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
Calculate molar conversions, concentration conversions, and spectrophotometric conversions.
www.fermentas.com/reviewer/app?page=Calculator&service=page
Formulas for conversion and concentration determination for oligonucleotides and DNA
GeneInfinity
PCR Primer Design Guides
PCR Primer Design Guidelines at PremierBiosoft
www.premierbiosoft.com/tech_notes/PCR_Primer_Design.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
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
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
AiO: a program integrating Oligo ordering/Data Base and DNA/Protein programs.
http://132.187.163.191/aio/text.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