Bioassay Development: Droplet Digital PCR (ddPCR)

Droplet Digital PCR (ddPCR) is a refinement of the well established RTqPCR and qPCR technology for the specific identification and amplification of target gene sequences for quantitation. While qPCR has been the benchmark for evaluation of cDNA and gDNA levels of target genes for decades, it does have some significant liabilities with regards to the potential for artifact when evaluating subtle differences in gene expression (<2-fold). The ddPCR process minimizes this risk by a partitioning of a traditional qPCR reaction into tens of thousands of nano liter droplets, which allows the mathematical determination of the number of target molecules in a given sample based on Poisson Distribution estimation. The increased confidence in ddPCR data has led to applications in Absolute Quantitation without standard, copy number variation (CNV) as well as rare mutation and sequence detection. Additionally, ddPCR is quickly becoming the go-to technology for evaluation of Vector Genome copy number in production of Adeno Associated Virus (AAV) and Lentivirus vectors and therapeutics and nucleic acid residuals in R&D/cGxP manufacturing workflows.

Highlights:

• BioRad QX200 AutoDG System with Automated Plate Preparation
• Custom reagent design, testing and optimization as well as Commercially Available probe/primer assays
• Process Residual Assays, AAV and Lentiviral genome titers in both process samples for AAV/Lenti production as well as tissue/biofluid distribution assessment for potency assays.
• Sample preparation from Tissues, Cells, Biofluids and FFPE with QC.

References and Links:

Minimum Information for the Publication of Quantitative Real-Time PCR Experiments (MIQE Guidelines): Clin Chem. 2009 Apr;55(4):611-22. doi: 10.1373/clinchem.2008.112797. Epub 2009 Feb 26.