Oligonucleotides & Primer Design
Melting Temperature
Wallace Method
Tm (in °C) = 2(A+T) + 4(C+G)
(A+T) - the sum of the A and T residues in the oligonucleotide,
(C+G) - the sum of G and C residues in the oligonucleotide.
DNA sequence between 14 and 18 nucleotides
GC content Method
Tm (in °C) = 81.5°C + 16.6°C x log10[Na+K] + 0.41°Cx(%GC) – 675/N
- 0.62x(%formamide) - (%mismatch)
N - number of bases, between 8 and 100 bp
%GC - percentage GC content
%formamide - percentage formamide
Salt concentration in M, at least 10 mM
Dimers and Hairpin structures
Tm calculaton and search for hairpin and dimer structure
www.idtdna.com/analyzer/Applications/OligoAnalyzer/
This tool is used to calculate the thermodynamic properties of an oligonucleotide sequence. Melting point, hybridization free energy, probable oligonucleotide self-dimers and hairpin structures are identified and displayed.
www.fermentas.com/reviewer/app?page=OligoProperties&service=page
This tool is used to identfy probable oligonucleotide duplexes for two sequences.
www.fermentas.com/reviewer/app?page=DuplexAnalysis&service=page
Calculators & convertors
Formulas for conversion and concentration determination for oligonucleotides and DNA
GeneInfinity
Calculate molar conversions, concentration conversions, and spectrophotometric conversions.
www.fermentas.com/reviewer/app?page=Calculator&service=page
Oligonucleotide Properties Calculator
http://www.basic.northwestern.edu/biotools/OligoCalc.html
PCR Primer Design Tools
Primer3 is a widely used program for designing PCR primers (PCR = "Polymerase Chain Reaction"). PCR is an essential and ubiquitous tool in genetics and molecular biology. Primer3 can also design hybridization probes and sequencing primers.
biotools.umassmed.edu/bioapps/primer3_www.cgi
Tool for designing primers for PCR or sequencing
www.idtdna.com/Scitools/Applications/Primerquest/
A software tool to manage target sequences and generate PCR primers
http://www.proteinstrukturfabrik.de/ORFprimer/index.html
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.
http://www.ncbi.nlm.nih.gov/tools/primer-blast/
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).
http://pride.molgen.mpg.de/pride.html
GenomePRIDE is primer design program that designs PCR primers or long oligos on an annotated sequence.
http://pride.molgen.mpg.de/genomepride.html
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.
http://perlprimer.sourceforge.net/
FastPCR is an integrated tool for automatic and manual PCR primers or probe design, alignment and any repeat searching.
http://www.biocenter.helsinki.fi/bi/Programs/fastpcr.htm
AutoPrime allows to rapidly design primers for real-time PCR measurement of eukaryotic 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.
http://www.autoprime.de/AutoPrimeWeb
ExonPrimer is a Perl script that helps to design intronic primers for the PCR amplification of exons.
http://ihg2.helmholtz-muenchen.de/ihg/ExonPrimer.html
Genome-scale oligonucleotide design for microarrays
http://berry.engin.umich.edu/oligoarray2_1/
Oligo Design 1.7 is a package of tools for manipulation of DNA sequences. It is primarily designed for the manipulation of relatively short sequences such as PCR primers and oligonucleotide probes. A key feature is the ability to compute oligo melting temperatures using several literature models.
http://www.enme.umd.edu/bioengineering/
This server performs intelligent design of oligonucleotides for DNA microarrays.
http://www.cbs.dtu.dk/services/OligoWiz/
OligoPicker is to help selecting up to five oligo probes for each of the DNA sequences you provided for microarray spotting.
http://pga.mgh.harvard.edu/oligopicker/
Promide is a collection of command-line tools for Probe selection and Microarray Design.
http://oligos.molgen.mpg.de/
PRIMEGENS (PRIMEr Design Using GEN Specific Fragments) is a computer program to select gene-specific fragments and then design primer pairs for PCR amplifications.
http://compbio.ornl.gov/structure/primegens/
Oligodb is a web-based system for interactive design of oligo DNA for transcription profiling (hybridization) of human genes.
http://oligodb.charite.de/
The CBS ProbeWiz server predicts optimal PCR primer pairs for generation of probes for cDNA arrays.
http://www.cbs.dtu.dk/services/DNAarray/probewiz.php
Blast Integrated Oligonucleotide Selection Accelerator Package
http://biosap.sourceforge.net/
AiO (All in One) is a program for Windows, that combines typical DNA/protein features such as plasmid map drawing, finding of ORFs, translate, backtranslate and high quality printing with a number of databases. These databases allow the management of oligonucleotides, oligonucleotide-manufacturers, restriction enzymes, structural DNA and program users in a multi-user/multi-group environment.
http://132.187.163.191/aio/text.html
Primer design for multiple DNA species and complexe gene amplifications
DOSSER (DNA Oligonucleotide Sequence Set Editor with Regular expressions) is software for designing combinatorial sequence sets containing short DNA oligonucleotides. A combinatorial sequence set is a set of DNA sequences where all sequences have the same length and are unique.
folk.uio.no/haavarne/DOSSER/
Based on the assumption that genes with related function from different organisms show high sequence similarity, degenerate primers can be designed from sequences of homologues genes. GeneFisher is an interactive web-based program for designing degenerate primers. The procedure leads to isolation of genes in a target organism using multiple alignments of related genes from different organisms. The term "gene fishing" refers to the technique where PCR is used to isolate a postulated but unknown target sequence from a pool of DNA.
bibiserv.techfak.uni-bielefeld.de/genefisher2/
Multi Rev Trans accepts a protein alignment and uses a codon usage table to generate a graph that can be used to find regions of minimal degeneracy at the nucleotide level. Each position in the resulting DNA sequence is shown as a group of four horizontal bars, each representing the probability that the position is occupied by a particular base. If one of the bars is longer than the "highlight" value you specify, the group of bars is highlighted. Use Multi Rev Trans to design PCR primers for amplifying genes that encode similar proteins.
bio.dfci.harvard.edu/Tools/SMS/multi_rev_trans.html
Primaclade is 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.
www.umsl.edu/services/kellogg/primaclade.html
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.
http://blocks.fhcrc.org/help/CODEHOP/CODEHOP_help.html
In-silico PCR
In-Silico PCR searches a sequence database with a pair of PCR primers; search on many genomes available.
http://genome.ucsc.edu/cgi-bin/hgPcr
VPCR 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.
http://elanor.sci.muni.cz/cgi-bin/vpcr2.cgi
PCR Primer Design Guides
PCR Primer Design Guidelines at PremierBiosoft
www.premierbiosoft.com/tech_notes/PCR_Primer_Design.html
http://dorakmt.tripod.com/genetics/realtime.html
http://www.staff.uni-mainz.de/lieb/additiva.html
Designed by OCTAVIAN HENEGARIU, Yale University
http://info.med.yale.edu/genetics/ward/tavi/PCR.html
Primer Databases
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 Symbol, PrimerBank ID or Keyword (gene description) or you can blast your gene sequence against the primerbank Sequence DB.
http://pga.mgh.harvard.edu/primerbank/
The Quantitative PCR Primer Database (QPPD) provides information about primers and probes that can be used to quantitate human and mouse mRNA by reverse transcription polymerase chain reaction (RT–PCR) assays. All data has been gathered from published articles, cited in PubMed.
http://web.ncifcrf.gov/rtp/gel/primerdb/
RTPrimerDB is a public database for primer and probe sequences used in real-time PCR assays employing popular chemistries (SYBR Green I, Taqman, Hybridisation Probes, Molecular Beacon) to prevent time-consuming primer design and experimental optimisation, and to introduce a certain level of uniformity and standardisation among different laboratories.
http://medgen.ugent.be/rtprimerdb/
This database provides qRT PCR primers for 99.96% human RefSeq sequences.
http://primerdepot.nci.nih.gov/
The GSC Resequencing Primer Database stores PCR and sequencing primers designed to amplify and sequence annotated genes. Currently, primers have been designed for more than fourteen-thousand known or predicted human genes.
http://genome.wustl.edu/activity/med_seq/primer_db.cgi
The NCBI Probe Database is a public registry of nucleic acid reagents designed for use in a wide variety of biomedical research applications, together with information on reagent distributors, probe effectiveness, and computed sequence similarities.
http://www.ncbi.nlm.nih.gov/sites/entrez?db=probe