Y-STR Multiplex Assay Development


Participants: Richard Schoske, Peter M. Vallone, and John M. Butler


Project Timeframe: June 2000 to May 2003


Purpose: Y-chromosome short tandem repeat (Y-STR) markers have a number of applications in human identity testing including typing the perpetrator of sexual assault cases without differential extraction and tracing paternal lineages for missing persons investigations. In order for Y-STR systems to become more widely accepted within the forensic DNA typing community, robust multiplex assays are required. We have focused on the design and development of new multiplexes for typing STRs located on the Y-chromosome for human identification purposes.


Progress: In order to improve the power of discrimination for Y-chromosome tests, we developed strategies for rapidly preparing multiplex PCR assays that utilize both four and five dye chemistries for detection and permit simultaneous amplification of 20 or more Y-chromosome STR markers in a single reaction. An important design aspect of our multiplex assays is that PCR product sizes are kept under 350 bp in order to ensure a greater success with testing degraded DNA samples. During primer design, efforts were to avoid any homology with X-chromosome sequences. Primers have been redesigned from previously published work with these Y-STR markers in order to make them more compatible in a multiplex amplification. In addition, allele ranges for each of the Y-STR markers have been well characterized in a diverse set of world population samples. Our Y-STR multiplex assays paved the way for future release of the PowerPlex Y (released September 2003 by Promega Corporation) and Yfiler (released December 2004 by Applied Biosystems) commercial kits.


Publications or Presentations Resulting From This Project:

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


Schoske, R., Vallone, P.M., Ruitberg, C.M., 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.


Schoske, R. (2003) The design, optimization and testing of Y chromosome short tandem repeat megaplexes. PhD dissertation, American University, 270 pp.


Butler, J.M., Schoske, R., Vallone, P.M. Highly multiplexed assays for measuring polymorphisms on the Y-chromosome. (2003) Progress in Forensic Genetics 9 (Brinkmann, B. and Carracedo, A., eds.), Elsevier Science: Amsterdam, The Netherlands, International Congress Series 1239, pp. 301-305.


Schoske, R., Vallone, P.M., Kline, M.C., Redman, J.W., Butler, J.M. (2004) High-throughput Y-STR typing of U.S. populations with 27 regions of the Y chromosome using two multiplex PCR assays, Forensic Sci. Int. 139: 107-121.


Butler, J.M. (2005) Constructing STR multiplex assays. Methods in Molecular Biology: Forensic DNA Typing Protocols (Carracedo, A., ed.), Humana Press: Totowa, New Jersey, 297: 53-66. [preprint]


Schoske, R., Butler, J.M., Vallone, P.M., Kline, M.C., Prinz, M., Redd, A.J., Hammer, M.F. (2001) Development of Y STR megaplex assays. Proceedings of the Twelve International Symposium on Human Identification 2001, Promega Corporation. http://www.promega.com/geneticidproc/ussymp12proc/contents/butler.PDF


John Butler talk at 19th Congress of the International Society of Forensic Genetics (Munster, Germany), August 30, 2001, "Highly multiplexed assays for measuring polymorphisms on the Y-chromosome"


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Last updated: 06/19/2007


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.