Here is a fairly comprehensive list with a brief description of FREE software available:
ExonPrimer
http://ihg.gsf.de/ihg/ExonPrimer.htmlExonPrimer is a Perl script that helps to design intronic primers for the PCR amplification of exons. The script needs a cDNA and the corresponding genomic sequence as input. It aligns these sequences using Blat and designs PCR primers to amplify each exon using Primer3. The positions of the exons are deduced from the alignment of the genomic and the cDNA sequences. Insertions/deletions up to 6 base pairs are bridged by postprocessing. Exons with small introns in-between are combined. Exons smaller than 20-25 bp will not be recognized. The user can define the maximum exon size. Exons larger than this size will be divided into several parts.
The poly-A tail of the cDNA should be clipped to allow the alignment of the cDNA and the genomic DNA sequence. The genomic sequence must be longer than the cDNA sequence. Otherwise the design of primers for the first and/or last exon is not possible.
Download of the human genome sequence with all SNPs masked by N's. Using this sequence, one can avoid primers to be positioned across SNPs.
ExonPrimer is also available in the UCSC Genome Browser for the human genome assemblies hg16 (July 2003) and hg17 (May 2004). One can find a link to ExonPrimer in the 'Quick Links to Tools and Databases' section of the known genes details page.
Autoprime
http://www.autoprime.de/AutoPrimeWebAutoPrime allows to rapidly design primers for real-time PCR measurement of eucaryotic expression. Such primers are usually selected so that they will amplify cDNA generated from mRNA but will not yield a product on the genomic DNA. This is achieved by taking advantage of the eucaryotic splicing system for mRNA. Either primer pairs are selected so that one of the primers matches the exon-exon border sequence that is not present in the genomic sequence or the pair is designed by placing each primer in a different exon so that a genomic product would include a long intronic sequence. AutoPrime provides these capabilities on top of the primer3 primer-design software.
CODEHOP: COnsensus-DEgenerate Hybrid Oligonucleotide Primers
http://blocks.fhcrc.org/blocks/codehop.htmlThe CODEHOP program designs PCR (Polymerase Chain Reaction) primers from protein multiple-sequence alignments. The program is intended for cases where the protein sequences are distant from each other and degenerate primers are needed.
The multiple-sequence alignments should be of amino acid sequences of the proteins and be in the Blocks Database format Proper alignments can be obtained by different methods.
The result of the CODEHOP program are suggested degenerate sequences of DNA primers that you can use for PCR. You have to choose appropriate primer pairs, get them synthesized and perform the PCR.
References:
"Consensus-degenerate hybrid oligonucleotide primers for amplification of distantly-related sequences" by T.M. Rose, E.R. Schultz, J.G. Henikoff, S. Pietrokovski, C.M. McCallum and S. Henikoff, Nucleic Acids Research, 26(7):1628-1635.
http://blocks.fhcrc.org/blocks/help/CODEHOP/manuscript.htmlMethPrimer - Design Primers for Methylation PCRs
http://www.urogene.org/methprimer/MethPrimer is a program for designing bisulfite-conversion-based Methylation PCR Primers. Currently, it can design primers for two types of bisulfite PCR: 1) Methylation-Specific PCR (MSP) and 2) Bisulfite-Sequencing PCR (BSP) or Bisulfite-Restriction PCR.
Input Sequence: A DNA sequences in any format. No editing is required before input.
Output: MethPrimer returns results in both text and graphic view.
Reference:
Li LC and Dahiya R. MethPrimer: designing primers for methylation PCRs. Bioinformatics. 2002 Nov;18(11):1427-31.
PerlPrimer - open-source PCR primer design
http://perlprimer.sourceforge.net/PerlPrimer is a free, open-source GUI application written in Perl that designs primers for standard PCR, bisulphite PCR, real-time PCR (QPCR) and sequencing. It aims to automate and simplify the process of primer design.
PerlPrimer's current features include the following:
Calculation of possible primer-dimers
Retrieval of genomic or cdna sequences from Ensembl
(including both sequences automatically for QPCR)
Ability to BLAST search primers using the NCBI server or
a local server
Results can be saved or optionally exported in a
tab-delimited format that is compatible with most
spreadsheet applications.
ORF and CpG island detection algorithms
Ability to add cloning sequences to primers, automatically
adjusted to be in-frame
QPCR primer design without manual intron-exon boundary
entry
PerlPrimer calculates primer melting temperature using J. SantaLucia's extensive nearest-neighbour thermodynamic parameters. To adjust for the salt conditions of the PCR, PerlPrimer uses the empirical formula derived by von Ahsen, et al. (2001) and allows the user to specify the concentration of Mg2+, dNTPs and primers, or use standard PCR conditions. The result is a highly accurate prediction of primer melting temperature, giving rise to a maximum yeild of product when amplified.
PerlPrimer is written in Perl and Perl/Tk. In addition, for QPCR functionality PerlPrimer uses the open-source Spidey executable from NCBI, and restriction enzyme data from the REBASE project is used when adding cloning sites. The program is designed to be cross-platform compatible and has been developed and tested on both Microsoft Windows and GNU/Linux-based operating systems. Users have also reported success using the program under Mac OS X.
Reference:
Marshall OJ. PerlPrimer: cross-platform, graphical primer design for standard, bisulphite and real-time PCR. Bioinformatics 2004 20(15):2471-2472
http://bioinformatics.oxfordjournals.org/cgi/screenpdf/20/15/2471Web Primer
http://seq.yeastgenome.org/cgi-bin/web-primerUses & Input
The user must choose a purpose for the primers to be designed. Current choices are limited to sequencing and PCR. Sequencing primers will be evenly spaced along the DNA. PCR primers will be at the ends of the DNA selected in a region of DNA the length of which is user defined.
DNA Source
The user must also enter either a locus name or an actual DNA sequence; either will then act as the source of DNA from which the primers are chosen. For the Locus field, any standard ORF name (i.e. YBL004W) and many gene names (i.e. BUD4, TUB2) are allowed. If an entry in this field is ambiguous (matching more than one entry in the database) a list of matching entries is created and the user is requested to choose one. An example of this is YJL12 which matches ORFs YJL120W through YJL129C. If these ORFs have gene names associated with them this name is present as well (i.e. YJL125C=GCD14). Entries which match MORE than 25 ORFs will not be processed and the user is requested to refine the entry. The DNA sequence may include numbers but should NOT include letter characters (other than the DNA sequence).
PCR primers
There are many factors which influence the success of a pair of primers. Some of the properties of primers which can affect the outcome of PCR include: the GC / AT ratio, length, melting temperature, and the extent of annealing between primers. The location of a primer also heavily influences its usefulness. All of these variables are able to be influence by the user and are further described below.
Sequencing Primers
Sequencing primers are also highly customizable. Potential valid primers are evenly spaced along the DNA of interest starting at each 5' end. The user is allowed to specify the area of DNA to be sequenced, how many strands to order sequencing primers for, which strand to order primers for, the approximate distance between primers, the length and percent GC content of the primers, and the maximum self annealing of the primers.
OligoMaster
http://www.oligomaster.co.uk/OligoMasterOligoMaster is the ideal tool to save you time, space and money. OligoMaster is a multi-user, multi-platform oligonucleotide cataloguing application to help you manage and organise your oligonucleotide collection. With OligoMaster, you form a database from your collection of oligonucleotides, allowing you to quickly and easily retrieve essential information about specific oligonucleotide(s) before running an experiment.
OligoMaster gives you the possibility of choosing between keeping your oligonucleotides private from others or sharing them with other users of the database. Common oligonucleotides such as sequencing primers do not need to be entered by each user of the database. This unique sharing feature allows all the users of the same database to view shared oligonucleotides, considerably improving the collaborative work of your research group.
Primer3
http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgiReference:
Steve Rozen and Helen J. Skaletsky (2000) Primer3 on the WWW for general users and for biologist programmers. In: Krawetz S, Misener S (eds) Bioinformatics Methods and Protocols: Methods in Molecular Biology. Humana Press, Totowa, NJ, pp 365-386
Source code available at
http://fokker.wi.mit.edu/primer3/.PrimerX
http://bioinformatics.org/primerx/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.
PrimerX can design mutagenic primers pairs based on two different kinds of sequence data. One option is for you to enter a mutation in your template DNA sequence, so that the desired base pair insertions, deletions, or substitutions are incorporated. This is recommended for generating specific SNPs and indels. The other is to enter a mutation within the protein sequence encoded by your template DNA, in which case PrimerX generates mutagenic primers based on all possible DNA sequences that can encode the desired mutation, taking into account codon degeneracy. This is recommended for changing a specific amino acid residue into another.
In addition to this, PrimerX can characterize primers that you have designed. Here, you only need to enter a mutagenic primer sequence and the number of mismatched bases, and PrimerX will compute and report back its reverse complement, GC content, melting temperature, etc.
ELXR v2.0
http://mutation.swmed.edu/ex-lax/Identifies the genomic contig containing your query checking in LocusLink annotation or via highest scoring BLAST sequence alignment to genomic nucleotide databases.Aligns query to genomic sequences using the Sim4 program. Exon flanking PCR/sequencing primers are designed according to certain parameters using the Primer3 program
Applications:
Scenario 1: Design primers to amplify exons from genes in multiple individuals to detect sequence variations
Scenario 2: Run multiple potential splice variant EST or cDNA sequences through ELXR to examine alternative splicing at the sequence level by comparing exon-annotated genomic sequences from ELXR output
Scenario 3: Use the genomic flanking sequence option to control the amount of uptream sequence is returned in ELXR output to evaluate sequence features in gene promoters.
PDA Primer Design Assistant
http://dbb.nhri.org.tw/primer/Input format Single Sequence in fasta format or ASCII (Pure text) will be allowed. But the multiple sequences pasted or uploaded into PDA must be in fasta format. Sequence(s) input or upload The example (fasta format) file and the other one (text format) file are here. Primer length: Too long a primer length may mean that even high annealing temperatures are not enough to prevent mismatch pairing and non-specific priming. The appropriate length of primer we suggest is between 18 -25 bps. PCR product size: The size is between 150 - 600 bps. This kind of sequence length is suitable for sequencing and further applications.
The Primer Generator
http://www.med.jhu.edu/medcenter/primer/primer.cgiThe 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.
Reference:
The Primer Generator: A Program that Facilitates the Selection of Oligonucleotides for Site-Directed Mutagenesis
BioTechniques 1999; 26:660-668
http://www.med.jhu.edu/medcenter/primer/manuscript.htmlFastPCR
http://www.biocenter.helsinki.fi/bi/Programs/fastpcr.htmFastPCR is universal software for everyday using, for manipulation and analysis sequences, PCR primer and probe design, alignment and any repeat searching.
FastPCR is a free software for Microsoft Windows and is based on a new approach in the design of PCR primers for standard and long PCRs, inverse PCR, direct amino acid sequence degenerate PCR, multiplex PCR, in silico PCR and group-specific PCR (common primers for multiple sequences), single primering PCR, automatically SSR loci detection and direct PCR primers design; for sequence alignments, clustering and any kind repeat sequence, MITE elements and SSR loci searching; restriction enzyme analysis.
FastPCR software can simultaneously work with multiple nucleic acid or protein sequences (up to 100,000). The multiplex PCR primers design and "in silico" PCR are also supported. The FastPCR program is an ideal software for personal databases homology searches which are similar to the basic local alignment search tool (BLAST) algorithm (a segment-to-segment alignment principle similar to DIALIGN). The program includes various bioinformatics tools and supports the clustering of sequences. A new repeats search theory was developed and applied to the program, which makes the accomplishment of all DNA repeat types searches fast and powerful.
FastPCR software has several specific, ready-to-use templates for many PCR and sequencing applications:
Standard, inverse, long, real-time PCR analysis - identification of the optimal primers for PCR, hybridisation, or sequencing;
Multiplex PCR primers design - fast primer design with a cross-dimer test for high sensitive multiplex PCRs;
Group-specific PCR primers design - design of universal PCR primers for all sequences (there is no need for a multiple DNA alignment); Unique PCR primers design - design of specific PCR primers for each sequence; Degenerate PCR - primers design directly on an amino acid sequence; Automatically SSR loci detection and direct PCR primers design; In silico PCR - prediction of probable PCR products and search of potential mismatching location of the specified primer(s); TaqMan and Molecular Beacons probes and other probes design;
LUX (self-quenched) primers design for quantitative PCR;
Self-Reporting DNA/DNA primers for qPCR analysis;
Primer Secondary structure analysis - self-dimer and cross-dimer primer analyses; primer alignment and melting temperature calculation; False priming analysis - (the secondary (non-specific) binding test) primers checking for multiple annealing sites using sequence alignment algorithms;
Primer quality report - a unique way for PCR efficiency determination; Comprehensive primer report - comprehensive pairs and individual primers analysis (Tm and dimer detection);
Multitask PCR primers and probes design - simultaneously design primers or probe with different parameters and for different targets within the same sequence; interaction different tasks.
The FastPCR software provides also a wide array of bioinformatics and bioanalysis tools, such as:
Repeats search: Invert, Direct, Simple and others; MITE elements search; LTR-retrotransposons search; SSR (Simple Sequence Repeat) locus search two, three, four or five perfect and imperfect core motif; Clustering sequences (BLAST2 related alignment); Sequence alignment using universal degenerated code Complement, reverse and reverse-complement strand modification; Consensus sequence for two or mores sequences;
Low complexity analysis for discover SSR loci, introns-exons, S/MARs (Scaffold/Matrix Attachment Regions) or else;
Extraction of specific fragments from a larger sequence;
DNA-to-Protein translation and Protein-to-DNA reverse translation; Calculation of the annealing temperature of PCR (for unknown PCR products); Database analysis tools; Restriction analysis; Pair Sequences Similarity calculation the similarity for entered sequences in alignment format like MEGA or GCG/MSF and also non alignment raw formatted sequences.
TGGE-Star
Optimization of mutation screening systems with temperature gradient gel electrophoresis and PCR
http://www.charite.de/bioinf/tgge/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.
Primer Design (PRIDE 2.0)
http://pride.molgen.mpg.de/pride.htmlPRIDE 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).
Features:
highly automated minimal set of user-defined parameters
ability to check for secondary binding sites within vector or repetitive elements fast computation of primers also on complicated sequences (e.g. high GC-content) already designed primers are recognized visualization of primers by specific tags
primers are named uniquely according to the template they are designed on flexible naming convention by using regular expressions additional tools for displaying and removal of groups of annotation tags
Primer Design for DNA-arrays/chips (GenomePRIDE 1.0)
http://pride.molgen.mpg.de/genomepride.htmlGenomePRIDE is primer design program that designs PCR primers or long oligos on an annotated sequence.
The program is tightly integrated into the Staden package (GAP4).
Features:
high record of success
computation of PCR primers and/or oligos
fast computation
defined fragment length
homologies are analyzed
visualization (based on the Staden package)
design of primers in a specific frame
Primer Bank
PCR Primers for Gene Expression Detection and Quantification
http://pga.mgh.harvard.edu/primerbank/PrimerBank is a public resource for PCR primers. These primers are designed for gene expression detection or quantification (real-time PCR). PrimerBank contains over 306,800 primers covering most known human and mouse genes. There are several ways to search for primers: GenBank Accession, NCBI protein accession, NCBI Gene ID, Gene SymbolNew!, PrimerBank ID or Keyword (gene description) or you can blast your gene sequence against the primerbank Sequence DBNew!.
The primer design algorithm has been extensively tested by real-time PCR experiments for PCR specificity and efficiency. We have tested 26,855 primer pairs that correspond to 27,681 mouse genes by Real Time PCR followed by agarose gel electrophoresis and sequencing of the PCR products. The design success rate is 82.6% (22,187 successful primer pairs) based on agarose gel electrophoresis.
Oligo2002 ver 1.07 by Sung-Hun Bae
http://www.bioinformatics.vg/bioinformatics_tools/oligo2002.shtmlJaMBW Chapter 3.1.9
Oligo Calculator
http://members.aol.com/_ht_a/lucatoldo/myhomepage/JaMBW/3/1/9/index.htmlIDT SciTools OligoAnalyzer 3.0
http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/Default.aspxVPCR 2.0
http://elanor.sci.muni.cz/cgi-bin/vpcr2.cgiVPCR 2.0 searches the specified database for matches to the primers. If matches are found within 10000 bases, a PCR simulation model predicts amplification. Calculated PCR products are displayed within a minute.
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