Wubin Qu , Zhiyong Shen and Chenggang Zhang .
MPprimer employs the widely used primer design program Primer3 [Rozen, et al. 2000] and primer specificity evaluation program MFEprimer [Qu, et al. 2009] to design and evaluate the candidate primers, followed with rigorous primer dimer examination to exclude unsuitable primers. A graph expanding algorithm derived from greedy algorithm was used to determine the optimal primer set combinations (PSCs) for multiplex PCR. In addition, MPprimer provides a virtual electrophotogram to help users choose the best PSC. In conclusion, MPprimer is a valuable tool for designing specific, no dimer formation, amplicons size constrained PSCs for multiplex PCR experiments.
To examine the performance of MPprimer, we designed a primer set combination (10 primers) for multiplex PCR analysis of five mouse genes (beta-actin, B2m, Pgk1, Rpl13a and GAPDH). Firstly, input the five mouse genes sequences in the input box. Secondly, specify the product size for each sequences. Here, we specify the 851-1000, 650-720, 400-520, 101-200 and 201-300 for the five mouse genes ( Fig. 1 ). Thirdly, set the "primer size" as following: Min: 20 Opt: 25 Max: 30. Other settings use the default values. At last, click "Pick primers" to submit. Some seconds later, MPprimer will output the results as Fig. 2 and Fig. 3 shows. Fig. 2 shows the summary results of 15 PSCs. We select the fifth PSC for experimental validation. Fig. 3 is the detailed information of PSC 5, including primer sequence, Tm, product size, etc. Fig. 4 is the virtual electrophotogram of the PSC 5. Fig. 5 is the real electrophotogram of our experimental results. Fig. 4 perfectly predict the mobility of amplicons of PSC5 as Fig. 5 showed.
Fig. 1 Specify the product size for each sequence
Fig. 2 PSC 5 was selected for experimental validation
Fig. 3 Primer details of PSC 5 . Select all the primer sets for virtual electrophoresis
Fig. 4 Virtual electrophoresis for five primer sets of PSC 5
Fig. 5 Experimental validation of PSC 5. The electrophotogram was matched perfectly with the virtual electrophotogram. Lane 0 and Lane 9 show the DNA marker of DL2000 (TaKaRa); PCR results of DL2000 size marker, Lane 1 to 5 shows the results of standard PCR with one primer set (not primer set combination) in the PSC list; Lane 6 shows the multiplex PCR results with equal mixture of the five primer set combination. Lane 7 shows the results of multiplex PCR with partial combination (two primer sets) of the five primer sets and Lane 8 shows the results of multiplex PCR with three primer sets combinations. All products had the expected length, and there was no visible unspecific amplicons. The conditions for all of the PCR analyses are the same as: first denaturing at 94 °C for 5 min; 36 cycles for denaturing at 94 °C for 30 s; annealing at 58 °C for 45 s; extension at 72 °C for 50 s; final extension at 72 °C for 5 min.
Designing the multiplex PCR primers for the DMD gene is a good example to evaluate the performance of MPprimer. For the 79 exons of DMD gene, we have successfully used MPprimer to design the multiplex PCR primers for 20×, 20×, 20×, 14×, and 5× plex PCR reactions in five tubes in one reaction. For DMD exon deletion test, users need to utilize high-resolution techniques such as SDS-PAGE or HPLC to separate the amplicons of nearly close size. In this case, the parameter MinBS (for minimal binding space) is not the critical factor anymore for consideration. Therefore, for designing multiplex PCR primers for the exons of DMD gene, we use the MinBS value of “10 bp” to replace the default “2 mm” mobility distance which was usually used in the agarose gel electrophoresis. To simplify the primer design in MPprimer, we have now added the MinBS option in the home page of MPprimer web service . We thank the review for this good suggestion because passing this examination has made the MPprimer service more flexible and useful.
Additionally, the settings of “ PRODUCT SIZE RANGE ” are very important in multiplex PCR primer design in MPprimer, especially for this kind of 20× plex PCR primers design. In other words, if the user set the suitable “ PRODUCT SIZE RANGE ”, they can successfully design reliable multiplex PCR primers with MPprimer. Although MPprimer can automatically assign “ PRODUCT SIZE RANGE ” for each of the template sequences, it still needs users' modification in some cases. We also add this information in the “ User guide ” page of MPprimer web service.
Following is the primer design process of 79 exons of the DMD gene:
Fig. 1 . Preparation of DNA template sequence of exons of the DMD gene for MPprimer design
Fig. 2 Set the parameters for DNA template sequences of exons of the DMD gene
Fig. 3 Specific the parameters “Target region” and “ PRODUCT SIZE RANGE ”
Fig. 4 . MPprimer output for exon templates of DMD gene (1)
Fig. 5 MPprimer output for exon templates of DMD gene (2)
Fig. 6 MPprimer output for exon templates of DMD gene (3)
Fig. 7 MPprimer output for exon templates of DMD gene (4)