title: Validation of male-specific, 12-locus fluorescent short tandem repeat (STR) multiplex.
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 microL; (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 1mM (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 degrees C did not generate artifacts or locus dropout and most laboratories observed full amplification with the annealing temperature increased by 2 degrees C and significant locus dropout with a 4 degrees 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 >or = 125 pg of male template with partial and/or complete profiles observed using 30-62.5 pg of DNA; (k) analysis of < or = 500 ng of female DNA did not yield amplification products; (l) the minor male component of a male/female mixture was observed with < or =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.
authors.affiliation: Promega Corporation, Genetic Analysis Research and Development, Madison, WI 53716, USA. benjamin.krenke@promega.com
authors.forename: Benjamin E
authors.initials: BE
authors.lastname: Krenke
authors.forename: Lori
authors.initials: L
authors.lastname: Viculis
authors.forename: Melanie L
authors.initials: ML
authors.lastname: Richard
authors.forename: Mechthild
authors.initials: M
authors.lastname: Prinz
authors.forename: Scott C
authors.initials: SC
authors.lastname: Milne
authors.forename: Carll
authors.initials: C
authors.lastname: Ladd
authors.forename: Ann Marie
authors.initials: AM
authors.lastname: Gross
authors.forename: Tanis
authors.initials: T
authors.lastname: Gornall
authors.forename: J Roger H
authors.initials: JR
authors.lastname: Frappier
authors.forename: Arthur J
authors.initials: AJ
authors.lastname: Eisenberg
authors.forename: Charles
authors.initials: C
authors.lastname: Barna
authors.forename: Xavier G
authors.initials: XG
authors.lastname: Aranda
authors.forename: Michael S
authors.initials: MS
authors.lastname: Adamowicz
authors.forename: Bruce
authors.initials: B
authors.lastname: Budowle
mesh_terms: D000818:Animals; D041322:Chromosomes, Human, Y; D016172:DNA Fingerprinting / Q000592:standards*; D017931:DNA Primers; D005260:Female; D005819:Genetic Markers; D005838:Genotype; D006801:Humans; D008297:Male; D016133:Polymerase Chain Reaction / Q000379:methods / Q000592:standards*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019849:Sex Determination Processes; D020080:Tandem Repeat Sequences; D013696:Temperature
chemical_list: D017931:DNA Primers; D005819:Genetic Markers