Optimization of crude sample qPCR with KAPA PROBE FORCE

With growing demands for high-throughput gene expression and genotyping analyses, real-time PCR applications must enable greater levels of detection and process efficiency.  DNA isolation presents a considerable obstacle when optimizing and streamlining such workflows.  Silica membrane-based column purification methods have long been employed but impose additional time, cost, and sample loss.  Low-cost, semi-crude extraction methods, such as sodium hydroxide and Proteinase K, reduce workflow time; however, carryover contamination and inhibition may lead to inconsistencies.  KAPA Express Extract, a novel thermostable protease and buffer system, offers a solution for crude PCR, allowing for the extraction of PCR-ready DNA in as little as 15 minutes. 

Wild-type enzymes as such as Taq and Tth are not suitable for high-throughput amplification directly from crude samples.  Through directed evolution and optimization of buffer composition, we have developed a novel, highly processive third-generation DNA polymerase, KAPA PROBE FORCE.  This enzyme is not only capable of crude sample amplification directly from plant, tissue, and blood samples, but also possesses the 5'-3' exonuclease activity required for probe-based quantitative PCR. The unique characteristics of this evolved enzyme result in robust amplification across a wide range of sample types and methods of extraction and purification.  

Here, we provide key parameters of optimization of KAPA PROBE FORCE assays with particular focus on crude sample qPCR.  Using experimental data, a troubleshooting workflow is presented for a range of challenging sample types.