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Primer Design

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These are a collection of primer design sites.

Primer Prediction and Analysis programs

[info] The PCR Jump Station
[info] GeneFisher
[info] GeneWalker
[info] CyberGene
[info] Web Primer
[info] Primer Design
[info] Primer3
[info] Web Primers
[info] POLAND - melting profiles of double stranded DNA
[info] CODEHOP - PCR primers designed from protein multiple sequence alignments
[info] NetPrimer
[info] Cassandra - Recognition of protein-coding segments for PCR
[info] GenePrimer - Computational support of gene identification experiments
[info] rawprimer - a tool for selection of PCR primers

Primer Design Criteria

[info] Alkami Biosystems
[info] Choosing Primers for sequencing
[info] Design of Primers for Automated Sequencing

Detailed information on the above options

The PCR Jump Station
The ultimate Web page for information and links on all aspects of the Polymerase Chain Reaction (PCR).

GeneFisher processes aligned or unaligned sequences. GeneFisher comes with a built in alignment tool that feeds the results directly into the next step. You have the option of looking at a graphical representation of the alignments and the consensus sequence that is really useful for a variety of tasks, not just primer design. At this point you can reject the alignment and adjust the parameters to try again or you can continue to the primer design step.

GeneWalker helps you designing your primers. It is a powerful tool, yet it is based on the philosophy of ease to use. GeneWalker allows you to work with two primer sequences simultaneously. You can also toggle between reversed complementary primer sequences. Basic functions includes calculation of primer secondary structures, primer annealing properties as well as primer dimers. GeneWalker also helps you to easily connect to EMBLs databases.

CyberGene is a company that provides commercial oligonucleotide synthesis, DNA sequencing, genotyping and bioinformatics services. They also offer this free primer design utility to help you test oligos before you spend time and money synthesising unsuitable ones.

Web Primer
An application that designs primers for PCR or sequencing purposes. The user must choose the purpose for which the primers will be used, and either specify a locus name or enter a sequence.

Primer Design
A free primer design utility, from the EMBL.

Primer3 picks primers for PCR reactions, according to the conditions specified by the user. Primer considers things like melting temperature, concentrations of various solutions in PCR reactions, primer bending and folding, and many other conditions when attempting to choose the optimal pair of primers for a reaction. All of these conditions are user-specifiable, and can vary from reaction to reaction.

Web Primers
Web Primers is a tool for graphically displaying primer sites along a target sequence. You are presented with an image showing the relative positions of the selected primers, their direction, and a separate list of the primers themselves.

POLAND - melting profiles of double stranded DNA
The Poland server will calculate the thermal denaturation profile of double stranded RNA or DNA based on sequence input and parameter settings in this form. Calculation is based on D. Poland's algorithm in the implementation described by G. Steger.

Calculations can be done for oligonucleotides (>15 bases) or long double strands (>50 bases), respectively.

CODEHOP - PCR primers designed from protein multiple sequence alignments
The 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.

NetPrimer combines the latest primer design algorithms with a web-based interface allowing the user to analyze primers over the Internet. All primers are analyzed for melting temperature using the nearest neighbor thermodynamic theory to ensure accurate Tm prediction. Primers are analyzed for all secondary structures including hairpins, self-dimers, and cross-dimers in primer pairs. This ensures the availability of the primer for the reaction as well as minimizing the formation of primer dimer. The program eases quantitation of primers by calculating primer molecular weight and optical activity. To facilitate the selection of an optimal primer, each primer is given a rating based on the stability of its secondary structures. A comprehensive analysis report can be printed for individual primers or primer pairs.

Cassandra - Recognition of protein-coding segments for PCR
Traditionally, the use of gene predictions in experimental practice amounts to construction of oligonucleotide probes or PCR primers and a biologist is forced to guess the primer on the top of not very reliable prediction. CASSANDRA is a program designed for recognition of coding segments, that is, segments situated within protein-coding DNA regions of higher eukaryotes, without direct recognition of complete genes. It produces a list of candidate segments that is virtually guaranteed to contain a given number of probes or primers situated within exons. The list of candidate segments is can be optimized by different parameters that depend on particular experiment that will utilize these segments.

At present the program is optimized for the following two problems:

GenePrimer - Computational support of gene identification experiments
This software implements an algorithm for experimental gene identification by multiple PCR amplifications.

Since current algorithms for gene recognition make mistakes, biologists have to perform experimental gene identification to eliminate errors in predictions. Conventional approaches amount to `guessing' PCR primers on top of unreliable gene predictions and frequently lead to wasting experimental efforts. An algorithm which eliminates the need of gene verification in some cases is the Las Vegas algorithm for gene recognition. We bypass the unreliable gene prediction step and propose an approach geared towards experimental gene identification. The algorithm locates a set of PCR primers which relatively uniformly cover the exons and can be used for RT-PCR and further sequencing of (unknown) mRNA.

rawprimer - a tool for selection of PCR primers
This page provides an interface to a PCR primer selection program based on xprimer.

It is designed for selection of sets of primers along very large queries, all with a relatively narrow Tm range. It is also useful in more traditional PCR applications.

This version of xprimer produces no graphics. It is set up to use human and dog repeat files and several species models. The query sequence (raw sequence with no header - only agct in either case are significant) can be dragged into the textarea in the interface.

Alkami Biosystems

Choosing Primers for sequencing
Information provided by the DNA sequencing facility at the University of Chicago Cancer Research Centre.

Design of Primers for Automated Sequencing
Guide to designing primers from the DNA sequencing core facility at the University of Michigan.

Any Comments, Questions? Support@hgmp.mrc.ac.uk