Development of the AmpF?STR SEfiler PCR amplification kit: a new multiplex containing the highly discriminating ACTBP2 (SE33) locus

Abstract

The AmpFISTR SEfiler kit co-amplifies 11 short tandem repeat loci including SE33 in a single multiplex. After establishing the optimum in primer titration studies, the primer concentrations of all loci in the multiplex were chosen such that the heterozygote peak height ratios of each of the loci were balanced. The combined primer set was then tested to determine the robustness of the multiplex under various conditions. Different MgCl(2) concentrations were evaluated to establish the optimum concentration for the multiplex. The amplification of the various loci in the multiplex was tested at several annealing temperatures (55-63 degrees C). Additionally, DNA from primates, non-primates and microorganisms were amplified to investigate the specificity of the kit. The stability of the AmpFISTR SEfiler kit was determined by addition of hematin, to simulate inhibition, and the use of degraded DNA. Population studies revealed a probability of identity of 6.47x10(-15) for African Americans and 7.46x10(-14) for US Caucasians. To assess the ability of the multiplex to analyze forensic samples, testing on blood, oral swabs and mixtures was performed. Based on the various studies, it was determined that the AmpFISTR SEfiler PCR amplification kit can be used to successfully analyze a variety of forensic, databasing and paternity samples.

Characterisation of variant alleles in the STR systems D2S1338, D3S1358 and D19S433

“…The mixture was incubated for 30 min at 56°C and then boiled for 8 min. D3S1358, D2S1338 and D19S433 were amplified using commercially available kits (AmpFlSTR SGMplus [7] and SEfiler [8], Applied Biosystems, Darmstadt, Germany; genRES MPX-3 [9], SERAC, Bad Homburg, Germany) and analyzed on ABI PRISM 310 and 3100 Genetic Analyzers with Genotyper 2.5.2 (Applied Biosystems). For sequencing, the samples were amplified using published primer sequences ( [10]; Genbank/GDB accession nos.…”

Development and validation of I-DNA1: a 15-Loci multiplex system for identity testing

“…They include a growing number of STR loci [5][6][7][8][9][10][11][12]. Moreover, multiplex polymerase chain reactions (PCRs) have been developed, which produce small amplicons to increase the likelihood of success in the analysis of highly degraded DNA samples [13][14][15][16][17][18][19][20].…”

Short amplicon STR multiplex for stain typing

“…The amplicon length, the number of loci to be harmonized in a one-tube reaction, and the different sensitivities of the fluorescent dyes have an influence [5][6][7][8][9][10].…”