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MPprimer: a program for reliable multiplex PCR primer design

Zhiyong Shen1, Wubin Qu1, Wen Wang1, Yiming Lu1, Yonghong Wu1, Zhifeng Li1, Xingyi Hang1, Xiaolei Wang2, Dongsheng Zhao2 and Chenggang Zhang1*

Author Affiliations

1 Beijing Institute of Radiation Medicine, State Key Laboratory of Proteomics, Beijing 100850, PR China

2 Beijing Institute of Health Administration and Medical Information, Beijing 100850, PR China

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BMC Bioinformatics 2010, 11:143  doi:10.1186/1471-2105-11-143

Published: 18 March 2010

Abstract

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 2× to 5× 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 20×, 20×, 20×, 14×, and 5× 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.