Amplicon

Software for designing PCR primers on aligned DNA sequences. A tool for molecular biologists.

Introduction
Oligonucleotide (primer) design for polymerase chain reaction (PCR) experiments is an important stage in successfully obtaining desired PCR products (amplicons). PCR primer design may be assisted by software that helps the user assess features of the oligonucleotides that determine how they may interact with each other and with target and non-target DNA sequences in a solution. The design of PCR primers where multiple target sequences may be present in a solution is a difficult problem. This situation has not been extensively investigated and is difficult to address with software solutions, especially given a surprising lack of knowledge relating to Tm calculation of mismatched oligonucleotide and target sequences.

Amplicon is a tool for designing PCR primers where groups of related DNA sequences can be assessed in aligned form. This is especially useful for designing 'group-specific' PCR primer sets that produce amplicons from a targer group of species, but not from other groups of species. Amplicon assists in group-specific PCR primer desing primarily by being a visualisation tool for alignments from which the user can select regions that may be group-specific primer binding sites. Other useful features that are not common in other PCR primer design software are handling of gapped sequences and degenerate sites in aligned DNA. Group-specific PCR primer sets are useful in a wide variety of situation where environmental DNA is being examined and the diversity or identity of some of the DNA sequences within a sample need to be determined. The software is described briefly in Jarman (2004) and the ideas behind group-specific PCR primer design and some reasons for needing it are described in Jarman et al. (2004).

Amplicon can import aligned DNA sequences produced by ClustalW or ClustalX (Thompson et al., 1997); or aligned sequences in MEGA (Kumar et al., 1997) or Fasta format. Primer analyses can be saved to text files for editing in any text editor or word processor. Alignments that have been altered in Amplicon can be saved in Fasta, MEGA, .aln, PAUP (Swofford, 2003) or Phylip (Felsenstein, 1993) formats.

Implementation
Amplicon is written in Python 2.3 and utilises the Tkinter graphics module version 8.4. Python scripts should run on any supported platform (www.python.org). An executable for Windows created using Thomas Heller's Py2exe (http://starship.python.net/crew/theller/py2exe/) is also available. Amplicon is currently beta software, meaning that it is expected to work reasonably well, but may still have bugs. The current release (version b09, July 2004) has only been tested by the author under Windows.

I have finally got hold of a Mac to test Amplicon on (September 2005) and am pleased to find that MacOSX.4.1 has Tkinter pre-installed, which will make Amplicon run relatively easily. I hope to produce a Mac version soon, although I would be grateful if anyone else would try this for me as I am trying to launch another software project for using DNA as a taxonomic identification tool (DNAID) and this is consuming a lot of time.

Bug reports, suggestions or requests for features can be directed to simon.jarman@aad.gov.au.

Downloads
The Amplicon script and Win32 executable can be downloaded from http://sourceforge.net/projects/amplicon/. A manual is included with each package.

References
Felsenstein J (1993) PHYLIP (Phylogeny Inference Package) version 3.5c. Distributed by the author. Department of Genetics, University of Washington, Seattle.

Jarman SN (2004) Amplicon: software for designing PCR primers on aligned DNA sequences. Bioinformatics 20: 1644-1645.

Jarman SN, Deagle BE, Gales NJ (2004) Group-specific polymerase chain reaction for DNA-based analysis of species diversity and identity in dietary samples. Molecular Ecology 13: 1313-1322.

Kumar S, Tamura K, Jakobsen IB & Nei M (2001) MEGA2: Molecular Evolutionary Genetic Analysis Software. Bioinformatics 17: 1244-1245.

Swofford DL (2003) PAUP*. Phylogenetic Analysis Using Parsimony (*and Other Methods). Version 4. Sinauer Associates, Sunderland, Massachusetts.

Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research 25: 4876-4882.