Highly multiplex PCR assays by coupling the 5'-flap endonuclease activity of Taq DNA polymerase and molecular beacon reporters.

ABSTRACT: Real-time PCR is the most utilized nucleic acid testing tool in clinical settings. However, the number of targets detectable per reaction are restricted by current modes. Here, we describe a single-step, multiplex approach capable of detecting dozens of targets per reaction in a real-time PCR thermal cycler. The approach, termed MeltArray, utilizes the 5'-flap endonuclease activity of Taq DNA polymerase to cleave a mediator probe into a mediator primer that can bind to a molecular beacon reporter, which allows for the extension of multiple mediator primers to produce a series of fluorescent hybrids of different melting temperatures unique to each target. Using multiple molecular beacon reporters labeled with different fluorophores, the overall number of targets is equal to the numbe

SUBMITTER: Huang Q 

PROVIDER: S-EPMC8892341 | biostudies-literature | 2022 Mar

REPOSITORIES: biostudies-literature