Design and Validation of Precision dPCR assays

Design and Validation of Precision dPCR assays

Abstract
Digital PCR has become the method of choice for many applications that was previously performed by qPCR. This involves for example absolute quantification, copy number variations and rare sequence variant detection. Taking primers and probes designed for qPCR and apply them in dPCR may work in some cases and in certain applications, but for more challenging applications, like the analysis of rare mutations, usually new designs are required. In some cases, using new design strategies such as the Two-tailed PCR may lead to improvements. Differences in design strategies, especially for rare mutations, will be discussed as well as validation parameter to evaluate new designs. Also, the specific dPCR instrument and its mastermix with unique composition may affect the optimal assay and run conditions. At TATAA Biocenter we have the Digital LightCycler (Roche), the QX200 (Bio-Rad), The Qiaquity (Qiagen), the AbsoluteQ (Thermo Fisher Scientific), and the Naica (Stilla Technologies). Aspects of the different instruments will be discussed.