February 16, 2021
MPprimer: a program for reliable multiplex PCR primer design
BACKGROUND: Multiplex PCR, defined as the simultaneous amplification of multiple regions of a DNA template or multiple DNA templates using more than one primer set (comprising a forward primer and a reverse primer) in one tube, has been widely used in diagnostic applications of clinical and environmental microbiology studies. However, primer design for multiplex PCR is still a challenging problem and several factors need to be considered. These problems include mis-priming due to nonspecific binding to non-target DNA templates, primer dimerization, and the inability to separate and purify DNA amplicons with similar electrophoretic mobility.
RESULTS: A program named MPprimer was developed to help users for reliable multiplex PCR primer design. It employs the widely used primer design program Primer3 and the primer specificity evaluation program MFEprimer to design and evaluate the candidate primers based on genomic or transcript DNA database, followed by careful examination to avoid primer dimerization. The graph-expanding algorithm derived from the greedy algorithm was used to determine the optimal primer set combinations (PSCs) for multiplex PCR assay. In addition, MPprimer provides a virtual electrophotogram to help users choose the best PSC. The experimental validation from 2x to 5x plex PCR demonstrates the reliability of MPprimer. As another example, MPprimer is able to design the multiplex PCR primers for DMD (dystrophin gene which caused Duchenne Muscular Dystrophy), which has 79 exons, for 20x, 20x, 20x, 14x, and 5x plex PCR reactions in five tubes to detect underlying exon deletions.
CONCLUSIONS: MPprimer is a valuable tool for designing specific, non-dimerizing primer set combinations with constrained amplicons size for multiplex PCR assays.
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Multiplex PCR primer design is complicated. In addition to the design software, a lot of configuations also required, such as SNP database for primer binding site prediction. So, for end users, a simple one-step program can’t design proper primers for their applications. Therefore, I try to develop application-specific one-stop service server for end users.
Currently, I released a server for multiplex PCR primer design for target enrichment following next-generation sequencing (NGS), please test here: https://mfeprimer3.igenetech.com/muld.
Other applications, for example, methlation, pathogen, ARMS, I also have well tested programs on local server. Before I released to public, I can provide service for non-profit users, please email me.