Ribonucleotide tag nucleic acid detection
AssigneesCentre National de Genotypage, Roche Molecular Systems, Inc.
InventorsDavid H. Gelfand, Ivo Glynne Gut, Keith A. Bauer, Florence Mauger
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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.
Claims10
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.
Dependencies are inferred from the claim text.