Resurrecting the Dodo: Positive control DNA for species identification

Positive controls are necessary standards for inclusion in forensic tests. When working as expected they demonstrate that methods have been applied correctly, and therefore results can be interpreted with confidence. However, the requirement for positive controls can also introduce problems. For species identification in wildlife DNA forensic testing, it is possible that the DNA sequence of the case sample will be a 100% match to the positive control. Whilst clear results for negative controls will indicate that cross-contamination is unlikely, it would be preferable to have a positive control that will not appear in casework. In addition, for many endangered species, obtaining positive control DNA for species-specific testing can be problematic. Here we present a simple method to use artificially generated positive control DNA from the extinct Dodo, Raphus cucullatus, for four species-identification tests run routinely in UK wildlife forensic casework.     

A review of low template STR analysis in casework using the DNA SenCE post-PCR purification technique

LGC has developed a method for analysing low-level DNA samples called DNA SenCE (Sensitive Capillary Electrophoresis) based on post-PCR treatment of standard 28-cycle SGMplus PCR product and demonstrated to be equally effective at enhancing profiles as 34-cycle PCR. The method has been validated and accredited and used in casework since July 2007. Inherent in the method is the initial generation of a standard 28-cycle SGMplus profile so a direct comparison of standard and DNA SenCE results for all casework is possible. Here we review DNA SenCE casework, reporting the magnitude of peak enhancement and stochastic effects seen in the DNA SenCE profiles.     

Automated processing of forensic casework samples using robotic workstations equipped with nondisposable tips: contamination prevention

An automated process has been developed for the analysis of forensic casework samples using TECAN Genesis RSP 150/8 or Freedom EVO liquid handling workstations equipped exclusively with nondisposable tips. Robot tip cleaning routines have been incorporated strategically within the DNA extraction process as well as at the end of each session. Alternative options were examined for cleaning the tips and different strategies were employed to verify cross-contamination. A 2% sodium hypochlorite wash (1/5th dilution of the 10.8% commercial bleach stock) proved to be the best overall approach for preventing cross-contamination of samples processed using our automated protocol. The bleach wash steps do not adversely impact the short tandem repeat (STR) profiles developed from DNA extracted robotically and allow for major cost savings through the implementation of fixed tips. We have demonstrated that robotic workstations equipped with fixed pipette tips can be used with confidence with properly designed tip washing routines to process casework samples using an adapted magnetic bead extraction protocol.     

Analysis of ancient human DNA and primer contamination: one step backward one step forward

The analysis of DNA from archaeological human remains is plagued by a unique set of methodological problems concerning contamination with modern exogenous DNA. Through an original approach, we propose complementary methods to identify all potential sources of contamination and complete guidelines for the validation of ancient human sequences. The study presented was conducted on non-European human samples (Polynesian and Amerindian) which were collected with all precautions during excavation. This permitted us to distinguish without ambiguity authentic and contaminant sequences. The samples' origins and histories were perfectly known, allowing us to trace all potential contamination sources and to determine the efficiency of precautions followed during all steps of the study. The data obtained confirm that precautions taken during sampling effectively prevent contamination. However, we demonstrate that human contamination can also be introduced during genetic analyses even if all precautions are strictly followed. Indeed, numerous human contaminations were detected in template-PCR products and negative controls, resulting in a striking diversity of contaminant mitochondrial DNA sequences. We argue that this contamination partly derives from the primers. To our knowledge, no previous experiment has been performed to investigate primers as a possible source of human contamination despite the fact that this specific type of contamination poses a real problem in terms of validating ancient human DNA studies. Finally, we confirm that the detection of contaminants in negative controls is clearly related to the number of PCR cycles used. This study enhances our understanding of contamination processes and confirms that, in reality, an absolutely contamination-free situation cannot be obtained. As a consequence, we propose improvements to the guidelines usually followed in the field in order to take the highly probable contamination of PCR reagents, including primers, into account.     

Identification and separation of DNA mixtures using peak area information

We introduce a new methodology, based upon probabilistic expert systems, for analysing forensic identification problems involving DNA mixture traces using quantitative peak area information. Peak area is modelled with conditional Gaussian distributions. The expert system can be used for ascertaining whether individuals, whose profiles have been measured, have contributed to the mixture. It can also be used to predict DNA profiles of unknown contributors by separating the mixture into its individual components. The potential of our probabilistic methodology is illustrated on case data examples and compared with alternative approaches. The advantages are that identification and separation issues can be handled in a unified way within a single probabilistic model and the uncertainty associated with the analysis is quantified. Further work, required to bring the methodology to a point where it could be applied to the routine analysis of casework, is discussed.     

The best possible result from the minimum available

In accordance with the Italian DNA legislation (DPR 7 April 2016, n. 87) a number of markers lower than seven are not considered usable for inclusion in the Italian forensic DNA database. For this reason, if the forensic DNA analysis performed in our laboratory do not provide acceptable results for a number greater than or equal to seven, the profile is not indicated in the final report. Thus, having indications about the possible success of an analysis before executing it, is a crucial point in the validation process of the accreditated method used in our laboratory.To achieve this goal, the quantification of extracts before typing plays a fundamental role. Especially when touched objects need to be examined tens or hundreds of nanograms may be present, but also very few or no cell can be present on the object. As such, quantification of every sample can ensure the maximum efficiency and prevent repeat analyses, over-amplified samples or completely useless examination.Quantifiler® Trio DNA quantification kit was validated in our laboratory according the guidelines approved by the ENFSI and always used before STR amplification of forensic casework DNA samples. Our attention has focused in particular on the definition of a minimum threshold at which it is useless to carry out DNA typing defining correlation of the negative results of the quantification by the absence of genetic profiles, as a result of DNA typing. Moreover, the validation of the Savant™ SPD131DDA SpeedVac™ Concentrator to get the maximum possible yield from DNA extracts was also investigated.     

Implementation and Validation of the Teleshake Unit for DNA IQ™ Robotic Extraction and Development of a Large Volume DNA IQ™ Method

Abstract: Automated platforms used for forensic casework sample DNA extraction need to be versatile to accommodate a wide variety of sample types, thus protocols frequently need modification. In this study, DNA IQ? methods previously developed for the Biomek^® 2000 Automation Workstation were adapted for the Teleshake Unit using normal volumes and all deepwell extraction, and a large volume DNA IQ? method developed. DNA purification without detectable contamination of adjacent reagent blanks is reported in the extraction of tissue samples containing several micrograms of DNA. Sensitivity and contamination studies demonstrated similar performance with the manual organic extraction method for bloodstain dilution samples. Mock casework samples demonstrated the effectiveness of the Teleshake and Teleshake large volume methods. Because of the performance and increased versatility of the DNA IQ? extraction with these modifications, the Teleshake Unit has been implemented in both normal and large volume automated DNA extractions at the Virginia Department of Forensic Science.     

An investigation of the rigor of interpretation rules for STRs derived from less than 100 pg of DNA

By increasing the PCR amplification regime to 34 cycles, we have demonstrated that it is possible routinely to analyse <100 pg DNA. The success rate was not improved (without impairing quality) by increasing cycle number further. Compared to amplification of 1 ng DNA at 28 cycles, it was shown that increased imbalance of heterozygotes occurred, along with an increase in the size (peak area) of stutters. The analysis of mixtures by peak area measurement becomes increasingly difficult as the sample size is reduced. Laboratory-based contamination cannot be completely avoided, even when analysis is carried out under stringent conditions of cleanliness. A set of guidelines that utilises duplication of results to interpret profiles originating from picogram levels of DNA is introduced. We demonstrate that the duplication guideline is robust by applying a statistical theory that models three key parameters - namely the incidence of allele drop-out, laboratory contamination and stutter. The advantage of the model is that the critical levels for each parameter can be calculated. This information may be used (for example) to determine levels of contamination that can be tolerated within the strategy employed. In addition we demonstrate that interpreting one banded loci, where allele dropout could have occurred, using LR=1/2f(a) was conservative provided that the band was low in peak area. Furthermore, we demonstrate that an apparent mis-match between crime-stain and a suspect DNA profile does not necessarily result in an exclusion. The method used is complex, yet can be converted into an expert system. We envisage this to be the next step.     

AmpFlSTR profiler Plus short tandem repeat DNA analysis of casework samples, mixture samples, and nonhuman DNA samples amplified under reduced PCR volume conditions (25 microL)

As part of the validation of the AmpFlSTR Profiler Plus short tandem repeat (STR) system, under reduced polymerase chain reaction (PCR) volume conditions (i.e., 25 microL), a total of 275 casework samples were processed. Examples of profiles are presented along with amplification conditions to improve the odds of obtaining balanced and complete profiles for samples showing partial results or profiles with a descending slope. Data collected and used to develop our interpretation guidelines are included. From the mixture studies, full profiles were obtained for minor contributors, using 2 ng of DNA, with ratios of 10:1 or 1:20 and using 1 ng of DNA, with ratios of 10:1 and 1:8. The specificity of the Profiler Plus amplification reaction performed in 25 microL was examined and confirmed using a large spectrum of nonhuman DNAs. This report supports the use of the AmpFlSTR Profiler Plus STR system for casework DNA typing under reduced PCR volume conditions.     

Automated PCR setup for forensic casework samples using the Normalization Wizard and PCR Setup robotic methods

Human genome, pharmaceutical and research laboratories have long enjoyed the application of robotics to performing repetitive laboratory tasks. However, the utilization of robotics in forensic laboratories for processing casework samples is relatively new and poses particular challenges. Since the quantity and quality (a mixture versus a single source sample, the level of degradation, the presence of PCR inhibitors) of the DNA contained within a casework sample is unknown, particular attention must be paid to procedural susceptibility to contamination, as well as DNA yield, especially as it pertains to samples with little biological material. The Virginia Department of Forensic Science (VDFS) has successfully automated forensic casework DNA extraction utilizing the DNA IQ(trade mark) System in conjunction with the Biomek 2000 Automation Workstation. Human DNA quantitation is also performed in a near complete automated fashion utilizing the AluQuant Human DNA Quantitation System and the Biomek 2000 Automation Workstation. Recently, the PCR setup for casework samples has been automated, employing the Biomek 2000 Automation Workstation and Normalization Wizard, Genetic Identity version, which utilizes the quantitation data, imported into the software, to create a customized automated method for DNA dilution, unique to that plate of DNA samples. The PCR Setup software method, used in conjunction with the Normalization Wizard method and written for the Biomek 2000, functions to mix the diluted DNA samples, transfer the PCR master mix, and transfer the diluted DNA samples to PCR amplification tubes. Once the process is complete, the DNA extracts, still on the deck of the robot in PCR amplification strip tubes, are transferred to pre-labeled 1.5 mL tubes for long-term storage using an automated method. The automation of these steps in the process of forensic DNA casework analysis has been accomplished by performing extensive optimization, validation and testing of the software methods.     

Development of a one-tube extraction and amplification method for DNA analysis of sperm and epithelial cells recovered from forensic samples by laser microdissection

Laser microdissection can be used in forensic casework to isolate specific cell types from mixtures of biological samples. Extraction of DNA from selected cells is still required prior to STR amplification. Because of the relatively pristine nature of the recovered cells, laser microdissection is more sensitive than more traditional methods of DNA analysis, theoretically resulting in DNA profiles from less cellular material. A one-tube extraction and amplification method minimises loss of DNA through liquid transfers and reduces the potential for contamination events occurring. In this paper, the development of a one-tube method for the effective extraction of DNA from laser microdissected sperm and epithelial cells is described. The performance of the in-house method was compared to that of a commercial DNA extraction kit for extraction of DNA from sperm and the downstream compatibility with STR amplification was determined for both sperm and epithelial samples. Full Identifiler™ profiles after 28 amplification cycles were obtained from as few as 15 epithelial cells and 30 sperm.     

Application of the BioMek 2000 Laboratory Automation Workstation and the DNA IQ System to the extraction of forensic casework samples

Robotic systems are commonly utilized for the extraction of database samples. However, the application of robotic extraction to forensic casework samples is a more daunting task. Such a system must be versatile enough to accommodate a wide range of samples that may contain greatly varying amounts of DNA, but it must also pose no more risk of contamination than the manual DNA extraction methods. This study demonstrates that the BioMek 2000 Laboratory Automation Workstation, used in combination with the DNA IQ System, is versatile enough to accommodate the wide range of samples typically encountered by a crime laboratory. The use of a silica coated paramagnetic resin, as with the DNA IQ System, facilitates the adaptation of an open well, hands off, robotic system to the extraction of casework samples since no filtration or centrifugation steps are needed. Moreover, the DNA remains tightly coupled to the silica coated paramagnetic resin for the entire process until the elution step. A short pre-extraction incubation step is necessary prior to loading samples onto the robot and it is at this step that most modifications are made to accommodate the different sample types and substrates commonly encountered with forensic evidentiary samples. Sexual assault (mixed stain) samples, cigarette butts, blood stains, buccal swabs, and various tissue samples were successfully extracted with the BioMek 2000 Laboratory Automation Workstation and the DNA IQ System, with no evidence of contamination throughout the extensive validation studies reported here.     

Simplified low-copy-number DNA analysis by post-PCR purification

Frequently, evidentiary items contain an insufficient quantity of DNA to obtain complete or even partial DNA profiles using standard forensic gentotyping techniques. Such low-copy-number (LCN) samples are usually subjected to increased amplification cylces to obtain genetic data. In this study, a 28-cycle polymerase chain reaction (PCR) was used to evaluate various methods of post-PCR purification for their effects on the sensitivity of fluorophore-based allelic detection subsequent to capillary electrophoretic separation. The amplified product was purified using filtration, silica gel membrane, and enzyme mediated hydrolysis purification techniques and evaluated for their effect on fluorescent allelic signal intensity. A purification method was selected and its effect on fluorescent allelic signal intensity was compared with that of the unpurified PCR product. A method of post-PCR purification is described which increases the sensitivity of standard 28-cycle PCR such that profiles from LCN DNA templates (<100 pg DNA) can be obtained. Full DNA profiles were consistently obtained with as little as 20 pg template DNA without increased cycle number. In mock case type samples with dermal ridge fingerprints, genetic profiles were obtained by amplification with 28 cycles followed by post-PCR purification whereas no profiles were obtained without purification of the PCR product. Allele dropout, increased stutter, and sporadic contamination typical of LCN analysis were observed; however, no contamination was observed in negative amplification controls. Post-PCR purification of the PCR product can increase the sensitivity of capillary electrophoresis to such an extent that DNA profiles can be obtained from <100 pg of DNA using 28-cycle amplification.     

A systematic analysis of PCR contamination.

In light of the strict legal scrutiny surrounding DNA typing at this time, it has become necessary to systematically address the issue of PCR contamination. To precisely define the parameters affecting PCR contamination under casework analysis conditions, PCR amplification reactions were intentionally compromised by employing sub-standard laboratory technique and by introducing secondary sources of DNA. The PCR parameters considered for potential sources of contamination include amplification set-up, amplification product handling, aerosol DNA and storage. In addition, analyst technique was evaluated by modifying or eliminating standard safeguards. Under the circumstances normally encountered during casework analysis, PCR contamination was never noted. Significantly, using the dot blot detection method, contamination was never observed when nanogram quantities of genomic DNA were mishandled or aerosolized. Contamination occurred only when amplification product was carelessly manipulated or purposefully sprayed near or directly into open tubes containing water or genomic DNA. Although standard precautions should be employed during PCR-based DNA typing, our data indicates that contamination during amplification procedures is not prevalent when detected by dot blot analysis.     

法医物证DNA自动化检验技术体系的研究

目的建立自动化工作站同步提取不同种类涉案法医生物检材DNA的新方法。方法选用TECAN Freedom EVO100．4、75—2型自动化提取、加样工作站,采用磁珠法及Chelex-100法对各类涉案生物检材进行DNA提取、PCR扩增、毛细管电泳检测其STR分型,进行比较测试。在“全国公安机关DNA数据库应用系统”中建立并应用实验室信息管理系统（LIMS）模拟实施规范化DNA检案。结果1552份各类检材,采用工作站-磁珠法提取DNA效果最佳,STR检测成功率为95％,工作站-Chelex法为88％;二者分别与其手工提取法比较,成功率无明显差异。92个样本同期检测,自动化工作站较手工操作DNA检案时间可缩减1．25倍。结论工作站域珠法提取涉案检材DNA,可获得满意的STR分型结果。应用LIMS管控,可有效防控污染,明显提高检案效率及鉴定质量。     

DNA mixtures in forensic casework: a 4-year retrospective study

Occasionally interpretation guidelines from validation studies are difficult to apply to real forensic casework, especially in the case of mixed samples. Exogenous contamination, an unknown number of contributors or unbalanced proportion of each one in the sample and a varied degree of degradation of the biological materials, contribute to the difficulties in the interpretation of sample profiles. In this paper we have reviewed all the mixed genetic STR profiles encountered in our laboratory over 4 years (1997-2000) and evaluated the problems in the interpretation of the results. From 1547 criminal cases with 2424 samples typed, 163 showed a mixed profile (6.7%). We have observed that occasionally, a mixture appeared in the same sample with one multiplex amplification kit (e.g. Blue) and not with another (e.g. Green). From our results, it can be suggested that technical characteristics of the different fluorochrome groups in the multiplexes override the molecular characteristics of each STR in their capacity to detect mixtures.     

Analysis of Mitochondrial DNA from a Burned,Ninhydrin‐Treated Paper Towel

Contact‐based evidence is likely to have limited quantities of DNA and may yield mixed profiles due to preexisting or contaminating DNA. In a recent arson investigation, a paper towel was collected and used as circumstantial evidence. The paper towel was partially burned and was likely set on fire with flammable liquid. As part of the investigation, the paper towel was treated with ninhydrin to visualize fingerprint evidence. Initial DNA analysis of two swabs was negative for short tandem repeat (STR) markers and revealed a mixture of mitochondrial DNA (mtDNA). Analysis of 13 additional cuttings yielded four more mixed profiles, but also two samples with a common single‐source profile. The single‐source mtDNA profile matched that of the primary suspect in the case. Thus, even if initial mtDNA analysis yields a mixed profile, a sampling strategy involving multiple locations can improve the chance of obtaining valuable single‐source mtDNA profiles from compromised evidence in criminal casework.     

The potential transfer of trace DNA via high risk vectors during exhibit examination

As the sensitivity and discriminative power of DNA technology progressively improves, the ability to utilise trace DNA as evidence in forensic casework similarly increases. Along with increased sensitivity comes the prospect of detecting contaminating DNA, complicating the interpretation of profiles. This study aimed to provide data into the occurrence and level of DNA potentially transferred between high risk vectors (scissors, forceps, gloves) and exhibits during the examination process in both light and heavy contamination/contact scenarios. DNA transfer was observed for all vectors in both transfer situations, with heavy conditions transferring more than light. Profiles with sufficient alleles to identify the origin were observed for each vector in both heavy and light scenarios, excluding forceps, where only a small number of alleles were transferred under light conditions,     

Applicability of DNA analysis on adhesive tape in forensic casework

Adhesive tape is commonly used in crimes and is often the subject of forensic evaluation. DNA analysis of adhesive tape can provide DNA profiles of suspects. The object of this study was to evaluate the applicability of DNA analysis on adhesive tape samples in forensic casework. We retrospectively reviewed all cases involving adhesive tape or similar items received by our institute for DNA analysis during the past 11 years. From 100 forensic cases reviewed, 150 adhesive tape samples were examined. A total of 98 DNA profiles were obtained from these samples. Sixty-two of the profiles provided feasible case-relevant information. In conclusion, DNA profiling of adhesive tape samples can be useful in a variety of forensic cases.     

SNP genotyping of forensic casework samples using the 52 SNPforID markers

The analysis of degraded DNA is one of the biggest challenges in forensic casework. SNPs, which can be amplified using small amplicons, have previously been successfully applied to the profiling of forensic evidence that could not be analyzed using conventional STRs. Here we selected the 52 SNPforID SNP markers, with amplicons that ranged in size from 59 bp to 115 bp, and used them to profile a range of casework samples from Malaysia. DNA degradation is a common problem in Malaysia due to the high temperatures and humidity. To carry out the study we modified the 52 SNPforID markers into four 13-plex SNaPshot assays to enable easier interpretation of profiles on the ABI PRISM^® 310 and 3500.Fifty-one crime samples comprising bloodstains on cloth, swabs, and a mat and 2 swabs of trace DNA from 10 crime scenes in Malaysia were profiled after DNA extraction using a phenol–chloroform method. The samples were also subjected to STR analysis using the Powerplex^® 16 system (Promega), which resulted in only 17 full profiles and 9 partial profiles; using SNPs, 36 full profiles and 5 partial profiles could be generated.