DETECTION OF PNA CLAMPING

摘要

This invention provides methods and systems for detecting sequence variants in a sample nucleic acid. Methods include PNA clamping/PCR using primers with binding moieties followed by capture and detection of amplicons on a solid support. Systems include PCR reagents with primers having binding moieties, a PNA clamping probe, a solid support capable of capturing the PCR amplicons incorporating the binding moieties, and a detector device.

主权项

1. A method of detecting a variant in a target nucleic acid of interest, the method comprising:
providing a first pair of PCR primer probes which allow formation of a PCR process product comprising a sequence covering a first region of interest in the target nucleic acid, wherein at least one of the primer probes comprises a binding moiety; providing a first PNA clamping probe fully complementary to a clamping target sequence in the nucleic acid target region of interest; admixing the primer probes and clamping probe with a putative target nucleic acid sample; performing a PCR amplification process in a reaction solution under hybridization conditions wherein the clamping probe hybridizes to a perfect complement clamping target sequence, but does not hybridize where there are one or more sequence variants in the clamping target sequence, thereby producing PCR amplicons comprising one or more primer probes only if the clamping target sequence includes one or more a sequence variants; providing one or more capture moieties on a solid support, which capture moieties are adapted to bind the binding moiety; contacting the solid support with the PCR reaction solution, thereby capturing PCR amplicons present in the solution; and, interrogating the solid support to determine the presence or absence of bound amplicons on the solid support;whereby the presence of a variant in the target sequence of interest is confirmed by detection of amplicons on the solid support.