Validation of a male-specific, 12-locus fluorescent short tandem repeat (STR) multiplex

Benjamin E. Krenke, Lori Viculis, Melanie L. Richard, Mechthild Prinz, Scott C. Milne, Carll Ladd, Ann Marie Gross, Tanis Gornall, J. Roger H. Frappier, Arthur J. Eisenberg, Charles Barna, Xavier G. Aranda, Michael S. Adamowicz, Bruce Budowle

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Abstract

Y chromosome-specific short tandem repeat (Y-STR) analysis has become another widely accepted tool for human identification. The PowerPlex ® Y System is a fluorescent multiplex that includes the 12 loci: DYS19, DYS385a/b, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438 and DYS439. This panel of markers incorporates the 9-locus European minimal haplotype (EMH) loci recommended by the International Y-STR User Group and the 11-locus set recommended by the Scientific Working Group on DNA Analysis Methods (SWGDAM). Described here are inter-laboratory results from 17 developmental validation studies of the PowerPlex ® Y System and include the following results: (a) samples distributed between laboratories and commercial standards produced expected and reproducible haplotypes; (b) use of common amplification and detection instruments were successfully demonstrated; (c) full profiles were obtained with standard 30 and 32 cycle amplification protocols and cycle number (24-28 cycles) could be modified to match different substrates (such as direct amplification of FTA ® paper); (d) complete profiles were observed with reaction volumes from 6.25 to 50 μL; (e) minimal impact was observed with variation of enzyme concentration; (f) full haplotypes were observed with 0.5-2x primer concentrations; however, relative yield between loci varied with concentration; (g) reduction of magnesium to 1 mM (1.5 mM standard) resulted in minimal amplification, while only partial loss of yield was observed with 1.25 mM magnesium; (h) decreasing the annealing temperature by 2-4°C did not generate artifacts or locus dropout and most laboratories observed full amplification with the annealing temperature increased by 2°C and significant locus dropout with a 4°C increase in annealing temperature; (i) amplification of individual loci with primers used in the multiplex produced the same alleles as observed with the multiplex amplification; (j) all laboratories observed full amplification with ≥125 pg of male template with partial and/or complete profiles observed using 30-62.5 pg of DNA; (k) analysis of ≤500 ng of female DNA did not yield amplification products; (l) the minor male component of a male/female mixture was observed with ≤1200-fold excess female DNA with the majority of alleles still observed with 10,000-fold excess female; (m) male/male mixtures produced full profiles from the minor contributor with 10-20-fold excess of the major contributor; (n) average stutter for each locus; (o) precision of sizing were determined; (p) human-specificity studies displayed amplification products only with some primate samples; and (q) reanalysis of 102 non-probative casework samples from 65 cases produced results consistent with original findings and in some instances additional identification of a minor male contributor to a male/female mixture was obtained. In general, the PowerPlex ® Y System was shown to have the sensitivity, specificity and reliability required for forensic DNA analysis.

Original languageEnglish
Pages (from-to)1-14
Number of pages14
JournalForensic Science International
Volume148
Issue number1
DOIs
StatePublished - 10 Feb 2005

Keywords

  • DNA typing
  • European minimal haplotype
  • Forensic science
  • Polymerase chain reaction (PCR)
  • PowerPlex
  • Short tandem repeat (STR)
  • Validation
  • Y chromosome

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    Krenke, B. E., Viculis, L., Richard, M. L., Prinz, M., Milne, S. C., Ladd, C., Gross, A. M., Gornall, T., Frappier, J. R. H., Eisenberg, A. J., Barna, C., Aranda, X. G., Adamowicz, M. S., & Budowle, B. (2005). Validation of a male-specific, 12-locus fluorescent short tandem repeat (STR) multiplex. Forensic Science International, 148(1), 1-14. https://doi.org/10.1016/j.forsciint.2004.07.008