Have you ever thought about the precision required to get an accurate quantitative readout from a multiplex PCR assay? Multiplex PCR enables targeted, parallel amplification of multiple targets, and it comes with a significant set of challenges, including optimizing the PCR conditions to achieve precise, quantitative results across all target loci. All primers must function under the same reaction conditions, each primer should anneal to its true target sequence, and non-specific amplification and primer-dimers should be minimal. Even the smallest variations in annealing kinetics can have a substantial impact on primer amplification efficiencies, producing biased PCR product libraries. When the observed frequency of each amplicon is not proportional to the original frequency of the input template, specific target templates can be under-amplified and undetectable.
Bias-free assays are critical for many studies – one such example is quantitative measurement of the frequency of specific immune receptor rearrangements. To achieve unbiased results in immune receptor amplification assays, researchers have invented a method that uses synthetic templates to optimize a multiplex PCR assay for sequencing TCRG (human T-cell receptor gamma locus) rearrangements in T cells (1). With these synthetic templates, they can use the multiplex PCR assay as a quantitative readout. Specifically, the team developed a synthetic analogue of a somatically rearranged immune receptor locus (human TCRG), generating a synthetic repertoire including every possible V/J combination (variable and joining gene segments).
Above: Schematic of the synthetic template described in the study, including a universal primer (UA), a template specific barcode (BC), TCRG V gene (V gene), synthetic template internal markers (IM1) (IM2), TCRG J gene (J gene), reverse universal primer (UB). Templates were designed to be 495 bp and allow for direct sequencing using either (a) universal adaptors without multiplex PCR, or (b) the multiplex PCR primer assays developed for this locus. This illustration by QIAGEN’s in-house designer represents the synthetic template as described by Figure 1 (1).
This synthetic template pool made it possible to exactly quantify the abundance of each synthetic template pre- and post-multiplex PCR, to optimize primer concentrations and to correct residual bias computationally by using experimentally derived normalization factors. Basically, this method shows that bias introduced by multiplex PCR amplification is purely a product of technical constraints which can be overcome by proper development of a multiplex PCR assay. As sequencing of multiplexed libraries moves toward clinical diagnostic applications, particularly in cases where quantitative B- or T-cell cancer clone tracking (haematological oncology) is needed for patient monitoring and treatment decisions, unbiased, quantitative multiplex PCR library preparation should be seen as a benchmark for the future.
Read more here about the multiplex PCR mastermix that the team used, and how QIAGEN addresses the challenges of multiplex PCR. To help you plan and optimize your multiplex PCR, we’ve created a special portal full of facts, insights and suggestions that will help you overcome every challenge. Visit the portal
Tackle the Challenges in qPCR
Remember to join our 2-Part Webinar series and learn how to overcome challenges in real-time PCR. These webinars will cover the following critical steps for efficient and precise gene expression studies using real-time PCR technology:
– Effect of RNA integrity on real-time PCR results – tips on how to achieve true RNA profiling suitable for real-time PCR studies
– Improved methods for cDNA synthesis, optimized for real-time PCR
– Real-time PCR analysis
Part 1 of the webinar series focuses on practical hints and new solutions for successful real-time PCR studies. Part 2 will focus on critical factors for successful multiplex real-time PCR.
The webinar takes place on September 15 and 28
We are looking forward meeting you there!
- Carlson, C.S. et al. (2013) Using synthetic templates to design an unbiased multiplex PCR assay. Nat. Commun. 4, 2680. Link