Nucleotide analogs for efficient DNA and RNA amplification

Inventors

Benner, Steven A • Yang, Zunyi • Kim, Myong Jung

Assignees

FIREBIRD DIAGNOTICS • FOUNDATION FOR APPLIED MOLECULAR EVOLUTION, INC.

Abstract

This invention covers processes that amplify multiple DNA and RNA targets, solving the “multiplexed PCR problem”, as well as supporting isothermal amplification and delivering other capabilities in multiplexed molecular analysis. It uses inventive DNA analogs.

Core Innovation

The invention provides a process for extending an oligonucleotide analog primer by template-directed polymerization. A template oligonucleotide having a portion substantially complementary to the oligonucleotide analog primer is contacted in aqueous solution with a DNA polymerase, an RNA polymerase, and/or a reverse transcriptase, and nucleoside triphosphates. The resulting mixture is incubated for a preselected time and a preselected temperature, and one or more nucleotide units in the oligonucleotide analog primer have, instead of a standard nucleobase, a substituted heterocycle structure integrated into the primer.

The substituted heterocycle is defined by an alkynyl or alkenyl linker, where Z attached to the alkynyl or alkenyl linker is H, CH3, an alkyl group, or an alkyl group carrying a tag, and R is the point of attachment to the remainder of the oligonucleotide analog primer. The document describes MODURA self-avoiding nucleotide analogs and emphasizes improved behavior in multiplexed DNA/RNA amplification by using these analogs as primer units. The improved pairing stabilizes target duplexes while preventing primer-primer interactions.

The described system is used for multiplexed DNA/RNA amplification, including PCR and isothermal amplification approaches, with examples including RT-PCR and recombinase polymerase amplification (RPA). The report indicates that the selected MODURA base analogs delay primer dimer formation and reduce off-target amplification while maintaining amplification performance. Documented characterization includes melting curve behavior, including sharp melting peaks for on-target performance, and reduced off-target Ct shifts in multiplex contexts.

Claims Coverage

The independent claim covers template-directed polymerization extension of an oligonucleotide analog primer containing substituted heterocycles (MODURA/self-avoiding units) under aqueous conditions with polymerase activity and nucleoside triphosphates. The claim includes several inventive refinements across dependent claims focused on heterocycle substituent selection, linker selection, and inclusion of additional analog primers containing selected heterocycles.

Across the claim set, the central coverage is extension of an oligonucleotide analog primer by template-directed polymerization in aqueous solution with polymerase activity, where the primer incorporates a substituted heterocycle defined via an alkynyl or alkenyl linker and a selectable Z substituent. Dependent claim refinements specify particular Z values and the use of particular heterocycles, including N-methylcytosine, N-ethylcytosine, 2-aminopurine, 2,6-diaminopurine, and hypoxanthine, either within the primer and/or in an additional analog contained in the mixture.

Stated Advantages

Documented Applications