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


The AmpFℓSTR 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 MgCl2 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°C). Additionally, DNA from primates, non-primates and microorganisms were amplified to investigate the specificity of the kit. The stability of the AmpFℓSTR 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 6.47×10−15 for African Americans and 7.46×10−14 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 AmpFℓSTR SEfiler PCR amplification kit can be used to successfully analyze a variety of forensic, databasing and paternity samples.

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The authors would like to thank the members of the Human Identification group at Applied Biosystems for their comments. The authors would specifically like to thank Dr. Dennis Reeder for his useful suggestions.

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Correspondence to Sulekha Rao Coticone.

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For research, forensic or paternity use only. Not for use in diagnostic procedures.

The PCR process is covered by patents owned by Roche Molecular Systems, Inc. and F. Hoffmann-La Roche Ltd.

Applied Biosystems, ABI PRISM, AmpFSTR, GeneScan, Genotyper, LIZ, MicroAmp, SGM Plus, and VIC are registered trademarks and FAM, Hi-Di, Identifiler, NED, PET, POP, POP-4, and ROX are trademarks of Applera Corporation or its subsidiaries in the US and certain other countries.

AmpliTaq Gold, GeneAmp, and QuantiBlot are registered trademarks of Roche Molecular Systems, Inc.

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Coticone, S.R., Oldroyd, N., Philips, H. et al. Development of the AmpFℓSTR SEfiler PCR amplification kit: a new multiplex containing the highly discriminating ACTBP2 (SE33) locus. Int J Legal Med 118, 224–234 (2004).

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  • SE33
  • PCR
  • Multiplex
  • Development
  • Forensic samples