MPprimer: a program for reliable multiplex PCR primer design

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 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.

Figure 1

Flowchart of MPprimer on primer design for multiplex PCR assay.

Figure 2

Schematic diagram showing the construction of PSC graph with N number of primer sets. PS: Primer set, comprising a forward and a reverse primer; PSs is the plural form of PS. PSC: Primer set combination, a group of PSs; PSCs is the plural form of PSC. O: Represents a PS, different colors represent different PSs which come from different DNA template sequences. --: An edge connecting two PSs indicates that they are compatible to work together in one tube without any interference or competition to produce nonspecific amplicons. N: Represents the number of DNA template sequences in multiplex PCR.

Figure 3

Diagram showing the experimental validation of the multiplex PCR by using the primer set combination (PSC) designed by MPprimer for five mouse genes (β-actin, B2m, Pgk1, GAPDH and Rp113a). The top panel showing the predicted (virtual) electrophoretogram of five conventional PCRs and a multiplex PCR by MPprimer. The middle and bottom panels showing the experimentally validated electrophoretogram. The plus sign indicates the use of the primer set.

Figure 4

Diagram showing the three letters "P", "C" and "R" by experimentally validated electrophoretogram using the primer set combination (PSC) designed by MPprimer for five mouse genes (β-actin, B2m, Pgk1, GAPDH and Rp113a). The three letters "P", "C" and "R" were shown in both a virtual electrophoretogram predicted by MPprimer (top panel) and the experimentally validated electrophoretogram (middle and bottom panels). The plus sign indicates the use of the primer set.