Design of PCR and Sequencing Primers and PCR reactions

• Design of Primers for Automated Sequencing
• PCR Bioinformatics and Web based primer design
• PCR links at Bioinformatics.Net
• Primer Design Workshop

Probe/ primer design software

Primer design webservers for PCR, mutagenesis and RNAi

• AntiSense Design - Design antisense primers at IDT
• AutoPrime - designs primers that are specific for expressed sequences (mRNA).
• BatchPrimer3 - High throughput web application for PCR and sequencing primer design
• CODEHOP - COnsensus-DEgenerate Hybrid Oligonucleotide Primers
• Exonprimer - Design primers for the amplification of exons with intronic primers
• Genefisher - Interactive PCR primer design
• MEDUSA  - A tool for automatic selection and visual assessment of PCR primer pairs (Karolinska)
• Methprimer - Design primers for methylation PCR
• mPrimer3 - modified Primer3
• MutScreener - Design primers for mutation screening (by PCR-direct sequencing)
• NetPrimer - Free primer design service of Premier Biosoft.
• Oligodb - a web-based system for interactive design of oligo DNA for transcription profiling (hybridization) of human genes. The oligodb system uses the human DNA-transcripts of ENSEMBL. Reference [PubMed][pdf]
• Osprey - Oligonucleotide Design Software for Sequencing and Gene Expression
• PCR suite - a collection of programs to search overlapping primers, genomic primers for exon amplification, SNP- and cDNA flanking primers
• PRIDE - The less automated webversion of PRIDE  (a.o. 50-70 mer oligo design)
• Primaclade - a web-based application that accepts a multiple species nucleotide alignment file as input and identifies a set of PCR primers that will bind across the alignment.
• Primer3 - a common used software for designing primers
• Primer3Plus - Use primer3 to pick primers for specific tasks
• PrimerQuest - Primer design at IDT
• Primer Generator - Automated generator of primers for site-directed mutagenesis
• PrimerStation - multiplex human PCR primer design site
• PrimerX - Automated design of primers for site-directed mutagenesis
• Primique - Automatic design of specific PCR primers for each sequence in a family
• Primo Unique - Primo Unique finds multiple primer pairs, each uniquely amplify one gene in a family.
• ProbeWiz Server - The CBS ProbeWiz WWW server predicts optimal PCR primer pairs for generation of probes for cDNA arrays. Reference [PubMed]
• PUNS - Primer-UniGene Selectivity Testing - compares primer sequences against the both the genome and transcriptome to assess the potential for multiple amplicons (Free registration required)
• RNAi Design - Design primers for RNAi at IDT
• ROSO - Software to design optimized oligonucleotide probes (size over 25 nucleotides) for microarrays
• Sirna - Target accessibility prediction and RNA duplex thermodynamics for rational siRNA design
• SNPbox - a modular software package that automates the design of PCR primers for large-scale amplification and sequencing projects in a standardized manner resulting in high quality PCR amplicons with a low failure rate.
• SNP Cutter SNP PCR-RFLP Assay Design. Primer design for restriction analysis of single nucleotide polymorphisms
• Soligo - Target accessibility prediction and rational design of antisense oligonucleotides and nucleic acid probes
• SOP3 - Selection of Oligonucleotide Primers for PCR and Pyrosequencing
• SPADS - Specific Primers & Amplicon Design Software for amplification of individual members of gene families

  PCR Primer design software for local installation

  Freely available

• Amplify - a freeware Macintosh program for simulating and testing polymerase chain reactions (PCRs).
• AmplifX - Software to test, manage and design your primers for Macintosh and Windows.
• Fast PCR - PCR primer design, DNA and protein tools, repeats and own database searches
• MEDUSA  - A tool for automatic selection and visual assessment of PCR primer pairs (Karolinska)
• Methyl Primer Express - free Applied Biosystems software to design high quality PCR primers for methylation mapping experiments.
• MutaPrimer - Designs primers for Stratagene's QuikChange site directed mutagenesis kits.
• OligoPicker - OligoPicker picks specific oligos by skipping regions with contiguous bases common in other sequences. In addition, oligo specificity is double-checked by NCBI BLAST. Sequence regions similar to non-coding RNAs are avoided because total RNA is often used for array hybridization. Low-complexity regions are also filtered out to maintain oligo specificity. Oligos and sequence regions that may form secondary structures are discarded since both the probes and the sequence target sites should be easily accessible for hybridization. Reference [PubMed]
• PRIMEGENS- PRIMEGENS (PRIMEr Design Using GEN Specific Fragments) is a computer program to select gene-specific fragments and then design primer pairs using Primer3 for PCR amplifications. Reference [PubMed]
• PrimerD - The primerD program implements a novel algorithm for the design of unique degenerate primer pairs.
• Primer3 - a common used software for designing primers for microarray construction.
• mPrimer3 - modified Primer3
• ProMide - ProMide is a collection of command-line tools for Probe selection and Microarray Design.
• STACKdb - The STACKdb, Sequence Tag Alignment and Consensus Knowledgebase, is generated by processing EST and mRNA sequences obtained from Genbank through a pipeline consisting of masking, clustering, alignment and variation analysis steps. The STACKdb database is created using tools called "stackPACK".

Not freely available or commercial packages

• AlleleID - For real time PCR based pathogen detection and bacterial identification. TaqMan probe design supported.
• Beacon Designer - Real time PCR primer and probe design for single tube and multiplex PCR assays.
• OligoChecker - An oligo database program which quickly checks which oligos available in a lab can be used on a given template (Shareware).
• PRIDE and GenomePRIDE  - (a.o. 50-70 mer oligo design)
• Visual OMP - multiplex primer and probe design optimized to reduce cross-hybridization between oligos and targets, an integrated folding engine for visualizing target and oligo structures, thermodynamics modeling, and built-in BLAST and ClustalW. Product of  DNA Software, Inc.

Microarray primer design webserver

• MAPHDesigner 1.2 design of primers and probes for genome copy number detection (MAPH/CGH microarrays)
• ROSO - Software to design optimized oligonucleotide probes (size over 25 nucleotides) for microarrays
• MEDIANTE - Freely accessible database of human and mouse RNG/MRC oligonucleotide probes for microarrays

   

Microarray primer design software for local installation

Freely available

• OligoArray2 - a free Java program that computes gene specific oligonucleotides for genome-scale oligonucleotide microarray construction. Reference [PubMed]
• OligoWiz Site - Download the OligoWiz Java client to access the CBS OligoWiz WWW server and predict optimal oligonucleotides for generation of spotted arrays.
• Probepicker - Featurama's Open Source Probepicker 0.7 for custom designed oligonucleotide microarrays
• Primer3 - a common used software for designing primers for microarray construction.

  Not freely available or commercial packages

• Array Designer 2 - Design hundreds of primers for DNA or oligonucleotide microarrays. Product of Premier Biosoft.
• PRIDE and GenomePRIDE  - (a.o. 50-70 mer oligo design)
• Sarani - Sarani Gold (Genome Oligo Designer) is a software for automatic large-scale design of optimal oligonucleotide probes for microarray experiments. Thousands of gene sequences can be analyzed together and best available oligonucleotide probes with uniform thermodynamic properties and minimal similarity to non-specific genes can be selected. Product of Strand Genomics.

In-silico PCR

• Genome tester - tests 1) whether PCR primers have excessive number of binding sites on template sequence and 2) how many PCR products would be amplified from the template DNA and where are they located.
• UCSC in-silico PCR - In-silico PCR on human genomic DNA at UCSC
• In-silico experiments with complete genomes - In-silico experiments (including PCR) on bacterial and lower eukaryotic genomes

Primer property calculators

• AutoDimer Rapid screen of previously selected multiplex PCR primers for primer-dimer and hairpin interactions in short DNA oligomers (< 30 nucleotides)
• MultiPLX 2.0 tool to analyze PCR primer compatibility and automatically finding optimal multiplexing (grouping) solution.
• OligoAnalyzer 3.0 at IDT
• Oligonucleotide Properties Calculator
• OligoTM 1.0 Program for calculation oligo melting temperature and GC content

PCR setup

• Optimase Protocolwriter- Transgenomic
• Optimase MasterMixCalculator- Transgenomic

Peter Taschner

Last modified: October 1, 2008