Ribonucleotide tag nucleic acid detection

Abstract

The present application provides polynucleotides comprising 5′-tails with sequence segments useful for the detection of target nucleic acid sequences, and methods for their use in detecting target nucleic acids. The polynucleotides are used to amplify a subsequence of a target nucleic acid in the presence of one or more ribonucleotides. The ribonucleotides are incorporated into amplification products at regular intervals complementary to the 5′-tail sequence segments. Cleavage of amplification products at the bond immediately 3′ to incorporated ribonucleotides produces detectably distinct fragments indicative of the presence or absence of a target nucleic acid.

What is claimed is: 
     
         1 . A reaction mixture comprising:
 a) a primer polynucleotide comprising a 5′ portion and a 3′ portion, the 5′ portion comprising two or more contiguous sequence segments, wherein each sequence segment comprises at least three nucleotide bases, and the 5′-end nucleotide base of each sequence segment is unique within one sequence segment and is the same in each sequence segment;   b) a nucleotide set comprising four different types of nucleotide bases, wherein three nucleotide bases are only in the form of deoxyribonucleotides (dNTPs), and the majority of bases of a fourth type is in the form of a ribonucleotide (rNTP), wherein the base of the ribonucleotide is complementary to the unique 5′ nucleotide base of each sequence segment of the primer polynucleotide; and   c) a polymerase comprising deoxyribonucleotide and ribonucleotide incorporating activities.   
     
     
         2 . The reaction mixture of  claim 1 , wherein each 5′ portion sequence segment is of equal mass. 
     
     
         3 . The reaction mixture of  claim 1 , wherein the 3′-end comprises a removable blocking moiety that substantially prevents extension of the polynucleotide unless removed by an exonuclease activity. 
     
     
         4 . The reaction mixture of  claim 3 , wherein the 3′-end of the polynucleotide comprises a 2′-terminator moiety. 
     
     
         5 . The reaction mixture of  claim 1 , wherein each sequence segment in the polynucleotide has the same nucleotide sequence. 
     
     
         6 . The reaction mixture of  claim 1 , wherein the polymerase comprises a single catalytic domain that comprises deoxyribonucleotide and ribonucleotide incorporating activities. 
     
     
         7 . The reaction mixture of  claim 1 , wherein the polymerase comprises first and second catalytic domains, wherein the first catalytic domain comprises deoxyribonucleotide incorporating activity and the second catalytic domain comprises ribonucleotide incorporating activity. 
     
     
         8 . The reaction mixture of  claim 1 , wherein at least 80% of fourth type of nucleotide base is in the form of a ribonucleotide (rNTP). 
     
     
         9 . The reaction mixture of  claim 1 , further comprising at least one target nucleic acid sequence. 
     
     
         10 . The reaction mixture of  claim 9 , further comprising a target nucleic acid amplicon generated with the primer polynucleotide, the amplicon comprising two or more contiguous amplicon segments complementary to the two or more contiguous sequence segments in the 5′ portion of the primer polynucleotide and comprising a ribonucleotide monophosphate (rNMP) incorporated as the 3′-end nucleotide base of each amplicon segment.