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Direct amplification of casework samples in fabrics using GlobalFiler® PCR Amplification Kit

  • P. Brito
    Correspondence
    Corresponding author.
    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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  • 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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  • 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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  • 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. 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. Anjos
    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 23, 2015DOI:https://doi.org/10.1016/j.fsigss.2015.09.193

      Abstract

      After a validation study developed in the Forensics Genetic and Biology Service of Coimbra, Portugal, we were able to demonstrate the ability of the GlobalFiler® PCR Amplification kit in direct amplification of reference samples with consistent and reproducible results. Since this kit is optimized for casework samples, we decided to test it on forensic samples, in particular on fabrics stains. The selected samples had already been processed according to the methodologies normally used for this type of samples included automated extraction, quantification and amplification, followed by capillary electrophoresis, resulting in complete profiles. To develop our study, samples were collected from regions of the fabrics that had previously shown/proven to have biological evidence. The chosen samples were directly amplified for the GlobalFiler® PCR Amplification kit, followed by capillary electrophoresis on the ABI PRISM® 3500 Genetic Analyzer. The results obtained with this methodology revealed full and well balanced genetic profiles. This study intends to show that in some cases this approach can be an alternative, when we are in the presence of biological stains on fabrics, that allows to obtain consistent results in a short period of time.

      Keywords

      1. Introduction

      The GlobalFiler® PCR Amplification kit has proven to be an effective tool on forensic genetics routine for direct amplification of reference samples [
      • Martín P.
      • et al.
      Improving DNA data exchange: validation studies on a single 6 dye STR kit with 24 loci.
      ], as it was possible to demonstrate in validation studies performed in our laboratory. Consistent and reproducible results showed that this kit is quite robust, proving to be tolerant to large amounts of DNA input, as well as to overcome the effect of some PCR inhibitors. Since this kit is optimized for casework samples, we decided to test it in the direct form with casework samples, in particular on fabrics stains.

      2. Materials and methods

      To carry out this work, different kinds of samples were selected from diverse types of investigations, in particular alleged sexual assaults and homicides. This selection resulted in different sorts of samples, including underwear, pants, curtains, nightgowns, shirts, socks and sheets, among other pieces, also covering various types of textile materials. The selected samples were previously processed according to the routinely used procedures for this type of samples including automated extraction, quantification and amplification, followed by capillary electrophoresis, resulting in complete profiles. To develop our study, samples were collected from stains on fabrics that had previously proven to have biological evidence. For those with the possible presence of semen, UV light (≈450 nm) was used to delimit the stain of the biological fluid. The chosen samples were directly amplified with GlobalFiler® PCR Amplification kit, adding 1,0 mm punch of each sample in a reaction volume of 25 μl, in the Veriti® Thermal Cycler (Life Technologies) according to the recommendations of the manufacturer [

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

      ]. Capillary electrophoresis was performed on the ABI PRISM® 3500 Genetic Analyzer.

      3. Results

      In the analyzed cases, it was possible to obtain genetic profiles for most samples. In the following pictures are shown two examples of investigations that resulted in genetic profiles.

      4. Discussion and conclusion

      After analyzing the results it was observed that most of the samples resulted in genetic profiles. In the sexual assault case (Fig. 1A), it was possible to obtain a well balanced mixed DNA profile, with two contributors (Fig. 2A). The obtained results were in agreement with the previously achieved profiles, where it was possible to distinguish the profile of the victim and the offender. In the case of alleged homicide (Fig. 1B), it was possible to obtain a single-source profile on the offender's garment, identified as the victim's profile (Fig. 2B), according to the results previously obtained. The achieved results for the presented cases reflect the results obtained for most of the studied samples. However, in some cases where it was expected a mixed DNA profile, only the victim’s profile was obtained. This may be due to the fact that the whole mixture stain was used for the DNA extraction. There were also situations where it was not possible to obtain a genetic profile. For this reason may have contributed the fact that in certain types of fabric, with less compact and/or coarser fibers, it was not easy to make a punch of it, not ensuring sufficient amount of DNA for amplification. After all, this approach could be an important alternative in certain investigations when in the presence of visible stains, allowing to obtain results in a short period of time.
      Figure thumbnail gr1
      Fig. 1Examples of evidence used in the study. (A) Nightgown of a suspected sexual assault case with presumed semen stains. (B) Shirt of a homicide suspect with visible bloodstains.
      Figure thumbnail gr2
      Fig. 2Electropherograms from direct amplification of casework samples with GlobalFiler® PCR Amplification kit. (A) Genetic profile mixture of two individuals. (B) Single-source genetic profile.

      Conflict of interest

      None.

      References

        • 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.