Locating DNA within fingermarks using fluorescent in situ detection; a collaboration between ESR and Flinders University

ABSTRACT

A pilot study on the detection of DNA in fingermarks using fluorescent in situ detection after different time periods since hand washing was undertaken by Flinders University. Collaboration was sought to show the ability to obtain similar results within different laboratories. The Institute of Environmental Science and Research (ESR) was involved in this inter-laboratory study in collaboration with Flinders University. The newly developed method involves the use of Diamond Nucleic Acid dye for the staining of fingermarks (20× concentration in 75% ethanol) using a hand-held microscope (Dino-Lite Edge Digital Microscope). Fingermarks were deposited by volunteers onto glass slides at varying time intervals after hand washing (2, 5, 15, 30, 60 and 180 minutes). The amount of cellular debris was calculated by counting the fluorescent dots present in three fields of view and estimating the amount of transferred cellular material for each fingermark. This article will outline the results obtained from ESR and how they compare with results already collated from Flinders University.     

Detection of latent DNA on tape-lifts using fluorescent in situ detection

Tape-lifts can be used for the removal of a range of evidence types without damaging the substrate. In addition to loosely adhering material such as hairs and fibres, tape-lifts can be used to remove latent cellular material on clothing that had originated from the wearer’s body or mouth. Common forensic practice is to examine the entire tape-lift in a non-targeted approach. An innovative field of research uses DNA binding dyes for the detection of latent DNA. This research describes the use of these DNA binding dyes as a DNA-targeted screening tool for tape-lifts. The individual cells can be visualized allowing the relevant area of the tape to be removed for subsequent DNA typing. Full single source DNA profiles were generated that matched the expected control sample after staining tape-lifts with two different dyes. Direct amplification was undertaken from tape-lifts of samples where biological material had been deposited over 12 months previously, from which a DNA profile matching the donor was obtained. The process outlines an effective means to visualize the presence of cellular material from which STR profiles can be generated allowing a targeted approach to be performed.     

Rapid genotyping of 25 autosomal STRs in a Japanese population using fluorescent universal primers containing locked nucleic acids

Amplification of fluorescently labeled products is one of the most popular methods for genotyping genetic variations. Two-step amplification using fluorescent universal primers simultaneously produces multiple targeted fragments labeled with fluorescent dyes, and this strategy is applicable to large-scale, cost-effective genotyping. In this study, we developed a fast PCR-based, multiple short tandem repeat (STR) genotyping method using fluorescent universal primers containing locked nucleic acids (LNAs). Four amplification reactions, each assaying six or seven markers and using 0.5–1.0 ng of genomic DNA, produced obvious Fam-labeled peaks in all 26 loci tested (25 autosomal STRs and amelogenin). The overall amplification time was 37 min. Moreover, fluorescent signals for the 25 STRs obtained from LNA-containing primers were 1.5–9.0 fold higher compared to those from non-LNA primers. Using genomic DNA from 120 Japanese individuals, 16 out of the 25 STRs had observed heterozygosity greater than 0.7. Some of these 25 STRs also had high discriminatory power, similar to that of the 13 core STRs in the Combined DNA Index System dataset. The probability of incorrectly assigning a match based on the accumulated matching probability for these 25 STRs is 1.2 × 10⁻²², and their combined use can provide robust information for Japanese forensics.     

DNA profiles from matchsticks

ABSTRACT

Matchsticks may be found in forensic investigations, and here we report on a method to generate informative DNA profiles from a matchstick. Matchsticks were struck or held emulating striking, cellular material was removed using a tape-lift method and DNA profiles were generated from a small portion of the tape-lift using either direct PCR amplification or post-extraction PCR. Performing an extraction from the tape-lift prior to PCR resulted in 62% of profiles being informative, compared with 86% held and 97% struck matchsticks that underwent direct PCR. Incorporation of a tape-lift method into matchstick analysis allows effective collection of cellular material for the generation of genetic information from the striker of the matchstick.     

Discrimination of haplotype in mitochondrial DNA mixtures using LNA-mediated PCR clamping

Locked nucleic acid (LNA) has been widely used for various genetic analyses, and has many benefits, in terms of the specificity or sensitivity of amplification, because LNA-containing primers/probes form more stable duplexes with template DNA than probes lacking LNA. Here, we developed a new method for discriminating HV1 haplotypes from mitochondrial DNA (mtDNA) mixtures by applying PCR clamping using LNA. PCR clamping is based on the selective inhibition of amplification using LNA-containing probes, which can discriminate single-nucleotide differences. Before designing probes, we selected 171 sequences with single-nucleotide variations from the HV1 region, and evaluated the specificity of LNA-containing probes for them by predicting Tm values. The differences of Tm between mismatched and exactly matched probe–template duplexes depended markedly on the type of LNA nucleotides for discriminating single-nucleotide differences, and the cytosine LNA nucleotide at the site of variations in the probes was most effective to discriminate these differences. For mixture analysis, each probe targeted one or two variations (16209C, 16217C, 16257A/16261T, 16297C/16298C, 16304C, 16362C, or 16362T) that are particularly common in the Japanese population, and seven designed probes completely inhibited the amplification of exactly matched templates. We prepared mixed samples by mixing DNA from two individuals at a ratio of 1:9, 1:4, 1:1, 4:1, or 9:1, and then performed Sanger sequencing analysis after PCR clamping with each probe. Our method distinguished each haplotype at lower ratios from two-person mixtures, and enabled sensitive detection at 12 pg of total DNA including 600 copies of mtDNA. Moreover, we analyzed three-person mixtures with representative sequences, and detected the minor haplotype of one individual present at a rate of 10% by adding two selected probes. The ability to discriminate haplotypes in mixed samples by using LNA-mediated PCR clamping indicates the potential value of mtDNA analysis in criminal investigations.     

Ribosomal DNA as target for the assessment of DNA degradation of human and canine DNA

The assessment of DNA amount and DNA integrity can support forensic DNA analysis, in particular of problematic traces such as single telogen hairs where STR typing success is often hampered by low amounts and strong degradation of nuclear DNA. Common strategies consist of quantitative polymerase chain reaction (qPCR)-based analysis of the abundance of a short versus a long nuclear amplicon, the latter prone to DNA degradation. To increase sensitivity, commercial qPCR solutions rest on amplification of multi-copy DNA sequences. Here we show that ribosomal DNA (rDNA) sequences are well suited for the same purpose. Because rDNA sequences are present in high copy number in most eukaryotic species, qPCR strategies can easily be adapted to non-human species. In this paper, we establish qPCR-based assays for human or dog DNA, respectively, which allow for sensitive analysis of DNA amounts and DNA degradation. We show that the human system can be applied to DNA of single telogen hairs, where STR typing success correlates with measured amounts and integrity of the DNA. By adapting the system to dog rDNA sequences we found that single telogen dog hairs often displayed less DNA degradation than human telogen hairs, in most cases allowing for successful STR typing. Thus, qPCR-based analysis of rDNA represents a cost-effective, highly sensitive strategy to assess DNA amount and integrity that can be adapted to hairs or other traces from various animal species.     

Concordance study between the ParaDNA® Intelligence Test,a Rapid DNA profiling assay,and a conventional STR typing kit (AmpFlSTR® SGM Plus®)

The ParaDNA^® Intelligence Test enables STR profiling directly from human biological samples and evidence items collected from crime scene in 75 min. Designed for non-expert use this system allows DNA information to be available to investigators before it would typically be available from a laboratory. The ParaDNA Intelligence Test system amplifies D3S1358, D8S119, D16S539, D18S1358 and TH01 STR loci and the gender typing locus amelogenin and detects the alleles present with HyBeacon^® probes. Individual DNA samples from 381 UK Caucasian individuals were analysed using AmpFlSTR^® SGM Plus^® and the ParaDNA Intelligence Test with the derived STR profiles compared. Here we describe the high level of concordance demonstrated between the two systems and discuss this with reference to allele frequencies and the discriminatory power offered by the ParaDNA Intelligence Test.     

DNA reviews: the national DNA database of the United Kingdom

The national DNA database in United Kingdom has now been operational for over 10 years. This review looks at the history and development of this investigative resource. From the development of commercial DNA profiling kits to the current statistics for matches obtained in relation to criminal investigation in the United Kingdom, before moving onto discussing potential future direction that national DNA databases might take, including international collaboration on a European and global scale.     

A high volume extraction and purification method for recovering DNA from human bone

DNA recovery, purity and overall extraction efficiency of a protocol employing a novel silica-based column, Hi-Flow^® (Generon Ltd., Maidenhead, UK), were compared with that of a standard organic DNA extraction methodology. The quantities of DNA recovered by each method were compared by real-time PCR and quality of DNA by STR typing using the PowerPlex^® ESI 17 Pro System (Promega Corporation, Madison, WI) on DNA from 10 human bone samples. Overall, the Hi-Flow method recovered comparable quantities of DNA ranging from 0.8 ng ± 1 to 900 ng ± 159 of DNA compared with the organic method ranging from 0.5 ng ± 0.9 to 855 ng ± 156 of DNA. Complete profiles (17/17 loci tested) were obtained for at least one of three replicates for 3/10 samples using the Hi-Flow method and from 2/10 samples with the organic method. All remaining bone samples yielded partial profiles for all replicates with both methods. Compared with a standard organic DNA isolation method, the results indicated that the Hi-Flow method provided equal or improved recovery and quality of DNA without the harmful effects of organic extraction. Moreover, larger extraction volumes (up to 20 mL) can be employed with the Hi-Flow method which enabled more bone sample to be extracted at one time.     

Development of a multiplex assay for detection of autosomal and Y-chromosomal STRs,assessment of the degradation state of mitochondrial DNA and presence of mitochondrial length heteroplasmies

The current focus in most routine forensic casework is detection of autosomal or gonosomal Short Tandem Repeats (STRs). With increasing degradation, STR analysis tends to be less successful up to complete failure. For challenging samples such as telogen hair roots and shafts, touch DNA samples or skeletal remains, mitochondrial DNA (mtDNA) analysis provides a powerful tool. Determination of DNA quantity is an important part in the casework workflow. Several ready-to-use kits are commercially available for nuclear DNA targets. However, quantification of mtDNA targets requires the establishment of an in-house method. Some assays even contain assessment of degradation, which alleviates the choice of target enrichment for sequencing through medium or small amplicons. As Sanger-type Sequencing (STS) still remains the golden standard in many laboratories, identification of heteroplasmies in C-tract regions prior to the sequencing reaction is advantageous. Firstly, primer selection can be expanded with primers binding near the C-tract and secondly, determination of the dominant variant is straightforward. All those quantity (nuclear and mtDNA) and quality (degradation and length heteroplasmies) evaluations usually require at least two separate reactions. Therefore, the aim of this project was the combination of all these targets in one multiplex assay using capillary electrophoresis to spare valuable sample extract. Amplification of representative autosomal and Y-chromosomal STRs allows estimate of success of (Y-)STR analysis. Simultaneously, five length heteroplasmies in the mitochondrial control region are targeted as well as three conservative regions of differing fragment lengths for assessment of the mitochondrial degradation state. Based on the outcome of this assay, forensic examiners can decide if STR analysis may be suitable. In case of absent STR peaks, appropriate proceeding of mtDNA sequencing can be determined.     

Sequential multiplex amplification (SMA) of genetic loci: A method for recovering template DNA for subsequent analyses of additional loci

A method called Sequential Multiplex Amplification (SMA) has been developed whereby a limited amount of DNA extracted from a sample can be reutilized for several single polymerase chain reaction (PCR) amplifications. The method involves recovery of genomic template DNA by microfiltration of PCR-amplified samples. Up to 5 different loci have been typed, each in a single system PCR-based assay, beginning with a test quantity of 5 ng template DNA. Genotypes of the DNA donors were compared with those obtained from individual amplifications and shown to be identical. This could be a useful technique for typing a number of loci from a limited amount of DNa and to recover template DNA from samples previously subjected to PCR. Obviously, when small quantities of template DNA are available, this technique can prove quite useful.     

A proof-of-principle study on implementing polymerase chain displacement reaction (PCDR) to improve forensic low-template DNA analysis

Polymerase chain reaction (PCR) plays an important role in forensic DNA analysis. However, the amplification of low-template DNA (LTDNA) samples usually encounters unsatisfactory results for the limited efficiency of PCR, which would interfere with the subsequent profile interpretation. Polymerase chain displacement reaction (PCDR) is a highly-efficient technique characterized by combining PCR and strand displacement reaction into a single PCDR cycle. This study explored the feasibility of PCDR for improving forensic LTDNA analysis. STR markers commonly used in forensic genetics were subjected to PCDR amplification and capillary electrophoresis detection. The results of singleplex reactions indicated that PCDR surpassed original PCR in efficiency for STR amplification. The average peak height of alleles in PCDR profiles was linearly correlated to the number of outer primers adopted for initiating the strand displacement process. Further, we assessed the multiplexing potential of PCDR by incorporating 17 STRs included in the expanded CODIS core loci and Amelogenin gene into a multiplex PCDR system. For pristine DNA templates ranged from 200 pg to 12.5 pg, the multiplex PCDR system consistently exhibited higher allele peak height as well as less allele dropout compared to the multiplex PCR references. Meanwhile, a significant reduction of stutter ratio was extensively observed in PCDR profiles. We also tested mock casework samples to verify the practical ability of multiplex PCDR for LTDNA detection. With DNA input varying from 48.1 pg to 6.6 pg, the multiplex PCDR system consistently obtained more allelic information than multiplex PCR methods. Our data collectively suggested that it is feasible to apply PCDR in forensic LTDNA analysis.     

DNA analysis of fingernail debris using different multiplex systems: a case report

A 55-year-old male nurse was accused of having introduced his fingers by force into the anus of a 20-year-old female patient. Debris from the fingernails of the suspect recovered 2 days after the incident was analysed with the VNTR locus D1S80, the triplex PCR system AmpFlSTR Blue kit, the AmpFlSTR Profiler kit and the pentaplex system genRES MPX. The D1S80 singleplex reaction revealed indications of DNA from the victim in the fingernail debris of the left hand. Using the AmpFlSTR Blue kit and AmpFlSTR Profiler, DNA alleles of the victim were found at four additional loci, while allelic drop-out was observed at five other loci. Only the pentaplex kit genRES MPX revealed alleles at all loci which could be assigned to the victim. Calculation of likelihood ratios resulted in a value of 1.4 × 10⁵ using the combination of the multiplex systems AmpFlSTR Blue kit and AmpFlSTR Profiler and 2.8 × 10⁸ for the genRES MPX kit. This case demonstrates the high sensitivity of the new genRES MPX kit and that DNA profiling of fingernail debris is possible despite a time lapse of 2 days between the incident and recovery of the evidential material. Received: 30 March 2000 / Accepted: 19 September 2000     

Estimating drop-out probabilities in forensic DNA samples: a simulation approach to evaluate different models

Allele drop-out is a well known phenomenon that is primarily caused by the stochastic effects associated with low quantity or low quality DNA samples. Recently, new interpretation models that employ the use of logistic regression have been utilised in order to estimate the probability of drop-out. The model parameters are estimated using profiles from samples of extracted DNA diluted to low template levels in order to induce drop-out. However, we propose that this approach is over-simplistic, because several sources of variability are not taken into account in this generalised model. For example, in real-life, small (discrete) crime-stains are analysed where cells are (or were) intact. The integrity of the paired chromosomes of the diploid cell is preserved. In extracted DNA that is diluted to low template levels, we argue that the paired-chromosome integrity is lost. This directly affects the outcome of the logistic model. To date, current experimentation procedures are more akin to haploid cells and thus, different logistic models are needed for haploid and diploid cells. In order to simplify the methodology to estimate the multiple logistic regressions, we propose the use of a simulation model of the entire process associated with the analysis of STR loci, as a supplement to the purely experimental approach to support the validation of new methods. We illustrate with an evaluation of some features of the logistic model proposed by Gill et al., 2009 [12] and discuss alternative models.     

Developmental validation of the ParaDNA® Intelligence System—A novel approach to DNA profiling

DNA profiling through the analysis of STRs remains one of the most widely used tools in human identification across the world. Current laboratory STR analysis is slow, costly and requires expert users and interpretation which can lead to instances of delayed investigations or non-testing of evidence on budget grounds. The ParaDNA^® Intelligence System has been designed to provide a simple, fast and robust way to profile DNA samples in a lab or field-deployable manner. The system analyses 5-STRs plus amelogenin to deliver a DNA profile that enables users to gain rapid investigative leads and intelligent prioritisation of samples in human identity testing applications. Utilising an innovative sample collector, minimal training is required to enable both DNA analysts and nonspecialist personnel to analyse biological samples directly, without prior processing, in approximately 75 min. The test uses direct PCR with fluorescent HyBeacon^® detection of STR allele lengths to provide a DNA profile. The developmental validation study described here followed the Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines and tested the sensitivity, reproducibility, accuracy, inhibitor tolerance, and performance of the ParaDNA Intelligence System on a range of mock evidence items. The data collected demonstrate that the ParaDNA Intelligence System displays useful DNA profiles when sampling a variety of evidence items including blood, saliva, semen and touch DNA items indicating the potential to benefit a number of applications in fields such as forensic, military and disaster victim identification (DVI).     

Semi-automatic DNA profiling in a Hungarian Romany population using the STR loci HumVWFA31, HumTH01, HumTPOX, and HumCSF1PO

A population study of Hungarian Romanies was carried out for the STR loci HumVWFA31, HumTH01, HumTPOX, and HumCSF1PO. After multiplex PCR amplification semi-automatic DNA profiling was performed using an ALF DNA sequencer. At the loci investigated there was little and no evidence for departures from Hardy-Weinberg expectations and linkage equilibrium, respectively. The allele sizing accuracy of the ALF DNA sequencer was increased to a high level (99.97% on average) by applying external and internal markers. Allele frequency distributions of the STR loci, with one exception, were significantly different between the Romany and other Hungarian population databases. On the other hand, however, only small differences in frequencies of individual phenotypes were found. Received: 30 December 1996 / Received in revised form: 11 March 1997     

Rapid PCR of STR markers: Applications to human identification

Multiplex PCR with fluorescently labeled primers has been an essential method for the amplification of short tandem repeats used in human identify testing. Within the STR workflow of extraction, quantitation, amplification, separation, and detection, multiplex PCR is commonly identified as the bottleneck in the process. The time requirement of up to three hours to complete 28–30 cycles of multiplex PCR for STR genotyping is the greatest amount of time required for a single step within the process. The historical use of commercially available thermal cyclers and heat stable polymerases may have given the impression that large multiplex will always require long PCR cycling times to ensure that all of the varying sized targets (typically 100–400 bp) can be amplified in a balanced manner throughout the multiplex. However, with the advent of improved polymerases and faster thermal cyclers the time required for the amplification of large STR multiplexes is no longer on the order of three hours, but as little as 14 min. Faster amplification times can be performed while retaining the balance and integrity of large multiplex PCRs by implementation of alternate polymerases and thermal cyclers. With the reduction in PCR cycling times there has also been an impact on the development of integrated and microfluidics devices which employ the use of reduced or rapid thermal cycling protocols as part of their integration. Similarly, PCR inhibitor resistant polymerases can also reduce overall STR processing times for reference samples by eliminating the need for DNA extraction and purification that is additionally implemented within the development of integrated DNA typing devices.     

Exploring tapelifts as a method for dual workflow STR amplification

Although a version of direct PCR is implemented in forensic laboratories for reference material, its incorporation into workflow for the analysis of touch DNA, as a form of latent DNA, from casework exhibits is not. In addition to concerns about increased sensitivity causing more complex mixtures or the generation of more genetic data implicating an individual superfluous to the context of the alleged event, the complete use of the collected sample in the PCR as template has meant that there is no possibility for data reproducibility when needed. Here it is proposed that the use of tapelifts in touch DNA collection can facilitate replicate direct PCR analysis from a single sample allowing the sample to be re-tested. If all portions of the tapelift result in profiles with allelic and likelihood ratio concordance, these sub-samples may be accepted as technical replicates, thus meeting any accreditation guideline requirements. Furthermore, we assess the use of a single tapelift for both direct PCR and extraction-based PCR workflows to illustrate the potential for benefits of both systems to be facilitated. DNA was deposited by three donors onto six substrates with five sample replicates of each condition. Separation of each tapelift into three portions for three direct PCRs ensued using VeriFiler™ Plus. Separation of single tapelifts into three direct PCRs showed no statistical difference in donor allele calls or RFU, or subsequent LRs associated with their profiles. Comparison of profiles within the single tapelift showed more similarity, with high mixture-to-mixture match likelihoods, than when these sub-samples were compared with profiles generated from other samples. This allows each sub-sample taken from the tapelift to be considered as technical replicates. For dual workflow facilitation assessment, one donor deposited DNA through touch onto six substrates with five research replicates of each. Separation of single tapelifts into two portions, one for direct PCR and the retention and use of the remaining portion for extraction and subsequent PCR, showed no significant difference in allelic yield and subsequent donor comparison LRs. Comparison of deconvoluted profiles produced from a single tapelift showed high mixture-to-mixture match likelihoods, supporting DNA donor concordance. This indicates that removing a portion of a tapelift for direct PCR amplification, while processing the remainder through standard processes, allows increased sensitivity through direct PCR while offering the preparation of an eluate suitable for repeated analyses.     

Searching for first-degree familial relationships in California's offender DNA database: validation of a likelihood ratio-based approach

A validation study was performed to measure the effectiveness of using a likelihood ratio-based approach to search for possible first-degree familial relationships (full-sibling and parent–child) by comparing an evidence autosomal short tandem repeat (STR) profile to California's ∼1,000,000-profile State DNA Index System (SDIS) database. Test searches used autosomal STR and Y-STR profiles generated for 100 artificial test families. When the test sample and the first-degree relative in the database were characterized at the 15 Identifiler^® (Applied Biosystems^®, Foster City, CA) STR loci, the search procedure included 96% of the fathers and 72% of the full-siblings. When the relative profile was limited to the 13 Combined DNA Index System (CODIS) core loci, the search procedure included 93% of the fathers and 61% of the full-siblings. These results, combined with those of functional tests using three real families, support the effectiveness of this tool. Based upon these results, the validated approach was implemented as a key, pragmatic and demonstrably practical component of the California Department of Justice's Familial Search Program. An investigative lead created through this process recently led to an arrest in the Los Angeles Grim Sleeper serial murders.     

Performance of a next generation sequencing SNP assay on degraded DNA

Forensic DNA casework samples are often of insufficient quantity or quality to generate full profiles by conventional DNA typing methods. Polymerase chain reaction (PCR) amplification of short tandem repeat (STR) loci is inherently limited in samples containing degraded DNA, as the cumulative size of repeat regions, primer binding regions, and flanking sequence is necessarily larger than the PCR template. Additionally, traditional capillary electrophoresis (CE) assay design further inherently limits shortening amplicons because the markers must be separated by size. Non-traditional markers, such as single nucleotide polymorphisms (SNPs) and insertion deletion polymorphisms (InDels), may yield more information from challenging samples due to their smaller amplicon size. In this study, the performance of a next generation sequencing (NGS) SNP assay and CE-based STR, mini-STR, and InDel assays was evaluated with a series of fragmented, size-selected samples. Information obtained from the NGS SNP assay exhibited higher overall inverse random match probability (1/RMP) values compared to the CE-based typing assays, with particular benefit for fragment sizes ≤150 base pairs (bp). The InDel, mini-STR, and NGS SNP assays all had similar percentages of loci with reportable alleles at this level of degradation; however, the relatively fewer number of loci in the InDel and mini-STR assays results in the NGS SNP assay having at least nine orders of magnitude higher 1/RMP values. In addition, the NGS SNP assay and three CE-based assays (two STR and one InDel assay) were tested using a dilution series consisting of 0.5 ng, 0.1 ng, and 0.05 ng non-degraded DNA. All tested assays showed similar percentages of loci with reportable alleles at these levels of input DNA; however, due to the larger number of loci, the NGS SNP assay and the larger of the two tested CE-based STR assays both resulted in considerably higher 1/RMP values than the other assays. These results indicate the potential advantage of NGS SNP assays for forensic analysis of degraded DNA samples.