AutoDimer: Software Developed to Enable Rapid Multiplex PCR Design


Participants: Peter M. Vallone and John M. Butler


Project Timeframe: 2000-2005


Purpose: To make a NIST-developed software tool for primer design freely available to the DNA diagnostic community.


Progress: The ability to rapidly screen short DNA oligonucleotides for non-specific interactions is essential for developing nucleic acid-based diagnostic tests.  When designing primers or probes for multiplex applications such as polymerase chain reaction (PCR), primer extension, and specific hybridization events, having the means to efficiently intercompare the primers/probes is useful.  The Web-based primer selection program Primer3 currently selects PCR primers for generating a single amplicon.  The selected forward and reverse primer sequences are screened for potential primer-dimer and intramolecular hairpin formation.  Algorithms for screening complementarity between short DNA oligomers have been described.  Various free and commercially available software packages for the selection of DNA probes and PCR primers employ variations on these basic algorithms for determining complementarity.  The primary purpose of AutoDimer is to screen sets of pre-selected PCR primer pairs (ranging from 2 up to 1,000 primer sequences) for potential cross reactivity.  The program performs primer-primer intercomparisons while evaluating interactions according to traditional Watson-Crick base pairing rules.  The results can be visually inspected or saved to a text file.  The information output consists of a visual component along with a score that represents the degree of interaction.  Predictions of the transition melting temperature (Tm) and free energy of melting (ΔG) are calculated for each of the potential primer-primer interactions.


The AutoDimer is available at .  Approximately 1 year after making the software publicly available, nearly 2,000 researchers had downloaded it.  AutoDimer has also been developed into a Web-based tool with the help of National Institute of Justice (NIJ) funding and the expertise of a software contactor (iSYS LLC). This web-based tool is now available at AutoDimer has enabled the NIST Human Identity Testing Project Team to develop a number of useful multiplex PCR assays to aid the human identity testing community and this tool is now being made available to others (see



Publications or Presentations Resulting from this Project:

Vallone, P.M., and Butler, J.M. (2004) AutoDimer: a screening tool for primer-dimer and hairpin structures. Biotechniques 37(2): 226-231.

Vallone, P.M., Just, R.S., Coble, M.D., Butler, J.M., and Parsons, T.J. (2004) A multiplex allele-specific primer extension assay for forensically informative SNPs distributed throughout the mitochondrial genome. Int. J. Legal Med. 118: 147-157.

Schoske, R., Vallone, P.M., Ruitberg, C.M., and Butler, J.M. (2003) Multiplex PCR design strategy used for the simultaneous amplification of 10 Y chromosome short tandem repeat (STR) loci. Anal. Bioanal. Chem. 375: 333-343.


Butler, J.M., Schoske, R., Vallone, P.M., Kline, M.C., Redd, A.J., and Hammer, M.F. (2002) A novel multiplex for simultaneous amplification of 20 Y chromosome STR markers. Forensic Sci. Int. 129: 10-24.



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Last updated: 06/27/2007


Disclaimer: This project was supported by National Institute of Justice Grant Number 2003-IJ-R-029, which is an interagency agreement between NIJ and the NIST Office of Law Enforcement Standards, awarded by the National Institute of Justice, Office of Justice Programs, US Department of Justice. Points of view in this document are those of the authors and do not necessarily represent the official position or policies of the US Department of Justice. Certain commercial equipment, instruments and materials are identified in order to specify experimental procedures as completely as possible.  In no case does such identification imply a recommendation or endorsement by the National Institute of Standards and Technology nor does it imply that any of the materials, instruments or equipment identified are necessarily the best available for the purpose.