- Email: [email protected]
Investigation into the usefulness of DNA profiling of earprints
Available online at www.sciencedirect.com
Science and Justice 47 (2007) 155 – 159
Investigation into the usefulness of DNA profiling of earprints E.A.M. Graham, V.L. Bowyer, V.J. Martin, G.N. Rutty ⁎ Forensic Pathology Unit, University of Leicester, Robert Kilpatrick Building, Leicester Royal Infirmary, Leicester, LE2 7LX, UK Accepted 25 September 2007
Abstract DNA profiling of biological trace evidence has been used for many years. The application of this technique specifically to the DNA profiling of earprints has not to date been thoroughly investigated. This report presents the results of 60 earprints collected from three healthy adult volunteers under controlled laboratory conditions. DNA profile analysis revealed that high levels of non-donor alleles are observed when earprints are collected for DNA profiling. The source of these non-donor alleles is investigated and the impact that their presence within the profile may have on the use of this technique is discussed. © 2007 Forensic Science Society. Published by Elsevier Ireland Ltd. All rights reserved. Keywords: Forensic science; Earprint; DNA profiling; Contamination; Low copy number
1. Introduction The potential use of earprints for individual identification remains a contentious issue within the forensic arena. Throughout history the ear has been touted as an individuality marker akin to the much utilised fingerprint [1]. Early attempts to develop the use of earprints were discontinued due to the overwhelming success of fingerprinting. Despite the estimation that earprints are reported to be found at up to 15% of crime scenes [2] the use of earprints did not re-emerge as a potentially useful forensic tool until the late 1980s after the publication of Iannarelli's work [3]. The use of earprint evidence has to date been hampered by a lack of peer-reviewed published evidence that is required to give statistical support to the assertion that each earprint is as individual to its creator as the fingerprint [4]. Several groups are aiming to investigate the uniqueness of the earprint [5, 6] and systems of automated analysis [2,5] to provide this much needed support. Until these developments have taken place, the use of the earprint's unique morphology is overridden by recovery of biological material deposited within it for DNA profiling [7]. It has long been recognised that DNA profiling can be undertaken on trace amounts of biological material. DNA ⁎ Corresponding author. Tel.: +44 116 252 3221; fax: +44 116 252 3174. E-mail address: [email protected] (G.N. Rutty).
profiles have successfully been produced from fingerprints [8– 10], saliva found on drinking vessels [11], shoe insoles [12] bed sheets [13] and even single buccal cells [14]. Investigation of such traces, and indeed any biological material that results in the recovery of less than 100 pg DNA has been termed low copy number (LCN) projects [15]. The ability to produce DNA profiles from such minute traces has the potential to provide intelligence to investigations where previously no leads could be pursued [16] making this method of analysis attractive to the investigation of earprints while their uniqueness is debated. There are a number of concerns associated with DNA profiling of low copy number samples. A defining nature of ‘trace’ biological samples is that the exact origin is unknown. The collected material could have been deposited by the victim, perpetrator or an innocent individual at a time before the crime has taken place [15,17]. The contamination of trace samples by scene investigators is also a concern [18]. There is also the possibility that the recovered biological sample is present via secondary or even tertiary transfer, in which case the sample donor need not have personally visited the physical location of sample recovery for their DNA profile to be linked to the scene [19]. Despite the pitfalls, the benefits of DNA profiling of biological traces have been highlighted by the potential identification of perpetrators of manual strangulation [20,21], that could not be achieved by any other means. This paper investigates whether information derived from earprint
1355-0306/$ - see front matter © 2007 Forensic Science Society. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.scijus.2007.09.006
156
E.A.M. Graham et al. / Science and Justice 47 (2007) 155–159
analysis or DNA profiling of earprints is more reliable for forensic identification of the earmark donor. 2. Materials and methods
Telephone usage before swabbing was recorded for each individual. Telephones were classed as either single or multiple user handsets, according to location and usual usage patterns, nine telephone earpieces, consisting of five single and four multiple user types were swabbed to assess background levels of DNA.
2.1. Ethical approval and sample collection 2.4. Template cleaning Local ethical permission was granted for the recruitment of healthy adult volunteers for donation of biological material in the form of buccal swabs and earprints to the Forensic Pathology Unit, University of Leicester, UK, for use in this research (LREC, 04/Q2501/127). Written informed consent was obtained from ten healthy adult volunteers for this project. Participants were asked to complete a short questionnaire to provide a brief history of activities involving the ear that might influence DNA profiling, such as washing and telephone usage. A buccal swab was collected from each participant for production of reference profiles for comparison to earprint DNA profiles. The epithelial shedder status of each volunteer was determined by the method described by Lowe et al. [19]. This involved asking each participant to grip a sterile 50 ml universal tube for 10 s, 15 min after washing their hands. A good shedder was defined by the recovery of 90–100% of their DNA profile and a poor shedder by recovery of b 90% of their STR profile after 34 PCR cycles. Additionally, reference DNA profiles were generated for all laboratory staff for recognition of operator contamination, should it occur. 2.2. Reproducibility of earprints Three volunteers representing a good shedder, and two poor shedders, as determined by the method above, were asked to participate in a study to assess the consistency of DNA profiles obtained from a series of ten earprints collected over a one month time period. Six glass templates measuring approximately 10 cm × 10 cm were produced specifically for use in this investigation. Earprints were generated by pressing the ear against a template for a period of 5 s. Biological material was collected from each template by repeated swabbing of the whole earprint using a sterile cotton swab moistened with sterile H2O. A total of 60 earprints were collected during this investigation, ten earprints from the left ear and ten earprints from the right ear of all three volunteers. A template control swab was also collected before each earprint was deposited to monitor the occurrence of any carry-over contamination on the glass templates. 2.3. Sources of unknown DNA profile components Preliminary results presented at the 17th meeting of the International Association of Forensic Sciences, Hong Kong 2005 [22] indicated that unknown origin, non-donor alleles were often observed in DNA profiles produced after swabbing of earprints. It was hypothesised that these non-donor alleles could be present on the ear due to close physical contact between individuals such as a hug, or by use of multi-user telephones. In order to test this theory the telephone (dominant) ear of ten volunteers was swabbed.
Each volunteer was assigned two glass plates, one for each ear. These templates were to be re-used multiple times for collection of earprints on different days. Great care was taken to avoid carryover of biological material previously deposited on each glass template. After swabbing of each earprint the templates were hand washed in 1% Alconox® solution before being soaked overnight in 10% sodium hypochlorite. Each template was then thoroughly washed with sterile distilled water before re-use. 2.5. Swab processing and DNA profile generation DNA extraction was performed on all swabs using the QIAamp DNA micro kit-swab protocol (Qiagen, West Sussex, UK). DNA was eluted in 60 μl buffer AE and was stored at − 20 °C. Negative control extractions were processed in parallel with each batch of swabs extracted. DNA profiling was carried out on 1/12th extracted DNA using the AmpFlSTR® SGM Plus® PCR Amplification kit (Applied Biosystems, Foster City, CA) in a final reaction volume of 12.5 μl. A LCN protocol of 34 PCR cycles was used for shedder status experiments, all recovered earprints and negative control samples; a standard 28 cycle amplification was used for buccal swabs and DNA recovered directly from the ear [23]. Negative control extractions along with negative and positive PCR controls were amplified along with each batch of DNA extracts. PCR products were separated and visualised on an ABI PRISM® 377 DNA Sequencer (Applied Biosystems, Foster City, CA). Fragment sizing was carried out using GeneScan® software version 2.1 (Applied Biosystems, Foster City, CA) and allele designation was carried out using Genotyper® software version 3.7 (Applied Biosystems, Foster City, CA). 2.6. DNA profile interpretation It was anticipated that low copy number and possibly mixed DNA profiles would be generated during this investigation. The advice of numerous key DNA profile interpretation papers [15,16,23–30] was considered. DNA profile interpretation was carried out with prior knowledge of the donor's DNA profile. The following guidelines were used by the authors for interpretation of the results: 1. DNA profile interpretation was carried out independently by two DNA analysts. 2. All peaks above 50 RFU were considered as potential alleles. 3. Peaks occurring in ‘stutter’ positions that were less than 20% of the major allele peak height were discounted. 4. Only peaks observed in both PCR amplifications were considered as originating from the template DNA. All peaks
E.A.M. Graham et al. / Science and Justice 47 (2007) 155–159 Table 1 DNA profile interpretation results for 60 earprints (DO = drop-out) Right Donor Non- Locus ear alleles donor DO alleles
Allele Left Donor Non- Locus Allele DO ear alleles donor DO DO alleles
Earprint donor 1 (19 alleles) — good shedder, dominant ear = right 1 0 0 10 0 1 15 0 2 2 0 0 10 0 2 9 9 5 3 10 0 2 5 3 4 4 6 4 0 0 10 0 4 13 0 2 5 5 0 6 2 5 12 4 3 6 0 0 10 0 6 8 9 6 7 6 0 5 3 7 9 6 2 8 15 0 1 2 8 19 0 0 9 3 0 7 3 9 8 0 4 10 16 1 0 1 10 14 0 0
0 1 3 2 1 2 6 0 3 4
Earprint donor 2 (20 alleles) — medium shedder, dominant ear = right 1 0 0 10 0 1 3 0 7 2 1 0 9 1 2 0 1 10 3 16 2 1 2 3 17 0 1 4 1 7 9 1 4 0 0 10 5 4 5 7 2 5 0 0 10 6 0 0 10 0 6 5 0 6 7 8 3 5 2 7 6 7 4 8 10 1 3 4 8 2 0 9 9 5 0 6 3 9 0 0 10 10 16 0 1 2 10 0 0 10
3 0 1 0 0 3 4 0 0 0
Earprint donor 3 (19 alleles) — poor shedder, dominant ear = right 1 2 0 8 2 1 0 0 10 2 0 0 10 0 2 2 0 8 3 3 1 9 1 3 0 1 10 4 0 0 10 0 4 10 0 4 5 5 0 6 2 5 1 1 9 6 2 5 8 1 6 9 0 5 7 12 3 1 5 7 7 2 5 8 13 0 1 4 8 9 1 2 9 10 2 3 2 9 5 0 5 10 8 0 4 4 10 2 0 8
0 2 0 1 1 1 2 6 4 2
occurring in a single amplification were disregarded as spurious alleles. 5. Homozygosity at an STR locus was only considered if a single allele was observed with a peak area in excess of 10,000 RFU.
157
If a mixture was evident after completion of DNA profile interpretation, the corresponding template control swab was processed to ensure that the non-donor DNA had not entered the experiment before earprint deposition. 3. Results 3.1. Reproducibility of earprints The results of 60 earprints collected from the left and right ears of three volunteers are given in Table 1. The results are expressed as number of donor alleles and number of non-donor alleles observed. Information is also provided to illustrate the nature of allele/locus drop-out observed. A full donor DNA profile was observed only once, and this full profile was generated from the earprint of the ‘good shedder’ (left ear: print 8). Of the remaining 59 earprints, 20 revealed evidence of nondonor DNA and in seven of these DNA profiles (bold in Table 1) the number of non-donor alleles present after analysis exceeds the number of donor alleles. In two of these seven cases (donor 2, left: 2 and donor 3, left: 3) only one allele was observed after DNA analysis and interpretation. 3.2. Sources of non-donor alleles DNA mixtures were observed from swabs taken from all four multi-user telephones in this study. Of the reportedly single-user handsets, a full single DNA profile was obtained from one earpiece. Of the four remaining single-user telephones, three DNA profiles showed evidence of only one individual's DNA being present, one did however show evidence of a DNA mixture being present. Upon further inspection it was revealed that this particular telephone, although present at an individual desk, was used by more than one person. The results of DNA profiling are given in Table 2. A full donor DNA profile was obtained from all ten dominant ear swabs. Of these 10, 3 DNA profiles were contaminated with non-donor alleles at 3 loci, one at 5 loci, two at 8 loci and four showed no evidence of non-donor alleles. The results of DNA profiling were compared to information provided by each
Table 2 Alleles observed after DNA extraction of swabs taken from the earpiece of nine telephones STR loci
Multi-user telephones (consensus profile)
D3S1358 VWA D16S539 D2S1338 AMEL D8S1179 D21S11 D18S51 D19S433 TH01 FGA
15,16,17 14,14 11,11 F X,X 13,13 28,29,30 F 13,14 6,7,9.3 F
F = Complete locus drop-out.
15,18 14,15,16,17,18 9,11,14 19,20 X,X 12,13,14 30,30 F 14,14 7,8 F
15,16,18 16,17,18 F F X,X 14,14 32.2,32.2 F 13,14 F F
Single-user telephones (consensus profile) 15,16,17,18 17,18 9,11,13,14 17,20 X,X 14,14 28,29,32 F 14,14 9.3,9.3 22,22
F 14,16 10,10 F X,X 12,13 31,31 F 13,14,15 7,9.3 F
F 16,18 9,11 F X,Y 15,16 F F 13,15.2 F F
16,18 18,18 11,12 22,22 X,X 13,14 30,32.2 13,16 12,14 7,9.3 20,21,22
15,16 16,17 11,11 23,24 X,X 10,14 31,34.2 F 14,14 9.3,9.3 22,24
F 16,17,18 11,12 17,17 X,X 13,14 F F 12,13,14 7,9.3 F
158
E.A.M. Graham et al. / Science and Justice 47 (2007) 155–159
volunteer regarding the recent history of the swabbed ear. We observed that non-donor DNA alleles were present on individuals who had both used and not used public telephones. Of the six individuals from whom non-donor alleles were observed, two were recorded as having no physical contact with other individuals in the 24 h period before swabbing and had not recently used multi-user telephones. All negative extraction and PCR control samples showed no evidence of contamination. The source of the non-donor alleles in these samples remained undetermined. The non-donor alleles observed in the four remaining cases had either had recent contact with non-sampled individuals or had recently used a multi-user telephone. 4. Discussion Preliminary work carried out within our unit demonstrated that DNA profiles could be generated from swabbed earprints deposited on several surface types (i.e. wood, plastic, PVC and glass) and that no difference in DNA recovery was observed between a series of four earprints deposited in succession [22]. It was, however, discovered during this initial research period that DNA transferred to the templates was not adequately removed from the templates using a standard bleaching protocol, using diluted commercial bleach, approximately 2.5% sodium hypochlorite. It was hypothesised that oils were transferred along with cellular material during contact between template and the ear and that these oils were providing a protective barrier between remaining cellular material or free DNA and the bleach solution. The stability of DNA on bones surfaces after treatment with 6% sodium hydroxide has been noted by other groups [31]. To combat the hypothesised ‘oil’ problem an additional wash, using strong detergent, was added to the cleaning protocol and the concentration of sodium hypochlorite was increased to 10%. A control swab was taken by swabbing the entire template surface before each earprint was deposited to monitor possible contamination through this route. Contamination of all templates was observed once during this project, when the bleach soak solution was not changed for 2 weeks. These contaminated samples were disregarded and re-collection was undertaken. The bleach solution used for soaking the templates was then changed every week during the collection period to ensure optimal cleaning was achieved. Due to the template swabbing controls and the inclusion of negative extraction and PCR controls in every batch of extractions and amplifications we are confident that all alleles present in each DNA profile were derived from the earprint and were not due to contamination of the template or laboratory induced sources. This series of experiments was carried out in a controlled environment, with measures taken at every stage to ensure that any DNA amplified during each PCR reaction was derived from the earprint itself and not from any other source. After each deposition the entire earprint was collected onto the cotton swab, completely destroying the earprint. Despite this immediate processing a full DNA profile was obtained from only one of 60 swabbed earprints and the presence of one or more non-donor alleles was, however,
observed in one-third of all DNA profiles produced from swabbed earprints. In order to verify the ear as the source of non-donor alleles, the dominant ear of ten volunteers was directly swabbed and processed. Of the ten swabs collected, six showed evidence of non-donor alleles, two of these showing mixtures at eight out of ten STR loci. We hypothesised that the source of non-donor DNA present on the ear could be from the earpiece of multi-user handsets. Swabbing and processing of four multi-user telephone earpieces showed high background levels of DNA that could not be attributed to any single user. We believe that transfer of biological material between telephone earpieces and user's ears definitely contributes to the observation of non-donor DNA from swabbed earprints. Another source of non-donor alleles appears to be close physical contact with other individuals. The observation of raised background levels of non-self DNA has also been observed on the neck surface of individuals with partners (unpublished observation). This high level of background non-donor DNA recovered from earprints is extremely concerning for the use of this evidence in criminal investigation and has been highlighted in a recent publication [32]. The question that must be asked is, if this level of non-donor DNA is observed in a controlled investigation, using DNA-free templates, how much non-donor DNA will be recovered from an earprint taken from an uncontrolled environment such as the crime scene? This question is particularly pertinent when examining the Court of Appeal ruling in the case of R. vs. Dallagher [32], which has resulted in the use of earprints for individual identification being disregarded as inaccurate and has led to a review of many other cases involving earprint evidence [7]. In this case, the first of its kind in the United Kingdom, the original conviction in 1998 at Leeds Crown Court was based on the individualisation of a series of earprints discovered on the window of the murder victim. The credibility of earprint evidence was questioned due to the lack of peer-reviewed publications and an appeal was granted on these grounds [7]. A low copy number DNA profile consisting of a single allele was produced from a swab taken from one area of the earprint lifts taken in connection with the case. This single allele was not consistent with the DNA profile of the suspect Mark Dallagher. The weight of this DNA evidence was enough to overrule the comparably un-reviewed technique of earprint examination and Mr Dallagher was acquitted on 25th July 2002 [32]. Although the theoretical application of DNA profiling to earprints could be foreseen by the successful application to other similar traces such as fingerprints [8–10], specific investigation had not been undertaken. This report provides the background research of DNA profiling from earprints that was absent during the Dallagher Court of Appeal ruling. Our work shows that LCN DNA profiling produced a ‘DNA profile’ from a swabbed earprint, consisting of a single non-donor allele on two occasions during this controlled experiment demonstrating that on occasions single non-donor alleles only can be amplified from swabbed earprints. This result was not dependent on the shedder status and should not be considered as proof that an individual did not deposit a questioned earprint.
E.A.M. Graham et al. / Science and Justice 47 (2007) 155–159
5. Recommendations While this report highlights the potential problems of DNA profiling of earprints, the investigation of earprint characteristics continues to grow in strength. Recent publications documenting the analysis of 400 [2] and 1229 [5] earprints illustrate that individualisation of earprints may not only be possible, but is potentially automatable and reliable [33]. As swabbing of earprints for DNA profiling is presently a destructive technique, we recommend that accurate documentation and archiving of all earprints investigated in relation to criminal activity is considered before swabbing for DNA profiling. This approach will allow for the maximum amount of information to be gained from this evidence type and will provide a back-up for the possibility of DNA profiling failing altogether or resulting in a complicated mixture, which may contain more non-donor alleles that are potentially unrelated to the investigation than related donor alleles. References [1] A. Abbas, G.N. Rutty, Forensic web watch, Journal of Clinical Forensic Medicine 10 (2003) 129–131. [2] G.N. Rutty, A. Abbas, D. Crossling, Could earprint identification be computerised? An illustrated proof of concept paper, International Journal of Legal Medicine 119 (2005) 335–343. [3] A.V. Iannarelli, Ear Identification, Paramount Publishing Company, Fremont, California, 1989. [4] I.C. Champod, I.W. Evett, B. Kuchler, Earmarks as evidence: a critical review, Journal of Forensic Sciences 46 (2001) 1275–1284. [5] I. Alberink, A. Ruifrok, Performance of the FearID earprint identification system, Forensic Science International 166, (2–3) (2 March 2007) 145–154. [6] L. Meijerman, S. Sholl, F. De Conti, M. Giacon, C. van der Lugt, A. Drusini, P. Vanezis, G. Maat, Exploratory study on classification and individualisation of earprints, Forensic Science International 140 (2004) 91–99. [7] A.P. Broeders, Of earprints, fingerprints, scent dogs, cot deaths and cognitive contamination—a brief look at the present state of play in the forensic arena, Forensic Science International 159 (2006) 148–157. [8] R.A. van Oorschot, M.K. Jones, DNA fingerprints from fingerprints, Nature 387 (1997) 767. [9] M.M. Schulz, W. Reichert, Archived or directly swabbed latent fingerprints as a DNA source for STR typing, Forensic Science International 127 (2002) 128–130. [10] F. Alessandrini, M. Cecati, M. Pesaresi, C. Turchi, F. Carle, A. Tagliabracci, Fingerprints as evidence for a genetic profile: morphological study on fingerprints and analysis of exogenous and individual factors affecting DNA typing, Journal Forensic Science 48 (2003) 586–592. [11] J. Abaz, S.J. Walsh, J.M. Curran, D.S. Moss, J. Cullen, J.A. Bright, G.A. Crowe, S.L. Cockerton, T.E. Power, Comparison of the variables affecting the recovery of DNA from common drinking containers, Forensic Science International 126 (2002) 233–240. [12] J.A. Bright, S.F. Petricevic, Recovery of trace DNA and its application to DNA profiling of shoe insoles, Forensic Sciences International 145 (2004) 7–12. [13] S.F. Petricevic, J.A. Bright, S.L. Cockerton, DNA profiling of trace DNA recovered from bedding, Forensic Science International 159 (1) (25 May 2006) 21–26.
159
[14] I. Findlay, A. Taylor, P. Quirke, R. Frazier, A. Urquhart, DNA fingerprinting from single cells, Nature 389 (1997) 555–556. [15] P. Gill, Application of low copy number DNA profiling, Croatian Medical Journal 42 (2001) 229–232. [16] A.D. Kloosterman, P. Kersbergen, Efficacy and limits of genotyping low copy number (LCN) DNA samples by multiplex PCR of STR loci, Journal of Social Biology 197 (2003) 351–359. [17] R.A. Wickenheiser, Trace DNA: a review, discussion of theory, and application of the transfer of trace quantities of DNA through skin contact, Journal of Forensic Sciences 47 (2002) 442–450. [18] G.N. Rutty, A. Hopwood, V. Tucker, The effectiveness of protective clothing in the reduction of potential DNA contamination of the scene of crime, International Journal of Legal Medicine 117 (2003) 170–174. [19] A. Lowe, C. Murray, J. Whitaker, G. Tully, P. Gill, The propensity of individuals to deposit DNA and secondary transfer of low level DNA from individuals to inert surfaces, Forensic Science International 129 (2002) 25–34. [20] R. Radtkey, L. Feng, M. Muralhidar, M. Duhon, D. Canter, D. DiPierro, S. Fallon, E. Tu, K. McElfresh, M. Nerenberg, R. Sosnowski, Rapid, high fidelity analysis of simple sequence repeats on an electronically active DNA microchip, Nucleic Acids Research 28 (2000) E17. [21] G.N. Rutty, An investigation into the transference and survivability of human DNA following simulated manual strangulation with consideration of the problem of third party contamination, International Journal of Legal Medicine 116 (2002) 170–173. [22] V.L. Martin, E.A.M. Graham, G.N. Rutty, DNA profiling from earprints, 17th Meeting of the International Association of Forensic Sciences, 2005, p. A0454, Hong Kong. [23] J.P. Whitaker, E.A. Cotton, P. Gill, A comparison of the characteristics of profiles produced with the AMPFlSTR SGM Plus multiplex system for both standard and low copy number (LCN) STR DNA analysis, Forensic Sciences International 123 (2001) 215–223. [24] T.M. Clayton, J.P. Whitaker, R. Sparkes, P. Gill, Analysis and interpretation of mixed forensic stains using DNA STR profiling, Forensic Science International 91 (1998) 55–70. [25] I.W. Evett, P.D. Gill, J.A. Lambert, Taking account of peak areas when interpreting mixed DNA profiles, Journal of Forensic Sciences 43 (1998) 62–69. [26] P. Gill, B. Sparkes, J.S. Buckleton, Interpretation of simple mixtures of when artefacts such as stutters are present — with special reference to multiplex STRs used by the Forensic Science Service, Forensic Science International 95 (1998) 213–224. [27] P. Gill, R. Sparkes, C. Kimpton, Development of guidelines to designate alleles using an STR multiplex system, Forensic Science International 89 (1997) 185–197. [28] P. Gill, R. Sparkes, R. Pinchin, T. Clayton, J. Whitaker, J. Buckleton, Interpreting simple STR mixtures using allele peak areas, Forensic Science International 91 (1998) 41–53. [29] P. Gill, J. Whitaker, C. Flaxman, N. Brown, J. Buckleton, An investigation of the rigor of interpretation rules for STRs derived from less than 100 pg of DNA, Forensic Science International 112 (2000) 17–40. [30] B. Leclair, C.J. Fregeau, K.L. Bowen, R.M. Fourney, Systematic analysis of stutter percentages and allele peak height and peak area ratios at heterozygous STR loci for forensic casework and database samples, Journal of Forensic Science 49 (2004) 968–980. [31] B.M. Kemp, D.G. Smith, Use of bleach to eliminate contaminating DNA from the surface of bones and teeth, Forensic Science International 154 (2005) 53–61. [32] R.v. Dallagher, Court of Appeal, Vol. EWCA Crim 1903, 2002. [33] L. Meijerman, Inter- and Intra-Individual Variation in Earprints, Barge's Anthropologica, Leiden, 2006.
Science and Justice 47 (2007) 155 – 159
Investigation into the usefulness of DNA profiling of earprints E.A.M. Graham, V.L. Bowyer, V.J. Martin, G.N. Rutty ⁎ Forensic Pathology Unit, University of Leicester, Robert Kilpatrick Building, Leicester Royal Infirmary, Leicester, LE2 7LX, UK Accepted 25 September 2007
Abstract DNA profiling of biological trace evidence has been used for many years. The application of this technique specifically to the DNA profiling of earprints has not to date been thoroughly investigated. This report presents the results of 60 earprints collected from three healthy adult volunteers under controlled laboratory conditions. DNA profile analysis revealed that high levels of non-donor alleles are observed when earprints are collected for DNA profiling. The source of these non-donor alleles is investigated and the impact that their presence within the profile may have on the use of this technique is discussed. © 2007 Forensic Science Society. Published by Elsevier Ireland Ltd. All rights reserved. Keywords: Forensic science; Earprint; DNA profiling; Contamination; Low copy number
1. Introduction The potential use of earprints for individual identification remains a contentious issue within the forensic arena. Throughout history the ear has been touted as an individuality marker akin to the much utilised fingerprint [1]. Early attempts to develop the use of earprints were discontinued due to the overwhelming success of fingerprinting. Despite the estimation that earprints are reported to be found at up to 15% of crime scenes [2] the use of earprints did not re-emerge as a potentially useful forensic tool until the late 1980s after the publication of Iannarelli's work [3]. The use of earprint evidence has to date been hampered by a lack of peer-reviewed published evidence that is required to give statistical support to the assertion that each earprint is as individual to its creator as the fingerprint [4]. Several groups are aiming to investigate the uniqueness of the earprint [5, 6] and systems of automated analysis [2,5] to provide this much needed support. Until these developments have taken place, the use of the earprint's unique morphology is overridden by recovery of biological material deposited within it for DNA profiling [7]. It has long been recognised that DNA profiling can be undertaken on trace amounts of biological material. DNA ⁎ Corresponding author. Tel.: +44 116 252 3221; fax: +44 116 252 3174. E-mail address: [email protected] (G.N. Rutty).
profiles have successfully been produced from fingerprints [8– 10], saliva found on drinking vessels [11], shoe insoles [12] bed sheets [13] and even single buccal cells [14]. Investigation of such traces, and indeed any biological material that results in the recovery of less than 100 pg DNA has been termed low copy number (LCN) projects [15]. The ability to produce DNA profiles from such minute traces has the potential to provide intelligence to investigations where previously no leads could be pursued [16] making this method of analysis attractive to the investigation of earprints while their uniqueness is debated. There are a number of concerns associated with DNA profiling of low copy number samples. A defining nature of ‘trace’ biological samples is that the exact origin is unknown. The collected material could have been deposited by the victim, perpetrator or an innocent individual at a time before the crime has taken place [15,17]. The contamination of trace samples by scene investigators is also a concern [18]. There is also the possibility that the recovered biological sample is present via secondary or even tertiary transfer, in which case the sample donor need not have personally visited the physical location of sample recovery for their DNA profile to be linked to the scene [19]. Despite the pitfalls, the benefits of DNA profiling of biological traces have been highlighted by the potential identification of perpetrators of manual strangulation [20,21], that could not be achieved by any other means. This paper investigates whether information derived from earprint
1355-0306/$ - see front matter © 2007 Forensic Science Society. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.scijus.2007.09.006
156
E.A.M. Graham et al. / Science and Justice 47 (2007) 155–159
analysis or DNA profiling of earprints is more reliable for forensic identification of the earmark donor. 2. Materials and methods
Telephone usage before swabbing was recorded for each individual. Telephones were classed as either single or multiple user handsets, according to location and usual usage patterns, nine telephone earpieces, consisting of five single and four multiple user types were swabbed to assess background levels of DNA.
2.1. Ethical approval and sample collection 2.4. Template cleaning Local ethical permission was granted for the recruitment of healthy adult volunteers for donation of biological material in the form of buccal swabs and earprints to the Forensic Pathology Unit, University of Leicester, UK, for use in this research (LREC, 04/Q2501/127). Written informed consent was obtained from ten healthy adult volunteers for this project. Participants were asked to complete a short questionnaire to provide a brief history of activities involving the ear that might influence DNA profiling, such as washing and telephone usage. A buccal swab was collected from each participant for production of reference profiles for comparison to earprint DNA profiles. The epithelial shedder status of each volunteer was determined by the method described by Lowe et al. [19]. This involved asking each participant to grip a sterile 50 ml universal tube for 10 s, 15 min after washing their hands. A good shedder was defined by the recovery of 90–100% of their DNA profile and a poor shedder by recovery of b 90% of their STR profile after 34 PCR cycles. Additionally, reference DNA profiles were generated for all laboratory staff for recognition of operator contamination, should it occur. 2.2. Reproducibility of earprints Three volunteers representing a good shedder, and two poor shedders, as determined by the method above, were asked to participate in a study to assess the consistency of DNA profiles obtained from a series of ten earprints collected over a one month time period. Six glass templates measuring approximately 10 cm × 10 cm were produced specifically for use in this investigation. Earprints were generated by pressing the ear against a template for a period of 5 s. Biological material was collected from each template by repeated swabbing of the whole earprint using a sterile cotton swab moistened with sterile H2O. A total of 60 earprints were collected during this investigation, ten earprints from the left ear and ten earprints from the right ear of all three volunteers. A template control swab was also collected before each earprint was deposited to monitor the occurrence of any carry-over contamination on the glass templates. 2.3. Sources of unknown DNA profile components Preliminary results presented at the 17th meeting of the International Association of Forensic Sciences, Hong Kong 2005 [22] indicated that unknown origin, non-donor alleles were often observed in DNA profiles produced after swabbing of earprints. It was hypothesised that these non-donor alleles could be present on the ear due to close physical contact between individuals such as a hug, or by use of multi-user telephones. In order to test this theory the telephone (dominant) ear of ten volunteers was swabbed.
Each volunteer was assigned two glass plates, one for each ear. These templates were to be re-used multiple times for collection of earprints on different days. Great care was taken to avoid carryover of biological material previously deposited on each glass template. After swabbing of each earprint the templates were hand washed in 1% Alconox® solution before being soaked overnight in 10% sodium hypochlorite. Each template was then thoroughly washed with sterile distilled water before re-use. 2.5. Swab processing and DNA profile generation DNA extraction was performed on all swabs using the QIAamp DNA micro kit-swab protocol (Qiagen, West Sussex, UK). DNA was eluted in 60 μl buffer AE and was stored at − 20 °C. Negative control extractions were processed in parallel with each batch of swabs extracted. DNA profiling was carried out on 1/12th extracted DNA using the AmpFlSTR® SGM Plus® PCR Amplification kit (Applied Biosystems, Foster City, CA) in a final reaction volume of 12.5 μl. A LCN protocol of 34 PCR cycles was used for shedder status experiments, all recovered earprints and negative control samples; a standard 28 cycle amplification was used for buccal swabs and DNA recovered directly from the ear [23]. Negative control extractions along with negative and positive PCR controls were amplified along with each batch of DNA extracts. PCR products were separated and visualised on an ABI PRISM® 377 DNA Sequencer (Applied Biosystems, Foster City, CA). Fragment sizing was carried out using GeneScan® software version 2.1 (Applied Biosystems, Foster City, CA) and allele designation was carried out using Genotyper® software version 3.7 (Applied Biosystems, Foster City, CA). 2.6. DNA profile interpretation It was anticipated that low copy number and possibly mixed DNA profiles would be generated during this investigation. The advice of numerous key DNA profile interpretation papers [15,16,23–30] was considered. DNA profile interpretation was carried out with prior knowledge of the donor's DNA profile. The following guidelines were used by the authors for interpretation of the results: 1. DNA profile interpretation was carried out independently by two DNA analysts. 2. All peaks above 50 RFU were considered as potential alleles. 3. Peaks occurring in ‘stutter’ positions that were less than 20% of the major allele peak height were discounted. 4. Only peaks observed in both PCR amplifications were considered as originating from the template DNA. All peaks
E.A.M. Graham et al. / Science and Justice 47 (2007) 155–159 Table 1 DNA profile interpretation results for 60 earprints (DO = drop-out) Right Donor Non- Locus ear alleles donor DO alleles
Allele Left Donor Non- Locus Allele DO ear alleles donor DO DO alleles
Earprint donor 1 (19 alleles) — good shedder, dominant ear = right 1 0 0 10 0 1 15 0 2 2 0 0 10 0 2 9 9 5 3 10 0 2 5 3 4 4 6 4 0 0 10 0 4 13 0 2 5 5 0 6 2 5 12 4 3 6 0 0 10 0 6 8 9 6 7 6 0 5 3 7 9 6 2 8 15 0 1 2 8 19 0 0 9 3 0 7 3 9 8 0 4 10 16 1 0 1 10 14 0 0
0 1 3 2 1 2 6 0 3 4
Earprint donor 2 (20 alleles) — medium shedder, dominant ear = right 1 0 0 10 0 1 3 0 7 2 1 0 9 1 2 0 1 10 3 16 2 1 2 3 17 0 1 4 1 7 9 1 4 0 0 10 5 4 5 7 2 5 0 0 10 6 0 0 10 0 6 5 0 6 7 8 3 5 2 7 6 7 4 8 10 1 3 4 8 2 0 9 9 5 0 6 3 9 0 0 10 10 16 0 1 2 10 0 0 10
3 0 1 0 0 3 4 0 0 0
Earprint donor 3 (19 alleles) — poor shedder, dominant ear = right 1 2 0 8 2 1 0 0 10 2 0 0 10 0 2 2 0 8 3 3 1 9 1 3 0 1 10 4 0 0 10 0 4 10 0 4 5 5 0 6 2 5 1 1 9 6 2 5 8 1 6 9 0 5 7 12 3 1 5 7 7 2 5 8 13 0 1 4 8 9 1 2 9 10 2 3 2 9 5 0 5 10 8 0 4 4 10 2 0 8
0 2 0 1 1 1 2 6 4 2
occurring in a single amplification were disregarded as spurious alleles. 5. Homozygosity at an STR locus was only considered if a single allele was observed with a peak area in excess of 10,000 RFU.
157
If a mixture was evident after completion of DNA profile interpretation, the corresponding template control swab was processed to ensure that the non-donor DNA had not entered the experiment before earprint deposition. 3. Results 3.1. Reproducibility of earprints The results of 60 earprints collected from the left and right ears of three volunteers are given in Table 1. The results are expressed as number of donor alleles and number of non-donor alleles observed. Information is also provided to illustrate the nature of allele/locus drop-out observed. A full donor DNA profile was observed only once, and this full profile was generated from the earprint of the ‘good shedder’ (left ear: print 8). Of the remaining 59 earprints, 20 revealed evidence of nondonor DNA and in seven of these DNA profiles (bold in Table 1) the number of non-donor alleles present after analysis exceeds the number of donor alleles. In two of these seven cases (donor 2, left: 2 and donor 3, left: 3) only one allele was observed after DNA analysis and interpretation. 3.2. Sources of non-donor alleles DNA mixtures were observed from swabs taken from all four multi-user telephones in this study. Of the reportedly single-user handsets, a full single DNA profile was obtained from one earpiece. Of the four remaining single-user telephones, three DNA profiles showed evidence of only one individual's DNA being present, one did however show evidence of a DNA mixture being present. Upon further inspection it was revealed that this particular telephone, although present at an individual desk, was used by more than one person. The results of DNA profiling are given in Table 2. A full donor DNA profile was obtained from all ten dominant ear swabs. Of these 10, 3 DNA profiles were contaminated with non-donor alleles at 3 loci, one at 5 loci, two at 8 loci and four showed no evidence of non-donor alleles. The results of DNA profiling were compared to information provided by each
Table 2 Alleles observed after DNA extraction of swabs taken from the earpiece of nine telephones STR loci
Multi-user telephones (consensus profile)
D3S1358 VWA D16S539 D2S1338 AMEL D8S1179 D21S11 D18S51 D19S433 TH01 FGA
15,16,17 14,14 11,11 F X,X 13,13 28,29,30 F 13,14 6,7,9.3 F
F = Complete locus drop-out.
15,18 14,15,16,17,18 9,11,14 19,20 X,X 12,13,14 30,30 F 14,14 7,8 F
15,16,18 16,17,18 F F X,X 14,14 32.2,32.2 F 13,14 F F
Single-user telephones (consensus profile) 15,16,17,18 17,18 9,11,13,14 17,20 X,X 14,14 28,29,32 F 14,14 9.3,9.3 22,22
F 14,16 10,10 F X,X 12,13 31,31 F 13,14,15 7,9.3 F
F 16,18 9,11 F X,Y 15,16 F F 13,15.2 F F
16,18 18,18 11,12 22,22 X,X 13,14 30,32.2 13,16 12,14 7,9.3 20,21,22
15,16 16,17 11,11 23,24 X,X 10,14 31,34.2 F 14,14 9.3,9.3 22,24
F 16,17,18 11,12 17,17 X,X 13,14 F F 12,13,14 7,9.3 F
158
E.A.M. Graham et al. / Science and Justice 47 (2007) 155–159
volunteer regarding the recent history of the swabbed ear. We observed that non-donor DNA alleles were present on individuals who had both used and not used public telephones. Of the six individuals from whom non-donor alleles were observed, two were recorded as having no physical contact with other individuals in the 24 h period before swabbing and had not recently used multi-user telephones. All negative extraction and PCR control samples showed no evidence of contamination. The source of the non-donor alleles in these samples remained undetermined. The non-donor alleles observed in the four remaining cases had either had recent contact with non-sampled individuals or had recently used a multi-user telephone. 4. Discussion Preliminary work carried out within our unit demonstrated that DNA profiles could be generated from swabbed earprints deposited on several surface types (i.e. wood, plastic, PVC and glass) and that no difference in DNA recovery was observed between a series of four earprints deposited in succession [22]. It was, however, discovered during this initial research period that DNA transferred to the templates was not adequately removed from the templates using a standard bleaching protocol, using diluted commercial bleach, approximately 2.5% sodium hypochlorite. It was hypothesised that oils were transferred along with cellular material during contact between template and the ear and that these oils were providing a protective barrier between remaining cellular material or free DNA and the bleach solution. The stability of DNA on bones surfaces after treatment with 6% sodium hydroxide has been noted by other groups [31]. To combat the hypothesised ‘oil’ problem an additional wash, using strong detergent, was added to the cleaning protocol and the concentration of sodium hypochlorite was increased to 10%. A control swab was taken by swabbing the entire template surface before each earprint was deposited to monitor possible contamination through this route. Contamination of all templates was observed once during this project, when the bleach soak solution was not changed for 2 weeks. These contaminated samples were disregarded and re-collection was undertaken. The bleach solution used for soaking the templates was then changed every week during the collection period to ensure optimal cleaning was achieved. Due to the template swabbing controls and the inclusion of negative extraction and PCR controls in every batch of extractions and amplifications we are confident that all alleles present in each DNA profile were derived from the earprint and were not due to contamination of the template or laboratory induced sources. This series of experiments was carried out in a controlled environment, with measures taken at every stage to ensure that any DNA amplified during each PCR reaction was derived from the earprint itself and not from any other source. After each deposition the entire earprint was collected onto the cotton swab, completely destroying the earprint. Despite this immediate processing a full DNA profile was obtained from only one of 60 swabbed earprints and the presence of one or more non-donor alleles was, however,
observed in one-third of all DNA profiles produced from swabbed earprints. In order to verify the ear as the source of non-donor alleles, the dominant ear of ten volunteers was directly swabbed and processed. Of the ten swabs collected, six showed evidence of non-donor alleles, two of these showing mixtures at eight out of ten STR loci. We hypothesised that the source of non-donor DNA present on the ear could be from the earpiece of multi-user handsets. Swabbing and processing of four multi-user telephone earpieces showed high background levels of DNA that could not be attributed to any single user. We believe that transfer of biological material between telephone earpieces and user's ears definitely contributes to the observation of non-donor DNA from swabbed earprints. Another source of non-donor alleles appears to be close physical contact with other individuals. The observation of raised background levels of non-self DNA has also been observed on the neck surface of individuals with partners (unpublished observation). This high level of background non-donor DNA recovered from earprints is extremely concerning for the use of this evidence in criminal investigation and has been highlighted in a recent publication [32]. The question that must be asked is, if this level of non-donor DNA is observed in a controlled investigation, using DNA-free templates, how much non-donor DNA will be recovered from an earprint taken from an uncontrolled environment such as the crime scene? This question is particularly pertinent when examining the Court of Appeal ruling in the case of R. vs. Dallagher [32], which has resulted in the use of earprints for individual identification being disregarded as inaccurate and has led to a review of many other cases involving earprint evidence [7]. In this case, the first of its kind in the United Kingdom, the original conviction in 1998 at Leeds Crown Court was based on the individualisation of a series of earprints discovered on the window of the murder victim. The credibility of earprint evidence was questioned due to the lack of peer-reviewed publications and an appeal was granted on these grounds [7]. A low copy number DNA profile consisting of a single allele was produced from a swab taken from one area of the earprint lifts taken in connection with the case. This single allele was not consistent with the DNA profile of the suspect Mark Dallagher. The weight of this DNA evidence was enough to overrule the comparably un-reviewed technique of earprint examination and Mr Dallagher was acquitted on 25th July 2002 [32]. Although the theoretical application of DNA profiling to earprints could be foreseen by the successful application to other similar traces such as fingerprints [8–10], specific investigation had not been undertaken. This report provides the background research of DNA profiling from earprints that was absent during the Dallagher Court of Appeal ruling. Our work shows that LCN DNA profiling produced a ‘DNA profile’ from a swabbed earprint, consisting of a single non-donor allele on two occasions during this controlled experiment demonstrating that on occasions single non-donor alleles only can be amplified from swabbed earprints. This result was not dependent on the shedder status and should not be considered as proof that an individual did not deposit a questioned earprint.
E.A.M. Graham et al. / Science and Justice 47 (2007) 155–159
5. Recommendations While this report highlights the potential problems of DNA profiling of earprints, the investigation of earprint characteristics continues to grow in strength. Recent publications documenting the analysis of 400 [2] and 1229 [5] earprints illustrate that individualisation of earprints may not only be possible, but is potentially automatable and reliable [33]. As swabbing of earprints for DNA profiling is presently a destructive technique, we recommend that accurate documentation and archiving of all earprints investigated in relation to criminal activity is considered before swabbing for DNA profiling. This approach will allow for the maximum amount of information to be gained from this evidence type and will provide a back-up for the possibility of DNA profiling failing altogether or resulting in a complicated mixture, which may contain more non-donor alleles that are potentially unrelated to the investigation than related donor alleles. References [1] A. Abbas, G.N. Rutty, Forensic web watch, Journal of Clinical Forensic Medicine 10 (2003) 129–131. [2] G.N. Rutty, A. Abbas, D. Crossling, Could earprint identification be computerised? An illustrated proof of concept paper, International Journal of Legal Medicine 119 (2005) 335–343. [3] A.V. Iannarelli, Ear Identification, Paramount Publishing Company, Fremont, California, 1989. [4] I.C. Champod, I.W. Evett, B. Kuchler, Earmarks as evidence: a critical review, Journal of Forensic Sciences 46 (2001) 1275–1284. [5] I. Alberink, A. Ruifrok, Performance of the FearID earprint identification system, Forensic Science International 166, (2–3) (2 March 2007) 145–154. [6] L. Meijerman, S. Sholl, F. De Conti, M. Giacon, C. van der Lugt, A. Drusini, P. Vanezis, G. Maat, Exploratory study on classification and individualisation of earprints, Forensic Science International 140 (2004) 91–99. [7] A.P. Broeders, Of earprints, fingerprints, scent dogs, cot deaths and cognitive contamination—a brief look at the present state of play in the forensic arena, Forensic Science International 159 (2006) 148–157. [8] R.A. van Oorschot, M.K. Jones, DNA fingerprints from fingerprints, Nature 387 (1997) 767. [9] M.M. Schulz, W. Reichert, Archived or directly swabbed latent fingerprints as a DNA source for STR typing, Forensic Science International 127 (2002) 128–130. [10] F. Alessandrini, M. Cecati, M. Pesaresi, C. Turchi, F. Carle, A. Tagliabracci, Fingerprints as evidence for a genetic profile: morphological study on fingerprints and analysis of exogenous and individual factors affecting DNA typing, Journal Forensic Science 48 (2003) 586–592. [11] J. Abaz, S.J. Walsh, J.M. Curran, D.S. Moss, J. Cullen, J.A. Bright, G.A. Crowe, S.L. Cockerton, T.E. Power, Comparison of the variables affecting the recovery of DNA from common drinking containers, Forensic Science International 126 (2002) 233–240. [12] J.A. Bright, S.F. Petricevic, Recovery of trace DNA and its application to DNA profiling of shoe insoles, Forensic Sciences International 145 (2004) 7–12. [13] S.F. Petricevic, J.A. Bright, S.L. Cockerton, DNA profiling of trace DNA recovered from bedding, Forensic Science International 159 (1) (25 May 2006) 21–26.
159
[14] I. Findlay, A. Taylor, P. Quirke, R. Frazier, A. Urquhart, DNA fingerprinting from single cells, Nature 389 (1997) 555–556. [15] P. Gill, Application of low copy number DNA profiling, Croatian Medical Journal 42 (2001) 229–232. [16] A.D. Kloosterman, P. Kersbergen, Efficacy and limits of genotyping low copy number (LCN) DNA samples by multiplex PCR of STR loci, Journal of Social Biology 197 (2003) 351–359. [17] R.A. Wickenheiser, Trace DNA: a review, discussion of theory, and application of the transfer of trace quantities of DNA through skin contact, Journal of Forensic Sciences 47 (2002) 442–450. [18] G.N. Rutty, A. Hopwood, V. Tucker, The effectiveness of protective clothing in the reduction of potential DNA contamination of the scene of crime, International Journal of Legal Medicine 117 (2003) 170–174. [19] A. Lowe, C. Murray, J. Whitaker, G. Tully, P. Gill, The propensity of individuals to deposit DNA and secondary transfer of low level DNA from individuals to inert surfaces, Forensic Science International 129 (2002) 25–34. [20] R. Radtkey, L. Feng, M. Muralhidar, M. Duhon, D. Canter, D. DiPierro, S. Fallon, E. Tu, K. McElfresh, M. Nerenberg, R. Sosnowski, Rapid, high fidelity analysis of simple sequence repeats on an electronically active DNA microchip, Nucleic Acids Research 28 (2000) E17. [21] G.N. Rutty, An investigation into the transference and survivability of human DNA following simulated manual strangulation with consideration of the problem of third party contamination, International Journal of Legal Medicine 116 (2002) 170–173. [22] V.L. Martin, E.A.M. Graham, G.N. Rutty, DNA profiling from earprints, 17th Meeting of the International Association of Forensic Sciences, 2005, p. A0454, Hong Kong. [23] J.P. Whitaker, E.A. Cotton, P. Gill, A comparison of the characteristics of profiles produced with the AMPFlSTR SGM Plus multiplex system for both standard and low copy number (LCN) STR DNA analysis, Forensic Sciences International 123 (2001) 215–223. [24] T.M. Clayton, J.P. Whitaker, R. Sparkes, P. Gill, Analysis and interpretation of mixed forensic stains using DNA STR profiling, Forensic Science International 91 (1998) 55–70. [25] I.W. Evett, P.D. Gill, J.A. Lambert, Taking account of peak areas when interpreting mixed DNA profiles, Journal of Forensic Sciences 43 (1998) 62–69. [26] P. Gill, B. Sparkes, J.S. Buckleton, Interpretation of simple mixtures of when artefacts such as stutters are present — with special reference to multiplex STRs used by the Forensic Science Service, Forensic Science International 95 (1998) 213–224. [27] P. Gill, R. Sparkes, C. Kimpton, Development of guidelines to designate alleles using an STR multiplex system, Forensic Science International 89 (1997) 185–197. [28] P. Gill, R. Sparkes, R. Pinchin, T. Clayton, J. Whitaker, J. Buckleton, Interpreting simple STR mixtures using allele peak areas, Forensic Science International 91 (1998) 41–53. [29] P. Gill, J. Whitaker, C. Flaxman, N. Brown, J. Buckleton, An investigation of the rigor of interpretation rules for STRs derived from less than 100 pg of DNA, Forensic Science International 112 (2000) 17–40. [30] B. Leclair, C.J. Fregeau, K.L. Bowen, R.M. Fourney, Systematic analysis of stutter percentages and allele peak height and peak area ratios at heterozygous STR loci for forensic casework and database samples, Journal of Forensic Science 49 (2004) 968–980. [31] B.M. Kemp, D.G. Smith, Use of bleach to eliminate contaminating DNA from the surface of bones and teeth, Forensic Science International 154 (2005) 53–61. [32] R.v. Dallagher, Court of Appeal, Vol. EWCA Crim 1903, 2002. [33] L. Meijerman, Inter- and Intra-Individual Variation in Earprints, Barge's Anthropologica, Leiden, 2006.