STR Analysis of Artificially Degraded DNA—Results of a Collaborative

Forensic Science International 139 (2004) 123–134 STR analysis of artificially degraded DNA—results of a collaborative European exercise Peter M. Schneidera,*, Klaus Bendera, Wolfgang R. Mayrb, Walther Parsonc, Bernadette Hosted, Ronny Decortee, Jan Cordonnierf, Daniel Vanekg, Niels Morlingh, Matti Karjalaineni, C. Marie-Paule Carlottij, Myriam Sabatierk, Carsten Hohoffl, Hermann Schmitterm, Werner Pflugn, Rainer Wenzelo, Dieter Patzeltp,Ru¨diger Lessigq, Peter Dobrowolskir, Geraldine O’Donnells, Luciano Garafanot, Marina Doboszu, Peter de Knijffv, Bente Mevagw, Ryszard Pawlowskix, Leonor Gusmaõy, Maria Conceicao Videz, Antonio Alonso Alonsoa1, Oscar Garcıá Fernańdeza2, Pilar Sanz Nicola´sa3, Ann Kihlgreena4, Walter Ba¨ra5, Verena Meiera6, Anne Teyssiera7, Raphael Coquoza8, Conxita Brandta9, Ursula Germanna10, Peter Gilla11, Justine Halletta12, Matthew Greenhalgha13 aInstitut fu¨r Rechtsmedizin, Universita¨t Mainz, Mainz, Germany bKlinische Abteilung fu¨r Blutgruppenserologie, Universita¨t Wien, Wien, Austria cInstitut fu¨r Gerichtliche Medizin, Universita¨t Innsbruck, Innsbruck, Austria dInstitut National de Criminalistique, Bruxelles, Belgium eLaboratory for Forensic Genetics and Molecular Archaeology, Leuven, Belgium fChemiphar, Brugge, Belgium gInstitute of Criminalistics, Prague, Czech Republic hDepartment of Forensic Genetics, Institute of Forensic Medicine, University of Copenhagen, Copenhagen, Denmark iNational Bureau of Investigation, Vantaa, Finland jInstitut de Recherche Criminelle, Rosny-Sous-Bois, France kLaboratoire de Police Scientifique, Toulouse, France lInstitut fu¨r Rechtsmedizin, Universita¨tMu¨nster, Germany mBundeskriminalamt, Wiesbaden, Germany nLKA Baden-Wu¨rttemberg, Stuttgart, Germany oLKA Rheinland-Pfalz, Mainz, Germany pInstitut fu¨r Rechtsmedizin, Universita¨tWu¨rzburg, Wu¨rzburg, Germany qInstitut fu¨r Rechtsmedizin, Universita¨t Leipzig, Leipzig, Germany rLKA Sachsen, Dez. DNA-Analytik, Dresden, Germany sForensic Science Laboratory, Dublin, Ireland tReparto Carabinieri Investigazioni Scientifiche, Parma, Italy uIstituto di Medicina Legale Universita` Cattolica, Roma, Italy vForensics Laboratory, University of Leiden, Leiden, The Netherlands wRettsmedisinsk Institutt, University of Oslo, Oslo, Norway xInstitute of Forensic Medicine, Medical University of Gdansk, Gdansk, Poland yIPATIMUP, Universidade do Oporto, Oporto, Portugal zInstituto de Medicina Legal, Universidade de Coimbra, Coimbra, Portugal a1Instituto National de Toxicologia, Madrid, Spain a2Area de Laboratorio Ertzaina, Bilbao, Spain a3Instituto National de Toxicologia, Sevilla, Spain * Corresponding author. Present address: Institute of Legal Medicine, Am Pulverturm 3, D-55131 Mainz, Germany. Tel.: þ49-6131-3932687; fax: þ49-6131-3933183. E-mail address: [email protected] (P.M. Schneider). 0379-0738/$ – see front matter # 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.forsciint.2003.10.002 124 P.M. Schneider et al. / Forensic Science International 139 (2004) 123–134 a4National Laboratory of Forensic Science, Linko¨ping, Sweden a5Institut fu¨r Rechtsmedizin, Universita¨tZu¨rich, Zu¨rich, Switzerland a6Instiut fu¨r Rechtsmedizin, Universita¨t Basel, Basel, Switzerland a7Institut Universitaire de Me´decine Le´gale, Universite´ de Gene`ve, Gene`ve, Switzerland a8Laboratoire AMS, Lausanne, Switzerland a9Institut Universitaire de Me´decine Le´gale, Lausanne, Switzerland a10Institut fu¨r Rechtsmedizin, Universita¨t St. Gallen, St. Gallen, Switzerland a11Forensic Science Service Headquarters, Birmingham, UK a12LGC, Teddington Middlesex, UK a13Orchid Bioscience Europe, Abingdon, UK Received 21 May 2003; received in revised form 2 October 2003; accepted 3 October 2003 Abstract Degradation of human DNA extracted from forensic stains is, in most cases, the result of a natural process due to the exposure of the stain samples to the environment. Experiences with degraded DNA from casework samples show that every sample may exhibit different properties in this respect, and that it is difficult to systematically assess the performance of routinely used typing systems for the analysis of degraded DNA samples. Using a batch of artificially degraded DNA with an average fragment size of approx. 200 bp a collaborative exercise was carried out among 38 forensic laboratories from 17 European countries. The results were assessed according to correct allele detection, peak height and balance as well as the occurrence of artefacts. A number of common problems were identified based on these results such as strong peak imbalance in heterozygous genotypes for the larger short tandem repeat (STR) fragments after increased PCR cycle numbers, artefact signals and allelic drop-out. Based on the observations, strategies are discussed to overcome these problems. The strategies include careful balancing of the amount of template DNA and the PCR cycle numbers, the reaction volume and the amount of Taq polymerase. Furthermore, a careful evaluation of the results of the fragment analysis and of automated allele calling is necessary to identify the correct alleles and avoid artefacts. # 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Short tandem repeat; Alleles; PCR degradation 1. Introduction Experience with degraded DNA from casework samples shows that every sample may exhibit different properties in Genetic analysis of short tandem repeat (STR) loci is this respect, and that it is difficult to systematically assess the currently the most powerful and widely used method to performance of routinely used typing systems to analyze identify the contributor of biological samples collected in the degraded DNA samples. To learn more about the efficiency context of criminal investigations, and the identification of of STR systems, a standardized reference sample of human remains. The successful application includes a vari- degraded DNA in sufficient amounts would be quite helpful, ety of substrates such as blood, saliva, epithelial cells, hair and could also be used for validation studies of new STR roots and even compact bone samples [1,2]. However, when typing systems [5–7]. Therefore, a collaborative exercise on more problematic samples such as old stains or decomposed degraded DNA was planned in the process of the ‘‘Standar- tissue are subjected to STR typing, failure to obtain repro- dization of DNA Profiling Methods in the European Union’’ ducible results may occur due to degradation of high mole- (STADNAP) network, and participation was offered to 50 cular weight DNA [3]. forensic DNA laboratories across Europe. The intention of In crime case investigations, degradation of human this exercise was to better understand the different para- DNA usually is the result of a natural process resulting meters influencing STR typing results obtained from from the exposure of the stain or tissue samples to the degraded DNA as well as to learn more about the strategies environment. Light, humidity, elevated temperatures as applied by the participating laboratories to optimize the well as bacterial and fungal contaminations followed by typing of problematic samples. the growth of these microorganisms lead to physical, chemical and biochemical degradation of genomic 2. Materials and methods DNA. Once the average DNA fragment length is reduced to sizes smaller than 300 bp, loss of genetic information 2.1. Production of degraded DNA samples may occur due to the lack of suitable template DNA and the subsequent failure of frequently used STR typing Batches of high molecular weight genomic DNA were methods [4]. prepared from two human cell lines, HepG2 and P118, with P.M. Schneider et al. / Forensic Science International 139 (2004) 123–134 125 male and female genotypes, respectively. The DNA samples 2.3. Data analysis were degraded under standardized conditions to an average fragment length of less than 200 bp using a combination of The submitted data were recorded for each STR locus physical and biochemical methods, i.e. sonication and treat- based on the peak height (in relative fluorescence units, rfu) ment with DNAse I. Based on previous experiences from a for each allele observed. To allow a comparison between smaller exercise among the STADNAP partner laboratories laboratories, the peak heights were grouped into three using a single degraded DNA sample (data not shown) it was categories: strong signal: >150 rfu, low signal: 150– intended to achieve a slightly higher degree of degradation in 30 rfu, very low or no signal: <30 rfu. Furthermore, two sample B compared to sample A. The degradation process was categories of data were defined based on the exercise closely monitored to control the resulting fragment sizes as instructions (see above): ‘‘standard’’ PCR conditions using well as the suitability to obtain partial results by multiplex 0.25–2.5 ng degraded DNA and 28 PCR cycles, and STR typing. It was observed that the final fragment size ‘‘enhanced’’ PCR conditions using 0.5–5 ng DNA and distribution heavily depends on the DNA quality and con- 28–36 PCR cycles. Unexpected results, artefacts or incorrect centration, the reaction conditions and volumes, as well as the alleles were recorded separately for each locus and were not properties and the concentration of the DNAse I digestion. included in the peak height analysis. Batch to batch reproducibility could only be achieved by repeatedly testing the degraded DNA using STR analysis [8]. 3. Results and discussion 2.2. Exercise design Results were received from 38 laboratories from 17 Two aliquots of 20 ml each

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