucleic acid sample comprising at least one target nucleic acid, wherein the open circle probe comprises a 5′ end and a 3′ end;   adding an effective amount of ATP to a solution of DNA ligase, thereby charging the DNA ligase to form a DNA ligase-AMP intermediate;   depleting ATP from the solution of DNA ligase-AMP intermediate;   ligating the 3′ end of the open circle probe to the 5′ end of the open circle probe using the solution of DNA ligase-AMP intermediate; and   detecting the ligated product.   
     
     
         35 . The method of  claim 34  wherein ATP depletion is accomplished by adding an effective amount of apyrase to catalytically convert ATP in solution to ADP and/or AMP. 
     
     
         36 . The method of  claim 34  wherein ATP depletion is accomplished by adding an effective amount of hexokinase to catalytically convert ATP in solution to ADP and/or AMP. 
     
     
         37 . The method of  claim 34 , wherein the DNA ligase is T4 DNA ligase. 
     
     
         38 . The method of  claim 35 , wherein the effective amount of apyrase is about 10 mU apyrase per μl of ATP depletion mix. 
     
     
         39 . The method of  claim 36  wherein the effective amount of hexokinase is about 0.4 U hexokinase per μl of ATP depletion mix. 
     
     
         40 . The method of  claim 34  wherein the effective amount of ATP is between 200 and 500 mM in a ligase charging mix. 
     
     
         41 . The method of  claim 34  wherein the effective amount of ATP is between 100 and 1000 mM in a ligase charging mix. 
     
     
         42 . The method of  claim 34  wherein the effective amount of ATP is between 0.1 and 200 mM in a ligase charging mix. 
     
     
         43 . The method of  claim 34  wherein the effective amount of ATP is between 10 and 100 mM in a ligase charging mix. 
     
     
         44 . The method of  claim 34  wherein the interrogation probed includes a detectable label. 
     
     
         45 . The method of  claim 34  wherein the step of detecting the ligated product is carried out by staining. 
     
     
         46 . The method of  claim 34  wherein the step of detecting the ligated product is carried out by staining with SYBR green or ethidium bromide.