Multi-template polymerase chain reaction

Abstract

PCR is a formidable and potent technology that serves as an indispensable tool in a wide range of biological disciplines. However, due to the ease of use and often lack of rigorous standards many PCR applications can lead to highly variable, inaccurate, and ultimately meaningless results. Thus, rigorous method validation must precede its broad adoption to any new application. Multi-template samples possess particular features, which make their PCR analysis prone to artifacts and biases: multiple homologous templates present in copy numbers that vary within several orders of magnitude. Such conditions are a breeding ground for chimeras and heteroduplexes. Differences in template amplification efficiencies and template competition for reaction compounds undermine correct preservation of the original template ratio. In addition, the presence of inhibitors aggravates all of the above-mentioned problems. Inhibitors might also have ambivalent effects on the different templates within the same sample. Yet, no standard approaches exist for monitoring inhibitory effects in multitemplate PCR, which is crucial for establishing compatibility between samples.

Scaling up: A guide to high‐throughput genomic approaches for biodiversity analysis

“…Target enrichment/amplification from mixed communities using PCR has been referred to as mixed-template or multitemplate PCR (Kalle, Kubista, & Rensing, 2014). PCR-coupled DNA metabarcoding is sensitive to the initial mixed-template PCR, including PCR cocktail composition, primers and cycling conditions.…”

Enzymological description of multitemplate PCR—Shrinking amplification bias by optimizing the polymerase–template ratio

“…Multitemplate PCR involves single-tube amplification of two or more homologous templates, typically with one primer pair, whereas single-tube amplification of several non-homologous sequences with distinct primer pairs is termed multiplex PCR (Kalle et al, 2014). Our conclusions are relevant for both cases.…”

Evaluation of novel multiplex qPCR assays for diagnosis of pathogens associated with the bovine respiratory disease complex

“…For a total of nine pathogens the Pneumo4B and Pneumo4V multiplex qPCR assays can be run at the same time under the same PCR program, with benefits in costs and processing time for a diagnostic laboratory compared to multiple singleplex qPCR assays run in parallel. However, the efficiency and sensitivity of a multiplex PCR can be compromised due to primer and probe sets interfering, or the competition for the components in the reaction (Kalle et al, 2014;Parker et al, 2015). In this study, the commercially-available multiplex qPCR Pneumo4B and Pneumo4V assays demonstrated that their efficiencies for seven of these target pathogens (93-106% and 91-104%, respectively) were comparable to those of published singleplex qPCR assays (84.2-101.8%) (Kishimoto et al, 2017).…”