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Multiplex PCR design strategy used for the simultaneous amplification of 10 Y chromosome short tandem repeat (STR) loci

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Abstract

The simultaneous amplification of multiple regions of a DNA template is routinely performed using the polymerase chain reaction (PCR) in a process termed multiplex PCR. A useful strategy involving the design, testing, and optimization of multiplex PCR primer mixtures will be presented. Other multiplex design protocols have focused on the testing and optimization of primers, or the use of chimeric primers. The design of primers, through the close examination of predicted DNA oligomer melting temperatures (T m) and primer–dimer interactions, can reduce the amount of testing and optimization required to obtain a well-balanced set of amplicons. The testing and optimization of the multiplex PCR primer mixture constructed here revolves around varying the primer concentrations rather than testing multiple primer combinations. By solely adjusting primer concentrations, a well-balanced set of amplicons should result if the primers were designed properly. As a model system to illustrate this multiplex design protocol, a 10-loci multiplex (10plex) Y chromosome short tandem repeat (STR) assay is used.

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Acknowledgements

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 or the US Department of Defense nor does it imply that any of the materials, instruments, or equipment identified are necessarily the best available for the purpose. The work described here was funded by National Institute of Justice (NIJ) research grants 1997–LB–VX–0003 to John Butler through the NIST Office of Law Enforcement Standards. Richard Schoske is a PhD candidate in chemistry at American University under Professor Jim Girard and is funded by the United States Air Force, through the Air Force Institute of Technology. The technical assistance of Margaret Kline is greatly appreciated for extracting and providing the DNA samples used in this study. The authors also thank David Duewer for his helpful review of this manuscript.

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Authors and Affiliations

  1. Department of Chemistry, American University, 4400 Massachusetts Ave. NW, Washington DC , 20016, USA

    Richard Schoske

  2. Biotechnology Division, National Institute of Standards and Technology, 100 Bureau Drive, Mail Stop 8311, Gaithersburg, MD 20899 , USA

    Richard Schoske, Pete M. Vallone, Christian M. Ruitberg & John M. Butler

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  1. Richard Schoske
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  2. Pete M. Vallone
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  3. Christian M. Ruitberg
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  4. John M. Butler
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Correspondence to Richard Schoske.

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Schoske, R., Vallone, P.M., Ruitberg, C.M. et al. Multiplex PCR design strategy used for the simultaneous amplification of 10 Y chromosome short tandem repeat (STR) loci. Anal Bioanal Chem 375, 333–343 (2003). https://doi.org/10.1007/s00216-002-1683-2

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  • DOI: https://doi.org/10.1007/s00216-002-1683-2

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