Notes: In 1991, the remains of murdered Emperor Nicholas II, Empress Alexandra and three of their five children were discovered. Until recently, the remains of the two other children were never found. In July of 2007 human bone fragments were discovered at a second grave site in the Ural region of Russia. The authors performed DNA typing to determine if these remains were those of the two missing children. Bone fragments and teeth were subjected to mitochondrial and nuclear DNA typing. DNA was quantitated using the Plexor® HY System. Nuclear DNA analysis was performed, in part, using the PowerPlex® S5 System. A comparison of mitochondrial DNA sequences from remains in the first and second graves and from maternal reference samples confirmed that the remains constituted a family with a "Queen Victoria" mitotype (Empress Alexandra was the granddaughter of Queen Victoria). Y-STR analysis of both sets of remains was performed, and the results confirmed that the Y-STR haplotypes of the two sets of male remains matched, and this haplotype matched that of several descendants from an unbroken paternal lineage of Nicholas I, father of Nicholas II. The mitochondrial and Y-STR haplotypes and autosomal STR profile also matched those obtained from a bloodstained shirt that Nicholas II was wearing during an assassination attempt. (3967)

Notes: The authors describe the developmental validation of the Plexor^® HY System, a quantitative PCR assay that simultaneously quantifies total human and male DNA. Validation studies examined: (1) human specificity, (2) sensitivity, (3) quantitation of degraded DNA, (4) impact of inhibitors, (5) male/female mixture and Y-assay male specificity, (6) reproducibility and concordance and (7) population studies. (3969)