Bioinformatic tools

Database search

• NCBI - BLAST at NCBI
• EBI  -  BLAST at EBI

Pairwise alignment

• MCALIGN2 [sequences must be entered in FASTa format]
• GGSEARCH, GLSEARCH to compare two sequences [enter sequences as Genbank Accesion Number or in FASTA format]

Multiple sequence alignment

• CLUSTALX (graphical version) or CLUSTALW (comand-line version) - [DNA or protein seqeunces can be submitted in 7 formats: Pearson (FASTA), EMBL/UniProtKB/Swiss-Prot, ALN/ClustalW, NBRF/PIR, GDE, GCG/MSF, RSF.
• FSA Multiple Alignment Server [enter DNA, RNA, or protein sequences in FASTA or text-only format]
• TCoffee [submit DNA, RNA, or protein sequences in FASTA format]

Visualization

• PyMOL

Other

• BioEdit - sequence alignment editor
• CINEMA 5 - Colour INteractive Editor for Multiple Alignments

Primer design

• Basic concepts about primer design
• PCR and primer design. Concepts

Primer design webservers for PCR, mutagenesis and RNAi (source: http://www.humgen.nl/primer_design.html)

• AntiSense Design - Antisense primers
• AutoPrime - Primers specific for expressed sequences (mRNA)
• BatchPrimer3 - High throughput web application for PCR and sequencing primer design
• CODEHOP - COnsensus-DEgenerate Hybrid Oligonucleotide Primers
• Exonprimer - Amplification of exons with intronic primers
• Genefisher - Interactive PCR primer design
• Methprimer - Design primers for methylation PCR
• mPrimer3 - Modified Primer3
• MutScreener - Primers for mutation screening (by PCR-direct sequencing)
• 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.
• 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
• ProbeWiz Server - The CBS ProbeWiz WWW server predicts optimal PCR primer pairs for generation of probes for cDNA arrays. Reference [PubMed]
• RNAi Design - Design primers for RNAi at IDT
• 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
• SPADS - Specific Primers & Amplicon Design Software for amplification of individual members of gene families

PCR Primer design software for local installation (Freely available)

• 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".

Microarray primer design

• MAPHDesigner 1.2 design of primers and probes for genome copy number detection (MAPH/CGH microarrays)

Microarrray 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.

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 3.1 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