Dynamic multiplex PCR: A pioneering approach to rapid, cost-effective, and high-quality microsatellite fragment analysis

This study introduces and validates dynamic multiplex PCR, a newly developed multiplex PCR method designed to overcome the inherent limitations of conventional multiplexing, particularly the challenges associated with heterogeneous primer melting temperatures (Tm) among multiple primer pairs in a single reaction. While traditional protocols rely on a single, fixed annealing temperature (Ta) per cycle, the dynamic multiplex PCR approach applies four sequential annealing temperatures (52°C, 54°C, 57°C, and 60°C) within each individual PCR cycle. This novel intra-cycle multi-temperature strategy was evaluated using an 8-plex microsatellite panel in European sea bass (Dicentrarchus labrax) samples and compared against three established protocols: multiplex touchdown PCR and two conventional PCR setups at fixed temperatures of 54°C and 57°C. The performance of the methods was assessed through a dual-platform quantitative analysis. Amplification yields were first quantified via densitometric analysis of agarose gel electrophoresis, while capillary fragment analysis was utilized to evaluate peak height, total peak area, quality scores, and fragment sizing precision. Sizing accuracy was rigorously determined as the absolute deviation between observed fragment sizes and assigned allele values. Statistical evaluations were conducted using a General Linear Model (GLM) and ANOVA framework to examine the effects of the PCR method, operator, locus, and biological sample on amplification performance and genotyping reliability. The results demonstrate that dynamic multiplex PCR significantly outperforms both conventional and multiplex touchdown PCR protocols by providing more balanced and robust amplification across all eight loci. By accommodating diverse primer requirements within a single cycle, this method eliminates the need for extensive trial-and-error optimization, thereby substantially reducing the consumption of laboratory consumables and total processing time. These findings establish dynamic multiplex PCR as a highly reliable, cost-effective, and reproducible multiplex PCR approach, offering a significant methodological advancement for high-quality multi-locus genotyping and fragment analysis workflows.