Abstract
Forensic genetics tends to be an experimental, diverse, and revolutionary area of science, and the proportion of information gathered from the tiniest remnants of DNA keeps expanding. DNA profiles are complex, and propositions are uncertain; therefore, legal professionals consistently seek novel investigative blueprints by relying on reliable forensic resources and judicial admissions of guilt. Researchers do not rely solely on visible and bulk amounts of conventional biological samples such as semen, saliva, and blood to generate a DNA profile. However, some sample sizes are so small and invisible that coming from such a brief encounter or transfer can appear seemingly useful. This small amount of DNA is known as “touch DNA” or “abandoned DNA,” which refers to “any amount of human tissue capable of DNA analysis and separated from a targeted individual’s person inadvertently or involuntarily, but not by police coercion.” The existing level of understanding pertaining to the cellular makeup of touch residues and the potential source of trace DNA contained therein is inadequate. The objective of the present review study is to conclude that how non-intimate skin-cell DNA accidentally or indirectly transfers from a targeted person under certain scenarios affects the potential results and could also be a reasonable explanation as conclusive evidence in the criminal justice system.
Introduction
Forensic genetics primarily focuses on non-coding regions of DNA, often referred to as “junk DNA,” which constitute approximately 0.1% of the total genome. These regions are highly polymorphic and play a crucial role in generating a unique genetic blueprint for each individual. Biological samples serve as valuable evidence for identification purposes, facilitating the comparison of DNA profiles. Since 1984, DNA analysis has been a reliable, valid, and confidential method in criminal trials, aiding in both convicting offenders and exonerating the wrongly accused. The process of DNA analysis involves extracting genetic material from biological specimens retrieved from a crime scene, analyzing specific genetic markers, and comparing them to potential suspects’ profiles. This results in the generation of a unique genetic identifier, commonly known as a DNA profile, which can be matched against forensic samples to establish identity. 1
Challenges in DNA Profiling
The occurrence of heterogeneous or incongruous genetic profiles within a DNA specimen can lead to inaccurate forensic conclusions. Several factors contribute to such discrepancies, including external contamination, genetic mosaicism, and hematopoietic stem cell transplantation.
External contamination occurs when DNA samples are mishandled or improperly stored, leading to contamination by laboratory personnel or previous samples and potentially causing mismatched profiles. 2
Genetic mosaicism refers to an individual having multiple genetic profiles due to mutations or alterations affecting certain cells or tissues. 2
Hematopoietic stem cell transplantation involves transferring stem cells from a donor to a recipient, replacing the recipient’s bone marrow and producing new leukocytes that contain the donor’s genetic information. This can result in mixed DNA profiles, leading to potential false inclusions or exclusions in forensic investigations. 2
Evolution of DNA Evidence: The Introduction of Touch DNA
Initially, forensic DNA analysis relied exclusively on bodily fluids (e.g., blood, semen, and saliva) and hair samples to establish genetic profiles. However, in 1997, Australian forensic scientists Roland Van Oorschot and Maxwell Jones revolutionized the field with their Nature publication, “DNA fingerprints from fingerprints.” Their findings demonstrated that DNA could be recovered from traces left behind by a mere handshake. 3
“Trace DNA” or “touch DNA” refers to biological material containing minimal DNA amounts, presumed to be left behind through physical contact. The expansion of touch DNA applications has significantly broadened forensic investigations, aiding in solving crimes such as theft, homicide, armed robbery, sexual assault, and clandestine laboratory cases. It has been detected on various objects, including tools, fabrics, vehicles, doorknobs, countertops, weapons, food items, glass, skin, paper, cables, and stones. 4
Touch DNA plays a crucial role in cases of sexual assault, including non-consensual groping, fondling, and touching over or under clothing. The collection of touch DNA can help identify perpetrators in such cases. 1 Other commonly tested items for touch DNA include toothbrushes, phones, facemasks, toothpicks, cigarette butts, facial wipes, tissues, and lip prints. 4
Touch DNA in India: Case Studies
India witnessed the potential impact of touch DNA in forensic investigations during the high-profile Aarushi-Hemraj double murder case. Due to the lack of standard procedures, high costs, and regulatory frameworks, authorities rejected the use of touch DNA. The parents of the victim, the Talwar couple, emphasized the significance of low copy number (LCN) or touch DNA testing in proving their innocence. However, investigative agencies relied on conventional evidence, considering touch DNA too speculative at the time. 5
Conversely, in the 2013 Bodh Gaya blast case, the National Investigation Agency successfully employed touch DNA to identify a terrorist disguised as a monk. DNA samples obtained from the suspect’s discarded clothing were compared with those of arrested suspects a year later, narrowing down the list of potential perpetrators. This marked a milestone in the use of touch DNA as crucial circumstantial evidence in India. 6
Nomenclature and Ambiguities in Touch DNA
Touch DNA is also referred to as contact DNA or abandoned DNA, encompassing minute genetic traces obtained through limited interaction, such as conversation, perspiration, or “wearer DNA” from fabric. 7
Kawecki 7 defines touch DNA as “any amount of human tissue capable of DNA analysis and separated from a targeted individual’s person inadvertently or involuntarily, but not by police coercion.” Humans shed approximately 400,000 epithelial cells daily, 8 primarily composed of keratinocytes, which lose their structural components and nuclei before being naturally removed from the skin’s surface. 9 Nucleated cells from different body areas can transfer to the hands or be shed through sweat ducts. 10
A major misconception is that touch DNA is always obtained from a touched surface. In reality, it can also be airborne or transferred indirectly. Additionally, it is often mistaken as synonymous with low-template DNA (LT-DNA), LCN-DNA, and trace DNA. 7 Trace DNA generally refers to biological samples containing less than 100 picograms of DNA, 7 while LCN-DNA refers to methods requiring increased amplification cycles due to low DNA quantity. 7
Systematic Methodology of Touch DNA
The successful development of a touch DNA profile requires rigorous procedures in forensic genetics laboratories. These include retrieval, isolation, measurement, and short tandem repeat (STR) amplification. However, touch DNA does not establish the biological origin of evidence. Three key factors impact touch DNA formation:
Duration of contact with the object Degree of force applied during contact Number of individuals who have handled the object
Transfer of Non-intimate Touch DNA
Touch DNA transfer occurs in two primary ways:
Direct transfer (Primary transfer): Cellular material is directly transferred through physical contact, speaking, coughing, or sneezing.8, 11, 12
Indirect transfer (Secondary transfer): DNA is transferred via an intermediary object rather than direct contact. 3
The recovery and persistence of touch DNA depend on several unpredictable factors, making its transfer highly variable.
Factors Influencing Touch DNA Recovery
Shedding characteristics: Some individuals, known as “good shedders,” naturally deposit higher amounts of DNA upon contact. Factors such as gender, age, and dermatological conditions (e.g., dandruff, eczema, and psoriasis)13, 14 affect shedding rates. Studies suggest that men are generally better shedders than women, and younger males shed more DNA than older individuals. However, individuals can alternate between good, intermediate, and poor shedding states over time, and hand washing can significantly reduce DNA deposition. Research indicates that children are typically good shedders, while adults and elderly individuals exhibit lower shedding tendencies. 15
Contact type: Increased contact time and pressure result in higher touch DNA deposition.
Individual habits: Frequent self-touching (e.g., nose, ears, and hair) contributes to DNA transfer, a phenomenon termed “loading DNA fingers.” 16
Surface texture and area: Rough surfaces, such as wood, can trap cells more effectively than smooth surfaces such as glass. However, smooth, non-porous surfaces tend to retain more DNA due to increased convection rates during contact. Larger surface areas also facilitate greater DNA transfer. DNA recovery varies across different materials, with brass-plated metals yielding the highest recovery rates. 17
Duration of contact and time between deposition and recovery: The yield of DNA decreases over time, particularly when exposed to environmental factors. Though a minimum of two seconds of contact is sufficient for successful profiling, DNA yield is higher from recently touched surfaces. Proper storage can mitigate degradation, with studies showing minimal loss after 12 weeks under controlled conditions. 18 However, DNA recovery significantly decreases over extended periods, especially in harsh environmental conditions.
Environmental factors: DNA samples in forensic investigations are often exposed to environmental stressors such as heat, humidity, ultraviolet (UV) light, and chemicals, leading to degradation. High temperatures and UV exposure accelerate DNA breakdown, while humidity and contaminants can further compromise sample integrity. 19 Proper handling and storage conditions are crucial to preserving DNA for forensic analysis.
Non-intimate Touch DNA Collection Methodologies
Touch DNA collection is often referred to as a “blind search” because the analyst does not have prior knowledge of whether epithelial cells or touch DNA are present in a given area. To ensure reliable forensic results and prevent contamination or degradation of the samples, systematic and validated collection and sampling methodologies should be employed. The selection of a collection method depends largely on the type and condition of the surface being sampled. Despite the widespread use of innovative touch DNA evidence collection techniques, there remains no standardized protocol for touch DNA collection. The initial step in trace sample collection involves identifying suitable regions for sampling. Trace samples on surfaces are typically not visible to the naked eye, making their collection challenging. Traditional laboratory methods for low copy DNA specimens were originally optimized for high-copy-number DNA evidence.
The “standard swab” technique is frequently employed due to factors such as cost, expertise requirements, and laboratory-specific validation procedures. Wet/dry double cotton swabbing expands upon the single swab technique by incorporating a second sterile swab to collect residue from the damp surface after an initial moist swabbing. The swabs are then combined before extraction. This method is widely applied to non-absorbent surfaces.20, 21 Castella and Mangin 21 examined 1,739 forensic cases and found that the double swab technique was significantly more effective than single swabbing. The mean DNA yield was 0.494 ng/µl with double swabbing compared to only 0.312 ng/µl with single swabbing. However, a recent review of sampling techniques suggests that the double swab method does not always enhance touch DNA recovery. 22
Despite the effectiveness of cotton swabbing, a considerable amount of trace DNA is lost within the cotton fibers, leading researchers to explore alternative materials. Self-saturating mini-papules, IsohelixTM, Dacron, Rayon, FLOQSwabsTM, Bode SecurSwabTM, and nylon or polyester-tipped swabs, have been recommended to reduce sample dispersion and entrapment. To mitigate DNA loss, many swabs are now designed without an internal absorbent core. 23
A significant breakthrough in touch DNA recovery involved soaking fired cartridge cases and bullets in tissue lysis buffer. 24 A study of 616 criminal cases using this method recovered usable genetic profiles in 163 cases (26.5%). Prasad et al. 25 evaluated this methodology across different ammunition types and confirmed its suitability for DNA recovery.
Hansson et al. 26 compared the Scene Safe FASTTM minitape, manufactured in the UK, with cotton, flocked, and foam swabs. Their results demonstrated that minitape achieved the highest trace DNA recovery. Similarly, Sessa et al. 27 analyzed DNA retrieval from brassieres worn by female volunteers using dry swabbing, tape lifting, and cutting techniques. Although no significant differences were found among these methods, the ‘‘cut-out’’ technique exhibited the highest DNA recovery, whereas adhesive tape sampling was statistically significant for ‘‘wearer’’ findings. The most recent handler of an item was often the primary contributor to its DNA profile, even after brief contact. This observation remained unchanged regardless of the identities of the handler and wearer.
To collect trace DNA from large surface areas, researchers have suggested filtered tips and vacuum-based techniques.28–30 The Mac Vac system employs a “mini hurricane” effect under the sampling head, created by spray and vacuum forces. The solution collects DNA content into a sterile container, dislodging epithelial cells, touch DNA, or contact DNA from porous or rough surfaces. This enhances forensic evidence collection capabilities (
Comte et al. 31 compared four swab types under controlled and quasi-operational conditions, including a reference swab (Prionics cardboard evidence collection kit) used by police forensic units, and three challenger swabs (COPAN 4N6FLOQSwabs™, Puritan fast and automated bacterial MINI typing by arbitrarily primed PCR (FAB-MINI-AP), and Sarstedt Forensic Swab). COPAN 4N6FLOQSwabs™ proved most efficient, yielding significantly more touch DNA from collars, steering wheels, and screwdrivers than the reference swab.
Fluorescent probes and laser microdissection techniques have been investigated to differentiate between trace DNA contributors. Anslinger et al. 32 and Vandewoestyne et al. 33 explored whether these methods could distinguish between male and female cells or two individuals of the same gender within mixed samples.
Non-intimate Touch DNA Extraction
Efficient DNA extraction is essential for isolating DNA while removing cellular debris, excess reagents, and polymerase chain reaction (PCR) inhibitors that can affect downstream applications such as restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD), variable number of tandem repeats (VNTR) and single nucleotide polymorphism (SNP) analysis. 34 These DNA markers are distributed across the human genome and exhibit high polymorphism, making them ideal for forensic DNA profiling and human identification. In forensic contexts, genotype and gene frequency data help estimate match probabilities in inclusion cases, and many ethnic groups now have published STR allele frequency data. 35
Each extraction procedure begins with cellular lysis, followed by deproteinization and DNA recovery. Extraction methods vary in deproteinization intensity and DNA fragment size retention. Many laboratory protocols fail to recover all extracted DNA, with losses of up to 75% observed in Chelex and organic extraction methods. 4 Substrate type and extraction technique significantly impact yield.
Forensic DNA extraction methods include solution-based, column-based, magnetic bead-based, and commercially available kits. 36 Common forensic touch DNA extraction kits include Chelex® 100, the QIAamp® DNA Blood Mini Kit, the QIAamp® DNA Investigator Kit, QIAsymphony®, the DNA Investigator® Kit, the DNA IQ™ system, and the PrepFiler® Express BTA.
Non-intimate Touch DNA Quantification
DNA quantification determines the DNA concentration necessary for downstream applications such as sequencing, PCR, and cloning. Proper quantification assists in approximating DNA quantity and evaluating profile relevance. However, in cases where touch DNA recovery is low, it may not always be possible to obtain a minimum viable DNA portion.3, 10, 19 Quantifiler® Duo DNA Quantification Kits and the Applied Biosystems® 7500 Fast Real-Time PCR System are commonly used for touch DNA quantification. 37
As forensic DNA techniques advance, the demand for precise trace DNA quantification increases. 38 If DNA quantities are insufficient, laboratories may struggle to generate viable DNA profiles, even after processing. 39 To ensure accurate low-level quantification, two key frameworks are recommended: (a) Targeting an amplicon of a similar base-pair size as intended for subsequent analysis (99–214 bp in major commercial kits), and (b) maximizing the dynamic range of standard curves at low concentrations (Quantifiler Duo, 2008; Qiagen, 2012). 40
Non-intimate Touch DNA Amplification
Following extraction and quantification, DNA amplification enables forensic analysts to generate millions of copies from minimal DNA samples for qualitative and quantitative analysis. Direct PCR amplification has gained preference over STR amplification because it retains DNA typically lost during extraction and purification. 41
ISO 17025 accreditation bodies have validated direct PCR for limited forensic applications. Despite its cost-effectiveness and time efficiency, direct PCR consumes the entire sample, making it unsuitable for re-examination in defense testing. 42 To ensure reliable touch DNA amplification, laboratories must use validated kits such as Identifier® Plus and GlobalFiler®, which effectively amplify LT-DNA. 43
Precautions for Minimizing Contamination
Crime scene investigators must take precautions during evidence collection to prevent contamination. Rutty (2001) advised restricting crime scene access and maintaining an exclusion database of investigator fingerprints. Wearing crime scene suits, gloves, facemasks, and overshoes minimizes contamination risks, whereas actions such as scratching, sneezing, coughing, eating, or talking increase the likelihood of contamination. 44
Issues Related to Touch DNA
Touch DNA analysis faces challenges such as non-reproducibility, undefined interpretation guidelines, and unvalidated mixture analysis results. 38 Compliance with forensic sample collection, preservation, and analysis standards can mitigate errors in forensic investigations. 45 Addressing issues such as stochastic effects, allele dropout, and stutter effects is crucial to prevent false conclusions. An exclusion database of investigators’ DNA and fingerprints can further reduce wrongful convictions. Laboratories analyzing touch DNA must meet stringent quality standards, and policymakers must ensure ethical regulations balance forensic advancements with constitutional rights. 46
Footnotes
Declaration of Conflicting Interests
The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Ethical Approval and Informed Consent
Not applicable.
Funding
The authors received no financial support for the research, authorship, and/or publication of this article.
