dPCR LNA Mutation Assay (200)

QIAGEN offers dPCR LNA Mutation Assays for the detection of individual sequence mutations selected from comprehensive curated databases such as COSMIC. These are duplex, hydrolysis probe-based assays that use LNA-enhanced primers and probes to increase specificity and sensitivity. One probe detects the mutant allele, and the other detects the wild-type allele, making digital PCR the most sensitive and reliable method for identifying a mutant DNA sequence in a wild-type background.

The assays are supplied in tube format as a ready-to-use, 30×-concentrated mix consisting of two primers and two probes for mutant and wild-type sequences, and are intended for use with the QIAcuity MasterMix. The sensitivity for detecting the target mutation is 0.1% in a wild-type background in a single well of a QIAcuity Nanoplate, and an even higher sensitivity can be achieved by splitting the reaction across multiple wells and combining the analysis. During product configuration, customers can choose between two fluorescence detection options — FAM + HEX or Atto 550 + ROX — for detecting mutant and wild-type targets, enabling multiplex analysis of two mutation targets in one well. The assay designs have been dPCR wet-lab tested, with the resulting data available.

To set up the dPCR experiment, an aliquot of genomic DNA isolated from fresh, frozen or fixed samples is added to the QIAcuity MasterMix together with the dPCR LNA Mutation Assay. Each reaction is analyzed in one or multiple wells of a QIAcuity Nanoplate, and after loading and sealing the nanoplate, the recommended cycling program is selected on the QIAcuity instrument. The QIAcuity Software Suite provides results as copies per microliter in the absolute quantification analysis and as calculated fractional abundance of the mutant target in the mutation analysis type. These assays are highly suited for the rapid and accurate identification of individual specific sequence mutations present in DNA from fresh, frozen or fixed samples.