Primer And Probe Design Tool

BACKGROUND INFORMATION: For sites describing PCR theory, as well as companies marketing PCR products you might want to begin by visiting Highveld. 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.
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)
BiSearch Primer Design and Search Tool - this is a useful tool for primer-design for any DNA template and especially for bisulfite-treated genomes. The ePCR tool provides fast detection of mispriming sites and alternative PCR products in cDNA libraries and native or bisulfite-treated genomes. (Reference: Arányi T et al. 2006. BMC Bioinformatics 7: 431).

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.

MFEprimer allows users to check primer specificity against genomic DNA and messenger RNA/complementary DNA sequence databases quickly and easily. This server uses a k-mer index algorithm to accelerate the search process for primer binding sites and uses thermodynamics to evaluate binding stability between each primer and its DNA template. Several important characteristics, such as the sequence, melting temperature and size of each amplicon, either specific or non-specific, are reported. (Reference: Qu W et al. 2012. Nucl. Acids Res. 40 (Web Server issue): W205-W208)

Primer Design and Search Tool

PrimerDesign-M - includes several options for multiple-primer design, allowing researchers to efficiently design walking primers that cover long DNA targets, such as entire HIV-1 genomes, and that optimizes primers simultaneously informed by genetic diversity in multiple alignments and experimental design constraints given by the user. PrimerDesign-M can also design primers that include DNA barcodes and minimize primer dimerization. PrimerDesign-M finds optimal primers for highly variable DNA targets and facilitates design flexibility by suggesting alternative designs to adapt to experimental conditions. (Reference: Yoon H & Leitner T. 2015. Bioinformatics 31:1472-1474).

RF-cloning (Restriction-free cloning) - is a PCR-based technology that expands on the QuikChange™ mutagenesis process originally popularized by Stratagene in the mid-1990s, and allows the insertion of essentially any sequence into any plasmid at any location. (Reference: Bond SR & Naus CC. 2012. Nucl. Acids Res 40(Web Server issue): W209-W213)

primers4clades - is a pipeline for the design of PCR primers for cross-species amplification of novel sequences from metagenomic DNA or from uncharacterized organisms belonging to user-specified phylogenetic lineages. It implements an extended CODEHOP strategy based on both DNA and protein multiple alignments of coding genes and evaluates thermodynamic properties of the oligonucleotide pairs, as well as the phylogenetic information content of predicted amplicons,computed from the branch support values of maximum likelihood phylogenies. Trees displayed on screen make it easy to target primers to interactively selected clades. (Reference: Contreras-Moreira B et al. 2009. Nucleic Acids Res. 37(Web Server issue):W95-W100).

TaxMan: Inspect your rRNA amplicons and taxa assignments - In microbiome analyses, often rRNA gene databases are used to assign taxonomic names to sequence reads. The TaxMan server facilitates the analysis of the taxonomic distribution of your reads in two ways. First, you can check what taxonomic names are assigned to the sequences produced by your primers and what taxa you will lose. Second, the produced amplicon sequences with lineages in the FASTA header can be downloaded. This can result in a much more efficient analysis with respect to run time and memory usage, since the amplicon sequences are considerably shorter than the full length rRNA gene sequences. In addition, you can download a lineage file that includes the counts of all taxa for your primers and for the used reference. (Reference: Brandt, B.W. et al. 2012. Nucleic Acids Research 40:W82-W87).

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. ).

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
OligoEvaluator (Sigma -Aldrich)
Oligo Calculation Tool(Genescript, U.S.A.) - allows modification

PCR primers based upon protein sequence:

If you has the protein sequence and want the DNA sequence the best sites are 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 Primaclade (Reference: Gadberry MD et al. 2005. Bioinformatics 21:1263-1264).

PCR and cloning:

AMUSER (Automated DNA Modifications with USER cloning) offers quick and easy design of PCR primers optimized for various USER cloning based DNA engineering. USER cloning is a fast and versatile method for engineering of plasmid DNA. This Web server tool automates the design of optimal PCR primers for several distinct USER cloning-based applications. It facilitates DNA assembly and introduction of virtually any type of site-directed mutagenesis by designing optimal PCR primers for the desired genetic changes. (Reference: Genee HJ et al. 2015. ACS Synth Biol. 4:342-349).

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.

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

PHUSER (Primer Help for USER ) - Uracil-Specific Exision Reagent (USER) fusion is a recently developed technique that allows for assembly of multiple DNA fragments in a few simple steps. PHUSER offers quick and easy design of PCR optimized primers ensuring directionally correct fusion of fragments into a plasmid containing a customizable USER cassette. The primers have similar annealing temperature (Tm). PHUSER also avoids identical overhangs, thereby ensuring correct order of assembly of DNA fragments. All possible primers are individually analysed in terms of GC content, presence of GC clamp at 3'-end, the risk of primer dimer formation, the risk of intra-primer secondary structures and the presence of polyN stretches. (Reference: Olsen LR et al. 2011. Nucl. Acids Res. 39 (Web Server issue): W61-W70)

Primerize is a Web Server for primer designs of DNA sequence PCR assembly. Primerize is optimized to reduce primer boundaries mispriming, is designed for fixed sequences of RNA problems, and passed wide and stringent tests. This efficient algorithm is suitable for extended use such as massively parallel mutagenesis library. (Reference: Tian, S., & Das, R. (2016) Quarterly Review of Biophysics 49(e7): 1-30).

Short interfering RNA (siRNA) design:

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. A variety of servers exist:
siRNA Target Finder (GenScript USA Inc.).

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

OligoWalk is an online server calculating thermodynamic features of sense-antisense hybidization. It predicts the free energy changes of oligonucleotides binding to a target RNA. It can be used to design efficient siRNA targeting a given mRNA sequence. (Reference: Lu ZJ & Mathews DH. 2008. Nucl. Acids Res. 36: 640-647).

VIRsiRNApred - a human viral siRNA efficacy prediction server (Reference: Qureshi A et al. 2013. J Transl Med. 11:305).

Dicer-substrate siRNAs (DsiRNAs) are chemically synthesized 27-mer duplex RNAs that have increased potency in RNA interference compared to traditional siRNAs.RNAi DESIGN (IDT Integrated DNA Technologies)

pssRNAit - Designing effective and specific plant RNAi siRNAs with genome-wide off-target gene assessment.

DSIR is a tool for siRNA (19 or 21 nt) and shRNA target design. (Reference: Vert JP et al. 2006. BMC Bioinformatics 7:520).

Imgenex siRNA retriever program has been designed to select siRNA encoding DNA oligonucleotides that can be cloned into one of the pSuppressor vectors. The input sequence can be directly accessed from a Genbank accession or sequence provided by the researcher.
siDRM is an implementation of the DRM rule sets for selecting effective siRNAs. The authors have performed an updated analysis using the disjunctive rule merging (DRM) approach on a large and diverse dataset compiled from siRecords, and implemented the resulting rule sets in siDRM, a new online siRNA design tool. siDRM also implements a few high-sensitivity rule sets and fast rule sets, links to siRecords, and uses several filters to check unwanted detrimental effects, including innate immune responses, cell toxic effects and off-target activities in selecting siRNAs. (
Reference: Gong W et al. 2008. Bioinformatics 24:2405-2406).

siMAX siRNA Design Tool (Eurofins Genomic, Germany) - is a proprietary developed software designed to help you selecting the most appropriate siRNA targeting your gene(s) of interest.

shRNA Designer (Biosettia Inc., USA) - Use this program to design shRNA oligos that are compatible with our SORT-A/B/C vectors. The design tool provides targets with the greatest chance of knocking down your gene. Please note, only one oligo is designed as it is palindromic.

siDESIGN Center (Horizon Discovery Ltd., UK) - is an advanced, user-friendly siRNA design tool, which significantly improves the likelihood of identifying functional siRNA. One-of-a-kind options are available to enhance target specificity and adapt siRNA designs for more sophisticated experimental design.

Realtime PCR primer design:

RealTimeDesign (Biosearch Technologies) - free but requires registration.

GenScript Real-time PCR (TaqMan) Primer Design - one can customize the potential PCR amplicon's size range, Tm (melting temperature) for the primers and probes, as well as the organism. You can also decide how many Primer/Probe sets you want the tool to return to you. It is possible to use a GenBank accession number as the template.

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)

PrimerQuest - (IDT, USA)

Introduction of 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.

PrimerX - can be uused to automate the design of mutagenic primers for site-directed mutagenesis. It is available in two flavours (a) Primer Design Based on DNA Sequence and (b) Primer Design Based on Protein Sequence

Primerize-2D - is designed to accelerate synthesis of large libraries of desired mutants through design and efficient organization of primers. The underlying program and graphical interface have been experimentally tested in our laboratory for RNA domains with lengths up to 300 nucleotides and libraries encompassing up to 960 variants. (Reference: Tian, S., & Das, R. (2017) Bioinformatics 33(9): 1405-1406).

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 Titration Calculator (Angel Herráez Cybertory: virtual molecular biology lab; Universidad de Alcalá, Spain) is a similar site.

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

PCR Optimization (Bioline, United Kingdom) - a lot of conditions

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.