Methods for temperature-mediated nested polymerase chain reaction

Inventors

Nelson, William M. • Armantrout, Kyle • Fecteau, Tracy Calvin

Assignees

Tetracore Inc

Abstract

Embodiments of present disclosure are directed to methods for amplifying nucleic acid, comprising two steps: a first step of preparing a reaction mixture comprising the target nucleic acid and a second step of processing the reaction mixture in a thermocycler. During a first phase of the processing step, the thermocycler may be configured to heat the reaction mixture to a first temperature and cool the reaction mixture to a second temperature repeatedly for a first plurality of cycles. During the first phase, fluorescence probes do not anneal to template strands and do not emit fluorescence signals. During a second phase of the processing step, the thermocycler may heat the reaction mixture to a third temperature and cool the reaction mixture to a fourth temperature repeatedly for a second plurality of cycles. During the second phase, fluorescence probes anneal to the template strands and are degraded by DNA polymerase to emit fluorescence signals for detection and/or quantification of the target nucleic acid. Methods for amplifying nucleic acid in accordance with the disclosure may be employed for nucleic acid amplification and detection in clinical and research settings.

Core Innovation

The invention provides a method for amplifying nucleic acid by preparing a reaction mixture comprising at least one target nucleic acid, a first set of primers, and a second set of primers. The reaction mixture is processed in a thermocycler using a first phase and a second phase with repeated heating and cooling cycles, where the first set of primers has melting temperatures around the second temperature and higher than those of the second set of primers.

During the second phase, the thermocycler processing heats the reaction mixture to a third temperature and cools it to a fourth temperature repeatedly for a second plurality of cycles, and the fourth temperature is gradually decreased over a predetermined number of cycles. The second set of primers is configured to have melting temperatures around the fourth temperature, and this two-phase temperature-mediated nested PCR workflow is described so that probes are prevented from annealing during the first phase and then anneal during the second phase.

The disclosed approach is further associated with real-time or quantitative PCR using cleavage-based probes that generate fluorescence for detection and quantification. Fluorescence is enabled in the second phase when probes anneal and are degraded by Taq polymerase, and the temperature profiles and primer melting temperature relationships are used to support specificity and primer-dimer reduction in the amplification workflow.

Claims Coverage

The independent claim provides a two-phase thermocycling method with a gradually decreased fourth temperature, where two primer sets are configured to have melting temperatures around different annealing temperatures, and the first primer set has higher melting temperatures than the second primer set. Dependent claims further introduce fluorescence probes and specify thermal relationships and numeric constraints on temperatures and probe characteristics.

Across the claim set, the core coverage is the two-phase thermocycling architecture with a second-phase fourth temperature that is gradually decreased, together with primer sets configured to match melting and annealing relationships, including the first primer set having higher melting temperatures than the second. Optional refinements include fluorescence-emitting probes configured by melting temperature, and RNA nucleotides defined as locked nucleic acids (LNA).

Stated Advantages

Documented Applications