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Direct amplification of reference samples with Globalfiler® PCR Amplification Kit

  • N. Gouveia
    Affiliations
    Forensics Genetic and Biology Service, Central Delegation, National Institute of Legal Medicine and Forensic Sciences, I.P., Coimbra, Portugal

    Faculty of Sciences, University of Lisbon, Lisbon, Portugal
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  • P. Brito
    Affiliations
    Forensics Genetic and Biology Service, Central Delegation, National Institute of Legal Medicine and Forensic Sciences, I.P., Coimbra, Portugal

    CENCIFOR, Forensic Sciences Centre, Coimbra, Portugal
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  • A. Serra
    Affiliations
    Forensics Genetic and Biology Service, Central Delegation, National Institute of Legal Medicine and Forensic Sciences, I.P., Coimbra, Portugal
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  • F. Balsa
    Affiliations
    Forensics Genetic and Biology Service, Central Delegation, National Institute of Legal Medicine and Forensic Sciences, I.P., Coimbra, Portugal

    CENCIFOR, Forensic Sciences Centre, Coimbra, Portugal
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  • L. Andrade
    Affiliations
    Forensics Genetic and Biology Service, Central Delegation, National Institute of Legal Medicine and Forensic Sciences, I.P., Coimbra, Portugal

    CENCIFOR, Forensic Sciences Centre, Coimbra, Portugal
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  • M.São Bento
    Affiliations
    Forensics Genetic and Biology Service, Central Delegation, National Institute of Legal Medicine and Forensic Sciences, I.P., Coimbra, Portugal

    CENCIFOR, Forensic Sciences Centre, Coimbra, Portugal
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  • P. Cunha
    Affiliations
    Forensics Genetic and Biology Service, Central Delegation, National Institute of Legal Medicine and Forensic Sciences, I.P., Coimbra, Portugal
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  • V. Bogas
    Affiliations
    Forensics Genetic and Biology Service, Central Delegation, National Institute of Legal Medicine and Forensic Sciences, I.P., Coimbra, Portugal

    CENCIFOR, Forensic Sciences Centre, Coimbra, Portugal
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  • V. Lopes
    Affiliations
    Forensics Genetic and Biology Service, Central Delegation, National Institute of Legal Medicine and Forensic Sciences, I.P., Coimbra, Portugal

    CENCIFOR, Forensic Sciences Centre, Coimbra, Portugal
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  • M.J. Porto
    Correspondence
    Corresponding author at: Forensic Genetics, National Institute of Legal Medicine and Forensic Sciences, Largo da Sé Nova, 3000-213 Coimbra, Portugal.
    Affiliations
    Forensics Genetic and Biology Service, Central Delegation, National Institute of Legal Medicine and Forensic Sciences, I.P., Coimbra, Portugal

    CENCIFOR, Forensic Sciences Centre, Coimbra, Portugal
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Published:September 17, 2015DOI:https://doi.org/10.1016/j.fsigss.2015.09.055

      Abstract

      The GlobalFiler® PCR Amplification system uses 6-dye fluorescent chemistry to enable multiplexing of 21 autosomal STRs, 1 Y-STR, 1 Y-indel and the sex-determining marker amelogenin. Validation studies were conducted to assess the performance of the GlobalFiler® casework kit for typing reference samples through a direct amplification. The findings support that the GlobalFiler® PCR Amplification Kit produces reliable results from blood and buccal reference DNA samples.

      Keywords

      1. Introduction

      The GlobalFiler® PCR Amplification system uses 6-dye fluorescent chemistry to enable multiplexing of 21 autosomal STRs, 1 Y-STR, 1 Y-indel and the sex-determining marker amelogenin. The kit contains all of the markers included in the global databases and an enhanced buffer system, designed to reduce amplification time, increase discrimination power and maximize information recovery [
      • Flores S.
      • et al.
      Internal validation of the GlobalFiler™ Express PCR Amplification Kit for the direct amplification of reference DNA samples on a high-throughput automated workflow.
      ,
      • Martín P.
      • et al.
      Improving DNA data exchange: validation studies on a single 6 dye STR kit with 24 loci.
      ,

      Applied Biosystems (2014). GlobalFiler® PCR Amplification Kit User Guide. Life Technologies.

      ]. It is divided into two separate kits: GlobalFiler® Express that is a kit specifically designed for processing reference DNA samples and GlobalFiler® which is optimized for use with the most challenging casework samples [

      Applied Biosystems (2014). GlobalFiler® PCR Amplification Kit User Guide. Life Technologies.

      ].
      In order to apply the same kit, GlobalFiler®, to reference and casework samples, validation studies were conducted to evaluate direct amplification in blood and buccal samples stored on FTA® cards.

      2. Materials and methods

      Eighteen blood and buccal DNA samples were collected on FTA® and FTA® indicating cards, respectively, and were allowed to dry at room temperature before using. For the validation study, 0.5, 0.75, 1 and 1.2 mm punches of each sample were added into the respective tube in a final reaction volume of 12.5 μL, consisting of 3.75 μL of GlobalFiler® Master Mix, 1.25 μL of GlobalFiler® Primer Set and 7.5 μL of low TE buffer (10 mM Tris–HCl, 0.1 mM EDTA, pH 8.0).
      Samples were amplified on a Veriti® 96-Well. Thermal cycling conditions consisted of an initial incubation step at 95 °C for 1 min, followed by 28 cycles of denaturation at 94 °C for 10 s and annealing/extension at 59 °C for 90 s. A final extension was set at 60 °C for 10 mins, followed by a final hold at 4 °C. Capillary electrophoresis was performed on the 3500 Genetic Analyzer according to manufacturer's instructions using GeneScan™ 600 LIZ® as internal size standard and HID files generated were analyzed with GeneMapper® ID-X Software v1.4.

      3. Results

      Electropherograms obtained from both types of reference samples using punches with a diameter equal or greater than 0.75 mm revealed some artifacts caused by the addition of too much DNA to the PCR reaction (Fig. 1). Such artifacts include background peaks that occur along the baseline, several split peaks and pronounced stutter peaks.
      Figure thumbnail gr1
      Fig. 1Example of an electropherogram of a 1.2 mm punch from a reference DNA sample on FTA® card amplified with the GlobalFiler® PCR Amplification Kit for 28 cycles.
      On the other hand, electropherograms obtained with a 0.5 mm punch did not evidence the typical artifacts related to the amplification of high quantities of DNA, showing genetic profiles more balanced in the same PCR conditions (Fig. 2).
      Figure thumbnail gr2
      Fig. 2Example of an electropherogram of a 0.5 mm punch from a reference DNA sample on FTA® card amplified with the GlobalFiler® PCR Amplification Kit for 28 cycles.

      4. Discussion and conclusion

      The ability to directly amplify preserved DNA on FTA® cards allows getting results faster and in a more efficient way [
      • Flores S.
      • et al.
      Internal validation of the GlobalFiler™ Express PCR Amplification Kit for the direct amplification of reference DNA samples on a high-throughput automated workflow.
      ]. According to manufacturer, a 1.2 mm punch disk contains approximately 5–20 ng of DNA [

      Applied Biosystems (2014). GlobalFiler® PCR Amplification Kit User Guide. Life Technologies.

      ]. Unwanted effects when amplifying high quantities of DNA can produce split peaks, noisy baseline and increased levels of stuttering and that was observed with punches of a larger diameter. Also the reduction of the reaction volume from 25 to 12.5 μL contributes to higher peak height intensity for the majority of STR markers [
      • Martín P.
      • et al.
      Improving DNA data exchange: validation studies on a single 6 dye STR kit with 24 loci.
      ].
      After analyzing the results, it was possible to verify that full and balanced profiles can be obtained from reference DNA samples with the GlobalFiler® PCR Amplification kit combining volume reduction with a 0.5 mm punch and 28 PCR cycles. Through this validation study, optimal settings were determined so that the GlobalFiler® casework kit can be used for direct amplification of DNA from common types of reference samples preserved on FTA® cards.

      Conflict of interest

      None.

      References

        • Flores S.
        • et al.
        Internal validation of the GlobalFiler™ Express PCR Amplification Kit for the direct amplification of reference DNA samples on a high-throughput automated workflow.
        Forensic Sci. Int. Genet. 2014; 10: 33-39
        • Martín P.
        • et al.
        Improving DNA data exchange: validation studies on a single 6 dye STR kit with 24 loci.
        Forensic Sci. Int. Genet. 2014; 13: 68-78
      1. Applied Biosystems (2014). GlobalFiler® PCR Amplification Kit User Guide. Life Technologies.