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Developmental validation of the AmpFℓSTR® SEfiler Plus™ PCR amplification kit: An improved multiplex with enhanced performance for inhibited samples
Available online at www.sciencedirect.com
Forensic Science International: Genetics Supplement Series 1 (2008) 121–122 www.elsevier.com/locate/FSIGSS
Research article
Developmental validation of the AmpF‘STR1 SEfiler PlusTM PCR amplification kit: An improved multiplex with enhanced performance for inhibited samples Julio J. Mulero *, Nicola J. Oldroyd, Michael T. Malicdem, Lori K. Hennessy Applied Biosystems, Human Identification Group, 850 Lincoln Centre Drive, Foster City, CA 94404, United States Received 6 September 2007; received in revised form 16 October 2007; accepted 25 October 2007
Abstract Since its introduction in 2002, the AmpF‘STR1 SEfilerTM kit has provided a highly discriminating DNA profiling option to German forensic laboratories by combining the widely used SGM Plus1 Kit loci with the SE-33 locus required for the German DNA Database. Whilst proving successful on database samples, laboratories using the SEfilerTM kit have reported the need for chemistry better able to handle the ever-increasing number of casework samples. The new AmpF‘STR1 SEfiler PlusTM kit contains the same loci and primer sequences as the SEfilerTM kit but uses improved synthesis and purification processes to minimize the presence of dye-labeled artifacts. Other improvements include modified PCR cycling conditions for enhanced sensitivity and a new buffer formulation that improves performance with inhibited samples when compared to the original SEfilerTM kit. Validation studies demonstrating the effectiveness of the multiplex are presented with emphasis on the models of inhibition and casework samples. # 2008 Elsevier Ireland Ltd. All rights reserved. Keywords: STR; AmpF‘STR1 SEfilerTM; Hematin; Humic acid
1. Introduction The new AmpF‘STR1 SEfiler PlusTM kit simultaneously coamplifies the seven loci of the European Standard Set (ESSL) (D3S1358, vWA, D8S1179, TH01, FGA, D21S11, and D18S51), the Amelogenin locus, the highly polymorphic SE33 (ACTBP2) locus, and the D2S1338, D16S539, and D19S433 loci. The developmental validation was performed according to the DNA Advisory Board (DAB) Quality Assurance Standards (DAB, 1998) and the revised guidelines from the Scientific Working Group on DNA Analysis Methods (SWGDAM, July 2003). 2. Materials and methods The SEfiler PlusTM PCR amplifications were performed in a reaction volume of 25 ml using the following PCR conditions: * Corresponding author. Tel.: +1 650 554 3405; fax: +1 650 554 2774. E-mail address: [email protected] (J.J. Mulero). 1875-1768/$ – see front matter # 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.fsigss.2007.10.010
95 8C for 11 min, followed by 30 cycles of denaturation at 94 8C for 20 s, annealing at 59 8C for 2 min, and extension at 72 8C for 1 min. A final extension was performed at 60 8C for 60 min. 3. Results and discussion DNA samples from crime scenes may contain inhibitors that can affect amplification of DNA samples. For example, heme compounds have been identified as PCR inhibitors in DNA samples extracted from bloodstains [1]. The effect of hematin on the amplification efficiency of the SEfilerTM and SEfiler PlusTM kits was examined by varying concentrations of hematin (0–55 mM) in the PCR. Overall inhibition of the SEfiler kit PCR reactions was observed as the concentration of hematin increased to 30 mM (Table 1). Complete PCR inhibition for the SEfilerTM kit was seen starting at 45 mM. However, informative profiles were reproducibly obtained with the SEfiler PlusTM kit up to 55 mM concentrations. Forensic evidence collected from soil may result in the coextraction of other soil components, such as humic acids (HA) which negatively interfere with DNA amplification processes
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J.J. Mulero et al. / Forensic Science International: Genetics Supplement Series 1 (2008) 121–122 Table 1 Amplification efficiency of control DNA 007 in the presence of increasing amounts of hematin (N = 3) Hematin (mM)
SEfiler PlusTM kit
SEfilerTM kit
0 30 45 55
1.0 1.0 0.96 0.07 0.64 0.39
1.0 0.17 0.14 0 0
A full profile consists of 24 amplification products (24/24 = 1.0). The values are expressed as the mean S.D. Thirty cycles of amplification were used for both kits.
Table 2 Amplification efficiency of control DNA 007 in the presence of increasing amounts of humic acid (N = 3) Humic acid (ng/ml)
SEfiler PlusTM kit
SEfilerTM kit
0 20 40 60
1.0 1.0 0.93 0.09 0.40 0.09
1.0 0 0 0
A full profile consists of 24 amplification products (24/24 = 1.0). The values are expressed as the mean S.D. Thirty cycles of amplification were used for both kits.
Acknowledgement The authors would like to thank our collaborator Dr. Peter Schneider for providing the casework data. Funding source TM
Fig. 1. Swab of automobile steering wheel. The SEfiler kit failed to amplify the D2S1338, SE33 and D18S51 loci while the SEfiler PlusTM kit recovered information from all loci. The circles identify the loci missing from the SEfilerTM kit profile but amplified by the SEfiler PlusTM kit.
[2]. The influence of increasing amounts of HA (0–60 ng/ml) on the amplification of DNA with the SEfilerTM and SEfiler PlusTM kits was examined. Complete inhibition of the SEfilerTM kit was observed as the concentration of HA reached 20 ng/ml (Table 2). Informative profiles were reproducibly achieved by the SEfiler PlusTM kit even at a HA concentration of 60 ng/ml. A performance comparison using DNA extracted from a swab of an automobile steering wheel demonstrates how the SEfiler PlusTM kit yields additional information when compared to the SEfilerTM kit (Fig. 1). In summary, testing of simulated and actual casework samples have demonstrated that the SEfiler PlusTM kit produces results which were not obtained using the current SEfilerTM kit.
The funding was provided by Applied Biosystems. The sponsor was involved in the study design; collection, analysis and interpretation of data; the writing of the manuscript and the decision to submit the manuscript for publication. Conflict of interest None. References [1] A. Akane, K. Matsubara, H. Nakamura, S. Takahashi, K. Kimura, Identification of the heme compound copurified with deoxyribonucleic acid (DNA) from bloodstains, a major inhibitor of polymerase chain reaction (PCR) amplification, J. Forensic Sci. 39 (1994) 362–372. [2] D. Sutlovic, M. Definis Gojanovic, S. Andelinovic, D. Gugic, D. Primorac, Taq polymerase reverses inhibition of quantitative real time polymerase chain reaction by humic acid, Croat. Med. J. 46 (4) (2005) 556–562.
Forensic Science International: Genetics Supplement Series 1 (2008) 121–122 www.elsevier.com/locate/FSIGSS
Research article
Developmental validation of the AmpF‘STR1 SEfiler PlusTM PCR amplification kit: An improved multiplex with enhanced performance for inhibited samples Julio J. Mulero *, Nicola J. Oldroyd, Michael T. Malicdem, Lori K. Hennessy Applied Biosystems, Human Identification Group, 850 Lincoln Centre Drive, Foster City, CA 94404, United States Received 6 September 2007; received in revised form 16 October 2007; accepted 25 October 2007
Abstract Since its introduction in 2002, the AmpF‘STR1 SEfilerTM kit has provided a highly discriminating DNA profiling option to German forensic laboratories by combining the widely used SGM Plus1 Kit loci with the SE-33 locus required for the German DNA Database. Whilst proving successful on database samples, laboratories using the SEfilerTM kit have reported the need for chemistry better able to handle the ever-increasing number of casework samples. The new AmpF‘STR1 SEfiler PlusTM kit contains the same loci and primer sequences as the SEfilerTM kit but uses improved synthesis and purification processes to minimize the presence of dye-labeled artifacts. Other improvements include modified PCR cycling conditions for enhanced sensitivity and a new buffer formulation that improves performance with inhibited samples when compared to the original SEfilerTM kit. Validation studies demonstrating the effectiveness of the multiplex are presented with emphasis on the models of inhibition and casework samples. # 2008 Elsevier Ireland Ltd. All rights reserved. Keywords: STR; AmpF‘STR1 SEfilerTM; Hematin; Humic acid
1. Introduction The new AmpF‘STR1 SEfiler PlusTM kit simultaneously coamplifies the seven loci of the European Standard Set (ESSL) (D3S1358, vWA, D8S1179, TH01, FGA, D21S11, and D18S51), the Amelogenin locus, the highly polymorphic SE33 (ACTBP2) locus, and the D2S1338, D16S539, and D19S433 loci. The developmental validation was performed according to the DNA Advisory Board (DAB) Quality Assurance Standards (DAB, 1998) and the revised guidelines from the Scientific Working Group on DNA Analysis Methods (SWGDAM, July 2003). 2. Materials and methods The SEfiler PlusTM PCR amplifications were performed in a reaction volume of 25 ml using the following PCR conditions: * Corresponding author. Tel.: +1 650 554 3405; fax: +1 650 554 2774. E-mail address: [email protected] (J.J. Mulero). 1875-1768/$ – see front matter # 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.fsigss.2007.10.010
95 8C for 11 min, followed by 30 cycles of denaturation at 94 8C for 20 s, annealing at 59 8C for 2 min, and extension at 72 8C for 1 min. A final extension was performed at 60 8C for 60 min. 3. Results and discussion DNA samples from crime scenes may contain inhibitors that can affect amplification of DNA samples. For example, heme compounds have been identified as PCR inhibitors in DNA samples extracted from bloodstains [1]. The effect of hematin on the amplification efficiency of the SEfilerTM and SEfiler PlusTM kits was examined by varying concentrations of hematin (0–55 mM) in the PCR. Overall inhibition of the SEfiler kit PCR reactions was observed as the concentration of hematin increased to 30 mM (Table 1). Complete PCR inhibition for the SEfilerTM kit was seen starting at 45 mM. However, informative profiles were reproducibly obtained with the SEfiler PlusTM kit up to 55 mM concentrations. Forensic evidence collected from soil may result in the coextraction of other soil components, such as humic acids (HA) which negatively interfere with DNA amplification processes
122
J.J. Mulero et al. / Forensic Science International: Genetics Supplement Series 1 (2008) 121–122 Table 1 Amplification efficiency of control DNA 007 in the presence of increasing amounts of hematin (N = 3) Hematin (mM)
SEfiler PlusTM kit
SEfilerTM kit
0 30 45 55
1.0 1.0 0.96 0.07 0.64 0.39
1.0 0.17 0.14 0 0
A full profile consists of 24 amplification products (24/24 = 1.0). The values are expressed as the mean S.D. Thirty cycles of amplification were used for both kits.
Table 2 Amplification efficiency of control DNA 007 in the presence of increasing amounts of humic acid (N = 3) Humic acid (ng/ml)
SEfiler PlusTM kit
SEfilerTM kit
0 20 40 60
1.0 1.0 0.93 0.09 0.40 0.09
1.0 0 0 0
A full profile consists of 24 amplification products (24/24 = 1.0). The values are expressed as the mean S.D. Thirty cycles of amplification were used for both kits.
Acknowledgement The authors would like to thank our collaborator Dr. Peter Schneider for providing the casework data. Funding source TM
Fig. 1. Swab of automobile steering wheel. The SEfiler kit failed to amplify the D2S1338, SE33 and D18S51 loci while the SEfiler PlusTM kit recovered information from all loci. The circles identify the loci missing from the SEfilerTM kit profile but amplified by the SEfiler PlusTM kit.
[2]. The influence of increasing amounts of HA (0–60 ng/ml) on the amplification of DNA with the SEfilerTM and SEfiler PlusTM kits was examined. Complete inhibition of the SEfilerTM kit was observed as the concentration of HA reached 20 ng/ml (Table 2). Informative profiles were reproducibly achieved by the SEfiler PlusTM kit even at a HA concentration of 60 ng/ml. A performance comparison using DNA extracted from a swab of an automobile steering wheel demonstrates how the SEfiler PlusTM kit yields additional information when compared to the SEfilerTM kit (Fig. 1). In summary, testing of simulated and actual casework samples have demonstrated that the SEfiler PlusTM kit produces results which were not obtained using the current SEfilerTM kit.
The funding was provided by Applied Biosystems. The sponsor was involved in the study design; collection, analysis and interpretation of data; the writing of the manuscript and the decision to submit the manuscript for publication. Conflict of interest None. References [1] A. Akane, K. Matsubara, H. Nakamura, S. Takahashi, K. Kimura, Identification of the heme compound copurified with deoxyribonucleic acid (DNA) from bloodstains, a major inhibitor of polymerase chain reaction (PCR) amplification, J. Forensic Sci. 39 (1994) 362–372. [2] D. Sutlovic, M. Definis Gojanovic, S. Andelinovic, D. Gugic, D. Primorac, Taq polymerase reverses inhibition of quantitative real time polymerase chain reaction by humic acid, Croat. Med. J. 46 (4) (2005) 556–562.