DESIGN PCR PRIMERS

BACKGROUND INFORMATION: For sites describing PCR theory, as well as companies marketing PCR products you might want to begin by visiting Horizon Press. For PCR techniques see PCRlink.com.  

There are several excellent sites for designing PCR primers:

 Primer3: WWW primer tool (University of Massachusetts Medical School, U.S.A.) – This site has a very powerful PCR primer design program permitting one considerable control over the nature of the primers, including size of product desired, primer size and Tm range, and presence/absence of a  3’-GC clamp.
GeneFisher - Interactive PCR Primer Design (Universitat Bielefeld, Germany) - a very good site allowing great control over primer design.
 PCR Now ( Computational Biology Group, PathoGene, Southwestern Medical Center, U.S.A. ) - created to design Real-Time Polymerase Chain Reaction (RT-PCR) primers for any number of user-defined coding sequences. Great control over primer properties. If you are interested in designing primers specific to published organismal or viral genes see the related site PathoGene.
 Primer3Plus - a new improved web interface to the popular Primer3 primer design program (Reference: A. Untergasser et al. 2007. Nucl. Acids Res. 35(Web Server issue):W71-W74)

Primer-BLAST was developed at NCBI to help users make primers that are specific to the input PCR template. It uses Primer3 to design PCR primers and then submits them to BLAST search against user-selected database. The blast results are then automatically analyzed to avoid primer pairs that can cause amplification of targets other than the input template.

 

RAPD-primer generator (J.Wöstemeyer, Institute of General Microbiology and Microbial Genetics, Germany)

Oligonucleotide physicochemical parameters

NetPrimer (Premier Biosoft International, U.S.A.) - In my opinion the best site since it provides one with Tm, thermodynamic properties and most stable hairpin & dimers.BUT it takes a while for the program to load.

dnaMATE - calculates a consensus Tm) for short DNA sequence (16-30 nts) using a merged method that is based on three different thermodynamic tables. The consensus Tm value is a robust and accurate estimation of melting temperature for short DNA sequences of practical application in molecular biology. Accuracy benchmarks using all experimental data available indicate that the consensus Tm prediction errors will be within 5 ºC from the experimental value in 89% of the cases. (Reference: A. Panjkovich et al. 2005. Nucl. Acids Res. 33: W570-W572.).

Melting: enthalopy, entropy and melting temperature (N. Le Novere, Pasteur Institute, Paris, France).

 OligoCalc: an online oligonucleotide properties calculator - (Reference: W.A. Kibbe. 2007. Nucl. Acids Res. 35(Web Server issue):W43-W46)
 OligoAnalyzer 3.1 (Integrated DNA Technologies, Inc )
 Mongo Oligo Mass Calculator v2.06
 Oligonucleotide Properties Calculator (University of North Carolina, U.S.A.)
 DNA Calculator (Sigma Genosys)
 Oligo Calculator (Dana-Farber Cancer Institute, U.S.A.)

PCR primers based upon protein sequence:

If you has the protein  sequence and  want the DNA sequence the best sites are Reverse Translate a Protein (Colorado State, U.S.A.), Protein to DNA reverse translation or Reverse Translation part of the Sequence Manipulation Suite .   If you are interested in changing a specific amino acid into another you should consult Reverse Translator (EMBL).  One other site is iCODEHOP (Reference: Boyce, R. et al. 2009.Nucl. Acids Res. 37 (Web Server issue): W222-228). The acronym is from COnsensus-DEgenerate Hybrid Oligonucleotide Primers, and is used to design primers based upon multiple sequence alignments.

PCR primers based upon multialignments:

PriFi - upload a file containing Fasta-formatted DNA sequences or alternatively a *.aln file, select the control one  wants over the primer design from an extensive list and press "Find primers in alignment." (Reference: J. Fredslund et al. 2005. Nuc. Acids Res. 33: W516-W520).

Genomic scale primers: (N.B. also see the JAVA page for additional downloadable programs)

The PCR Suite (Klinische Genetica, Erasmus MC Rotterdam, Netherlands) - this is a suite of four programs based upon Primer3 for genomic primer design.  All offer considerable control on primer properties:

Overlapping_Primers - creates multiple overlapping PCR products in one sequence.
Genomic_Primers - designs primers around exons in genomic sequence. All you need is a GenBank file containing your  gene.
SNP_Primers - designs primers around every SNP in a GenBank file.
cDNA_Primers - designs primers around open reading frames. Simply upload a GenBank file containing your genes.

MuPlex: multi-objective multiplex PCR assay design version 3 - designed for large-scale multiplex PCR assay design in an automated high-throughput environment, where high coverage is required. (Reference: J. Rachlin et al. 2005. Nucl. Acids Res. 33: W544-W547).

Overlapping primer sets:

 Two sites offer  software is based on the Primer3 program for design overlapping PCR primer pair sets - Multiple Primer Design with Primer 3 and Overlapping Primersets 

 GenoFrag - is a software package to design primers optimized for whole genome scanning by long-range PCR. It was developed for the analysis of Staphylococcus aureus genome plasticity by whole genome amplification in ~10 kb-long fragments. Site is in French. (Reference: N. Ben Zakour et al. 2004. Nucl. Acids Res. 32: 17-24).

Short interfering RNA (siRNA) design:

SiRNA Selector - Small interfering RNA (siRNA) guides sequence-specific degradation of the homologous mRNA, thus producing "knock-down" cells. siRNA design tool scans a target gene for candidate siRNA sequences that satisfy user-adjustable rules. The program evaluates siRNA functionality and specificity. (Reference: N. Levenkova et al. (2003) Bioinformatics 2004 20: 430-432). Other similar programs are  siRNA Target Designer  (Promega, U.S.A.); the similarly named  siRNA Target Finder(Ambion, U.S.A.) and siRNA Target Finder (GenScript USA Inc.).

siRNA Design Software - compares existing design tools, including those listed above. They also attempt to improve the MPI principles and existing tools by an algorithm that can filter ineffective siRNAs. The algorithm is based on some new observations on the secondary structure. (Reference: S. M. Yiu et al. (2004) Bioinformatics 21: 144-151).

Realtime PCR primer design:

 RealTimeDesign (Biosearch Technoloogies) - free but requires registration.

 QuantPrime - is a flexible program for reliable primer design for use in larger qPCR experiments. The flexible framework is also open for simple use in other quantification applications, such as hydrolyzation probe design for qPCR and oligonucleotide probe design for quantitative in situ hybridization. (Reference: S. Arvidsson et al. 2008. BMC Bioinformatics  9:465)

Introduction of silent mutations:

WatCut (Michael Palmer, University of Waterloo, Canada) - takes an oligonucleotide and introduces silent mutations in potential restriction sites such that the amino acid sequence of the protein is unaltered. 

When you are ready to set-up your PCR reaction see:

PCR Box Titration Calculator (Allotron Biosensor Corporation) - for figuring out the amounts of each reagent to use in a two-dimensional box titration for PCR.  For standard PCR reactions adjust volume, and change "row" and "column" number to "1", click on all the "top" or "bottom" and "done".

 PCR Reaction Mixture Setup (R. Kalendar, University of Helsinki, Finland) - very nice site (requires Java).

Primer presentation on the DNA sequence:

Sequence Extractor (Paul Stothard) - generates a clickable restriction map and PCR primer map of a DNA sequence (accepted formats are: raw, GenBank, EMBL, and FASTA) offering a great deal of control on output. Protein translations and intron/exon boundaries are also shown. Use Sequence Extractor to build DNA constructs in silico.

 

Updated October 30, 2012