MPprimer: a program for reliable multiplex PCR primer design

MPprimer

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A program for reliable multiplex PCR primer design

News: MPprimer-1.4 command-line version available. [Download] [Brief Introduction]

MPprimer: a program 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.

Citation: Zhiyong Shen*, Wubin Qu*, Wen Wang, Yiming Lu, Yonghong Wu, Zhifeng Li, Xingyi Hang, Xiaolei Wang, Dongsheng Zhao, Chenggang Zhang. MPprimer: a program for reliable multiplex PCR primer design. BMC Bioinformatics, 2010, 11:143.[Abstract] [PDF]

Input DNA templates for primer sets design (Compulsory).

Paste source sequence(s) as DNA templates for primer sets design for PCR in FASTA format below (5'->3', string of ACGTNacgtn -- other letters treated as N -- numbers and blanks ignored).
MPprimer also support for conventional PCR primer design by pasting one sequence.

Example query sequences

Input the targets region and choose the prefered product size ranges for each of the template DNA sequences (Optional).

Targets region: If one or more Targets region is specified then a legal primer pair must flank at least one of them. Detailed information please visit here.
Product size ranges: A list of optimal product size ranges for each of the template DNA sequence to choose from, for example: 50-100 101-200 201-300 301-400 401-500 501-600 601-700 701-1000. Visit here for detailed information. Alternatively, users can choose their prefered Product Size Ranges for MPprimer to replace the default settings.

Targets region: If one or more Targets region is specified then a legal primer pair must flank at least one of them. A Target might be a simple sequence repeat site (for example a CA repeat) or a single-base-pair polymorphism. The value should be a space-seprated list of

start-stop

pairs where start is the index of the first base of a Target, and stop is the index of the last base of a Target.

Product size ranges: A list of optimal product size ranges for each of the template DNA sequence to choose from, for example: 50-100 101-200 201-300 301-400 401-500 501-600 601-700 701-1000. Visit here for detailed information. Alternatively, users can choose their prefered Product Size Ranges for MPprimer to replace the default settings.

An example:	Target region:	Product Size Ranges:
Leave the following blank, MPprimer will automatically choose the proper "Target region" and "Product Size Range" for each of the template DNA sequence.
Template 1:	Target region:	Product Size Ranges:
Template 2:	Target region:	Product Size Ranges:
Template 3:	Target region:	Product Size Ranges:
Template 4:	Target region:	Product Size Ranges:
Template 5:	Target region:	Product Size Ranges:
Template 6:	Target region:	Product Size Ranges:

Primer specificity check settings (Optional).

The database is used to check the specificity of the PCR primer as described in MFEprimer paper(Qu et al., 2009, Bioinformatics, 25(2), 276-278). To be noticed that, for the Multiplex PCR reaction, the database here is not only used to check the specificity of the pair of primers (forward and reverse primer), but also to check the cross-reaction among primers, for example, to check the primer A (from template A) and primer B (from template B) whether has the ability to initial a PCR reaction. Although this is optional, it's strongly suggested that users should choose a database before picking the primers.

Select database:	MinBS	Word size for BLASTN	E-value for BLASTN

E-value is a parameter used in NCBI BLAST (Altschul SF et al. J Mol Biol. 1990 Oct 5;215(3):403-10). MFEprimer uses NCBI BLAST to check the sequence similarity between primers and the DNA templates. By using a larger E-value, BLAST would report more hits. As a result, MFEprimer would be more sensitive. CAUTION: MPprimer would collapse if the E-value is too large. The default value is recommended.

Word size is a parameter used in NCBI BLAST (Altschul SF et al. J Mol Biol. 1990 Oct 5;215(3):403-10). MFEprimer uses BLAST to check the sequence similarity between primers and the DNA templates. With smaller word size, BLAST would report more hits. As a result, MFEprimer would be more sensitive. CAUTION: MPprimer would collapse if the Word size is too small. The default value is recommended.

MinBS (Minimum Band Spacing): defined as the minimum migration distance of the amplicons in 1% agarose gel electrophoresis and should be greater than a certain distance so that the DNA bands can be easily distinguished by human naked eyes.

MinBS = min | position_i - position_j | (i != j) (1)

Where position_i and position_j represents the mobility of the two amplicons (i and j) amplified by the PSC. The formula used to predict the mobility of amplicons (In formula 1) was reported by Helling et al., 1974.

There are two different ways to set the MinBS:

One is the base pair number, i.e., "10 bp". In this case, formula (1) is not used anymore;
The other is the actual migration distance, i.e., "2 mm", as the formula (1) defined.

We recommended the users use the default setting for the MinBS option. However, for some specific usage, users can set the base pair number for the MinBS option. For example, use PAGE to instead the agarose gel when designing 10-plex or more than 10-plex PCR primers.

Primer pick settings (Optional).

These are the general primer picking conditions for each DNA sequence to generate candidate primers.

Primer Tm:	Min:	Opt:	Max:	Max 3' stability
Primer Size:	Min:	Opt:	Max:	Max self complementarity:
Primer GC%:	Min:	Opt:	Max:	Max 3' self complementarity:
Concentration of monovalent cations (usually KCl, mM)				Concentration of divalent cations (usually MgCl₂, mM)
Concentration of dNTPs (mM)				Annealing oligo concentration (nM)

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