Evaluation of a quadruplex short tandem repeat system (HUMVWA31/A, HUMD11S554, HUMAPOAI1, and HUMACTBP2 loci) for forensic identity testing, confident typing of complex alleles, and population databases

Aim. To evaluate a quadruplex short tandem repeat (STR) system based on the amplification of allelic products from HUMVWA31/A, HUMD11S554, HUMACTBP2, and HUMAPOAI1 loci for routine identity testing in forensic casework, and to provide details for the construction of a "multiplex allelic ladder" for confident typing of complex alleles from the quadruplex STR system and compiling a database for three representative populations living in Northern Norway (Norwegian, Saami, and Russian/Ukrainian). Methods. The constructed "multiplex allelic ladder" consisted of 44 regularly spaced and uniform alleles, spanning from 135 to 333 bp, and included 9 HUMVWA31/A alleles (TCTA repeat unit), 9 HUMD11S554 alleles (AAAG repeat unit), and 26 HUMACTBP2 alleles (AAAG repeat unit). Confident typing of selected complex alleles from the quadruplex STR system was secured only when they were compared with their matching ladder composed of the same repetitive unit as in our constructed "multiplex allelic ladder". Using different fluorescent dyes, PCR products from the quadruplex STR system were sized with constructed allelic ladder (red labeled) in one lane. Results. Population database was established for unrelated Norwegians (n=141), Saami (n=78), and Russians/Ukrainians (n=138). Combined paternity exclusions were 99.86%, 99.94%, 99.95% and the values of probability of match were 8.0x10^-8, 1.4x10^-8, and 1.1x10^-8, in Saami, Norwegians, and Russians/Ukrainians, respectively. Conclusion. We conclude that by applying our constructed allelic ladder a confident typing of alleles from our quadruplex STR system may be ensured. Further, the high efficiency of the selected loci makes this system a reliable tool for use in forensic casework.