Advances in Assay Stewardship: Understanding why the “Difference is in the Design” in PCR Assays

Introduction

Polymerase Chain Reaction (PCR) assays are the backbone of molecular biology, serving as a cornerstone in genetic research and diagnostics. As we dive deeper into the nuances in the design of a PCR assay, we will explore the key components involved in designing a comprehensive PCR assay, shedding light on the intricate process that paves the way for uniquely sensitive and specific assays.

PCR lab sceneUnderstanding the Science behind our kits at PCRAssays.com

The more than 200 PCR kits available at www.PCRassays.com have been designed with the power of DNA Software, the state-of-the-art software in multiplex primer and probe design. At PCRassays, our comprehensive in-silico modeling includes the necessary “playlists” which are composed of the most relevant and important NCBI accessions for all pathogen genomes of interest. Our kits play a pivotal role in simplifying the complex processes of variant coverage, providing wide access to researchers across various disciplines.

PCRAssays and Target Amplification of Relevant Variants

When designing an efficacious PCR assay, the primary goal is to amplify specific DNA targets. This involves careful selection of the target region within the genome of interest. However, which pathogen genomes should be included in the “inclusivity playlist” and which should be excluded against? The team at DNA Software has expertly curated “playlists” or pathogen databases if you will that allow for variant coverage while avoid false positives with the ThermoSleuth algorithm that detects all off-target effects and unintended hybridization.

Further Exploring the Integral Role of Primer Design

Primer design is a critical aspect of PCR assay development that is often overlooked and not given appropriate attention. Upstream of the design itself is the critical exercise of determining which pathogen accessions the primers will cover and which accession numbers to avoid. Remembering that Primers are short, single-stranded DNA sequences that flank the target region and act as the starting point for DNA synthesis, the design step is critical for the desired outcome of accurate detection. PCR kits often provide a generic set of primers, but in designing a custom assay, researchers must meticulously design primers tailored to the specific target sequence to ensure the accuracy and specificity of the amplification process. Not all methods are suitable for such specific design, particularly in multiplex which we further consider.

Optimizing Multiplex Design

PCR kits, including multiplex PCR kits, offer a variety of reaction conditions to suit different experimental needs. However, when designing a custom assay, researchers must optimize reaction conditions such as temperature, annealing times, and cycling parameters. This optimization process ensures that the PCR assay operates with maximum efficiency, yielding reliable and reproducible results. Researchers often take a trial-and-error approach to optimization of a multiplex, however, the fundamental models used to the design of the multiplex upstream ought to be considered. Namely a 2-state model as often used in free software can’t appropriately account for the free energy of the entire system. As our kits at www.pcrassays.com are powered by DNA Software, they have been designed using our proprietary multi-state coupled equilibrium which can measure the free energy of all components and the system as a whole which is unique advantage for well performing multiplexed assays. Multiplexed kits from PCRAssays allow for simultaneous target amplification with an LOD >10 molecules.

Customization with PCRAssays

The ability to customize your assay is a unique capability of our DNA Software solutions, client services team and the more than 200 assays currently offered at www.pcrassays.com. While standard PCR kits offer versatility for a range of applications, our custom PCRAssays provide researchers with the flexibility to essentially have on demand assays tailor made for their targets of interest. This includes the choice of targets and related variants, the organisms we intend to exclude and be certain not to amplify, all while customizing the preferred conditions in multiplex.

Ensuring Specificity and Sensitivity

A well-designed PCR assay must be both specific and sensitive. Specificity ensures that the assay amplifies only the target DNA sequence, while sensitivity ensures detection even in low-abundance samples. Our unique design paradigm enables for both the sensitivity and specificity as our solutions and models use a cloud-based approach to exhaustively interrogate all sequences for coverage while leveraging our ThermoSleuth algorithm to ensure desired amplification of intended targets.

Conclusion

In the world of molecular biology, designing a PCR assay is a meticulous process that requires a deep understanding of the target DNA, primer design principles, and optimization techniques. With over 200 kits from PCRAssays, including more than 50 multiplex PCR kits, we provide value to researchers with the tools needed to simplify and streamline this complex process. As advancements in technology continue to shape the field, the ability to customize PCR assays opens new avenues for research and diagnostics. So, whether unraveling the genetic code of a rare species or diagnosing genetic disorders, the design of your PCRAssay, empowered by DNA Software, remains at the forefront of scientific exploration.