Validation of the AmpFℓSTR^® SEfiler Plus™ PCR Amplification kit for forensic STR analysis

Article Outline

• Abstract
• 1. Introduction
• 2. Materials and methods
• 3. Results and discussion
• Conflict of interest
• References
• Copyright

Abstract 

Validation of the AmpFℓSTR^® SEfiler Plus™ PCR Amplification kit with 29 and 30 PCR cycles for forensic STR analysis demonstrated that the kit had fewer artefacts than the AmpFℓSTR^® SGM Plus™ kit (28 PCR cycles). The SEfiler Plus kit was more sensitive and devoid of colour artefacts, but showed more stutters, drop-ins, drop-outs and allelic imbalances.

Keywords: Short tandem repeats (STRs), AmpFℓSTR^® SEfiler Plus™ PCR Amplification kit, Forensic genetics

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1. Introduction 

We tested the AmpFℓSTR^® SEfiler Plus™ PCR Amplification kit (Applied Biosystems, AB) that amplifies the same STR regions as the AmpFℓSTR^® SGM Plus™ PCR Amplification kit and is supplemented with the highly polymorphic STR system SE33 [1], [2], [3]. The colour chemistry and the buffer system were improved. The SEfiler Plus kit was validated by the manufacturer for 30 PCR cycles compared to 28 cycles for the SGM Plus kit. We compared the two kits regarding sensitivity, stutters, drop-ins, allele balances, drop-outs and dye artefacts.

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2. Materials and methods 

We investigated 15 crime case samples with dye artefacts in SGM Plus. Between 200 and 500pg DNA was amplified with SGM Plus (28 PCR cycles) and SEfiler Plus (28 and 30 PCR cycles, respectively) (AB), analyzed with the AB 3130xl Genetic Analyzer (AB) followed by GeneScan^® 3.7 and Genotyper^® 3.7 (AB) software analysis. The results of the SE33 locus obtained with SEfiler Plus were not considered. Genotyper analysis was performed with an allele detection threshold of 50 RFUs. The presence of dye artefacts was assessed.

Between 150 and 200pg DNA from 35 biological trace samples with single-donor STR profiles was amplified with 29 and 30 SEfiler Plus amplification cycles, respectively. The numbers of alleles present and the peak heights were detected. The allele balances in heterozygous STR systems were measured as the ratio between the RFUs of the minor and the major alleles. The investigations of allele balances included all results above 20 RFUs. Homozygous systems were not considered.

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3. Results and discussion 

Amplifications of negative template controls with SGM Plus revealed several dye artefacts (data not shown). The peaks were wider than normal allele peaks but might mask true alleles. With SEfiler Plus, the background level was significantly lower and no artefact was present at 28 and 30 PCR cycles. With 28 PCR cycles, the amplification efficiency was lower with SEfiler Plus than with SGM Plus. With 30 PCR cycles, SEfiler Plus generally yielded reproducible STR profiles with stronger average RFU signals than those of SGM Plus (28 PCR cycles).

Fig. 1 shows that the balances of STR alleles were almost similar with SEfiler Plus at 29 and 30 PCR cycles. Fig. 2 shows an SEfiler Plus STR profile (30 PCR cycles) with two allelic drop-outs. The peak heights of the corresponding alleles were 393 RFUs and 590 RFUs, respectively.

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• Fig. 1. 

  Allele balances of heterozygous STR loci with the AmpFℓSTR^® SEfiler Plus™ PCR Amplification kit. Results from 35 single-donar trace samples with 150–200pg DNA signals above 20 RFUs in positions of common alleles were included.

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• Fig. 2. 

  Allelic drop-outs with the AmpFℓSTR^® SEfiler Plus™ PCR Amplification kit. Amplification of 50pg AmpFℓSTR^® Control DNA 007 (AB). The expected alleles D21S11-28 and D18S51 (circles) were not detected.

Table 1 shows the numbers of locus and allele drop-outs as well as the numbers of unexpected allele calls (stutters/drop-ins).

Table 1. Allelic drop-outs, stutters and drop-ins with the AmpFℓSTR^® SEfiler Plus™ PCR Amplification kit.

Results of 35 single-donor trace samples.

We have used the SEfiler Plus kit for approximately 6 months for STR typing of crime case samples. Our experience is that the sensitivity of the SEfiler Plus kit at 30 PCR cycles is more than enough for routine case work, and that the price paid by increased allelic imbalance, likelihood of stutters, and drop-ins is not worth the increased sensitivity. In the future, we will use the SEfiler Plus kit with 29 PCR cycles for routine crime case work.

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Conflict of interest 

None.

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References 

1. Coticone SR, Oldroyd N, Philips H, Foxall P. Development of the AmpFℓSTR SEfiler PCR amplification kit: a new multiplex containing the highly discriminating ACTBP2 (SE33) locus. International Journal of Legal Medicine. 2004;118:224–234
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2. Mulero JJ, Oldroyd NJ, Malicdem MT, Hennesey LK. Developmental validation of the AmpFℓSTR^® SEfiler Plus™ PCR Amplification kit: an improved multiplex with enhanced performance for inhibited samples. Forensic Science International: Genetics Supplement Series. 2008;1:121–122
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3. Schneider PM, Förster G, Diesch S, Schmitt C, Rothschild MA. Improved STR genotyping results from challenging casework samples in Germany using the AmpFℓSTR® SEfiler Plus™ PCR Amplification kit, Applied Biosystems. Forensic News. 2008;February http://marketing.appliedbiosystems.com/images/All_Newsletters/Forensic_Vol13/docs/53144_FN_Customer_Corner_c.pdf
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