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Mini review article, past, present, and future of dna typing for analyzing human and non-human forensic samples.

• 1 Department of Biological Sciences, Florida International University, Miami, FL, United States
• 2 International Forensic Research Institute, Florida International University, Miami, FL, United States

Forensic DNA analysis has vastly evolved since the first forensic samples were evaluated by restriction fragment length polymorphism (RFLP). Methodologies advanced from gel electrophoresis techniques to capillary electrophoresis and now to next generation sequencing (NGS). Capillary electrophoresis was and still is the standard method used in forensic analysis. However, dependent upon the information needed, there are several different techniques that can be used to type a DNA fragment. Short tandem repeat (STR) fragment analysis, Sanger sequencing, SNapShot, and capillary electrophoresis-single strand conformation polymorphism (CE-SSCP) are a few of the techniques that have been used for the genetic analysis of DNA samples. NGS is the newest and most revolutionary technology and has the potential to be the next standard for genetic analysis. This review briefly encompasses many of the techniques and applications that have been utilized for the analysis of human and nonhuman DNA samples.

Introduction

Forensic genetics applies genetic tools and scientific methodology to solve criminal and civil litigations ( Editorial, 2007 ). Locard’s Exchange Principle states that every contact leaves a trace, making any evidence a key component in forensic analysis. Biological evidence can comprise of cellular material or cell-free DNA from crime scenes, and as technologies improved, genetic methodologies were expanded to include human and non-human forensic analyses. Although these methodologies can be used for any genome, the prevalence of databases and standard guidelines has allowed human DNA typing to become the gold standard. This review will discuss the historical progression of DNA analysis techniques, strengths and limitations, and their possible forensic applications applied to human and non-human genetics.

Methodologies to Detect Genetic Differences in Humans Is the “Gold Standard”

“dna fingerprinting”: the beginning of human forensic dna typing.

“DNA fingerprinting” was serendipitously discovered in 1984 ( Jeffreys, 2013 ). What they found propelled DNA “fingerprinting,” or DNA typing, to the forefront in legal cases to become the “gold standard” for forensic genetics in a court of law. Jeffreys first used restriction enzymes to fragment DNA, a method in which restriction endonucleases (RE) enzymes fragment the genomic DNA, producing restriction fragment length polymorphisms (RFLP) patterns. Since each RE recognizes specific DNA sequences to enzymatically cut the DNA, then inherent differences between gene sequences, due to evolutionary changes, will produce different fragment lengths. If the enzyme site is present in one individual but has changed in a different individual, the fragment lengths, once separated and visualized, will differ. While this technique was useful for some studies, Jeffreys did not find it useful for his particular genetic studies. Subsequently when working with the myoglobin gene in seals, he discovered that a short section of that gene – a minisatellite – was conserved and when isolated and cloned could be used to detect inherited genetic lineages as well as individualize a subject. Fragment length separation by electrophoresis, followed by transfer to Southern blot membranes, hybridized with a specific or non-specific complementary isotopic DNA probe, allowed for DNA fragments visualization ( Jeffreys et al., 1985b ). Upon careful analysis, Jeffreys determined that the fragments represented different combinations of DNA repetitive elements, unique to each individual, and could be used to better identify individuals or kinship lineages ( Jeffreys et al., 1985b ). Jeffreys’ technology was used in several subsequent paternity, immigration, and forensic genetics cases ( Gill et al., 1985 ; Jeffreys et al., 1985a ; Evans, 2007 ). This was just the beginning of a whole new era in DNA typing.

Restriction Fragment Length Polymorphism (RFLP) Analysis: The Past

After Jeffreys’ discoveries, many DNA analyses methods involving electrophoretic fragment separation were discovered. Many were based on RFLP principles ( Botstein et al., 1980 ), e.g., amplified fragment length polymorphism (AFLP) ( Vos et al., 1995 ), and terminal restriction fragment length polymorphism (TRFLP) ( Liu et al., 1997 ). Others like length heterogeneity- polymerase chain reaction (LH-PCR) ( Suzuki et al., 1998 ) were based on intrinsic insertions and deletions of bases within specific genetic markers. Sanger sequencing ( Sanger and Coulson, 1975 ), and single-strand conformational polymorphism (SSCP) analysis ( Orita et al., 1989 ), while separated by electrophoresis, are theoretically based on single base sequence changes rather than insertions, deletions or RE site differences. While Jeffrey’s DNA fingerprinting method provided a very high power of discrimination, the main limitations were it was very time-consuming and required at least 10–25 ng of DNA to be successful ( Wyman and White, 1980 ). With these limitations, RFLP was not always feasible for forensic cases.

Short Tandem Repeat (STR) Analysis: The Present

The polymerase chain reaction (PCR) was discovered by Kary Mullis in 1985 and helped transform all DNA analyses ( Mullis et al., 1986 ). The current standard for human DNA typing is short tandem repeat (STR) analysis ( McCord et al., 2019 ). This method amplifies highly polymorphic, repetitive DNA regions by PCR and separates them by amplicon length using capillary electrophoresis. These inheritable markers are a series of 2–7 bases tandemly repeated at a specific locus, often in non-coding genetic regions. Forensic STRs are commonly tetranucleotide repeats ( Goodwin et al., 2011 ), chosen because of their technical robustness and high variation among individuals ( Kim et al., 2015 ). The combined DNA index system (CODIS) uses 20 core STR loci, expanded in 2017, and several commercial kits are available that contain these STRs ( Oostdik et al., 2014 ; Ludeman et al., 2018 ). After amplification, different fluorochromes on each primer set allow for visualization of STRs after deconvolution, creating a STR profile consisting of a combination of genotypes ( Gill et al., 2015 ). This method has become the gold standard for human forensics. Its greatest strength is the standardization of loci used by all laboratories and an extremely large searchable database of genetic profiles. However, some limitations and challenges are faced when dealing with highly degraded or low template DNA samples. To overcome these technical challenges, standardized mini-STR kits have been developed which use shorter versions of the core STRs and can be used in the same manner for forensic cases ( Butler et al., 2007 ; Constantinescu et al., 2012 ). Keep in mind, DNA typing of humans – a single species – is the gold standard because of (a) the concerted scientific effort to standardize loci to analyze, (b) the development of commercial kits that can produce the same results regardless of instrumentation or laboratory performing the work, (c) a compatible and very large database that provides allelic frequencies for all sub-populations of humans, (d) standardized statistical methods used to report the results and (e) many court cases that have accepted human DNA typing evidence in a court of law – setting the precedent for future cases to use DNA typing results.

Methodologies to Detect Genetic Differences in Non-Humans: Past and Present

Amplified fragment length polymorphism (aflp) analysis.

It was not long before scientists realized that non-human DNA could provide informative genetic evidence in forensic cases. Applications include bioterrorism, wildlife crimes, human identification through skin microorganisms, and so much more ( Arenas et al., 2017 ). Since large quantities of biological materials are frequently not found at crime scenes, successful RFLP analyses were unlikely. Combining restriction enzymes and PCR technology, a process known as AFLP analysis ( Vos et al., 1995 ), became a method for DNA fingerprinting using minute amounts of unknown sourced DNA. REs digest genomic DNA, then ligation of a constructed adapter sequence to the ends of all fragments allows the annealing of primers designed to recognize the adaptor sequences. Subsequent amplification generates many amplicons ranging in length when separated and visualized in an electropherogram or on a gel ( Vos et al., 1995 ; Butler, 2012 ). AFLP markers for plant forensic DNA typing have been used because it provides high discrimination, requires only small amounts of DNA and the method is reproducible, all forensically important characteristics ( Datwyler and Weiblen, 2006 ). For example, since most cannabis is clonally propagated, subsequent generations will have identical genetic profiles as seen with AFLP ( Miller Coyle et al., 2003 ), providing useful intelligence links back to the source population. But there are significant variation between cultivars and within populations, so not having a standard database representing the species’ diversity for statistical comparisons greatly limits the method’s applicability. Another forensic example of its use is differentiating between marijuana and hemp, two morphologically and genetically similar plants, one an illicit drug while the other is not. In this study, three populations of hemp and one population of marijuana were analyzed with AFLP producing 18 bands that were specific to hemp samples. Additionally, 51.9% of molecular variance occurred within populations indicating these polymorphisms were useful for forensic individualization ( Datwyler and Weiblen, 2006 ).

Terminal Restriction Fragment Length Polymorphism (TRFLP) Analysis

As a result of the anthrax letter attacks of 2001, microbial forensics came to the forefront ( Schmedes et al., 2016 ), a discipline that combines multiple scientific specialties – microbiology, genetics, forensic science, and analytical chemistry. One method used to compare microbial communities is TRFLP ( Liu et al., 1997 ; Osborn et al., 2000 ; Butler, 2012 ). With this method, the DNA is amplified using “universal,” highly conserved primer sequences shared across all organisms of interest, i.e., the 16S rRNA genes in bacteria and Archaea, and then uses REs to fragment the PCR products ( Table 1 ). Separated by capillary electrophoresis, only the fluorescently tagged terminal restricted fragments are visualized ( Mrkonjic Fuka et al., 2007 ), reducing the profile complexity and providing high discrimination. TRFLP has been used to characterize complex microbial communities for forensic applications by linking the similarity of the amplicon patterns generated from the intrinsic soil communities to the evidence from a crime scene ( Meyers and Foran, 2008 ; Habtom et al., 2017 ). This method does provide a distinct pattern reflective of the microbial community, useful for forensic genetics but the method does not provide any sequence information. Another limitation is no standardization of which primer pairs or REs are used, making direct comparisons between studies difficult. This lack of standardization also hinders the development of a database for species identification. Additionally, the method is time-consuming due to the additional step of restriction digestion and the possibility of incomplete enzymatic digestion can complicate the interpretation of results ( Osborn et al., 2000 ; Moreno et al., 2006 ).

Table 1. The basis of differentiation, advantages, and disadvantages of past and current technologies.

Length Heterogeneity-Polymerase Chain Reaction (LH-PCR)

Another methodology has been used to characterize microbial communities is length heterogeneity- polymerase chain reaction (LH-PCR) ( Suzuki et al., 1998 ). Universal primers complementary to highly conserved domains within genomes are used to amplify hypervariable sequences within specific sequence domains. The 16S/18S rRNA genes, the chloroplast genes or Internal Transcribed Spacer (ITS) regions are commonly used. This technique is based on the natural sequence length variation due to insertions and deletions of bases that occur within a domain ( Moreno et al., 2006 ). It has been used to characterize microbial communities for forensic soil applications where a correlation between geographic location and microbial profiles has proven to be more discriminating than elemental soil analysis ( Moreno et al., 2006 , 2011 ; Damaso et al., 2018 ). With LH-PCR, metagenomic DNA extracted from the soil is amplified using fluorescently labeled universal primers with amplicon peaks within the electropherogram representing the minimum diversity within the community. However, specific sequence information is not known as many peaks of the same size could represent more than one species, thereby masking the community’s actual taxonomic diversity. A recent study showed the intrinsic diversity of a microbial mat, masked by LH-PCR, could be further resolved by the inherent sequence differences using capillary electrophoresis-single strand conformational polymorphism (CE-SSCP) analysis ( Damaso et al., 2014 ) and confirmed by sequencing. The advantage of LH-PCR is it is a fast and reproducible method that can correlate geographical areas to microbial patterns with bioinformatics ( Damaso et al., 2018 ); but a soil database would need to be developed to be useful beyond specific geographical areas.

Methodologies to Detect Intersequence Variation: The Past and Present

Sanger sequencing and single nucleotide polymorphism (snp) variation.

The basis of genomic differentiation is the intrinsic order of base pairs within a region that can be evaluated by sequencing. Sanger sequencing has been the gold standard since the 1970s ( Sanger and Coulson, 1975 ). Sanger sequencing was termed the gold standard because of the ability for single base pair resolution allowing for full sequence information to be determined. Robust and extensive databases are also readily available for comparison, i.e., GenBank, to identify an organism. However, it does have some limitations such as the short length (<500–700 bp) and it cannot sequence mixtures of organisms, for example, without cloning, so it would not be useful for sequencing complex microbial communities without intense time, effort and cost.

Other approaches use the ability to identify intrinsic single base sequence variation using single nucleotide polymorphisms (SNPs) within four forensically relevant SNP classes: identity-testing, ancestry informative, phenotype informative, and lineage informative. SNPs are particularly useful when typing degraded DNA or increasing the amount of genetic information retrieved from a sample ( Budowle and van Daal, 2008 ; Goodwin et al., 2011 ). SNaPshot TM is a commercially available SNP kit that can identify known SNPs using single base extension (SBE) technology ( Daniel et al., 2015 ; Fondevila et al., 2017 ). Wildlife forensics has used SNaPshot TM to identify endangered or trafficked species that are illegally poached to support criminal prosecutions. Elephant species identification from ivory and ivory products ( Kitpipit et al., 2017 ) or differentiating wolf species from dog subspecies ( Jiang et al., 2020 ) are both examples of SNaPshot TM assays developed for wildlife forensics. By using species-specific SNPs, the samples could be identified. But yet again, the limitation becomes the need for species-specific reference databases and the monumental task of developing a robust database for each species. Human SNPs databases with allele frequencies, as seen in dbSNP, however, are available making their forensic application more feasible in some cases.

Next-Generation Sequencing: The Present

Massively parallel sequencing (MPS) or next-generation sequencing (NGS) allows for mixtures of genomes of any species to be sequenced in one analysis ( Ansorge, 2009 ). This technology can sequence thousands of genomic regions simultaneously, allowing for whole-genome, metagenomic sequencing or targeted amplicon sequencing ( Gettings et al., 2016 ). Various NGS technologies are available each using slightly different technologies to sequence DNA ( Heather and Chain, 2016 ). Verogen has developed kits explicitly for human forensic genomics using Illumina’s MiSeq FGx system ( Guo et al., 2017 ; Moreno et al., 2018 ). The FBI recently approved DNA profiles generated by Verogen forensic technology to be uploaded into the National DNA Index System (NDIS) ( SWGDAM, 2019 ), making it the first NGS technology approved for NDIS.

Short tandem repeat mixture deconvolution, degraded, low template samples, and even microbial community samples are just a few of the potential NGS applications for forensic genomics and metagenomics ( Borsting and Morling, 2015 ). In human STR analyses, the greatest challenge is mixture deconvolution. NGS technology presents an increased power of discrimination of STR alleles using the intrinsic SNPs genetic microhaplotypes – a combination of 2–4 closely linked SNPs within an allele ( Kidd et al., 2014 ; Pang et al., 2020 ). However, the acceptance of analyses programs to deconvolve mixtures has not been standardized to the same level as it has for STRs.

Microbes are the first responders to changes in any environment because they are rapidly affected by the availability of nutrients and their intrinsic habitats. This makes them excellent indicators for studies investigating post-mortem interval (PMI) or as an indicator of soil geographical provenance ( Giampaoli et al., 2014 ; Finley et al., 2015 ). In decaying organisms, shifts in epinecrotic communities or the thanatomicrobiome are becoming increasingly critical components in investigating PMI ( Javan et al., 2016 ). Sequencing of the thanatomicrobiome revealed the Clostridium spp. varied during different stages human decomposition, the “Postmortem Clostridium Effect” (PCE), providing a time signature of the thanatomicrobiome, which could only have been uncovered through NGS ( Javan et al., 2017 ). However, the lack of consensus in analyses techniques must be addressed before NGS methodologies can be introduced into the justice system ( Table 1 ).

Future Directions and Concluding Remarks

Forensic DNA typing has progressed quickly within a short timeframe ( Figure 1 ), which can be attributed to the many advancements in molecular biology technologies. As these techniques advance, forensic scientists will analyze more atypical forms of evidence to answer questions deemed unresolvable with traditional DNA analyses. For example, epigenetics and DNA methylation markers have been proposed to estimate age, determine the tissue type, and even differentiate between monozygotic twins ( Vidaki and Kayser, 2018 ). However, since epigenetic patterns are also influenced by environmental factors, they can be dynamic, and a number of confounding factors have the potential to affect predictions and must be taken into account when preparing prediction models (i.e., age estimation). Additionally, phenotype informative SNPs across the genome can infer physical characteristics like eye, hair, and skin color, even age, from an unknown source of DNA retrieved from a crime scene. But this technology could pose an “implicit bias” toward minorities, especially in “societies where racism and xenophobia are now on the rise” ( Schneider et al., 2019 ) if not ethically and judicially implemented. With the increased sensitivity of NGS, low biomass samples from environmental DNA (eDNA) – DNA from soil, water, air – can complement and enhance intelligence gathering or provenance in criminal cases. Pollen and dust are two types of eDNA recently explored for their future forensic potential ( Alotaibi et al., 2020 ; Young and Linacre, 2021 ). However, if used in criminal investigations where the eDNA collected has had interaction with other environments, there must be some protocol or quality control established to account for variability that is likely to occur. This makes the prudent validation of this type of DNA analysis, essential. Limitations also arise due to lack of a database for comparison of samples and statistical analyses to evaluate the strength of a match like in the analysis of human STR profiles.

Figure 1. Timeline of the evolution of DNA typing technologies from the 1970’s to the present.

DNA has long been the gold standard in human forensic analysis because of the standardization of DNA markers, databases and statistical analyses. It has laid the foundation for these promising new technologies that will significantly enhance intelligence gathering and species identification – human and non-human – in forensic cases. In order for these methodologies to be useful in criminal investigations, they must adhere to the legal standards such as the Frye or Daubert Standards which determines if an expert testimony or evidence is admissible in court. A method can be deemed acceptable if it follows forensic guidelines set by organizations such as NIST’s Organization Scientific Area Committees (OSAC), Society for Wildlife Forensic Sciences (SWFS), Scientific Working Group on DNA Analysis Methods (SWGDAM), and the International Society for Forensic Genetics (ISFG) ( Linacre et al., 2011 ) just to name a few. These committees provide the guidelines for validation, interpretation, and quality assurance, all necessary components for DNA analysis. The US Fish and Wildlife forensic laboratory has standardized protocols for crimes against federally endangered or threatened species 1 . However, the more common limiting factors in the development of standard guidelines of non-human forensic genetic analyses across different state laboratories are the lack of consensus in methodologies, supporting allelic databases and standardized statistical analyses. Addressing those issues could lay the foundation for non-human analyses to be on par with human analyses.

Author Contributions

DJ designed and wrote the manuscript. DM edited and contributed to the writing of the manuscript. Both authors contributed to the article and approved the submitted version.

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgments

We would like to acknowledge the invitation by the editors to contribute to this special edition. DJ was supported by the Florida Education Fund’s McKnight Doctoral Fellowship.

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Keywords : forensic genetics, DNA typing, metabarcoding, soil, microbes, minisatellites, next-generation sequencing

Citation: Jordan D and Mills D (2021) Past, Present, and Future of DNA Typing for Analyzing Human and Non-Human Forensic Samples. Front. Ecol. Evol. 9:646130. doi: 10.3389/fevo.2021.646130

Received: 25 December 2020; Accepted: 02 March 2021; Published: 22 March 2021.

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*Correspondence: DeEtta Mills, [email protected]

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Life and Death: New Perspectives and Applications in Forensic Science

Molecular Basis of Identification Through DNA Fingerprinting in Humans

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• Moumita Sinha 5 ,
• I. Arjun Rao 5 &
• Mitashree Mitra 6  

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DNA, the operating element of the genes, brings the coded notes of inheritance in every single surviving thing: animals, plants, bacteria, and other microorganisms. Within human beings, the information-bringing DNA arises in each cell having nucleus, including cells surrounding hair roots, spermatozoa, white blood corpuscles, and salivary cells. These would be the cells of utmost significance in forensic investigations. DNA testing has countless prospective advantages for civil and criminal justice; in spite of this, because of the likelihood for its mishandling or abuse, vital issues have been mentioned about trustworthiness, authenticity, and privacy. The methods of DNA testing are results of the innovation in molecular biology that is generating an increase of knowledge about human genetics. The greatly personal and complex info that can be developed by DNA testing involves firm and meticulous knowledge of genetic basis of testing methods.

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Sinha, M., Arjun Rao, I., Mitra, M. (2018). Molecular Basis of Identification Through DNA Fingerprinting in Humans. In: Dash, H., Shrivastava, P., Mohapatra, B., Das, S. (eds) DNA Fingerprinting: Advancements and Future Endeavors. Springer, Singapore. https://doi.org/10.1007/978-981-13-1583-1_7

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DNA fingerprinting in forensics: past, present, future

• Lutz Roewer 1  

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DNA fingerprinting, one of the great discoveries of the late 20th century, has revolutionized forensic investigations. This review briefly recapitulates 30 years of progress in forensic DNA analysis which helps to convict criminals, exonerate the wrongly accused, and identify victims of crime, disasters, and war. Current standard methods based on short tandem repeats (STRs) as well as lineage markers (Y chromosome, mitochondrial DNA) are covered and applications are illustrated by casework examples. Benefits and risks of expanding forensic DNA databases are discussed and we ask what the future holds for forensic DNA fingerprinting.

The past - a new method that changed the forensic world

'“I’ve found it! I’ve found it”, he shouted, running towards us with a test-tube in his hand. “I have found a re-agent which is precipitated by hemoglobin, and by nothing else”,’ says Sherlock Holmes to Watson in Arthur Conan Doyle’s first novel A study in Scarlet from1886 and later: 'Now we have the Sherlock Holmes’ test, and there will no longer be any difficulty […]. Had this test been invented, there are hundreds of men now walking the earth who would long ago have paid the penalty of their crimes’ [ 1 ].

The Eureka shout shook England again and was heard around the world when roughly 100 years later Alec Jeffreys at the University of Leicester, in UK, found extraordinarily variable and heritable patterns from repetitive DNA analyzed with multi-locus probes. Not being Holmes he refrained to call the method after himself but 'DNA fingerprinting’ [ 2 ]. Under this name his invention opened up a new area of science. The technique proved applicable in many biological disciplines, namely in diversity and conservation studies among species, and in clinical and anthropological studies. But the true political and social dimension of genetic fingerprinting became apparent far beyond academic circles when the first applications in civil and criminal cases were published. Forensic genetic fingerprinting can be defined as the comparison of the DNA in a person’s nucleated cells with that identified in biological matter found at the scene of a crime or with the DNA of another person for the purpose of identification or exclusion. The application of these techniques introduces new factual evidence to criminal investigations and court cases. However, the first case (March 1985) was not strictly a forensic case but one of immigration [ 3 ]. The first application of DNA fingerprinting saved a young boy from deportation and the method thus captured the public’s sympathy. In Alec Jeffreys’ words: 'If our first case had been forensic I believe it would have been challenged and the process may well have been damaged in the courts’ [ 4 ]. The forensic implications of genetic fingerprinting were nevertheless obvious, and improvements of the laboratory process led already in 1987 to the very first application in a forensic case. Two teenage girls had been raped and murdered on different occasions in nearby English villages, one in 1983, and the other in 1986. Semen was obtained from each of the two crime scenes. The case was spectacular because it surprisingly excluded a suspected man, Richard Buckland, and matched another man, Colin Pitchfork, who attempted to evade the DNA dragnet by persuading a friend to give a sample on his behalf. Pitchfork confessed to committing the crimes after he was confronted with the evidence that his DNA profile matched the trace DNA from the two crime scenes. For 2 years the Lister Institute of Leicester where Jeffreys was employed was the only laboratory in the world doing this work. But it was around 1987 when companies such as Cellmark, the academic medico-legal institutions around the world, the national police, law enforcement agencies, and so on started to evaluate, improve upon, and employ the new tool. The years after the discovery of DNA fingerprinting were characterized by a mood of cooperation and interdisciplinary research. None of the many young researchers who has been there will ever forget the DNA fingerprint congresses which were held on five continents, in Bern (1990), in Belo Horizonte (1992), in Hyderabad (1994), in Melbourne (1996), and in Pt. Elizabeth (1999), and then shut down with the good feeling that the job was done. Everyone read the Fingerprint News distributed for free by the University of Cambridge since 1989 (Figure  1 ). This affectionate little periodical published non-stylish short articles directly from the bench without impact factors and resumed networking activities in the different fields of applications. The period in the 1990s was the golden research age of DNA fingerprinting succeeded by two decades of engineering, implementation, and high-throughput application. From the Foreword of Alec Jeffreys in Fingerprint News , Issue 1, January 1989: 'Dear Colleagues, […] I hope that Fingerprint News will cover all aspects of hypervariable DNA and its application, including both multi-locus and single-locus systems, new methods for studying DNA polymorphisms, the population genetics of variable loci and the statistical analysis of fingerprint data, as well as providing useful technical tips for getting good DNA profiles […]. May your bands be variable’ [ 5 ].

Cover of one of the first issues of Fingerprint News from 1990.

Jeffreys’ original technology, now obsolete for forensic use, underwent important developments in terms of the basic methodology, that is, from Southern blot to PCR, from radioactive to fluorescent labels, from slab gels to capillary electrophoresis. As the technique became more sensitive, the handling simple and automated and the statistical treatment straightforward, DNA profiling, as the method was renamed, entered the forensic routine laboratories around the world in storm. But, what counts in the Pitchfork case and what still counts today is the process to get DNA identification results accepted in legal proceedings. Spectacular fallacies, from the historical 1989 case of People vs. Castro in New York [ 6 ] to the case against Knox and Sollecito in Italy (2007–2013) where literally DNA fingerprinting was on trial [ 7 ], disclosed severe insufficiencies in the technical protocols and especially in the DNA evidence interpretation and raised nolens volens doubts on the scientific and evidentiary value of forensic DNA fingerprinting. These cases are rare but frequent enough to remind each new generation of forensic analysts, researchers, or private sector employees that DNA evidence is nowadays an important part of factual evidence and needs thus intense scrutiny for all parts of the DNA analysis and interpretation process.

In the following I will briefly describe the development of DNA fingerprinting to a standardized investigative method for court use which has since 1984 led to the conviction of thousands of criminals and to the exoneration of many wrongfully suspected or convicted individuals [ 8 ]. Genetic fingerprinting per se could of course not reduce the criminal rate in any of the many countries in the world, which employ this method. But DNA profiling adds hard scientific value to the evidence and strengthens thus (principally) the credibility of the legal system.

The technological evolution of forensic DNA profiling

In the classical DNA fingerprinting method radio-labeled DNA probes containing minisatellite [ 9 ] or oligonucleotide sequences [ 10 ] are hybridized to DNA that has been digested with a restriction enzyme, separated by agarose electrophoresis and immobilized on a membrane by Southern blotting or - in the case of the oligonucleotide probes - immobilized directly in the dried gel. The radio-labeled probe hybridizes to a set of minisatellites or oligonucleotide stretches in genomic DNA contained in restriction fragments whose size differ because of variation in the numbers of repeat units. After washing away excess probe the exposure to X-ray film (autoradiography) allows these variable fragments to be visualized, and their profiles compared between individuals. Minisatellite probes, called 33.6 and 33.15, were most widely used in the UK, most parts of Europe and the USA, whereas pentameric (CAC)/(GTG) 5 probes were predominantly applied in Germany. These so-called multilocus probes (MLP) detect sets of 15 to 20 variable fragments per individual ranging from 3.5 to 20 kb in size (Figure  2 ). But the multi-locus profiling method had several limitations despite its successful application to crime and kinship cases until the middle of the 1990s. Running conditions or DNA quality issues render the exact matching between bands often difficult. To overcome this, forensic laboratories adhered to binning approaches [ 11 ], where fixed or floating bins were defined relative to the observed DNA fragment size, and adjusted to the resolving power of the detection system. Second, fragment association within one DNA fingerprint profile is not known, leading to statistical errors due to possible linkage between loci. Third, for obtaining optimal profiles the method required substantial amounts of high molecular weight DNA [ 12 ] and thus excludes the majority of crime-scene samples from the analysis. To overcome some of these limitations, single-locus profiling was developed [ 13 ]. Here a single hypervariable locus is detected by a specific single-locus probe (SLP) using high stringency hybridization. Typically, four SLPs were used in a reprobing approach, yielding eight alleles of four independent loci per individual. This method requires only 10 ng of genomic DNA [ 14 ] and has been validated through extensive experiments and forensic casework, and for many years provided a robust and valuable system for individual identification. Nevertheless, all these different restriction fragment length polymorphism (RFLP)-based methods were still limited by the available quality and quantity of the DNA and also hampered by difficulties to reliably compare genetic profiles from different sources, labs, and techniques. What was needed was a DNA code, which could ideally be generated even from a single nucleated cell and from highly degraded DNA, a code, which could be rapidly generated, numerically encrypted, automatically compared, and easily supported in court. Indeed, starting in the early 1990s DNA fingerprinting methods based on RFLP analysis were gradually supplanted by methods based on PCR because of the improved sensitivity, speed, and genotyping precision [ 15 ]. Microsatellites, in the forensic community usually referred to short tandem repeats (STRs), were found to be ideally suited for forensic applications. STR typing is more sensitive than single-locus RFLP methods, less prone to allelic dropout than VNTR (variable number of tandem repeat) systems [ 16 ], and more discriminating than other PCR-based typing methods, such as HLA-DQA1 [ 17 ]. More than 2,000 publications now detail the technology, hundreds of different population groups have been studied, new technologies as, for example, the miniSTRs [ 18 ] have been developed and standard protocols have been validated in laboratories worldwide (for an overview see [ 19 ]). Forensic DNA profiling is currently performed using a panel of multi-allelic STR markers which are structurally analogous to the original minisatellites but with much shorter repeat tracts and thus easier to amplify and multiplex with PCR. Up to 30 STRs can be detected in a single capillary electrophoresis injection generating for each individual a unique genetic code. Basically there are two sets of STR markers complying with the standards requested by criminal databases around the world: the European standard set of 12 STR markers [ 20 ] and the US CODIS standard of 13 markers [ 21 ]. Due to partial overlap, they form together a standard of 18 STR markers in total. The incorporation of these STR markers into commercial kits has improved the application of these markers for all kinds of DNA evidence with reproducible results from as less than three nucleated cells [ 22 ] and extracted even from severely compromised material. The probability that two individuals will have identical markers at each of 13 different STR loci within their DNA exceeds one out of a billion. If a DNA match occurs between an accused individual and a crime scene stain, the correct courtroom expression would be that the probability of a match if the crime-scene sample came from someone other than the suspect (considering the random, not closely-related man) is at most one in a billion [ 14 ]. The uniqueness of each person’s DNA (with the exception of monozygotic twins) and its simple numerical codification led to the establishment of government-controlled criminal investigation DNA databases in the developed nations around the world, the first in 1995 in the UK [ 23 ]. When a match is made from such a DNA database to link a crime scene sample to an offender who has provided a DNA sample to a database that link is often referred to as a cold hit. A cold hit is of value as an investigative lead for the police agency to a specific suspect. China (approximately 16 million profiles, the United States (approximately 10 million profiles), and the UK (approximately 6 million profiles) maintain the largest DNA database in the world. The percentage of databased persons is on the increase in all countries with a national DNA database, but the proportions are not the same by the far: whereas in the UK about 10% of the population is in the national DNA database, the percentage in Germany and the Netherlands is only about 0.9% and 0.8%, respectively [ 24 ].

Multilocus DNA Fingerprint from a large family probed with the oligonucleotide (GTG) 5 ( Courtesy of Peter Nürnberg, Cologne Center for Genomics, Germany ).

Lineage markers in forensic analysis

Lineage markers have special applications in forensic genetics. Y chromosome analysis is very helpful in cases where there is an excess of DNA from a female victim and only a low proportion from a male perpetrator. Typical examples include sexual assault without ejaculation, sexual assault by a vasectomized male, male DNA under the fingernails of a victim, male 'touch’ DNA on the skin, and the clothing or belongings of a female victim. Mitochondrial DNA (mtDNA) is of importance for the analyses of low level nuclear DNA samples, namely from unidentified (typically skeletonized) remains, hair shafts without roots, or very old specimens where only heavily degraded DNA is available [ 25 ]. The unusual non-recombinant mode of inheritance of Y and mtDNA weakens the statistical weight of a match between individual samples but makes the method efficient for the reconstruction of the paternal or maternal relationship, for example in mass disaster investigations [ 26 ] or in historical reconstructions. A classic case is the identification of two missing children of the Romanov family, the last Russian monarchy. MtDNA analysis combined with additional DNA testing of material from the mass grave near Yekaterinburg gave virtually irrefutable evidence that the two individuals recovered from a second grave nearby are the two missing children of the Romanov family: the Tsarevich Alexei and one of his sisters [ 27 ]. Interestingly, a point heteroplasmy, that is, the presence of two slightly different mtDNA haplotypes within an individual, was found in the mtDNA of the Tsar and his relatives, which was in 1991 a contentious finding (Figure  3 ). In the early 1990s when the bones were first analyzed, a point heteroplasmy was believed to be an extremely rare phenomenon and was not readily explainable. Today, the existence of heteroplasmy is understood to be relatively common and large population databases can be searched for its frequency at certain positions. The mtDNA evidence in the Romanov case was underpinned by Y-STR analysis where a 17-locus haplotype from the remains of Tsar Nicholas II matched exactly to the femur of the putative Tsarevich and also to a living Romanov relative. Other studies demonstrated that very distant family branches can be traced back to common ancestors who lived hundreds of years ago [ 28 ]. Currently forensic Y chromosome typing has gained wide acceptance with the introduction of highly sensitive panels of up to 27 STRs including rapidly mutating markers [ 29 ]. Figure  4 demonstrates the impressive gain of the discriminative power with increasing numbers of Y-STRs. The determination of the match probability between Y-STR or mtDNA profiles via the mostly applied counting method [ 30 ] requires large, representative, and quality-assessed databases of haplotypes sampled in appropriate reference populations, because the multiplication of individual allele frequencies is not valid as for independently inherited autosomal STRs [ 31 ]. Other estimators for the haplotype match probability than the count estimator have been proposed and evaluated using empirical data [ 32 ], however, the biostatistical interpretation remains complicated and controversial and research continues. The largest forensic Y chromosome haplotype database is the YHRD ( http://www.yhrd.org ) hosted at the Institute of Legal Medicine and Forensic Sciences in Berlin, Germany, with about 115,000 haplotypes sampled in 850 populations [ 33 ]. The largest forensic mtDNA database is EMPOP ( http://www.empop.org ) hosted at the Institute of Legal Medicine in Innsbruck, Austria, with about 33,000 haplotypes sampled in 63 countries [ 34 ]. More than 235 institutes have actually submitted data to the YHRD and 105 to EMPOP, a compelling demonstration of the level of networking activities between forensic science institutes around the world. That additional intelligence information is potentially derivable from such large datasets becomes obvious when a target DNA profile is searched against a collection of geographically annotated Y chromosomal or mtDNA profiles. Because linearly inherited markers have a highly non-random geographical distribution the target profile shares characteristic variants with geographical neighbors due to common ancestry [ 35 ]. This link between genetics, genealogy, and geography could provide investigative leads for investigators in non-suspect cases as illustrated in the following case [ 36 ]:

Screenshot of the 16169 C/T heteroplasmy present in Tsar Nicholas II using both forward and reverse sequencing primers ( Courtesy of Michael Coble, National Institute of Standards and Technology, Gaithersburg, USA ).

Correlation between the number of analyzed Y-STRs and the number of different haplotypes detected in a global population sample of 18,863 23-locus haplotypes.

Screenshot from the YHRD depicting the radiation of a 9-locus haplotype belonging to haplogroup J in Southern Europe.

In 2002, a woman was found with a smashed skull and covered in blood but still alive in her Berlin apartment. Her life was saved by intensive medical care. Later she told the police that she had let a man into her apartment, and he had immediately attacked her. The man was subletting the apartment next door. The evidence collected at the scene and in the neighboring apartment included a baseball cap, two towels, and a glass. The evidence was sent to the state police laboratory in Berlin, Germany and was analyzed with conventional autosomal STR profiling. Stains on the baseball cap and on one towel revealed a pattern consistent with that of the tenant, whereas two different male DNA profiles were found on a second bath towel and on the glass. The tenant was eliminated as a suspect because he was absent at the time of the offense, but two unknown men (different in autosomal but identical in Y-STRs) who shared the apartment were suspected. Unfortunately, the apartment had been used by many individuals of both European and African nationalities, so the initial search for the two men became very difficult. The police obtained a court order for Y-STR haplotyping to gain information about the unknown men’s population affiliation. Prerequisites for such biogeographic analyses are large reference databases containing Y-STR haplotypes also typed for ancestry informative single nucleotide markers (SNP) markers from hundreds of different populations. The YHRD proved useful to infer the population origin of the unknown man. The database inquiry indicated a patrilineage of Southern European ancestry, whereas an African descent was unlikely (Figure  5 ). The police were able to track down the tenant in Italy, and with his help, establish the identity of one of the unknown men, who was also Italian. When questioning this man, the police used the information retrieved from Y-STR profiling that he had shared the apartment in Berlin with a paternal relative. This relative was identified as his nephew. Because of the close-knit relationship within the family, this information would probably not have been easily retrieved from the uncle without the prior knowledge. The nephew was suspected of the attempted murder in Berlin. He was later arrested in Italy, where he had committed another violent robbery.

Information on the biogeographic origin of an unknown DNA could also be retrieved from a number of ancestry informative SNPs (AISNPs) on autosomes or insertion/deletion polymorphisms [ 37 , 38 ] but perhaps even better from so-called mini-haplotypes with only <10 SNPs spanning small molecular intervals (<10 kb) with very low recombination among sites [ 39 ]. Each 'minihap’ behaves like a locus with multiple haplotype lineages (alleles) that have evolved from the ancestral human haplotype. All copies of each distinct haplotype are essentially identical by descent. Thus, they fall like Y and mtDNA into the lineage-informative category of genetic markers and are thus useful for connecting an individual to a family or ancestral genetic pool.

Benefits and risks of forensic DNA databases

The steady growth in the size of forensic DNA databases raises issues on the criteria of inclusion and retention and doubts on the efficiency, commensurability, and infringement of privacy of such large personal data collections. In contrast to the past, not only serious but all crimes are subject to DNA analysis generating millions and millions of DNA profiles, many of which are stored and continuously searched in national DNA databases. And as always when big datasets are gathered new mining procedures based on correlation became feasible. For example, 'Familial DNA Database Searching’ is based on near matches between a crime stain and a databased person, which could be a near relative of the true perpetrator [ 40 ]. Again the first successful familial search was conducted in UK in 2004 and led to the conviction of Craig Harman of manslaughter. Craig Harman was convicted because of partial matches from Harman’s brother. The strategy was subsequently applied in some US states but is not conducted at the national level. It was during a dragnet that it first became public knowledge that the German police were also already involved in familial search strategies. In a little town in Northern Germany the police arrested a young man accused of rape because they had analyzed the DNA of his two brothers who had participated in the dragnet. Because of partial matches between crime scene DNA profiles and these brothers they had identified the suspect. In contrast to other countries, the Federal Constitutional Court of Germany decided in December 2012 against the future court use of this kind of evidence.

Civil rights and liberties are crucial for democratic societies and plans to extend forensic DNA databases to whole populations need to be condemned. Alec Jeffreys early on has questioned the way UK police collects DNA profiles, holding not only convicted individuals but also arrestees without conviction, suspects cleared in an investigation, or even innocent people never charged with an offence [ 41 ]. He also criticized that large national databases as the NDNAD of England and Wales are likely skewed socioeconomically. It has been pointed out that most of the matches refer to minor offences; according to GeneWatch in Germany 63% of the database matches provided are related to theft while <3% related to rape and murder. The changes to the UK database came in the 2012’s Protection of Freedoms bill, following a major defeat at the European Court of Human Rights in 2008. As of May 2013 1.1 million profiles (of about 7 million) had been destroyed to remove innocent people’s profiles from the database. In 2005 the incoming government of Portugal proposed a DNA database containing samples from every Portuguese citizen. Following public objections, the government limited the database to criminals. A recent study on the public views on DNA database-related matters showed that a more critical attitude towards wider national databases is correlated with the age and education of the respondents [ 42 ]. A deeper public awareness on the benefits and risks of very large DNA collections need to be built and common ethical and privacy standards for the development and governance of DNA databases need to be adopted where the citizen’s perspectives are taken into consideration.

The future of forensic DNA analysis

The forensic community, as it always has, is facing the question in which direction the DNA Fingerprint technology will be developed. A growing number of colleagues are convinced that DNA sequencing will soon replace methods based on fragment length analysis and there are good arguments for this position. With the emergence of current Next Generation Sequencing (NGS) technologies, the body of forensically useful data can potentially be expanded and analyzed quickly and cost-efficiently. Given the enormous number of potentially informative DNA loci - which of those should be sequenced? In my opinion there are four types of polymorphisms which deserve a place on the analytic device: an array of 20–30 autosomal STRs which complies with the standard sets used in the national and international databases around the world, a highly discriminating set of Y chromosomal markers, individual and signature polymorphisms in the control and coding region of the mitochondrial genome [ 43 ], as well as ancestry and phenotype inference SNPs [ 44 ]. Indeed, a promising NGS approach with the simultaneous analysis of 10 STRs, 386 autosomal ancestry and phenotype informative SNPs, and the complete mtDNA genome has been presented recently [ 45 ] (Figure  6 ). Currently, the rather high error rates are preventing NGS technologies from being used in forensic routine [ 46 ], but it is foreseeable that the technology will be improved in terms of accuracy and reliability. Time is another essential factor in police investigations which will be considerably reduced in future applications of DNA profiling. Commercial instruments capable of producing a database-compatible DNA profile within 2 hours exist [ 47 ] and are currently under validation for law enforcement use. The hands-free 'swab in - profile out’ process consists of automated extraction, amplification, separation, detection, and allele calling without human intervention. In the US the promise of on-site DNA analysis has already altered the way in which DNA could be collected in future. In a recent decision the Supreme court of the United States held that 'when officers make an arrest supported by probable cause to hold for a serious offense and bring the suspect to the station to be detained in custody, taking and analyzing a cheek swab of the arrestee’s DNA is, like fingerprinting and photographing, a legitimate police booking procedure’ (Maryland v. Alonzo Jay King, Jr.). In other words, DNA can be taken from any arrestee, rightly or wrongly arrested, as a part of the normal booking procedure. Twenty-eight states and the federal government now take DNA swabs after arrests with the aim of comparing profiles to the CODIS database, creating links to unsolved cases and to identify the person (Associated Press, 3 June 2013). Driven by the rapid technological progress DNA actually becomes another metric of quick identification. It remains to be seen whether rapid DNA technologies will alter the way in which DNA is collected by police in other countries. In Germany for example the DNA collection is still regulated by the code of the criminal procedure and the use of DNA profiling for identification purposes only is excluded. Because national legislations are basically so different, a worldwide system to interrogate DNA profiles from criminal justice databases seems currently a very distant project.

Schematic overview of Haloplex targeting and NGS analysis of a large number of markers simultaneously. Sequence data are shown for samples from two individuals and the D3S1358 STR marker, the rs1335873 SNP marker, and a part of the HVII region of mtDNA ( Courtesy of Marie Allen, Uppsala University, Sweden ).

At present the forensic DNA technology directly affects the lives of millions people worldwide. The general acceptance of this technique is still high, reports on the DNA identification of victims of the 9/11 terrorist attacks [ 48 ], of natural disasters as the Hurricane Katrina [ 49 ], and of recent wars (for example, in former Yugoslavia [ 50 ]) and dictatorship (for example, in Argentina [ 51 ]) impress the public in the same way as police investigators in white suits securing DNA evidence at a broken door. CSI watchers know, and even professionals believe, that DNA will inevitably solve the case just following the motto Do Not Ask, it’s DNA, stupid! But the affirmative view changes and critical questions are raised. It should not be assumed that the benefits of forensic DNA fingerprinting will necessarily override the social and ethical costs [ 52 ].

This short article leaves many of such questions unanswered. Alfred Nobel used his fortune to institute a prize for work 'in ideal direction’. What would be the ideal direction in which DNA fingerprinting, one of the great discoveries in recent history, should be developed?

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Article Contents

1. introduction, 2. 20th-century developments in paternity testing, 3. ‘what has one to observe [in paternity testing]… properly speaking everything’: the anthropological approach of margarete weninger, 4. matsukura toyoji and the ‘biological value’ of fingerprints, 5. fingerprint-based paternity tests on the eve of dna profiling: the case of 1980s and 1990s china, 6. general discussion and conclusion.

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Fingerprints and paternity testing: a study of genetics and probability in pre-DNA forensic science

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Daniel Asen, Fingerprints and paternity testing: a study of genetics and probability in pre-DNA forensic science, Law, Probability and Risk , Volume 18, Issue 2-3, June-September 2019, Pages 177–199, https://doi.org/10.1093/lpr/mgz014

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This article is a study of forensic science researchers’ attempts to develop paternity tests based on fingerprint patterning, a physical trait that is partially inherited. Pursued in different times and places—ranging from Austria to Japan to China and from the early 20th century to the 1990s—the projects under study represent an ongoing dialogue, carried out through decades of international scientific exchange, about how to extract genetic information from fingerprints and present this data as scientifically-valid evidence in courts of law. Over time, those who engaged in this work increasingly experimented with methods for presenting fingerprint-based evidence of paternity in quantifiable and even probabilistic terms. Fingerprint-based paternity tests remained an obscure area of forensic practice and were eventually overshadowed by advances in serology and DNA profiling. This unfamiliar corner of forensic science, nonetheless, can provide additional perspective on the history of statistical expertise and probabilistic reasoning in modern forensic science, including the application of Bayesian approaches. The larger body of 20th-century ‘dermatoglyphics’ knowledge out of which these tests emerged also continues to influence the foundation of scientific knowledge on which latent print examination is based today.

Since the start of the 21st century, discussions about the strengths and weaknesses of forensic disciplines and pathways for their reform have been deeply shaped by expectations and standards associated with DNA profiling ( Murphy, 2010 ). As stated in the report of the National Research Council of the National Academies: ‘Unlike many forensic techniques that were developed empirically within the forensic community, with little foundation in scientific theory or analysis, DNA analysis is a fortuitous byproduct of cutting-edge science’ ( National Research Council, 2009 , p. 99). Viewed in this way as a ‘model forensic discipline’ grounded in basic research, academically-validated methods, and probabilistic reasoning, DNA profiling has come to provide a general blueprint for what it might mean to give other forensic disciplines stronger scientific foundations ( Murphy, 2010 , p. 17; National Research Council, 2009 , pp. 128, 133, 139–140; Lynch et al. , 2008 , pp. 4–5, 306–310). Against this backdrop, the field of latent print evidence has seen a number of new developments. These include, for example, new procedures for formalizing the analysis of fingerprint minutiae, attempts to quantify error and formalize its presentation, and the development of models for using probabilistic reasoning to assess the value of latent fingermark evidence ( Champod et al. , 2016 ; Edmond et al. , 2014 ; Abraham et al. , 2013 ).

The present moment is not, in fact, the first time that academically-validated methods associated with scientific disciplines such as human genetics or even probabilistic reasoning have influenced forensic applications of fingerprinting. During the 20th century, these same elements coalesced around the interpretation of fingerprint evidence in another area of forensic practice—namely, paternity testing. It is well known that the 20th century saw a revolution in methods of paternity testing that was driven by a series of technical developments in serology, human genetics, and, subsequently, molecular biology (e.g. Patzelt, 2004 ). The history of fingerprinting intersects with this story via the skein of scientific concerns associated with ‘dermatoglyphics’, a prolific but obscure discipline concerned with the scientific study of skin ridge patterning on the fingers and palms. Before and after World War II, researchers in this field explored the possibility that fingerprint patterning could yield insights into human heredity, the origins and migrations of racially-defined populations, and even the presence of congenital conditions such as Down syndrome ( Cummins and Midlo, 1943 ; Cole, 2002 , pp. 97–118; Miller, 2002 , 2003 ; Asen, 2018 ).

It was in the context of this multifaceted scientific field that researchers working at various global sites attempted to develop paternity tests that could use fingerprint patterning to investigate the genetic relationship between a known biological parent, putative parent and child. Three such projects are examined in this article: (1) Austrian anthropologist Margarete Weninger’s (1896–1987) use of the early 20th-century methodology of ‘similarity diagnosis’ to incorporate fingerprint evidence into paternity testing; (2) Japanese medico-legal scientist Matsukura Toyoji’s (1906–1993) development of a paternity test based on the novel theory of fingerprint pattern genetics that he developed during the 1950s; and (3) Chinese medico-legal researcher Jia Jingtao (1927-) and his colleagues’ critical evaluation of Matsukura’s approach in 1980s and 1990s China, a context in which DNA profiling was just starting to see adoption. While pursued in very different times and places, these projects represent three points in an ongoing dialogue—carried out through decades of international scientific exchange—about how to extract genetic information from fingerprints and present this data as scientifically-valid evidence in courts of law. Those who engaged in this work increasingly came to experiment with methods for presenting fingerprint-based evidence of paternity in quantifiable and even probabilistic terms.

The research projects that are the focus of this article did not, in any simple or direct sense, lead to the forensic science of today. Over time, the considerable limitations involved in using fingerprint patterning as an object of genetic analysis became apparent. These included the difficulty of identifying the relevant phenotype to be analysed (whether it should be the fingerprint pattern-type, ridge count, or something else) and establishing the relative influence of genes and (prenatal) environment in the formation of these patterns. 1 Those who developed paternity tests necessarily put much time and effort into addressing these questions, if not attempting to resolve them. Over the decades, their work became part of the large body of research in dermatoglyphics that was devoted to investigating the genetics of fingerprint patterning ( Mavalwala, 1977 ; Cole, 2002 , pp. 99–103, 109–111, 117–118). These research efforts tended to confirm the partially-inherited nature of fingerprint patterning without conclusively identifying the underlying mechanisms of genetic inheritance. By the end of the 20th century, the epistemic weaknesses of fingerprint-based genetic analysis combined with the greater effectiveness of other techniques led to a general diminishing of interest in trying to use fingerprints to establish paternity.

While fingerprint-based paternity testing was at times utilized in judicial practice, it remained an obscure area of forensic practice and was eventually overshadowed by advances in serology and DNA profiling. This largely forgotten subfield of forensic science, nonetheless, can provide additional perspective on the history of statistical expertise and probabilistic reasoning in modern forensics, including the application of Bayesian approaches. 2 Such approaches have become increasingly prominent in various subfields of 21st-century forensic science, including latent print evidence ( Cole, 2017 ). Long before DNA profiling provided an impetus for the use of probabilistic reasoning in today’s forensic disciplines, it was serology (and the related discipline of population genetics) that served as an earlier ‘model forensic discipline’ whose approaches and methods had a tendency to migrate to (and influence standards of evidence in) other areas of forensic practice. This was the context in which Matsukura Toyoji, Jia Jingtao, and their colleagues attempted to work out the mechanisms underlying the genetic inheritance of fingerprint patterning and quantify the significance of this evidence through approaches such as Erik Essen-Möller’s formula for calculating Probability of Paternity (e.g. Hummel, 1981 ).

At a moment today when the scientific validation of latent print evidence is a pressing issue, it is worth considering how the relationship between fingerprinting and scientific knowledge has been understood in the past. Today’s discussions about what it means for fingerprinting to be ‘scientific’ tend to revolve around issues such as validity testing, the determination of error rates, and other ways of improving latent print examination (e.g. Haber and Haber, 2008 ). By contrast, the ‘science’ of fingerprints represented by dermatoglyphics was broader than forensic identification in scope, drew on the techniques of other scientific fields such as physical anthropology and population genetics, and, we will see, was defined by epistemic ambiguities from the start. As much as this older science of fingerprints might seem outdated today, there are points of continuity, discussed in the conclusion, that connect this story to the current field of latent print evidence. Examining this unfamiliar history can provide new perspectives on the multilayered nature of today’s knowledge about fingerprints and the different ways in which this knowledge has impacted forensic practice in modern times.

Much is at stake in the ability to determine with certainty that a child is or is not the offspring of particular biological parents. As historian Nara B. Milanich (2019) has shown, modern understandings of paternity in the Americas and Europe emerged at the intersection of varied political, legal, social and cultural concerns, not to mention through the involvement of experts of diverse disciplinary backgrounds who constructed paternity as a biological fact that could be investigated through scientific methods. While the practical applications and legal admissibility of paternity testing practices differ across different legal (and political) systems, the confirmation of biological parentage is, generally speaking, an area of applied scientific knowledge that impinges upon a broad range of legal, administrative and cultural concerns in modern societies. 3

A watershed moment in the history of paternity testing occurred in the first half of the 20th century with the discovery of the ABO blood groups as well as advances in knowledge of their heredity and distribution in different populations ( Schneider, 1983 , 1996 ). The resulting explosion in blood groups research, which was carried out at a truly international level, gave rise to new fields such as immunology and seroanthropology, and also provided forensic medicine with new methods for identifying individuals on the basis of blood type. The understanding that blood group factors were inherited in predictable ways that followed Mendelian laws also made it possible to establish with certainty which combinations of parents could yield a child of a certain blood type and which could not (Lattes, 1932, pp. 245–250). By the 1920s, parentage tests that relied upon this logic to exclude a putative parent were being used in Germany and Austria. Over the 1920s and 1930s, these methods were adopted in other continental European countries, even though there was still wide variation in the extent to which national legal systems were accepting of such evidence (Lattes, 1932, pp. 250–256; Schwidetzky, 1954 , p. 2; Schneider, 1983 , pp. 553–555).

Over subsequent decades, the use of blood-based paternity tests saw a number of further developments. Additional blood group systems were discovered—MN in the late 1920s, Rh in 1940, and so on, including additional sub-groups within the existing systems—and these were added to the battery of genetically-determined serological factors upon which parentage tests could be based ( Sussman, 1976 ). Applying knowledge of the highly variable human leukocyte antigen (HLA) system, which only started to develop in the 1950s, provided an additional set of powerful tools for excluding a putative parent ( Bryant, 1980 , pp. 110–118; Kaye and Ellman, 1979 ).

In this formula, x denoted the chance that the putative father and known biological mother would yield a child embodying the specific genetic makeup (blood types or other serological factors) of the child in question, whereas y denoted the chance of the known biological mother and a random man from the relevant population yielding a child with these genes ( Hummel, 1981 , , 1984 ; Sussman, 1976 , pp. 124–131). The value that resulted, W, was the ‘Probability of Paternity’, and it represented the likelihood of the putative father being the actual biological father weighed against the likelihood that he was not.

Since mid-century, this formula as well as other ways of expressing the likelihood of paternity such as the ‘Paternity Index’ (which is presented as a ratio rather than as a percentage) have been used in the legal systems of various countries. By the time that DNA profiling began to transform practices of forensic identification in the 1980s, paternity tests relying on the examination of blood and HLA factors were widely used, albeit not without controversy or misunderstandings of application or interpretation ( Kaye, 1989 ). Such tests were used not simply to exclude a putative parent, but also as the basis for a calculation of Probability of Paternity, Paternity Index, or other ways of calculating the likelihood of paternity ( Valentin, 1980 ; Litovsky and Schultz, 1998 ).

Paternity tests based on analysis of genetically-inherited serological factors were not the only ones that were used. Throughout the 20th century, forensic experts and law courts in various countries have also relied upon examination of a range of other physical and physiological traits—for example, physical resemblance of facial features or the ability to taste phenylthiocarbamide (PTC)—in such tests ( Milanich, 2019 , Chapter 5; Schwidetzky, 1954 ; Bryant, 1980 , pp. 18–27). An important theoretical foundation for such (non-serological) paternity tests was provided in the work of Hermann Werner Siemens (1891–1969) ( Schmuhl, 2008 , pp. 60–68; Teo and Ball, 2009 ). Siemens was a proponent of Nazi racist ideologies and eugenics who is conventionally viewed as a founding figure and systematizer of twin research in human genetics. By studying the relative variability of many different traits across monozygotic (single-egg) and fraternal twins, Siemens was able to identify certain traits that routinely appeared to be similar or identical in monozygotic twins but not in fraternal twins ( Siemens, 1927 ; Newman et al. , 1968 [1937], pp. 19–21; Schmuhl, 2008 , pp. 60–61). In order to diagnose the unknown zygosity of other pairs of twins, one could examine the similarities or differences in these specific traits—for example, hair and eye colour—which had been shown to appear with great regularity in monozygotes ( Newman et al. , 1968 [1937], 55–93). 4

Siemens’ ‘similarity diagnosis’ was influential not only in human genetics research at the time and after, but also in forensic parentage testing. This approach, which relied upon the comparison of multiple heritable traits, provided an opening for fingerprint patterning to become a viable source of evidence in such cases. It is important to remember that prior to the rise of human genetics based on molecular biology, the patterning of friction ridge skin was viewed as a physical trait worthy of genetic study due to its partially inherited nature, imperviousness to environmental influence, and convenience of use (e.g. Rife, 1953 , p. 389). This was the context in which researchers turned to fingerprints as one of the traits that could potentially be used in paternity tests.

We find one elaboration of this kind of approach in the work of Austrian anthropologist Margarete Weninger, a long-time faculty member of University of Vienna. Early on, Weninger became a member of the Working Group on Genetic Biology founded by her spouse Josef Weninger (1886–1959) at this school in the early 1930s. This group was dedicated to researching the genetic inheritance of various anatomical characteristics and also applying this knowledge in forensic appraisals of questioned parentage, which had been sought from the anthropology faculty since the mid-1920s ( Teschler-Nicola, 2007 , pp. 58–59; Schaumann and Plato, 1987 ). Weninger was also a participant in the Marienfeld Project, in which the members of the Working Group applied their various fields of expertise (Weninger’s was dermal patterning of the hands) to investigate the anthropological parameters and ethno-racial identity of a local German-speaking community in Romania ( Teschler-Nicola, 2007 ; Weninger, 1965a , p. 47). Following the end of the Nazi regime, which barred the Weningers from continuing their work due in part to the fact that Margarete Weninger was Jewish ( Teschler-Nicola, 2007 , pp. 70–71), Weninger went on to explore other subfields of dermatoglyphics research, including the inheritance of dermal patterning on palms and fingers within families and paternity testing ( Weninger, 1965a ).

Weninger’s favoured approach to paternity testing drew on the one that had been used by the Working Group on Genetic Biology in the 1930s—that is, the comparative examination of multiple anthropological traits across known parent, putative parent, and child ( Teschler-Nicola, 2007 , pp. 59, 63, 70). Weninger provided an overview of this approach at the XI International Congress of Genetics (The Hague, September 1963) in a symposium that was also attended by Norma Ford Walker (1893–1968) and Lionel Penrose (1898–1972), both of whom were important figures in the field of post-World War II dermatoglyphics ( Geerts, 1965 , pp. 973–1003; Weninger, 1965b ; Miller, 2002 ). Weninger began by distinguishing between traits such as fingerprint patterning that are genetically-influenced yet whose mechanism of inheritance is obscure, on the one hand, and blood groups, the only trait with ‘definitive mode of inheritance with discrete phenotypes’, on the other. While, as Weninger would note later on, ‘[it] is obvious that [paternity] exclusions on the basis of traits with known mode of inheritance are decisive’, one could in no way discount the value of other characteristics such as fingerprint patterning. Rather, such traits could provide useful evidence if one carried out ‘a detailed comparison of the similarities of the three probands that ought to include as many characteristics as possible ( polysymptomatic similarity diagnosis ) [italics in original]’ ( Weninger, 1965b , p. 992). Thus, in response to the rhetorical question ‘What has one to observe [in paternity testing]?’, Weninger’s response was ‘Properly speaking everything!’ (p. 995).

This was the most productive approach to the use of fingerprint patterning in paternity testing, Weninger suggested, because so many questions remained about its mode of genetic inheritance. As Weninger’s own survey of the existing literature showed, investigators had studied the genetic inheritance of various aspects of fingerprint patterning—ridge-counts, pattern-types, size of the fingerprint pattern, and so on—and these had yielded inconclusive results as well as limited value when it came to paternity testing. As should be clear by now, Weninger’s approach was not based on principles or methods associated with serology, which had become essential for paternity testing by mid-century. Weninger (1965b , p. 992) did acknowledge the exclusionary value of blood evidence, the rare human trait with ‘known mode of inheritance’. It was only when one had to rely upon traits of unknown genetic mechanism such as fingerprint patterning that ‘similarity diagnosis’ was called for. In such cases, it went without saying, blood-based parentage tests did not – indeed could not – provide a model for the very different kind of genetic material represented by fingerprints.

Weninger was not the only researcher with an interest in using fingerprint patterning as evidence of paternity. By the end of World War II, a number of others in continental Europe and elsewhere had also pursued this area of research ( Milanich, 2019 , Chapter 5; Lauer and Poll, 1930 ; Cummins and Midlo, 1943 , pp. 246–250). 5 This work proceeded alongside a large quantity of basic research that investigated various aspects of the genetic inheritance of fingerprint patterning. For example, an influential demonstration of the partial heritability of fingerprint patterning came from the work of geneticist Sarah B. Holt (d. 1986) of the Galton Laboratory (University College London) during the 1950s and 1960s. Holt (1968) investigated correlations between the Total Finger Ridge Count values (the total number of ridges observed on all ten fingerprints) of parents and children, monozygotic and dizygotic twins, other siblings, and unrelated persons. Holt found that the observed correlations matched the values that would be expected for a physical trait that was governed by the additive effect of multiple genes.

Such work tended to generate more questions than answers not only about the specific mechanisms that were involved in the genetic inheritance of these characteristics but even about the most productive ways in which to classify fingerprint patterns to facilitate genetic study (e.g. Cole, 2002 , pp. 109–111). Questions remained, for example, about whether the focus of such work should be inheritance of the pattern-type itself (arch, loop, whorl, and so on) or that of a quantitative value such as ridge counts ( Fig. 1 ). Even among the strongest proponents of dermatoglyphics, it was not unusual to find frank statements about how little was actually known. As Harold Cummins (1894–1976) and Charles Midlo, Tulane University anatomists who were early proponents of this field of study, concluded in the early 1940s: ‘Even in the present state of knowledge, dermatoglyphics can claim a place only as a minor accessory in cases of questioned paternity; there are as yet no laws of inheritance so firmly substantiated that they qualify for rule-of-thumb practice’ ( Cummins and Midlo, 1943 , p. 247).

Main types of fingerprint patterns with lines indicating method for counting ridges. Source: S.B. Holt (1968) . The Genetics of Dermal Ridges . Charles C Thomas · Publisher, Springfield, p. 20. Courtesy of Charles C Thomas Publisher, Ltd.

An important site for research on the genetics of fingerprint patterning had always been Japan. Since the early 20th century, Japanese researchers had pursued various areas of dermatoglyphics research, including prolific studies of racial variation and genetic inheritance ( Asen, 2018 , pp. 64–69). As much as European and American figures—Francis Galton (1822–1911) or Harold Cummins, for example—are traditionally viewed as the founding figures of the field of scientific fingerprint research, early 20th-century Japanese research in this field was just as considerable in quantity, coherence and international impact, so much so that it is difficult to imagine that the field of Anglophone dermatoglyphics knowledge could have developed in the way that it did without the data-sets or approaches provided by this research community (pp. 68-69, 70). Some of this research on fingerprints was carried out by academics working within Japan’s considerable early 20th-century infrastructure of medico-legal institutes, which emerged under the modernization of Japan’s legal and educational systems following the Meiji Restoration of 1868 ( Jia, 2000 , pp. 290–302). These institutions provided fertile ground for pursuing basic scientific research on various aspects of fingerprint patterning in addition to other problems in forensic science.

All of this provides the context in which Matsukura Toyoji, a prolific researcher and synthesizer of medico-legal knowledge (and professor of legal medicine at Tokushima University and subsequently Osaka University), 6 developed a new theory in the 1950s that was meant to explain the genetic inheritance of fingerprint patterning and provide the basis for a workable paternity test.

4.1 Defining the genetic mechanism of fingerprint pattern inheritance

Matsukura’s primary assumption was that the most important object of research when studying the genetic inheritance of fingerprint patterning was not the pattern-type of the fingerprint itself—for example, whorl, loop, or arch ( Matsukura, 1967 ; Jia, 1993a , pp. 573–578). Rather, it was the quantifiable degree to which the orientation of the pattern could be said to rotate around a central point—in other words, its degree of ‘winding’. Arches could be said to represent the least amount of winding, loops a moderate amount, and whorls the most, with three additional types (looping arch, whirling loop and whirling arch) reflecting intermediate degrees of winding between these pattern-types ( Matsukura, 1967 , pp. 228–233). 7 The degree of winding of each one of a person’s fingerprints could be expressed in a numerical value: arches were assigned a value of 6, loops 18, whorls 30, and so on. Matsukura designated the sum of these values for all 10 fingers as the ‘Biological Value’ (BV) of a person’s fingerprints ( Matsukura, 1967 , p. 233). A person’s BV could range from 0 to 300 depending on the configuration of pattern-types across all of the fingers. 8

Matsukura went even further, however, suggesting that the degree of winding, represented in quantitative terms as the BV, could be analysed as a physical character (phenotype) governed by alleles at four genetic loci ( Matsukura, 1967 , pp. 235–236). An individual who inherited a greater number of dominant factors at these loci could be expected to express more winding in their fingerprints, thus having more whorls. Individuals who inherited fewer dominant factors would have less winding, expressed in more arches. The entire observed range of human fingerprint pattern variation could thus be mapped onto nine distinct genotypes, each associated with a different number of dominant factors, ranging from zero (aabbccdd) to eight (AABBCCDD) across these four genetic loci ( Table 1 ).

Ranges of biological value and associated genotypes

Note: Dominant factors indicated by capital letters. Based on Yonemura (1981 , p. 129).

While all of this was, in Matsukura’s admission, ‘of course merely of a hypothetic nature’, the distribution of BV values that Matsukura observed in a sample of 1365 persons from the ‘general public’ matched the distribution that was theoretically expected under this four-loci genetic model, as did his survey of the distribution of BV genotypes among the parent–child groupings of 329 families ( Matsukura, 1967 , pp. 236–240). On this basis, Matsukura claimed to have discovered a new law describing the genetic inheritance of fingerprint patterning.

4.2 Applying the biological value in paternity tests

As a researcher in legal medicine, Matsukura was interested in using this theory to develop practical testing procedures for evaluating paternity claims in the legal context. In the 1950s and early 1960s, Matsukura (1964 ; 1965 ) himself handled at least 23 cases in which serological tests were supplemented with analysis of the BV values of the known parent, putative parent and child, as well as with examination of facial resemblance and in some cases other characteristics of fingerprint patterning. Other Japanese medico-legal experts also analysed fingerprint patterning in cases of questioned paternity during this period, sometimes using Matsukura’s method and at other times analysing the genetics of fingerprint patterning in other ways (e.g. Ueno, 1964 ; Shikata, 1964 ; Nanikawa et al. , 1990 ).

One of the ways in which Matsukura’s theory could be used in paternity tests was to exclude a putative father, especially for cases in which an exclusion could not be made on the basis of serological testing. The logic was as follows: given that a child’s BV was determined by the number of dominant genetic factors inherited from the parents at the four loci hypothesized by Matsukura, one could easily tell whether the genotypes of known biological mother and putative father contained the necessary genetic material to produce the BV observed in the child in question. To put it another way, there were limits to which combinations of parental BV genotypes could produce a child of a certain genotype, and an examiner could use this knowledge of the possible and impossible parent–child groupings to exclude a putative parent ( Table 2 ).

Possible and impossible parent–child genotype groupings

Note: This table only indicates parental combinations in which one parent is of genotype 1. A complete listing would include all possible combinations of parents (genotypes 0–8). Based on Matsukura (1967 , p. 242).

In one case that Matsukura (1967 , p. 261) handled, for example, a putative father could not be ruled out by serological testing, yet was excluded by fingerprint examination: according to Matsukura’s theory, the parental combination of genotypes 1 and 5 (BV values of 150 and 264, respectively) could not have yielded a child of genotype 6 (BV 288). In another case, this time involving two possible fathers, analysis of both MN blood factors and BV values established that one of the men could not possibly have been the true biological father ( Matsukura, 1967 , p. 262). This was because the first putative father (of genotype 5) could have yielded the child in question with the known biological mother (given that the parental combination of genotypes 5 and 3 could yield a child of genotype 6) whereas the second putative father (genotype 2) could not have.

When a putative father could not be ruled out in this way, Matsukura instead characterized the fingerprint evidence with a frequency-percentage (labelled ‘rate of appearance’) that was listed in the same table as the results of serological testing and other examinations that were carried out. In one case involving a known biological mother and putative father of genotypes 4 (BV 216) and 3 (BV 198) and child of genotype 7 (BV 300, equivalent of 10 whorls), Matsukura (1965 , p. 54) calculated that the frequency with which this particular child-genotype (7) would appear among this parental combination (genotypes 4 and 3) was the low value of 0.1%. In another case, this time involving a parental combination of genotypes 5 and 6 (BV values of 264 and 294) and child of genotype 6 (BV 276), the frequency was calculated as 38%. 9 These percentage values represented not a Probability of Paternity (in the way that this concept was used in serological testing), but rather simply the frequency with which one might expect to find a child of a certain BV genotype among parents of particular combinations of genotypes, according to Matsukura’s four-loci theory. Thus, the frequency (38%) obtained in the latter case simply indicated a grouping of parental and child genotypes that was much more likely to occur than the grouping encountered in the former case (0.1% frequency).

4.3 Probability of paternity

Over subsequent decades, other Japanese researchers went beyond Matsukura’s presentation of frequencies to develop more sophisticated methods for calculating the probability that a putative father was the biological father on the basis of an analysis of BV values. In doing so, they directly drew on methods that were being used at the time in serological paternity tests. Furuya Yoshito and Shintaku Kikue (1976) of Tokyo Medical and Dental University, for example, calculated all possible Probability of Paternity values for different groupings of BV genotypes of known mother, putative father, and child. These values were presented in an easy-to-use table that other examiners could use to find the relevant figure without having to carry out the calculations themselves. According to Furuya and Shintaku, these calculations were made ‘on the basis of Bayes’s theorem [ sic ]’. Undoubtedly, this referred to Essen-Möller’s formula. 10 A similar approach was followed by Yonemura Isamu (1981) , a medico-legal expert at the medical school of Shinshu University, who also used Essen-Möller’s formula to calculate the Probability of Paternity for BV values. Just like Furuya and Shintaku, Yonemura also presented this information in tables that could be consulted by examiners to find the relevant figure without carrying out the calculations.

We can see how this Bayesian approach to determining probabilities associated with Matsukura’s BV analysis might have been used through an elaboration that appeared in a Chinese textbook of forensic anthropology in the early 1990s, a context discussed further below. In explaining Matsukura’s method to Chinese readers, medico-legal expert Lin Ziqing used Furuya and Shintaku’s table of probabilities to resolve a hypothetical case involving a known mother, putative father, and child with the configuration of fingerprints indicated in Table 3 ( Jia, 1993a , pp. 581–582). Following Matsukura’s method, each of these pattern-types was assigned a value indicating its degree of winding (arches = 6, loops = 18, and so on). BV values were then calculated for each person (in this case, 96, 180 and 132 for mother, putative father and child), and this in turn formed the basis for determining each person’s genotype (0, 2 and 1). As Lin noted, it was not impossible for parents of genotypes 0 and 2 to yield a child of genotype 1, thus one could not exclude the putative father on this basis. Rather, inserting these values into the table of probabilities provided by Furuya and Shintaku would yield a Probability of Paternity of 66.168%, which did not allow for paternity to be confirmed or ruled out either way.

Finding probability of paternity: a hypothetical case

Note: Based on Jia (1993a , p. 581).

As Lin Ziqing noted, the highest Probability of Paternity that could be obtained on the basis of Matsukura’s method was 91.637%, which was the greatest value that appeared in Furuya and Shintaku’s table ( Jia, 1993a , pp. 580–581; Furuya and Shintaku, 1976 , p. 21). The significance of this percentage could be further elucidated, Lin noted, by translating it into language following the style of Konrad Hummel’s well-known ‘verbal predicates’ for Probability of Paternity values, which circulated widely (albeit in modified form) in the Japanese and Chinese forensic science literature of this period (e.g. Matsukura, 1974 , p. 375; Zheng, 1982 , p. 296; Jia, 1984 , p. 17). Thus, the highest level of certainty that one could obtain from Matsukura’s test might be characterized by the verbal predicate ‘likely the father’, a judgment associated with values falling within the range of 90–95%. Much like the procedures for calculating Probability of Paternity on which the work of Furuya and Shintaku and Yonemura were based, this method for translating numerical probabilities into language had also originated within the context of serological testing, only subsequently migrating into dermatoglyphics.

On this point, it is worth noting just how much Matsukura’s fingerprint-based approach to paternity testing was influenced by the more widely-used and authoritative field of serology. Much as in forensic uses of serology, Matsukura’s approach was based on an analysis of both the inheritance of genes within putative biological family groupings and the distribution of the same genes within the larger population. In the cases that Matsukura handled, the examination of fingerprints was used to supplement the testing of blood groups and other serological factors, which influenced how the fingerprint evidence was presented. In the work of Furuya and Shintaku as well as that of Yonemura, the influence was even more direct, resulting in the calculation of an actual Probability of Paternity on the basis of Essen-Möller’s formula. Even Furuya and Shintaku’s presentation of all possible Probability of Paternity values in an easily-consulted table utilized the exact same format that was used to provide such information in serological testing ( Hummel et al. , 1971 ; Lee, 1980 ). In all of these ways, serology provided a model for the use of fingerprint evidence in paternity tests.

One way to evaluate the legacy of Matsukura’s four-loci theory of fingerprint pattern inheritance is by examining its reception in 1980s and 1990s China. Following the end of the Maoist period and the initiation of the economic reforms of the late 1970s, China’s police and judicial organs saw rapid development, and this in turn facilitated an expansion of medico-legal practice, academic research in forensic science, and training programmes ranging from short-term courses to advanced graduate education ( Huang, 1997 ). These developments were buttressed by Chinese researchers’ new connections with other countries’ forensic experts, institutions, and knowledge, including those of Japan. This was the context in which Matsukura’s theory was introduced into China and critically evaluated by Chinese medico-legal researchers.

5.1 Paternity testing in post-Mao China

Paternity testing was one area of forensic practice that saw a resurgence during this period. By the late 1980s, Chinese medico-legal experts were assisting police and judicial officials in questioned paternity cases by testing various blood group systems (ABO, MN, P, Rh), serum protein systems, red cell enzyme systems, and HLA, not simply for exclusions but also to calculate the likelihood of paternity (commonly in the form of a Paternity Index value or Relative Chance of Paternity percentage) ( Zhao et al. , 1984 ; Zhang et al. , 1991 ; Yang et al. , 1991 ; Wang and Shen, 1994 ). By the early 1990s, Chinese medico-legal experts were starting to offer DNA profiling in cases involving questioned paternity, even though it was still not widely used at this point ( Lu, 1994 , p. 83; Sun et al., 2002 , p. 154).

As much as the testing of blood groups and HLA rapidly gained authority in post-Mao China, the examination of other physical and physiological traits also remained part of the repertoire of paternity testing. In describing the different traits that could be tested in such cases, early reform-era textbooks of legal medicine generally mentioned the examination of physical appearance, dermal ridge patterning, earwax type (wet or dry), ability to taste PTC, and other physical characteristics as yielding genetic evidence that could be used to supplement serological testing. One of these textbooks, edited by Li Baozhen (1986 , p. 261), noted that the ridged skin patterning of fingers, palms, and soles ‘has definite reference value’ in paternity tests because family members demonstrate ‘a definite resemblance’ that is determined by genetics. Another textbook, edited by Zheng Zhongxuan (1982 , p. 296), noted that examining characteristics such as fingerprint and palm patterning and facial resemblance in addition to serological testing could yield a ‘suitably reasonable judgment – that is to say, the accuracy provided by a combined probability obtained from different kinds of tests can improve the reliability of parentage appraisals’.

Beyond the discussions that appeared in textbooks, such methods were used in cases as a supplement to serological testing. In a case involving a dispute over child support handled by judicial authorities in Beijing in late 1986, for example, a range of methods were employed to attempt to establish paternity. 11 The plaintiff in the case, a Li Yinzhu, accused Qi Chuntian of avoiding his responsibility to provide child support for their son, Qi Ran, who had been born out of wedlock in late 1985. Qi denied being the father. Paternity testing in the case was handled by the medico-legal office of the Higher People’s Court of Beijing. The examiners began by investigating each person’s ability to have sexual intercourse and conceive a child, as well as the timeline of the pregnancy. Next they examined the fingerprints, palm patterning, ability to taste PTC, earwax, and physical appearance of mother, putative father, and child, thereby establishing that Qi Ran had ‘many characteristics that were similar to those of Qi Chuntian’. The examiners then conducted serological tests across 15 systems (including blood groups, serum proteins, red cell enzymes, and HLA), none of which ruled out Qi as the biological father.

In the end, the decisive metric was the 98.35% cumulative Probability of Exclusion of Non-Fathers, which indicated a very high likelihood that a man who was not the biological father would already have been excluded by the tests. On the basis of these tests, the court affirmed that Qi Chuntian was the biological father and ordered him to pay child support.

5.2 Jia Jingtao’s research on fingerprint genetics

Within a legal and academic-research context in which fingerprints had some degree of salience as evidence in questioned paternity cases, it is not surprising that Chinese researchers engaged with Matsukura’s theory of the genetic inheritance of fingerprint patterning. This evaluation of Matsukura’s work took place through the work of Jia Jingtao and his colleagues in the legal medicine department of China Medical University, one of the earliest schools to re-establish an educational program in legal medicine after the end of the Maoist period. Jia himself had joined the faculty of the medical school in the 1950s, having studied under Chen Dongqi (1912–2006), an expert in legal medicine who had completed his own medical education at the Japanese-administered Manzhou Medical College during the 1930s (this institution was subsequently absorbed by China Medical University). In the post-Mao period, this department became one of the first to offer doctoral training in legal medicine and Jia Jingtao oversaw the training and completion of at least eight doctorates from the late 1980s to mid-1990s ( Huang, 1997 , pp. 162–163).

During this period, Jia developed the department’s capabilities in both forensic serology and forensic anthropology, the latter being the sub-discipline within legal medicine under which his fingerprint-related research was carried out ( Jia, 1993b , p. 452). In forensic serology, Jia worked out procedures for calculating Probability of Paternity and Probability of Exclusion of Non-Fathers values (also known as ‘Exclusion Probability of Parentage’) on the basis of Chinese gene frequency data ( Jia, 1984 ; Jia and Song, 1986 ). Jia and his colleagues’ work on the genetics of fingerprint patterning followed its own progression.

In the mid-late 1980s, Jia and his colleagues Lin Ziqing and Song Hongwei (at the time a PhD student under Jia) carried out a survey of existing research on the inheritance of fingerprint patterning ( Lin et al. , 1987 ). Organizing their article around previous work on the inheritance of form, pattern-type, ridge count, and pattern direction (ulnar, radial or symmetrical) of fingerprints, they described the theories of Matsukura and others, with a heavy reliance on Japanese dermatoglyphics research. They concluded their review by questioning the validity of existing attempts to establish a ‘biological classification’ of fingerprint patterning, and suggested that these were without basis in biology and heavily influenced by ‘subjective factors’. Jia and his colleagues further acknowledged that the ‘mechanism of inheritance of fingerprints has still not been made clear’.

Jia and his colleagues also collected population data on the distribution of fingerprint ridge counts and pattern-types among Han Chinese living in Jilin province ( Lin and Jia, 1989a , c ). By this point, a considerable body of research on population-level fingerprint variation among China’s other ethnic groups had been conducted, and Jia and his colleagues drew on this literature in their own work. They viewed this work as foundational research that was relevant not only to methods of individual identification in policing and forensics (implicitly, for example, latent print examination), but also to anthropological study of the ‘origins and migrations of nationalities, the relations between different nationalities, and medico-legal parentage appraisals [italics added]’ ( Lin and Jia, 1989a , p. 366). In questioned paternity cases, possessing baseline data on dermatoglyphic variation within the general population would help an examiner to better evaluate the significance of any similarities and differences observed across the fingerprints of known mother, putative father and child. Population-level gene frequency data would also be necessary if one wanted to calculate Probability of Paternity, a concept that was clearly of interest to Jia and his colleagues in the fields of both serology and dermatoglyphics.

5.3 Evaluating the applicability of Matsukura’s theory for a Chinese population

Possessing data on the population-level distribution of fingerprint characteristics within China was also useful because it allowed Jia and his colleagues to test the applicability of Matsukura’s four-loci theory for a population that could, potentially, have a distribution of pattern-types (and thus genotypes) that was different from the one that Matsukura had studied when developing his theory in Japan. In response to this question, Lin Ziqing and Jia Jingtao (1989b ) published an article in the Journal of Forensic Medicine , a publication associated with the Chinese Ministry of Justice’s Academy of Forensic Science, detailing the results of their testing of Matsukura’s theory. As described in the article, Lin and Jia had surveyed the fingerprint pattern-types of 412 families (1662 people in total) in Jilin province, the same local population that had been the focus of their other work on the distribution of fingerprint characteristics. Each set of fingerprints in the sample was classified by pattern-type, BV, and genotype (0–8), according to Matsukura’s system.

Lin and Jia found that the observed distribution of pattern-types and genotypes only partially matched Matsukura’s data. For example, the Han Chinese population that they surveyed had more looping arches and whorls and fewer loops than had been found in most studies that used Japanese population samples, including Matsukura’s own work ( Lin and Jia, 1989b , p. 34). Expectedly, the distribution of BV genotypes (which was related to the distribution of pattern-types) also differed from that which Matsukura had observed in Japan. Lin and Jia also found that in 4.13% of families examined in their study, there were parent–child genotype groupings that should have been impossible according to Matsukura’s theory (pp. 33-34). As discussed above, the ranges of possible and impossible parent–child genotype groupings were crucial information that had allowed Matsukura to exclude putative fathers in the cases that he handled. This discrepancy thus had serious implications for the applicability of Matsukura’s four-loci theory for questioned paternity cases involving individuals identified as Han Chinese. It suggested that Matsukura’s paternity testing method was less suitable for China.

In the end, Jia and his colleagues managed to strike a not unoptimistic tone, despite the persistent uncertainties surrounding the genetics of fingerprint patterning. While the fact that this physical trait was influenced by genetics was beyond question, the mechanisms of this influence were simply still unclear. After providing a summary of various theories about the inheritance of fingerprint patterning in his textbook of forensic anthropology, Jia (1993a , pp. 521–522) concluded:

However, due to the complexity of the inheritance of fingerprints, a number of [research] achievements that have already been made have mostly remained at the stage of being hypotheses. Not only is it that the genetic loci determining the inheritance of fingerprints are still unclear, but that the genotypes along with their expression – that is, phenotypes – are still unable to be clearly established in the same way as are blood groups. Thus, we believe that the inheritance of dermal ridge features and their application in parentage appraisals still represent an important field with a pressing need for continuing diligent investigation.

Jia and his colleagues’ engagement with these issues did not end with their critique of Matsukura’s approach. Jia along with Lin Ziqing and Song Hongwei developed their own method for using fingerprint patterning in paternity tests, introducing their approach in an article published in an English-language supplement to the Journal of China Medical University as well as in a long section of Jia’s textbook of forensic anthropology ( Lin et al. , 1988 ; Jia, 1993a , pp. 582–597). Their approach involved calculating various values that described what they called the ‘Intimate Degree’ of fingerprints—that is, the degree to which a particular grouping of known biological mother, putative father and child demonstrates similarity across all individuals’ fingerprints going beyond that which would be expected among a grouping of random people. The results of such tests, as the authors explained, could be presented as a percentage value, either in the form of a Probability of Paternity or Probability of Exclusion of Non-Fathers. These were, of course, the very same concepts that were used to quantify the weight of evidence in questioned paternity cases involving serological testing.

By the time that Jia Jingtao and his colleagues put forward this last innovation in fingerprint-based paternity testing, the testing of blood groups and HLA had already become the norm in such cases, to be followed soon after by the ascent of DNA profiling. Subsequently, the idea that fingerprint patterning could serve as valid and useful evidence in paternity testing would lose whatever legitimacy it had enjoyed earlier in the 20th-century. It goes without saying that fingerprint-based paternity testing is not part of today’s discussions of forensic uses of fingerprinting, which focus on fingermark detection and source attribution. This section briefly discusses the decline of dermatoglyphics and then describes some points of continuity between this older field of knowledge and current uses of fingerprinting in forensic identification.

6.1 The decline of dermatoglyphics

As Simon A. Cole (2002 , pp. 111–117) has described, the scientific study of fingerprint patterning—out of which the discipline of dermatoglyphics emerged—began to decline in status early in the 20th century despite ‘small pockets of research’ that persisted for decades afterward. One of the reasons that this happened, Cole argues, is that police examiners distanced themselves from dermatoglyphics in order to construct fingerprints as ‘solely an individual identifier’ without any connection to a subject’s race, heredity, or other identifying personal characteristics. Doing so was meant to make their identification practices ‘seem less value-laden, more factual’ (pp. 100–101, 112–113) and, ostensibly, to separate police identification work from a body of research that was contradictory and inconclusive. By implication, not only was dermatoglyphics knowledge divorced from identification work, but over time it lost status and authority.

The subfield of dermatoglyphics concerned with paternity testing was peripheral, to be sure, but also persistent. The examples described in this article confirm the genuinely international scope of this field as well as its long lifespan: the paternity tests discussed above developed in disparate locations, ranging from continental Europe to East Asia, and over a period that spanned much of the 20th century, even continuing into the 1980s and 1990s. 12 The researchers who developed these techniques were not, as a rule, uninfluential or marginal figures (e.g. Cole, 2002 , p. 113). The fingerprint-related research that they carried out was developed in connection with other established academic fields. We have seen, for example, that Jia Jingtao applied his knowledge, experience and interest in forensic applications of serology to his research on fingerprints. Whatever the outcome of these efforts, in a certain sense they exemplify the kind of academically-grounded, experimentally-rigorous research process that is being called upon today as the basis for the production and validation of new forensic knowledge ( Cole, 2010 ).

This example, as well as the others discussed in this article, suggests a field of knowledge that was generally receptive to developments that were occurring in other scientific fields. Even as the collective enterprise of scientific fingerprint research was declining in importance, it was still evolving. At the same time, of course, the examples discussed above show that there were limits to this field’s potential for development and even effectiveness. Basic questions about the mode of inheritance of fingerprint patterning were never resolved satisfactorily despite the attention of generations of researchers. In the end, the deep changes that have occurred in genetics since the mid-late 20th century have not made fingerprints a more productive or valuable object of inquiry for studying human heredity. Rather, answers for the anthropological, genetic and medical questions posed by generations of dermatoglyphics researchers are now sought in molecular biology or elsewhere.

6.2 Afterlives of dermatoglyphics knowledge

Despite these shifts in the status of dermatoglyphics, today’s forensic science researchers continue to find value in certain parts of this older body of knowledge. It is not unusual, for example, to find discussions of the anatomy and physiology, embryology and even genetics of dermal ridge patterning in today’s literature on latent print evidence (e.g. National Institute of Justice, 2011 , Chapter 3). The authors of such works tend to present these topics as a way of explaining or validating the ‘uniqueness and persistence’ of finger ridge patterning. These principles are still viewed as foundational to latent print examination despite the fact, expounded by Cole (e.g. 2009) and others, that the claim of fingerprint uniqueness cannot in itself guarantee the accuracy or reliability of fingerprint examination methods or evidence. The report of the National Research Council (2009 , pp. 143–144), for example, included the following sentence: ‘Some scientific evidence supports the presumption that friction ridge patterns are unique to each person and persist unchanged throughout a lifetime.’ The footnote supporting this statement cited key authors of the 20th-century dermatoglyphics literature such as Harold Cummins and Charles Midlo as well as Sarah B. Holt.

A more sophisticated discussion is found in Fingerprints and Other Ridge Skin Impressions , by Champod et al. (2016 , pp. 1–31). This work covers similarly fundamental topics (for example, anatomy, morphogenesis, and genetics of friction ridge skin), but does so in order to illuminate the principles of ‘permanence’, ‘variability’, and ‘selectivity’ of fingerprint patterning, which are emphasized in lieu of ‘uniqueness’ (p. 27). These concepts support the authors’ use of a Bayesian approach to formalizing the forensic decision-making process and weighing the significance of latent print evidence through the use of likelihood ratios (pp. 33-126). Here too foundational authors of dermatoglyphics are cited, including Cummins and Midlo, Holt and others, and there is substantial use of the work of Michio Okajima, whose contributions to the earlier dermatoglyphics literature included studies on comparative dermatoglyphics and the embryology of dermal ridge patterning ( Biographical Sketch, 1994 ).

As a field concerned with a wide range of scientific concerns, the scope of dermatoglyphics was significantly broader than the forensic examination of latent fingermarks. Today, by contrast, it is the latter that has become the most important site for the application of scientific knowledge about fingerprints. Another manifestation of this shift in focus is the emphasis that is placed today on fingerprint minutiae, features that are relevant to latent print examination but that were not the focus of most of the 20th-century work on dermatoglyphics. As we have seen, earlier generations of researchers tended to view pattern-types, ridge counts, and other characteristics—not fingerprint minutiae—as being most relevant to the anthropological, genetic and forensic questions about which they were most concerned.

6.3 The problem of population-level fingerprint pattern variation

In paternity testing, the most salient question is the relationship between the members of a putative biological family unit. In such tests, fingerprint patterning was not used as evidence of individual identity, but rather of the genetic relationship pertaining to a specific group of individuals. We might say that in paternity testing the emphasis was placed on using fingerprints to investigate ‘collective identity’, to use Cole’s (2013 , p. 77) phrasing, rather than individual identification. 13 The focus was not on identifying one individual to the exclusion of others, but rather on establishing an individual’s association with a biological family unit and, in a certain sense, defining the parameters of that person’s genetic makeup. There were also instances in which the use or development of paternity testing procedures involved making claims about the distribution of fingerprint patterning at the level of populations . Matsukura (1967 , p. 237), for example, tested his theory of fingerprint pattern inheritance by surveying 1365 members of the ‘general public’. Jia and his colleagues tested the applicability of Matsukura’s theory by surveying individuals who were identified as members of China’s Han majority, a designation that followed the official system for classifying the country’s ethnic groups ( Lin and Jia, 1989b ).

Today researchers are also concerned with understanding fingerprint pattern variation at the level of populations rather than simply that of individuals. This issue has emerged, for example, in the development of methods for presenting latent fingermark evidence in probabilistic form. As part of this work, researchers are exploring ways of presenting such evidence as a likelihood ratio ‘comparing (a) the likelihood of observing a given fingermark considering that it originates from a particular person and (b) the likelihood of observing that fingermark considering that it originates from a random individual in a relevant population’, the latter requiring a ‘reference database’ of population-level data ( Neumann et al. , 2015 , p. 168; Neumann et al. , 2012 ). The issue of population-level variation in fingerprint patterning is also relevant for attempts to formalize the procedures for selecting fingerprint features (especially minutiae) for analysis, which also involves determining their relative value for making an identification ( Expert Working Group on Human Factors in Latent Print Analysis, 2012 , pp. 55–62). Evaluating the evidentiary value of fingerprint characteristics in this way involves determining the relative ‘rarity’ of different features in the larger population.

In support of this and other applications, researchers have already turned to the question of how frequently particular classes of fingerprint minutiae appear across the different fingers of individuals and in different human populations ( Fournier and Ross, 2016 ; Gutiérrez et al. , 2007 ; Gutiérrez-Redomero et al. , 2011 , , 2012 ; Dankmeijer et al. , 1980 ). 14 It seems likely that more research will be done in this area in the future. Both the 2009 report of the National Research Council and a 2012 report sponsored by the National Institute of Justice and National Institute of Standards and Technology have identified producing data on ‘the frequency of [fingerprint] features in different populations’ as an area of productive research ( National Research Council, 2009 , pp. 139–140; Expert Working Group on Human Factors in Latent Print Analysis, 2012 , p. 75). This work is meant to improve the evidentiary value of fingermarks discovered at crime scenes. Once again, the goal of current research is narrower in scope than that of the older field of dermatoglyphics, which was concerned with producing general anthropological knowledge about different human populations.

6.4 Conclusion

Looking back from the start of the 21st century, it is apparent that there are aspects of both continuity and change in the foundation of scientific knowledge that supports fingerprint identification. Researchers continue to study fingerprint patterning at the level of individuals and populations, in the process negotiating its meanings as both a signifier of individual identity and an indicator of broader socially-relevant categories ( Cole, 2007 , 2013 , 2018 ). New concepts of proof and statistical techniques (and, of course, technologies) continue to transform the base of knowledge underlying forensic uses of fingerprint patterning, much as they did throughout the 20th century. From this perspective, today’s attempts to apply scientific validation, population data, and Bayesian approaches to the field of latent print evidence should not be viewed as wholly unprecedented. Rather, they represent one more iteration of negotiations between fingerprinting, scientific disciplines, and probabilistic reasoning that have been evolving over decades.

This work was supported by the National Science Foundation [grant # 1654990].

1 As Fiona A. Miller (2002 , 2003 ) has shown, the fact that fingerprint and palm patterning is influenced by both genetics and prenatal environment made dermatoglyphics useful in the diagnosis of congenital conditions such as Down syndrome.

2 For more on the use of such approaches in the history of various forensic fields, see Taroni et al. (1998) .

3 For example, in 1980s and 1990s China, a context to which we will return below, paternity tests were used to resolve the following kinds of issues: questions of forensic evidence in rape or abduction cases, civil disputes involving divorce and child-support, cases involving the mistaken identification of infants in hospitals, and even the need to confirm biological parentage within the context of China’s ‘birth planning’ policies (commonly referred to as the One-Child Policy), which penalized couples for having more than a prescribed number of children. See Yang et al. (1991 , p. 166); Lu (1994 , p. 80); Wang and Shen (1994 , p. 243); Sun et al. (2002 , p. 149).

4 Anthropology and genetics researchers of the interwar and post-World War II periods also investigated fingerprint patterning as one of the traits that could be used to differentiate monozygotic from dizygotic twins (e.g. MacArthur, 1938 ; Newman et al. , 1968 [1937], pp. 62–64, 83–85, 87, 92–93).

5 Also see the entries of published articles on this topic listed in Mavalwala (1977) .

6 Matsukura’s authored and edited works included a book of tables of anatomical and physiological statistics of relevance to legal medicine, books on the medico-legal dimensions of medical malpractice, and overviews of legal medicine and its role in criminal investigation.

7 In conceptualizing fingerprint patterning in this way, Matsukura was building upon the work of medico-legal expert Hōjō Harumitsu (1898–1971), who had posited that the fingerprints of children might be expected to differ from those of their biological parents in certain predictable ways—namely, a given pattern-type (for example, an arch or loop) in the parent might transmute into a slightly different, albeit recognizably transitional pattern-type in the child. Under this theory, the focus of investigation shifted from the individual pattern-types themselves to the mutual relations between them, now conceptualized as part of an organic whole of genetically-influenced interactions. For an explanation of Hōjō’s theory, see Jia (1993a , pp. 570–572).

8 As Matsukura (1967 , p. 234) showed, each BV value tended to have certain characteristic configurations of fingerprint patterning that were associated with it. In 81.2% of cases, for example, those who had a BV of 282 could be expected to have one loop, one whirling loop, and eight whorls. The rest of the time (in 18.8% of cases), they could be expected to have three whirling loops and seven whorls.

9 Tables listing the frequencies with which each child-genotype was expected to occur for each combination of parents were included in Matsukura’s (e.g. 1967 , p. 239) published work. The frequencies that Matsukura presented in his cases at times coincide with and at times slightly differ from those provided in the published tables, suggesting that Matsukura was working with other tables of frequencies (or multiple such tables) over the 10+ year period in which the cases were handled.

10 For other examples from contemporary Japanese medico-legal literature in which the formula for calculating Probability of Paternity was presented as being derived from Bayes’ Theorem without mention of Essen-Möller, see Matsukura (1974 , p. 355); Yonemura (1981 , p. 128).

11 An account of the case was included in a collection of medico-legal appraisal cases compiled by China’s highest judicial authority, the Supreme People’s Court. See Fayi anli bianxuan zu (1988 , pp. 60–61).

12 For more on the considerable amount of dermatoglyphics research that has been carried out in East Asia throughout the 20th century, see Asen (2018) .

13 This issue is also addressed in Cole (2018), as well as Cole (2007), which explores the connections and tensions between ‘individualization’ and ‘racial categorization’ in the history of American fingerprinting.

14 For discussion of some of the pitfalls of using ‘race’ as a category for classifying populations in such research, see Cole’s (2018 , pp. 5–10) critique of Fournier and Ross’ (2016) study of fingerprint minutiae variation. Cole refutes the claim advanced by Fournier and Ross that one might be able to ‘predict the [racial] ancestry of an individual’ from an examination of fingerprint minutiae. By implication, Cole claims, ‘[the] limited practical significance of corroborating a fingerprint association with an ancestry analysis [a possibility raised by Fournier and Ross] suggests that dermatoglyphics may be a hammer in search of a nail’ (p. 8). Cole’s point is well-taken in regard to this particular way of using dermatoglyphics knowledge. At the same time, it is important to note that the kind of ‘predictive’ approach outlined by Fournier and Ross is one that has been unusual even among 20th-century dermatoglyphics researchers, who were much more interested in surveying fingerprint-pattern variation across racially-defined groups than they were in attempting to determine racially-defined identities in individuals (e.g. Asen, 2018 ).

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Zhao T. , Lu Y. , Dong J. , Yao L. , Bu K. , Zhang G. , Gu W. , Zheng S. , Liu Z. ( 1984 ). Shiyong xuexing jianding qinzi guanxi de chubu baogao [An initial report on the use of blood groups in the appraisal of parentage ]. Hereditas (Yichuan) , 6 , 4 , 18 – 20 (Chinese).

Zheng Z. ( 1982 ). Fayixue [Legal medicine] . Falü chubanshe , Beijing (Chinese) .

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DNA Profiling in Forensic Science: A Review

Jaya lakshmi bukyya.

1 Department of Oral Medicine and Radiology, Tirumala Institute of Dental Sciences, Nizamabad, Telangana, India

M L. Avinash Tejasvi

2 Department of Oral Medicine and Radiology, Kamineni Institute of Dental Sciences, Narketpally, Telangana, India

Anulekha Avinash

3 Department of Prosthodontics, Kamineni Institute of Dental Sciences, Narketpally, Telangana, India

Chanchala H. P.

4 Department of Pedodontics and Preventive Dentistry, JSS Dental College, Mysore, Karnataka, India

Priyanka Talwade

Mohammed malik afroz.

5 Department of Oral Surgery and Diagnostic Sciences, Oral Medicine, College of Dentistry, Dar Al Uloom University, Riyadh, Kingdom of Saudi Arabia

Archana Pokala

Praveen kumar neela.

6 Department of Orthodontics, Kamineni Institute of Dental Sciences, Narketpally, Telangana, India

T K. Shyamilee

7 Department of Oral Pathology, Private Practice, Hyderabad, Telangana, India

Vammi Srisha

8 Department of Oral Medicine and Radiology, Private Practice, Bangalore, Karnataka, India

DNA is present in most of the cells in our body, which is unique in each and every individual, and we leave a trail of it everywhere we go. This has become an advantage for forensic investigators who use DNA to draw conclusion in identification of victim and accused in crime scenes. This review described the use of genetic markers in forensic investigation and their limitations.

Introduction

Forensic identification is a universal method used to establish the veracity in the process of forensic investigation. Both criminalities and medico-legal identification are integrative parts of forensic identification, having probative value. The value of an identification method resides in the specialist's ability to compare traces left at the crime scene with traces found on other materials such as reference evidence. Through this procedure, one can compare traces of blood, saliva, or any biological sample left at the crime scene with those found on a suspect's clothes and with samples from the victim. Medico-legal identification is based on scientific methods or intrinsic scientific methods absorbed from other sciences, usually bio-medical sciences. Scientific progress in the last 30 to 40 years has highlighted and continues to highlight the role of the specialists in identification. Their role proves its significance in cases that have to do with civil, family, and criminal law, as well as in cases of catastrophes with numerous victims (accidents, natural disasters, terrorist attacks, and wars). Together with the discovery by Mullis in 1983 of the polymerase chain reaction (PCR), Sir Alec Jeffreys in the field of forensic genetics used this technique by studying a set of DNA fragments that proved to have unique characteristics, which were nonrecurring and intrinsic for each individual, the only exception being identical twins. Alec Jeffreys named these reaction products “genetic fingerprints.” 1 PCR procedure is correct as per the reference.

Brief History of Forensic Genetics

• In 1900, Karl Landsteiner distinguished the main blood groups and observed that individuals could be placed into different groups based on their blood type. This was the first step in development of forensic hemogenetics. 2
• 1915: Leone Lattes describes the use of ABO genotyping to resolve paternity case. 2
• 1931: Absorption–inhibition of ABO genotyping technique had been developed. Following on from this, various blood group markers and soluble blood serum protein markers were characterized. 2
• In the 1960s and 1970s: Developments in molecular biology, restriction of enzymes, Southern blotting, 3 and Sanger sequencing 4 enabled researchers to examine sequences of DNA.
• 1978: Detection of DNA polymorphisms using Southern blotting. 5
• 1980: First polymorphic locus was reported. 6
• 1983: A critical development in the history of forensic genetics came with the advent of PCR process that can amplify specific regions of DNA, which was conceptualized by Kary Mullis, a chemist; later he was awarded Nobel Prize in 1993. 7
• 1984: Alec Jeffrey introduced DNA fingerprinting in the field of forensic genetics, and proved that some regions in the DNA have repetitive sequences, which vary among individuals. Due to this discovery, first forensic case was solved using DNA analysis. 8

DNA Structure and Genome

DNA was first described by Watson and Crick in 1953, as double-stranded molecule that adopts a helical arrangement. Each individual's genome contains a large amount of DNA that is a potential target for DNA profiling.

DNA Structure

DNA is often described as the “blue print of life,” because it contains all the information that an organism requires in function and reproduction. The model of the double-helix structure of DNA was proposed by Watson and Crick. The DNA molecule is a polymer of nucleotides. Each nucleotide is composed of a nitrogenous base, a five-carbon sugar (deoxyribose), and a phosphate group. There are four nitrogenous bases in DNA, two purines (adenine and guanine) and two pyrimidines (cytosine and thymine). Each base is attracted to its complimentary base: adenine base always pairs with thymine base whereas cytosine base always pairs with guanine base ( Fig. 1 ). 9

Structure of DNA. Image courtesy: National Human Genome Research Institute.

Organization of DNA into Chromosomes

There are two complete copies of the genome in each nucleated human cell. Humans contain ∼3,200,000,000 base pairs (BPs) of information, organized in 23 pairs of chromosomes. There are 2 sets of chromosomes; 1 version of each chromosome is inherited from each parent with total of 46 chromosomes. 10 11 12

Classification of Human Genome 2

Based on the structure and function, Classification of Human Genome into following different types ( Fig. 2 ).

Classification of human genome.

• Coding and regulatory regions: The regions of DNA that encode and regulate protein synthesis are called genes. Approximately, a human genome contains 20,000 to 25,000 genes; 1.5% of the genome is involved in encoding for proteins.
• Noncoding: Overall, 23.5% of the genome is classified under genetic sequence but does not involve in enclosing for proteins; they are mainly involved with the regulation of genes including enhancers, promoters, repressors, and polyadenylation signals.
• Extragenic DNA: Approximately 75% of the genome is extragenic, of which 50% is composed of repetitive DNA and 45% of interspersed repeats. Four common types of interspersed repetitive elements are: (i) short interspersed elements, (ii) long interspersed elements, (iii) long terminal repeats, and (iv) DNA transposons. Tandem repeats consist of three different types: (i) satellite DNA, (ii) minisatellite DNA, and (iii) microsatellite DNA.

Genome and Forensic Genetics

DNA loci that are to be used for forensic genetics should have the following ideal properties:

• Should be highly polymorphic.
• Should be easy and cheap to characterize.
• Should be simple to interpret and easy to compare between laboratories.
• Should have a low mutation rate.

With recent advances in molecular biology techniques, it is possible to analyze any region with 3.2 billion BPs that make up the genome. 2

Biological Material

Three most important steps are collection, characterization, and storage.

Sources of Biological Evidence

Human body is composed of trillions of cells and most of them are nucleated cells, except for the red blood cells. Each nucleated cell contains two copies of individual's genome and can be used to generate a DNA profile. Usually, samples show some level of degradation but when the level of degradation is high, more cellular material is needed to produce a DNA profile. 13

Biological samples with nucleated cells are essential for forensic genetic profiling, such as: 14

• Liquid blood or dry deposits.
• Liquid saliva, semen, or dry deposits.
• Hard tissues like bone and teeth.
• Hair with follicles.

Collection and Handling of Material at the Crime Scenes

Whole blood is considered as one of the widely used source of DNA. It is preserved in an anticoagulant (ethylenediamine tetra acetic acid) and conserved at 4°C for 5 to 7 days initially. After this period, DNA samples are kept at –20°C for few weeks or at –80°C for longer periods of time. Epithelial cells collected from crime scenes are harvested with sterile brush or bud. After harvesting, they are wrapped in plastic envelope or paper envelope and kept in a dry environment at room temperature. 15 It is essential that proper care is taken, such as maintaining integrity of the crime scene, wearing face masks and full protective suits during the investigation of scene, 16 17 18 as inappropriate handling of the evidence can lead to serious consequences. In worst cases, cross-contamination leads to high level of sample degradation; this can confuse or avert the final result of evidence.

Characterization of DNA Analysis: Basic Steps 1

Analysis of DNA involves four basic steps, which are as follows ( Fig. 3 ):

Extraction of DNA.

• DNA extraction.
• DNA quantification.
• DNA amplification.
• Detection of the DNA-amplified products.

DNA Extraction

The first DNA extraction was performed by Friedrich Miescher in 1869. Since then, scientists have made progress in designing various extraction methods that are easier, cost-effective, reliable, faster to perform, and producing a higher yield. With the advent of gene-editing and personalized medicine, there has been an increase in the demand for reliable and efficient DNA isolation methods that can yield adequate quantities of high-quality DNA with minimal impurities.

There are various methods of extraction as mentioned below, though commonly used are Chelex-100 method, silica-based DNA extraction, and phenol–chloroform method.

• Chromatography-based DNA extraction method.
• Ethidium bromide–cesium chloride (EtBr-CsCl) gradient centrifugation method.
• Alkaline extraction method.
• Silica matrices method.
• Salting-out method.
• Cetyltrimethylammonium bromide (CTAB) extraction method.
• Phenol–chloroform method.
• Sodium dodecyl sulfate (SDS)-proteinase K method.
• Silica column-based DNA extraction method.
• Magnetic beads method.
• Cellulose-based paper method.
• Chelex-100 extraction method.
• Filter paper-based DNA extraction method.

Chromatography-Based DNA Extraction Method

Chromatography-based DNA extraction method is used to isolate DNA from any kind of biological material. 19 This method is divided into three different types:

• Size-inclusion chromatography: In this method, molecules are separated according to their molecular sizes and shape.
• Ion-exchange chromatography (IEC): In this method, solution containing DNA anion-exchange resin selectively binds to DNA with its positively charged diethylaminoethyl cellulose group. 20 This method is simple to perform when compared with other DNA extraction methods. 19
• This procedure is used for isolation of messenger ribonucleic acid (m-RNA).
• It is time-efficient.
• It yields a very good quality of nucleic acids. 21

EtBr-CsCl Gradient Centrifugation Method

In 1957, Meselson et al developed this method. 22 DNA is mixed with CsCl solution, which is then ultra-centrifuged at high speed (10,000–12,000 rpm) for 10 hours, resulting in separation of DNA from remaining substances based on its density. EtBr is incorporated more into nonsupercoiled DNA than supercoiled DNA molecules resulting in accumulation of supercoiled DNA at lower density, and location of DNA is visualized under ultraviolet (UV) light.

• This method is used to extract DNA from bacteria.

Limitations:

• Greater amount of material source is needed.
• Time-consuming.
• Costly procedure due to long duration of high-speed ultra-centrifugation.
• Complicated method. 23

Alkaline Extraction Method

First introduced by Birnboim and Doly in 1979, this method is used to extract plasmid DNA from cells. 24 Sample is suspended in NaOH solution and SDS detergent for lysis of cell membrane and protein denaturation. Potassium acetate is then added to neutralize the alkaline solution, which results in formation of precipitate. Plasmid DNA in the supernatant is recovered after centrifugation.

Limitation:

• Contamination of plasmid DNA with fragmented chromosomal DNA. 25

Silica Matrices Method

The affinity between DNA and silicates was described by Vogelstein and Gillespie in 1979. 26

Principle: Selective binding of negatively charged DNA with silica surface is covered with positively charged ions. DNA tightly binds to silica matrix, and other cellular contaminants can be washed using distilled water or Tris-EDTA. 27

Advantages:

• Fast to perform.
• Cost-efficient.
• Silica matrices cannot be reused.

Salting-Out Method

Introduced by Miller et al 55 in 1988, this method is a nontoxic DNA extraction method.

Procedure: Sample is added to 3 mL of lysis buffer, SDS, and proteinase K, and incubated at 55 to 65°C overnight. Next, 6 mL of saturated NaCl is added and centrifuged at 2,500 rpm for 15 minutes. DNA containing supernatant is transferred into fresh tube and precipitated using ethanol. 28

• This method is used to extract DNA from blood, tissue homogenate, or suspension culture.
• High-quality DNA is obtained.
• Reagents are nontoxic.28,29

Cetyltrimethylammonium Bromide (CTAB) Extraction Method

This method was introduced by Doyle et al in 1990. 30

Samples are added to 2% CTAB at alkaline pH. In a solution of low ionic strength, buffer precipitates DNA and acidic polysaccharides from remaining cellular components. Solutions with high salt concentrations are then added to remove DNA from acidic polysaccharides; later, DNA is purified using organic solvents, alcohols, phenols, and chloroform. 20

• Time-consuming method.
• Toxic reagents like phenol and chloroform are used.

Phenol–Chloroform Method

This method was introduced by Barker et al in 1998. 31 Lysis containing SDS is added to cells to dissolve the cell membrane and nuclear envelope; phenol–chloroform–isoamyl alcohol reagent is added in the ratio 25:24:1. 28 Both SDS and phenol cause protein denaturation, while isoamyl alcohol prevents emulsification and hence facilitates DNA precipitation. The contents are then mixed to form biphasic emulsion that is later subjected to vortexing. This emulsion separates into two phases upon centrifugation, upper aqueous phase, composed of DNA, and the lower organic phase, composed of proteins. Upper aqueous phase is transferred to fresh tube and the lower organic phase is discarded. These steps are further repeated until the interface between the organic and aqueous phase is free from protein. 31 Later, sodium acetate solution and ethanol are added in 2:1 or 1:1 ratio, followed by centrifugation for separation of DNA from the solution. The pellet is washed with 70% ethanol to remove excess salt from the DNA and subjected to centrifugation for removal of ethanol. The pellet is dried and suspended in an aqueous buffer or sterile distilled water.

• Used to extract DNA from blood, tissue homogenate, and suspension culture.
• Inexpensive.
• Gold standard method.
• Toxic nature of phenol and chloroform. 28

SDS-Proteinase K Method

It was first introduced by Ebeling et al in 1974. 32 For extraction of DNA, 20 to 50 µL of 10 to 20 mg/mL proteinase K is added. SDS is added to dissolve the cell membrane, nuclear envelope, and also to denature proteins. The solution is incubated for 1 to 18 hours at 50 to 60°C and then DNA can be extracted using the salting-out method or phenol–chloroform method. 33

Silica Column-Based DNA Extraction Method

In this method, 1% SDS, lysis buffer (3 mL of 0.2 M tris and 0.05 M EDTA), and 100 mg of proteinase K are added to sample and incubated at 60°C for 1 hour, and this mixture is added in a tube containing silica gel. To this, phenol–chloroform is added in the ratio of 1:1 and centrifuged for 5 minutes. This separates the organic phase containing proteins beneath the silica column while aqueous phase containing DNA above the gel polymerase, and then aqueous layer is transferred to the tube and dissolved in TE buffer.

• Increase in purity of extracted DNA.
• Silica gel prevents physical contact with toxic reagents.
• DNA yield is 40% higher than organic solvent-based DNA extraction method.34

Magnetic Beads Method

Trevor Hawkins filed a patent “DNA purification and isolation using magnetic particles” in 1998. 35

Magnetic nanoparticles are coated with DNA-binding antibody or polymer that has specific affinity to bind to its surface. 36 In this method, a magnetic field is created at the bottom of the tube using an external magnet that causes separation of DNA-bound magnetic beads from cell lysate. The supernatant formed is rinsed, and beads aggregated at the bottom can be eluted with ethanol precipitation method; and the magnetic pellet is incubated at 65°C to elute the magnetic particles from the DNA. 28

• Time taken is less than 15 minutes.
• Faster compared with other conventional methods.
• Little equipment is required.
• Less cost.19,37

Cellulose-Based Paper

It was first introduced by Whatman in 2000, who filed a patent titled “FTA-coated media for use as a molecular diagnostic tool.” Cellulose is a hydroxylated polymer with high binding affinity for DNA. Whatman FTA cards are commercially available as cellulose-based paper that is widely used for extraction of DNA. 38 They are impregnated with detergents, buffers, and chelating agents that facilitate DNA extraction. About 1 to 2 mm of sample area is removed with micro punch and further processed for downstream applications. 19 39

• Extraction of DNA using cellulose-based paper is fast.
• Highly convenient.
• Does not require laboratory expertise.
• Easy storage of sample.40

Chelex-100 Extraction Method

In 2011, Xlonghui et al 40 patented a DNA extraction method using Chelex-100. Chelex resin is used to chelate metal ions acting as cofactors for DNases. After incubating overnight, 5% Chelex solution and proteinase K are used to degrade the added DNases, which are later boiled in 5% Chelex solution to lyse the remaining cell membranes, and to denature both proteins and DNA. Also, 5% Chelex solution prevents DNA from being digested by DNases that remain after boiling, hence stabilizing the preparation. The resulting DNA can then be concentrated from the supernatant after centrifugation.

• Reduced risk of contamination.
• Use of single test tube.
• Isolated DNA can be unstable. 38

Filter Paper-Based DNA Extraction Method

This method was described by Ruishi and Dilippanthe in 2017. DNA extraction method using filter paper can be used to isolate DNA from plant sources. A spin plate composed of 96-well plate is used, with a hole 1 mm in diameter drilled into bottom of each well used, and each well containing a disk of 8 mm diameter Whatman FTA filter paper. Samples subjected to lysis buffer are filtered with centrifugation.

• Less cost. 41

DNA Quantification

After DNA extraction, an accurate measurement of the amount and quality of DNA extract is desirable. When the correct amount of DNA is added to PCR, it results in best quality within short duration of time. Adding less or more amount of DNA will results in a profile that is difficult or impossible to interpret. 40

Quantity of DNA that can be extracted from a sample depends on the type of model. Quantity of DNA in different biological samples is shown in Table 1 . 42

Classification of Quantification 43

DNA quantification can be classified as follows:

• Microscopic and macroscopic examination.
• Chemical and immunological methods.
• ○ PicoGreen homogenous microtiter plate assays.
• Intact vs degraded DNA–agarose gel electrophoresis.
• Human total autosomal DNA.
• Y chromosome DNA, mitochondrial DNA (mt-DNA), Alu repeat real-time PCR.
• Multiplex real-time PCR.
• End-point PCR DNA quantification and alternative DNA detection methods.
• RNA-based quantification.

Visualization on agarose gels

• It is relatively easy and quick method for assessing both quality and quantity of extracted DNA.
• Gives indication of size of extracted DNA molecules.

Disadvantages:

• Quantification is subjective.
• Total DNA obtained can be mixture of human DNA and microbial DNA and this can lead to overestimation of DNA concentration. 2

Ultraviolet Spectrometry

Spectrometry is commonly used for quantification of DNA in molecular biology but has not been widely adopted by the forensic community. Usually, DNA absorbs light maximally at 260 nm; this feature is used to estimate the amount of DNA extraction by measuring wavelengths ranging from 220 nm to 300 nm. With this method, it is possible to assess the amount of protein (maximum absorbance is 280 nm) and carbohydrate (maximum absorbance is 230 nm). If the DNA extract is clean, the ratio of absorbance should be between 1.8 and 2.0.

• Difficult to quantify small amounts of DNA.
• It is not human specific. 2

Fluorescence Spectrometry

EtBr or 4′,6 diamidino-2-phenylindole can be used to visualize DNA in agarose gels. In addition to staining agarose gels, fluorescent dyes can be used as an alternative to UV spectrometry for DNA quantification. PicoGreen dye is commonly used because it is specific for double-stranded DNA as it has the ability to detect little amount of DNA as 25 pg/mL.

Disadvantage:

• Nonhuman specific. 44

DNA Amplification

There are eight DNA- and RNA-based techniques, but PCR and reverse transcription-PCR have been the predominant techniques.

PCR is the commonly used method of amplification of DNA. PCR amplifies specific regions of DNA template; even a single molecule can be amplified to 1 billion fold by 30 cycles of amplification. 45

DNA amplification occurs in cycling phase, which consists of three stages.

• Denaturation.
• Extraction.

Normal range of PCR cycle is between 28 and 32; when DNA is very low, then cycles can be increased to 34 cycles. 46

Other methods are as follows: 47

• Nucleic acid sequence-based amplification method.
• Strand displacement amplification.
• Recombinase polymerase amplification.
• Strand invasion-based amplification.
• Multiple displacement amplification.
• Hybridization chain reaction.

After the amplification of DNA, the final step is detection of the DNA-amplified products.

Detection of the DNA-Amplified Products

The following methods are used in forensic human identification:

• Autosomal short-tandem repeat (STR) profiling
• Analysis of the Y chromosome
• Analysis of mt-DNA.
• Autosomal single-nucleotide polymorphism (SNP) typing.

Autosomal STR Profiling

STRs were discovered in 1980. Since then, they are considered as gold standard in human identification in forensics. STR or microsatellites are the most frequently genotyped to distinguish between individuals. STR consists of mononucleotide, dinucleotide, trinucleotide, tetranucleotide, pentanucleotide, and hexanucleotide repeats of which tetranucleotide repeats are used for genotyping. 2

STR profiling is used in paternity/maternity testing, rape perpetrators' identification, kinship testing, and disaster victim identification. 48

STR-based DNA analysis in forensic has been well accepted by professionals and population as an important tool in criminal justice and in human identification.

• The test is simple.
• Can be done rapidly. 49

Analysis of the Y Chromosome

Typically, biologically a male individual has 1 Y chromosome and contains 55 genes. Because of this unique feature, analysis of Y chromosome is done in crime cases. 50

Application of Y chromosome in forensic medicine: It is present only in males. Thus, in crime cases, the investigators expect to find Y chromosome at the crime scene. Also, when talking about male–female ratio in body fluid mixtures, such as sexual assault or rapes, by analyzing the Y-STR component, the investigators can obtain more information regarding the male component. It is well known that azoospermic or vasectomized rapists do not leave semen traces, and it is impossible to find spermatozoa on the microscopic examination. In such cases, the Y-STR profiling is very useful, offering information regarding the identity of the accused person. 50

Analysis of Mitochondrial DNA (mt-DNA)

mt-DNA is inherited from mother; thus all the members of a matrilineal family share the identical haplotype.

• mt-DNA has 200 to 1,700 copies per cell.
• Increased probability of survival when compared to nuclear DNA.

Applications:

• Analysis of biologic samples that are severely degraded or old.
• Samples with low amount of DNA (e.g., hair shafts). 51

Autosomal Single-Nucleotide Polymorphism Typing

SNP has a lower heterozygosity when compared with STRs. Advantage of SNP typing over STR is that the DNA template size can be as large as 50 BPs, compared with STRs that need a size of 300 BPs to obtain good STR profiling. 52 Due to this reason, SNP has become an important tool in analyzing degraded samples. Thus in the 2001 World Trade Center disaster, victims were identified using SNP typing. 53 54

Impact of Genetic Identification in Justice 1

Genetic testing using DNA has been widely applicable to the field of justice. This method is being used for the following:

• Identification of accused and confirmation of guilt.
• Exculpation of innocent ones.
• Identification of persons who commit crimes or serial killers.
• Identification of victims in disasters.
• Establishing consanguinity in complex cases.

Currently, the DNA genotyping of all types of microtraces or biological traces containing nucleated cells is possible if they are not entirely demolished, either chemically or by bacteria. The DNA analysis is an important tool in solving caseworks in forensic medicine, such as establishing the custody of a child through paternity or maternity tests, identifying victims from crimes or disasters, or exonerating innocent people convicted to prison.

Conflict of Interest None declared.

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• Crime, justice and law

Forensic Information Databases annual report 2022 to 2023

The report includes information on the National Fingerprint Database policing collections and the National DNA Database.

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The report highlights the continued value of fingerprints and DNA in solving crimes and the part these biometrics play in bringing offenders to justice, keeping the public safe and preventing harm to potential future victims.

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• TECHNOLOGY FEATURE
• 08 May 2024

Powerful ‘nanopore’ DNA sequencing method tackles proteins too

• Caroline Seydel 0

Caroline Seydel is a science writer in Los Angeles, California.

You can also search for this author in PubMed   Google Scholar

A nanopore sequencing device is typically used for sequencing DNA and RNA. Credit: Anthony Kwan/Bloomberg/Getty

With its fast analyses and ultra-long reads, nanopore sequencing has transformed genomics, transcriptomics and epigenomics. Now, thanks to advances in nanopore design and protein engineering, protein analysis using the technique might be catching up.

“All the pieces are there to start with to do single-molecule proteomics and identify proteins and their modifications using nanopores,” says chemical biologist Giovanni Maglia at the University of Groningen, the Netherlands. That’s not precisely sequencing, but it could help to work out which proteins are present. “There are many different ways you can identify proteins which doesn’t really require the exact identification of all 20 amino acids,” he says, referring to the usual number found in proteins.

In nanopore DNA sequencing, single-stranded DNA is driven through a protein pore by an electrical current. As a DNA residue traverses the pore, it disrupts the current to produce a characteristic signal that can be decoded into a sequence of DNA bases.

Proteins, however, are harder to crack. They cannot be consistently unfolded and moved by a voltage gradient because, unlike DNA, proteins don’t carry a uniform charge. They might also be adorned with post-translational modifications (PTMs) that alter the amino acids’ size and chemistry — and the signals that they produce. Still, researchers are making progress.

Water power

One way to push proteins through a pore is to make them hitch a ride on flowing water, like logs in a flume. Maglia and his team engineered a nanopore 1 with charges positioned so that the pore could create an electro-osmotic flow that was strong enough to unfold a full-length protein and carry it through the pore. The team tested its design with a polypeptide containing negatively charged amino acids, including up to 19 in a row, says Maglia. This concentrated charge created a strong pull against the electric field, but the force of the moving water kept the protein moving in the right direction. “That was really amazing,” he says. “We really did not expect it would work so well.”

Super-speedy sequencing puts genomic diagnosis in the fast lane

Chemists Hagan Bayley and Yujia Qing at the University of Oxford, UK, and their colleagues have also exploited electro-osmotic force, this time to distinguish between PTMs 2 . The team synthesized a long polypeptide with a central modification site. Addition of any of three distinct PTMs to that site changed how much the current through the pore was altered relative to the unmodified residues. The change was also characteristic of the modifying group. Initially, “we’re going for polypeptide modifications, because we think that’s where the important biology lies”, explains Qing.

And, because nanopore sequencing leaves the peptide chain intact, researchers can use it to determine which PTMs coexist in the same molecule — a detail that can be difficult to establish using proteomics methods, such as ‘bottom up’ mass spectrometry, because proteins are cut into small fragments. Bayley and Qing have used their method to scan artificial polypeptides longer than 1,000 amino acids, identifying and localizing PTMs deep in the sequence. “I think mass spec is fantastic and provides a lot of amazing information that we didn’t have 10 or 20 years ago, but what we’d like to do is make an inventory of the modifications in individual polypeptide chains,” Bayley says — that is, identifying individual protein isoforms, or ‘proteoforms’.

Molecular ratchets

Another approach to nanopore protein analysis uses molecular motors to ratchet a polypeptide through the pore one residue at a time. This can be done by attaching a polypeptide to a leader strand of DNA and using a DNA helicase enzyme to pull the molecule through. But that limits how much of the protein the method can read, says synthetic biologist Jeff Nivala at the University of Washington, Seattle. “As soon as the DNA motor would hit the protein strand, it would fall off.”

Nivala developed a different technique, using an enzyme called ClpX (see ‘Read and repeat’). In the cell, ClpX unfolds proteins for degradation; in Nivala’s method, it pulls proteins back through the pore. The protein to be sequenced is modified at either end. A negatively charged sequence at one end allows the electric field to drive the protein through the pore until it encounters a stably folded ‘blocking’ domain that is too large to pass through. ClpX then grabs that folded end and pulls the protein in the other direction, at which point the sequence is read. “Much like you would pull a rope hand over hand, the enzyme has these little hooks and it’s just dragging the protein back up through the pore,” Nivala says.

Source: Ref. 3

Nivala’s approach has another advantage: when ClpX reaches the end of the protein, a special ‘slip sequence’ causes it to let go so that the current can pull the protein through the pore for a second time. As ClpX reels it back out again and again, the system gets multiple peeks at the same sequence, improving accuracy.

Last October 3 , Nivala and his colleagues showed that their method can read synthetic protein strands of hundreds of amino acids in length, as well as an 89-amino-acid piece of the protein titin. The read data not only allowed them to distinguish between sequences, but also provided unambiguous identification of amino acids in some contexts. Still, it can be difficult to deduce the amino-acid sequence of a completely unknown protein, because an amino acid’s electrical signature varies on the basis of both its surrounding sequence and its modifications. Nivala predicts that the method will have a ‘fingerprinting’ application, in which an unknown protein is matched to a database of reference nanopore signals. “We just need more data to be able to feed these machine-learning algorithms to make them robust to many different sequences,” he says.

NatureTech hub

Stefan Howorka, a chemical biologist at University College London, says that nanopore protein sequencing could boost a range of disciplines. But the technology isn’t quite ready for prime time. “A couple of very promising proof-of-concept papers have been published. That’s wonderful, but it’s not the end.” The accuracy of reads needs to improve, he says, and better methods will be needed to handle larger PTMs, such as bulky carbohydrate groups, that can impede the peptide’s movement through the pore.

How easy it will be to extend the technology to the proteome level is also unclear, he says, given the vast number and wide dynamic range of proteins in the cell. But he is optimistic. “Progress in the field is moving extremely fast.”

Nature 629 , 492-493 (2024)

doi: https://doi.org/10.1038/d41586-024-01280-5

Sauciuc, A., Morozzo della Rocca, B., Tadema, M. J., Chinappi, M. & Maglia, G. Nature Biotechnol . https://doi.org/10.1038/s41587-023-01954-x (2023).

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Martin-Baniandres, P. et al. Nature Nanotechnol. 18 , 1335–1340 (2023).

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Motone, K. et al. Preprint at bioRxiv https://doi.org/10.1101/2023.10.19.563182 (2023).

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