UserGuideForMPprimer

Introduction

MPprimer is a program developed by CZlab for reliable multiplex PCR primer design. MPprimer employs the widely used primer design program Primer3 [Rozen, et al. 2000] and the primer specificity evaluation program MFEprimer [Qu, et al. 2009] 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. In addition, MPprimer provides a virtual electrophotogram to help users choose the best PSC. In short, MPprimer is a valuable tool for designing specific, no dimer formation and amplicons size constrained PSCs to improve the multiplex PCR experiments. However, as the most things in the world, MPprimer has its advantages and shortcomings. We list them both here to help users understand the advantages and limitations of MPprimer better and help users choose the proper tool for multiplex PCR primer design.

Advantages of MPprimer

1. Reliable primer design by Primer3: Primer3 [Rozen, et al. 2000] has been widely used for primer design for ordinary PCR. The quality of the primer designed by Primer3 is time-tested.
2. Primer specificity evaluation by MFEprimer: MFEprimer [Qu, et al. 2009] is a tool developed by us for evaluation of specificity of PCR primers. Only the specific primers could succeeded in the evaluation by MFEprimer.
3. Dimer checking: Dimer checking is essential in multiplex PCR primer design. To avoid primer dimerization, MPprimer checks each pair of primers in a PSC using a dimer checking program named PriDimerCheck, which is part of the PerlPrimer [Marshall, 2004] source code, with some modifications.
4. Constraint of electrophoretic mobility of the amplicons: In multiplex PCR, the electrophoretic migration distance of the amplicons in 1% agarose gel electrophoresis should be greater than a certain distance so that the DNA bands can be easily distinguished by the naked eye for purification, so we defined MinBS (minimum band spacing) as an essential condition in multiplex PCR primer design. The MinBS has two types of parameters in MPprimer. One is the base pair number and the other is the real distance in millimeter. The distance of the amplicon in agarose gel electrophoresis can be predicted by the formula reported by Helling, et al. 1974.

Disadvantages and limitations of MPprimer

1. Computing limitation of MPprimer web service: We recommended users who are familiar with Linux/Unix command-line tools using MPprimer command-line version for their multiplex PCR primer design. That's because MPprimer utilize the MFEprimer for evaluation of the specificity of primer, which is a very time and space consuming step in MPprimer. MPprimer would not return any result if the MPprimer running too long. In this case, users can reduce the number of template sequences and try again.
2. Not for degenerate primer design: We are very sorry to users who want to design degenerate primers. MPprimer do not support degenerate primer design currently.

Some suggestions

1. To new users: Firstly, MPprimer may have "too many" parameters to choose and may puzzle some users. However, Most of the parameters has the default values and users do not need to change them. Secondly, MPprimer is customized, so it's flexible. Users need to know the meanings of the parameters and test MPprimer several times. Thirdly, the settings of PRODUCT SIZE RANGE is very important in multiplex PCR primer design in MPprimer, that's because the MinBS should be satisfied. In other words, if the user set the proper PRODUCT SIZE RANGE, they usually can design the reliable multiplex PCR primers with MPprimer. Users should be awared that although MPprimer can assign “PRODUCT SIZE RANGE” for each of the template sequences, it still need users’ modification in some cases. Fourthly, we are happy to receive your emails if you have any questions or comments.
2. To advanced users: If you have specific needs and MPprimer can not meet them, please contact us. We will try our best to make MPprimer helpful for everyone.