Primer design tools

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This section contains tools useful for designing primers, summarized in a "Quick Links" section, with more detailed descriptions below.

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Primer design tools - Quick Links Edit

Primer3 PrimerQuest
Primer Premier FastPCR
AutoPrime - web-based primer design software PrimerX
OligoMaster PerlPrimer
Methprimer NetPrimer
Oligo2002 CODEHOP
The Primer Generator Primer Design Assistant
PROBEmerExon Locator and Extractor for Resequencing
TGGE-StarPrimer3 (UMass server)
QuantPrime - RT-qPCR primer design tool GETPrime- transcript- or gene-specific qPCR primer database

Primer design tools - Web Resources Descriptions in Detail Edit

DPL Forum: Too few categories!

Primer3 Picks decent primers from a DNA sequence.

PrimerQuest is a tool for the design of PCR primers, probes, and sequencing primers.

Primer Premier is a comprehensive primer design tool for multiplex, nested and degenerate primer design, by Premier Biosoft

GETPrime A gene- or transcript-specific primer database for quantitative real-time PCR. This user-friendly plateform uniquely combines and automates several features critical for optimal qPCR primer design. These include the consideration of all gene splice variants to enable either gene-specific (covering the majority of splice variants) or transcript-specific (covering one splice variant) expression profiling, primer specificity validation, automated best primer pair selection according to strict criteria and graphical visualization of the latter primer pairs within their genomic context. GETPrime primers have been extensively validated experimentally demonstrating their high quality and demonstrating high transcript specificity in complex samples. Until now, you could retrieve primers in a high-throughput fashion for all Homo sapiens, Mus musculus, Caenorhabditis elegans, Drosophila melanogaster and Danio rerio genes in assembled chromosomes annotated in the Ensembl database.

QuantPrime is an intuitive and user-friendly, fully automated tool for primer pair design in small- to large-scale real-time reverse transcription qPCR (also known as realtime qRT-PCR or RT-qPCR) analyses. QuantPrime can be used on the website or on a local computer; it offers design and specificity checking with highly customizable parameters and is ready to use with most publicly available eukaryotic transcriptomes, including all higher eukaryote model organisms and important plant crops, while benefiting from exon-intron border and splice variant information in available genome annotations. Experimental results with the model plant Arabidopsis thaliana, the crop Hordeum vulgare (barley) and the model green alga Chlamydomonas reinhardtii show success rates of designed primer pairs exceeding 96 %.

Downloadable PCR primer design software. This software can simultaneously work with multiple nucleic acid or protein sequences (up to 100,000). It is efficient for standard, long, inverse PCR, direct amino acid sequence degenerate PCR, multiplex PCR, automatically SSR loci detection and direct PCR primers design, and in silico PCR. Real-Time (quantitative) PCR: LUX primer and Self-Reporting DNA/DNA primers design. Long oligonucleotides can be design for microarray analysis and design dual-labelled oligonucleotides probes: TaqMan or Molecular Beacon and also native molecular beacon (short hairpins). The program includes various bioinformatics tools and supports the clustering of sequences. It can also perform sequence alignments, clustering and any kind repeat sequence searching. A new repeats search theory was developed and applied to the program, which makes the accomplishment of all kind repeat sequence searches fast and powerful.

AutoPrime - web-based primer design software
AutoPrime is freely accessible web-based primer design software which designs primers of high quality for Real-Time-Quantitative PCR. The software AutoPrime automates the task of generating primers for Real-Time-Quantitative-PCR experiments by combining the information from sequence databases (Ensembl) with primer design software (primer3). The software is able to cope with the high demand of RT-PCR validation experiments following the recent increase in microarray expression profiling studies. The software generates high quality primers more efficiently than possible by manual design methods.

Automated design of mutagenic primers for site-directed mutagenesis. PrimerX is a web-based program written to automate the design of mutagenic PCR primers for site-directed mutagenesis. Based on your input, PrimerX compares a template DNA sequence with a DNA or protein sequence that already incorporates the desired mutation. It then generates forward primer sequences by computing for all possible oligonucleotide sequences of appropriate length that encode this mutation and follow your specified constraints. Finally, PrimerX generates corresponding reverse primer sequences, and computes for other necessary information such as melting temperature and GC content for each primer pair

Database tool for organising and cataloguing oligonucleotides. OligoMaster is a multi-user, cross-platform oligonucleotide cataloguing application, designed to help you manage and organise your oligonucleotide collection. Free trial.
Database tool for organising and cataloguing oligonucleotides. OligoMaster is a multi-user, cross-platform oligonucleotide cataloguing application, designed to help you manage and organise your oligonucleotide collection. OligoMaster allows you to quickly and easily retrieve essential information about specific oligonucleotide(s). Oligonucleotides can be made private or can be shared with other users of the database. OligoMaster can run either in single workstation mode, or client-server mode, where multiple OligoMaster clients can connect to a single database over the network. In addition, OligoMaster has built-in automatic calculation of values, allowing you to enter a sequence and OD260 value and have all other values calculated from these. The calculation is entirely user scriptable, allowing you to substitute your preferred algorithms.

PerlPrimer v1.1.1
Free. Features include (I) Calculation of possible primer-dimers, (II) Retrieval of genomic or cdna sequences from Ensembl (including both sequences automatically for QPCR), (III) Ability to BLAST search primers using the NCBI server, (IV) Results can be saved or optionally exported in a tab-delimited format that is compatible with most spreadsheet applications, (V) ORF and CpG island detection algorithms, (VI) Ability to add cloning sequences to primers, automatically adjusted to be in-frame, (VII) QPCR primer design without manual intron-exon boundary entry.

MethPrimer - Design Primers for Methylation PCRs by Long-Cheng Li, M.D. of Urology Research Dept at VA Medical Center and UCSF San Francisco. The only free public methylation primer program available.

NetPrimer is a free web based primer analysis software.

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Version 1.07 calculates melting temperature, tendency to form hairpin loops or dimers, molecular weight, and base composition.

Degenerate PCR primers designed from protein multiple sequence alignments. COnsensus-DEgenerate Hybrid Oligonucleotide Primer PCR primer design. CODEHOPs are used in PCR amplification to isolate distantly related sequences encoding the conserved amino acid sequence. The primer design software and the CODEHOP PCR strategy have been utilized for the identification and characterization of new gene orthologs and paralogs in different plant, animal and bacterial species.<pubmed argument="CODEHOP">12824413</pubmed>

The Primer Generator
Generates primers for site-directed mutagenesis. The Primer Generator is an automated generator of primers for site-directed mutagenesis. The program analyzes the original nucleotide sequence and desired amino acid sequence and designs a primer that either has a new restriction enzyme site or is missing an old one. This allows for faster sorting out of mutated and non-mutated sequences.

Primer Design Assistant - a web-based primer design tool. Web interface primer design service combined with thermodynamic theory to evaluate the fitness of primers. A succinct user interface of PDA is accomplished by built-in parameters setting. Advanced options on GC content, dimer check and hairpin check are available. The option of covered region constrains the PCR product to cover a user-defined segment. PDA accepts single sequence query or multiple ones in FASTA format.

Beacon Designer Free Edition Free web based real time PCr primer analysis tool. analyzes SYBR® Green primers and TaqMan® probes for possible secondary structures such as dimers or hairpins.

Web-based software tool for selecting optimal DNA oligos. Selects optimal oligos for PCR applications and multiplex detection platforms including oligonucleotide microarrays and bead-based arrays. Given two groups of nucleic-acid sequences, a target group and a non-target group, the software identifies oligo sequences that occur in members of the target group, but not in the non-target group. To help predict potential cross hybridization, PROBEmer computes all near neighbors in the non-target group and displays their alignments. The software has been used to obtain genus-specific prokaryotic probes based on the 16S rRNA gene, gene-specific probes for expression analyses and PCR primers.

Designs PCR primers or oligos (50-70mers) used for DNA chips. Requires Solaris, Alpha or Linux and pre-installation of Staden package. GenomePRIDE is primer design program that designs PCR primers or long oligos on an annotated sequence. So far, GenomePRIDE was used for the design of DNA arrays/chips containing all ORFs of the following organisms: Bacillus subtilis, Schizosaccharomyces pombe, Drosophila melanogaster, Candida albicans. ORF specific primer pairs were designed with more than 97% success.

Large scale design of sequence primers especially for walking. PRIDE is a primer design program that automatically designs primers in single contigs or whole sequencing projects to extend the already known sequence and to double strand single-stranded regions. The program is fully integrated into the Staden package (GAP4) and accessible with a graphical user interface.

Exon Locator and Extractor for Resequencing
Maps cDNA to genomic sequence and designs primers for amplification and sequencing of exons and other targets. ELXR (Exon Locator and Extractor for Resequencing) was developed to accomplish the steps of exon identification and primer design in a fraction of the time consumed by manual methods. ELXR and ELXRdb, a database of pre-computed primer pairs for all human, mouse and rat RefSeq mRNA sequences mRNA are at ELXR has been integrated into the high-throughput SNP sequencing pipeline in a NHLBI Program for Genomic Applications at UT Southwestern (

PCR-primer design for TGGE/DGGE - optimization of mutation screening systems with temperature gradient gel electrophoresis and PCR. PCR combined with temperature gradient gel electrophoresis is a rapid and very sensitive screening method for point mutations. It may be used to identify exons or other DNA segments with abnormal electrophoretic mobility which are then subjected to DNA sequencing. Computer aided design of PCR primers for denaturing gradient gel electrophoresis and the careful choice of a suitable temperature range is the most important warranty for the success of the screening. The program was written, to facilitate the design of PCR primers fulfilling the requirements for a sensitive mutation screening. It supports the new concept of bipolar clamping which is important when mono-polar TGGE leads to fuzzy bands.

Primer3 (UMass server)
Picks decent primers from a DNA sequence


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