“Ancient” protocols for the crime scene?

Forensic Science International 131 (2003) 59±64

``Ancient'' protocols for the crime scene? Similarities and differences between forensic genetics and ancient DNA analysis C. Capellia,b,*, F. Tschentscherc, V.L. Pascalia a

Immunohematology Laboratory, Department of Forensic Medicine, Catholic University, Largo F. Vito, 1 00168 Rome, Italy b Department of Biology, University College London, 4, Stephenson Way, NW1 2HE London, UK c Institute for Human Genetics, University Clinic, Hufelandstr. 55, 45122 Essen, Germany Received 17 April 2002; received in revised form 28 September 2002; accepted 15 October 2002

Abstract We provide a short overview on some current issues in the ®elds of forensic genetics and ancient DNA (aDNA) analysis. We discuss about the existence of the possible points of contact between the two disciplines, in terms of open problems and the inherent approach to their solution. We mainly focus on the problem of results authentication, its theoretical and technical aspects. # 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Ancient DNA; PCR; Contamination; Authentication criteria

1. Introduction

2. Ancient DNA analysis

In the last two decades, DNA analysis has become effective, versatile and accessible to a larger community of scientists. This circumstance has not only affected the development of ®elds of genomics (such as gene mapping and functional analysis) but it has also stimulated novel ®elds of application. Ancient DNA (aDNA) analysis and forensic DNA pro®ling are, probably in equal parts, excellent examples of an unconventional way to use the information coming from DNA, with a potential for sharing methodologies and approaches. Here we wish to raise and discuss some critical points of the approaches followed in these two ®elds in their respective way to solve apparently common problems. We will try to ®nd possibly the common ground of problems and to underline differences where they exist. With this, we intend to open a debate on the still largely controversial issues which should deserve wider coverage than they have so far had in either ®elds.

Ancient DNA became the subject of systematic molecular analysis in the late 1980s. By the combined efforts of a group of Allan Wilson's scholars (Russell Higuchi in Berkeley, USA, Svante PaÈaÈbo at the University of Munich, Germany and Mark Stoneking at the Penn State University, USA), what could initially have seemed like an intellectual divertissement has generated an autonomous ®eld of modern molecular biology, then de®ned as `molecular archaeology' (MA) [1,2]. Starting from the simple idea of trying to extract and characterize deoxyribonucleic acid from archaeological and fossilised samples, molecular archaeology has found an innovative way to tackle ambitious goals, such as studying the genetic structure of extinct species and their relationship to contemporary species [3,4] and solving phylogenetic relationships within the genus Homo, throwing light on the origin of modern humans [5,6]. Relying on the molecular counterpart of some fundamental paradigms of modern biology (®rst of all, the existence of a Darwinian continuity in the evolution of animal genomes), MA contrives to offer an historical perspective to molecular investigation and a molecular insight into the natural history of the living species. As repeatedly noted, MA intertwines with a variety

* Corresponding author. Tel.: ‡39-6-35507031; fax: ‡39-6-35507033. E-mail address: [email protected] (C. Capelli).

0379-0738/03/$ ± see front matter # 2003 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 9 - 0 7 3 8 ( 0 2 ) 0 0 3 9 6 - 1

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C. Capelli et al. / Forensic Science International 131 (2003) 59±64

Table 1 Summary description of commonly occurring DNA biochemical modi®cations Biochemical reaction

DNA modification

Effect on DNA analysis

Oxidation

Several base modificationsa

Base misincorporation

Phosphodiester bond break N-glycosyl bond break Deamination

DNA fragmentation

Hydrolysis

a

Unextendable DNA strand

Base misincorporation Base misincorporation

See [13] for a more comprehensive description.

of disciplines such as population genetics, anthropology, archaeology, linguistics and palaeontology [3,6±11]. The invention of the polymerase chain reaction (PCR) de®nitely entailed the continuing rise of MA [1]. Landmarks of this new ®eld have been the DNA analyses of the Neandertal mtDNAs [5,7,8,12]. Rooted in a singularly vast scienti®c background, molecular archaeology has had to face with a formidable array of technical problems. Ancient DNA is chemically modi®ed and it is refractory to most of the current procedures of nucleic acid analysis. Long exposure to UV light, drastic temperature gradients, low/high pH and humidity conditions are the most important determinants of ancient DNA degradation causing several biochemical nucleotide modi®cations [13] (Table 1). Only occasionally, biological samples from the permafrost worldÐa far more stable environmentÐdo offer the analyst ancient samples in decent condition [3,9,14]. Such combination of technical dif®culties and multidisciplinary rooting has ignited exciting controversies over some major achievements of MA. One of the best known examples are the publication of DNA sequences alleged to originate from 120 to 135 million years old amber entombed insects [15] or Table 2 Criteria of authenticity

Separated workstation and labware Investigation of biochemical preservation Clean extract/PCR controls Cloning of PCR products DNA quantitation Phylogenetic test Independent reproduction of the results See main text for discussion.

Forensic genetics

Molecular archaeology

Yes

Yes

±

Yes

Yes ± Informative ± Yes

Yes Yes Yes Yes Recommended for human remains

obtained from a dinosaur bone [16]. Today, we know that these sequences were possibly the result of contamination [17,18] and most scientists date the theoretical limit for the survival of ampli®able DNA to about 100,000 years [19]. Similarly, retrieval of DNA from archaeological human remains has been often claimed in the past, but it has been seldom con®rmed by additional and more strict analyses [20,21]. These and other controversial reports have raised the urgent problem of authentication of the results. Precautions have eventually been adopted to certify results before publication (Table 2). 3. Forensic DNA analysis Almost at the same time, DNA technology has opened a new front in forensic science. Until 1985, all biochemical methods available to identify biological samples in criminal cases were of limited applicability. Conventional blood group and enzyme analysis (e.g. typing of ABO, Km, Gm, EsD, PGM1, AcP) had been used for decades, but they would invariably provide modest analytical power. The way to a new course of events was ®rst paved by the introduction of DNA restriction fragment length polymorphism (RFLP) analysis [22,23], which would have soon provided an incomparably higher discrimination power. However, the procedures of molecular typing were too laborious and required too large amounts of intact DNA to be routinely used in the dif®cult ®eld of `street' DNA sample analysis. Just as it happened with ancient DNA, the advent of PCR was the turning point in the crucial matter of analytical ef®ciency [24]. Since then, the ®eld of molecular identi®cation seemed to have acquired a virtually unlimited power of analysis, allowing forensic experts to address the most inaccessible sources of DNA evidences. Minute quantities of degraded DNA (such as for cigarette butts, ®ngerprints and bone remains) have since then become the target of `DNA pro®ling' (molecular analysis of multiple polymorphic sites) with startlingly high rates of success. On the other hand (and unsurprisingly), as long as forensic scientists would venture into the analysis of minute samples, the problem of result veri®cation has assumed an increasing importanceÐjust in the same way as it was happening in the MA circles. Moreover, molecular archeologists and forensic scientists have also shared projects aiming at the pro®ling of `historical' DNA sources [25,26]Ð thus strengthening the public perception that the two ®elds are somewhat contiguous. Developing a parallel between molecular archaeology and forensic genetics and speculating on the possible points of contact in their respective methodology are therefore relevant issues to address. In this paper, we will raise a number of such theoretical and experimental problems with a view to stimulate the debate among the two scienti®c communities.

C. Capelli et al. / Forensic Science International 131 (2003) 59±64

4. Analogies and differences Forensic genetics does in principle focus on modern DNA: both the nuclear and the mitochondrial genomes. Under these circumstances, the forensic analyst is not systematically faced with severe patterns of biochemical modi®cation. The opposite is true for aDNA. The universal and steady phenomenon of deep degradation is still currently screwing the ®eld of ancient DNA analysis to mitochondrial sequencesÐto a point that a sharp leap of this ®eld into the dimension of nuclear DNA analysis is today somewhat overdue [3,9]. All this makes a striking difference. On the other hand, severe fragmentation of the DNA molecule occurs in some forensic specimens and this makes them look like ancient genomes. This is a point in common. What would in principle seem a super®cial analogy is turned into a fundamental issue as both disciplines make wide use of PCR technology to overcome the problem of expanding the `surviving' integer template-molecule number. In fact, the two ®elds happen to toil on a number of experimental asperities: (a) the DNA molecules retrieved and to be copied are often degraded down to 100±300 bp or less [27]; (b) the amount of molecules per gram of specimen may be critically low (down to thousands and less) [28]; (c) very typical failures in molecular ampli®cation are met in both ®elds (essentially caused by biochemical modi®cations and/or the presence of PCR inhibitors); (d) authenticity of results is often jeopardized by a typical competition between the authentic sample (the one the investigator has an interest into) and a foreign genome (whatever else DNA, in the broadest sense) [28]; (e) very strict measures are needed to let the procedure copy the genome of interest, with tight controls placed at various steps of the procedure, in order to prevent miscopying; (f) there is a steady search for setting general criteria of authentication whose compliance can confer plausibility to the ®nal results (Table 2). The general criteria conferring authenticity and the methodology to adopt for ensuring credible results are probably the benchmark on which to test whether MA and forensics really have something to swap. 5. Authenticity/plausibility of results: the inner logic Both forensic and ancient DNA analysis make a strong point in guaranteeing the authenticity of their results. Authenticity is to be simply de®ned in terms of ®delity of the PCR product to the original DNA template (thought as the DNA speci®c for the biological specimen of interest). In both disciplines, a false result mostly arises by superimposition of a foreign DNA strain and a derangement of the correct biochemical copying procedure. However, differences in the approach to the result authentication derive from the distinctive goals pursued in the two ®elds. Ancient DNA analysis has been seldom called to compare samples of the same age in the same experiment. Thus, a

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cornerstone in the policy of its result authentication is in verifying that there is a realistic genetic distance between the molecule under analysis and a de®nite modern sample chosen as reference for the molecular comparison. Finding the appropriate degree of molecular analogy between a fossil sequence and its contemporary counterpart (with similarities pointing at the common origin and differences re¯ecting the time elapsed according to a `molecular clock' action) will then help to accept the fossil sequence as `authentic' and to rule out contamination by modern DNA. For example, in the case of the Neandertal mtDNA sequences, an average of 34.9 differences from a modern human sequence is found to suit the Neandertal-humans chrono-biological relatedness [5]. And this is the real benchmark for whatever test involving Neandertal sequence authenticity. In the ®eld of forensic identi®cation, contemporary DNA sources have to be compared in search of individual identities. Comparisons are generally developed between quasiunique genetic pro®les based on autosomal microsatellites. While comparing autosomal pro®les, similarities (allele shared by chance) between random individuals can of course occur, especially if the two individuals belong to the same ethnic group (as it is well known to population geneticists). But this feature is of no use for the process of result authentication, as we have seen it in ancient DNA analysis. So here there is no way to catch contamination in the act while looking at the results, and the authenticity game is made of a subtler and a more elusive character. Hints for unfaithful results must be retrieved from indirect evidence (typically, the erratic results obtained from a control DNA ampli®cation, the unexpected results from ampli®cation of a blank test tube). Additionally, false inclusions may not have the same chance to be detected. Usually, a false inclusion may occur when a well-preserved, high-concentrated sample is placed side-to-side in comparison to a poorly preserved, low-copy number sample (classically: the evidentiary and the reference sample), and the most fresh DNA is left to leap into the other test tube. An elementary countermeasure to avoid this is to process critical samples (such as very small stains, single hairs and bone remains) well ahead of their reference. Even better, the two DNAs can be processed by different laboratories and then the results compared. However, in these technical measures there is no intrinsic (i.e. logical) way to distinguish a true identity from a contamination. Only occasionally the formal structure of a test involving an identity inference can protect against incorrect inclusion. For example, when the specimen of a missing individual is compared to one/a couple of putative parents and autosomal pro®les are used, comparisons are brought over randomly assorted series of results (every allele of a genotype referring to either parent's). So the logic speaks for itself, as there is the slightest chance ever to ®nd pairs of genomes matching a third (however authentic or spurious) by mere chance. Unfortunately, the same cannot be for the case of genetic exclusion. Here, the overlapping of a fresh (foreign) DNA

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contaminating the reaction tube can never be logically arguedÐunless the foreign DNA strain is in the track record of the typing laboratory (assuming the uniqueness of each pro®le, one such ®nding would automatically betray a labware contamination). 6. Authentication by technical measures To confer authenticity to a result, forensic and the ancient DNA scientists have developed their own technical measures. Some of these overlap, others do not (Table 2). A point of major interest is whether, in this more practical aspect, there could be an advantage in exporting to one ®eld what is peculiar to the other. An evaluation of certain biochemical components outside the DNA molecule has been suggested as a way to guarantee the good state of DNA preservation in fossil and archaeological remains. Preliminary results have suggested that the degree of amino acid racemization (AAR) is somehow related to the capability to amplify DNA [14], and that ¯ash pyrolysis (FP) with gas chromatography and mass spectrometry might be useful to investigate fossil DNA preservation [29]. This cannot be in principle a criterion of results' authenticity. Of course, AAR and FP will be useful when series of specimens are available for analysis and good candidates have to be quickly foundÐwhile avoiding the waste of precious museum samples. All considered, costs and complications of the inherent technology should discourage the application of these tests to forensic genetics in those (rare) cases when AAR and FP could help (most forensic samples are unsuitable for both). As noted previously, the main problem in aDNAwork is the pre-PCR contamination by whole DNA or PCR products. For this reason, tight segregation should be kept between rooms where manipulation of DNA extracts occurs and where PCR ampli®cation takes place. The two workstations must have separate sets of materials and instruments and never share any material. Sometimes, the laboratory personnel itself better be separate. The basic out®t of an aDNA workstation includes extensive employment of disposable labware and coats, ®ltered tips, facial masks and UV lamps to irradiate the workingbench. During the analysis, several internal controls are introduced at every step of the procedure. These include extraction and PCR blanks, to intercept contaminant molecules originating from the sample environment or from the laboratory (because the most important source of contaminating DNA is human, analysis of animal species DNA is easier to perform and control). All these measures can be certainly shared by the forensic analysts. Besides keeping modern DNA at bay, aDNA analysis also has to face PCR artefacts. Although the normal result of DNA ampli®cation is an invariant replication of few template molecules, the polymerase can introduce nucleotide changes in the original template sequence during the ®rst ampli®cation cycles, due to both DNA damage and inappropriate enzyme activity [7]. These changes are spread through the reaction

products, whose ®nal composition will be a mixture of the original and the altered molecule, in proportion depending on how early the misincorporation has taken place. Cloning and sequencing a selection of PCR products could reveal these events in the form of different nucleotides at the same sequence position. Jumping PCR products are another example of PCR-introduced errors, detectable by clone-and-sequence protocols [30]. Low copy number molecules are often responsible for incorrect DNA typing, as for example when allelic dropout occurs. DNA quantitation [7,28] could be useful in order to calculate the number of molecules present, suggesting which cases require multiple repetitions of genotyping. On account of the existence of PCR errors, ancient sequences have to be ampli®ed and sequenced at least twiceÐor more if discrepancies persist. In addition, the whole procedure is very often applied to different DNA extracts of the same specimen. In turn, PCR amplicons are produced from different DNA extracts, to ensure complete reliability of the results. Alignment of the obtained sequenced clones permits the identi®cation of endogenous versus contaminant nucleotides. When uncertainty occurs at certain positions, additional PCR fragments covering such positions are ampli®ed, cloned and sequenced. Finally, the consensus sequence constructed with the information provided by the multiple clones is analysed to test for phylogenetic con®rmation. The occurrence of polymerase artefacts is normally not considered in the forensic context, because these have limited impact in the ®eld (for example, most identity tests, based on length differences, are insensitive to single nucleotide changes). However, this may not always be true. For example, PCR artefacts may affect extensive identity testing by mtDNA sequence analysis by occasionally altering the sequence and degraded/modi®ed DNA can indeed create artefacts even in replicating the correct template repeat number. To summarise thewhole issue, virtually all technical devices used in ancient DNA technology can be in principle used in forensic DNA analysis, and in fact they often areÐalthough in more or less relaxed versions. But molecular cloning of PCR templates is unlikely to be used as a way to discern PCR artefacts from true tissue heteroplasmy in a forensic context. Repetition of the experiment would rather discriminate between the heteroplasmy and random polymerase errors. 7. Reproducing the results in another laboratory? Reproduction by an independent laboratory has been recently felt as the ultimate test of authentication for an aDNA genotyping result. However, this countermeasure only guarantees against laboratory environmental contamination (because a foreign molecule circulation in the ®rst laboratory could never be present in another) and it does not protect against earlier contamination of the sample. Then, if other conditions of aDNA authenticity are veri®ed (for example, multiple clones from different ampli®ca-

C. Capelli et al. / Forensic Science International 131 (2003) 59±64

tion reactions consistently giving the same sequence, clean extraction/PCR controls, etc.), there should be no speci®c reason to always ask for a second opinion. Such an approach should nevertheless be followed whenever dealing with human remains. Reproducibility of results by a third party is a guarantee of objectivity in a forensic context, to a point that in court cases leaving a sample residue for a `second opinion' is basic routine. In fact thesame analysisisoften repeated by experts of opposite parties in a trial, or in another degree of the judiciary procedure. As shown by the examples we just mentioned above, the problem of result authentication is a much wide-open issue in forensic genetics. Moreover, this ®eld will hardly borrow signi®cant experience from the ®eld of aDNA, because the two disciplines refer to different principles. While moving from different positions on the subject of result authentication, forensic and ancient analysis of DNA can nonetheless see results iteration procedures as a common step in the process of authentication and they can both promote all measures to ensure reproducibilityÐpossibly by another laboratory. 8. Future aspects Forensic and ancient DNA analyses have so far extensively used the same molecular polymorphisms. First of all, mtDNA but also nuclear DNA markersÐan area in which MA aims to progress further [3,9]. Although there is no guarantee that the joint path will be a continuing perspective, no changes seem to be on the horizon. In fact, the single nucleotide polymorphisms and new technologies to genotype them (microarrays, or the equivalent) are seen as the next step in both disciplines [31,32]. Another interesting point is that the two disciplines would substantially bene®t from the introduction of a typing method, whatsoever, which can free scientists from the slavery of PCR pitfalls. There is therefore much of a reason to believe that MA and forensic science will walk in the same direction for a while longer. Acknowledgements We would like to thank Matthias Krings for comments on an earlier version of this work. C. Capelli is particularly indebted to Jim Wilson for precious advice and suggestions and Sonia Bortoletto for continuing support. F. Tschentscher would like to thank Petra BaÈrschneider. References [1] S. PaÈaÈbo, R.G. Higuchi, A. Wilson, Ancient DNA and the polymerase chain reaction: the emerging ®eld of molecular archeology, J. Biol. Chem. 264 (1989) 9709±9712.

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