Toward Analysis at the Point of Need: A Digital Microfluidic Approach to Processing Multi-Source Sexual Assault Samples.

Forensic case samples collected in sexual assaults typically contain DNA from multiple sources, which complicates short-tandem repeat (STR) profiling. These samples are typically sent to a laboratory to separate the DNA from sperm and non-sperm sources prior to analysis. Here, the automation and miniaturization of these steps using digital microfluidics (DMF) is reported, which may eventually enable processing sexual assault samples outside of the laboratory, at the point of need. When applied to vaginal swab samples collected up to 12 h post-coitus (PC), the new method identifies single-source (male) STR profiles. When applied to samples collected 24-72 h PC, the method identifies mixed STR profiles, suggesting room for improvement and/or potential for data deconvolution. In sum, an automated, miniaturized sample pre-processing method for separating the DNA contained in sexual assault samples is demonstrated. This type of automated processing using DMF, especially when combined with Rapid DNA Analysis, has the potential to be used for processing of sexual assault samples in hospitals, police offices, and other locations outside of the laboratory.

Successful application of modified crude DNA extraction from muscle tissues for various types of PCR amplifications.

BACKGROUND: One of the most challenging aspects of nucleic acid amplification tests is the extraction of genomic DNA. However, achieving satisfactory quality and quantity of genomic DNA is not always easy, while the demand for rapid, low-cost and less laborious DNA isolation methods is ever-increasing. METHODS AND RESULTS: We have developed a rapid (⁓2 min) crude DNA extraction method leading to direct-PCR that requires minimum reagents and laboratory equipment. It was developed by eliminating the time-consuming purification steps of DNA extraction, by processing the sample in optimized amounts of Taq KCl PCR buffer and DNARelease Additive/Proteinase K in only two minutes and carrying out amplification using conventional Taq DNA polymerase. The DNA preparation method was validated on muscle tissue samples from 12 different species as well as 48 cooked meat samples. Its compatibility was also successfully tested with different types of PCR amplification platforms extensively used for genetic analysis, such as simplex PCR, PCR-RFLP (Restriction Fragment Length Polymorphism), multiplex PCR, isothermal amplification, real-time PCR and DNA sequencing. CONCLUSIONS: The developed protocol provides sufficient amount of crude DNA from muscle tissues of different species for PCR amplifications to identify species-of-origin via different techniques coupled with PCR. The simplicity and robustness of this protocol make nucleic acid amplification assays more accessible and affordable to researchers and authorities for both laboratory and point-of-care tests.

Processing biological samples from simulated radiological terrorist events using Rapid DNA instruments.

Two commercially available portable Rapid DNA instruments were evaluated for their ability to process 1 µL and 10 µL saliva samples deposited on metal and plastic surfaces and contaminated with surrogates of cesium (Cs)-137, strontium (Sr)-90 and cobalt (Co)-60; radioactive materials potentially released during a nuclear weapon accident or a radiological dispersal device detonation. A comparable success rate was noted for both Rapid DNA instruments when considering the number of complete and balanced DNA profiles, the number of profiles with a minimum of 10 autosomal STR loci (out of 23 [FlexPlex™ 27] or 21 [GlobalFiler™ Express]), and the possibility to search a national DNA database in Canada and the United States. Cobalt had an adverse impact on the quality of the megaplex short tandem repeat (STR) DNA profiles derived on each instrument for two of the three contamination levels tested in this study, i.e., 0.05 M and 0.1 M as reflected by a reduced number of detected alleles and decreased profile peak heights. Strontium exhibited some adverse effect on the Rapid DNA results when used at the highest contamination level (0.1 M) whereas cesium had none. No new artifacts were observed in the Rapid DNA profiles of samples spiked with the non-radiogenic surrogates. Importantly, in the context of a radiological/nuclear (RN) event, the ANDE™ 6C offers the possibility to dispose of all radioactive materials associated with contaminated samples quickly using a chip on which all steps of the Rapid DNA process are performed whereas the RapidHIT™ ID accumulates radioactive materials for many days before disposal. An individual handling 25 samples in a week (5 per day) on the RapidHIT™ ID at a 30.5 cm distance with a 5 min exposure to the radioactive source estimated at every run would exceed the 0.042 µSv/5 min limit with gamma dose rates for Cs at 0.13 mSv and for Co at 3.8 mSv. Beta dose rates calculated for the surrogate isotopes at the three concentrations tested were also above the recommended radiation exposure limit of 1 mSv/yr (0.042 µSv/5 min). Various potential mechanisms of action behind the interference noted for Sr and Co at high concentrations are presented. These elements may play a role in the steps prior to PCR (at the DNA molecule by binding to bases or to phosphate groups), during PCR (at the DNA polymerase as cofactors for catalytic sites), or even during amplified DNA fragment detection (as fluorescence quenchers).

Comparative analysis of two Rapid DNA technologies for the processing of blood and saliva-based samples.

Rapid DNA technologies recently gained significant momentum as a means to generate DNA profiles faster than with standard laboratory workflows. Initially developed for the analysis of buccal reference samples, applications are being considered for other types of forensic samples. In this study, an identical set of 150 blood and saliva-based samples was processed using two different Rapid DNA technologies, the Applied BioSystems™ RapidHIT™ ID System using the RapidINTEL™ sample cartridge and the ANDE™ 6C Rapid DNA Analysis™ System using the I-Chip. A subset of samples were subjected to alternative collection methods or sample pre-treatments to determine the optimal strategy for each instrument. An equivalent sample set was also processed using a conventional DNA analysis workflow. The sensitivity range of the two Rapid DNA technologies was comparable based on blood and saliva dilution series, with both technologies able to generate full profiles from samples typically yielding 5-10 ng of DNA when processed using conventional DNA analysis. The brand of cotton swabs used for Rapid DNA analysis had an impact on the results for both systems. Differences were observed in success rate between the two systems when processing blood (on fabrics, FTA paper or hard surfaces) and saliva-based samples (drink containers, FTA paper, chewing gum, cigarette butt filter paper) and depended on the sample type. Importantly, deviating from the manufacturer's instructions for sample collection and pre-treatment was more detrimental to the ANDE 6C results. The quality of DNA profiles, as assessed using heterozygote peak height ratios, interloci balance and artifact presence, confirmed the results to be reliable and acceptable for single source samples. Profiling results were obtained when samples were reprocessed using the same Rapid DNA technology or conventional DNA analysis. Secondary analysis using a substitute software (GeneMapper ID-X v1.5) to recover additional genetic information was shown to be feasible. Finally, a comparison between the Applied Biosystems™ RapidHIT™ ID System Software v1.3.1 and v1.3.2 was also performed. Findings of this study could assist those interested in using Rapid DNA technology for blood or saliva-based samples, in various settings and for different applications.

Introducing a Rapid DNA Analysis Procedure for Crime Scene Samples Outside of the Laboratory-A Field Experiment.

Technological innovations enable rapid DNA analysis implementation possibilities. Concordantly, rapid DNA devices are being used in practice. However, the effects of implementing rapid DNA technologies in the crime scene investigation procedure have only been evaluated to a limited extent. In this study a field experiment was set up comparing 47 real crime scene cases following a rapid DNA analysis procedure outside of the laboratory (decentral), with 50 cases following the regular DNA analysis procedure at the forensic laboratory. The impact on duration of the investigative process, and on the quality of the analyzed trace results (97 blood and 38 saliva traces) was measured. The results of the study show that the duration of the investigation process has been significantly reduced in cases where the decentral rapid DNA procedure was deployed, compared to cases where the regular procedure was used. Most of the delay in the regular process lies in the procedural steps during the police investigation, not in the DNA analysis, which highlights the importance of an effective work process and having sufficient capacity available. This study also shows that rapid DNA techniques are less sensitive than regular DNA analysis equipment. The device used in this study was only to a limited extent suitable for the analysis of saliva traces secured at the crime scene and can mainly be used for the analysis of visible blood traces with an expected high DNA quantity of a single donor.

A Systematic Review on Commercially Available Integrated Systems for Forensic DNA Analysis.

This systematic review describes and discusses three commercially available integrated systems for forensic DNA analysis, i.e., ParaDNA, RapidHIT, and ANDE. A variety of aspects, such as performance, time-to-result, ease-of-use, portability, and costs (per analysis run) of these three (modified) rapid DNA analysis systems, are considered. Despite their advantages and developmental progress, major steps still have to be made before rapid systems can be broadly applied at crime scenes for full DNA profiling. Aspects in particular that need (further) improvement are portability, performance, the possibility to analyze a (wider) variety of (complex) forensic samples, and (cartridge) costs. Moreover, steps forward regarding ease-of-use and time-to-result will benefit the broader use of commercial rapid DNA systems. In fact, it would be a profit if rapid DNA systems could be used for full DNA profile generation as well as indicative analyses that can give direction to forensic investigators which will speed up investigations.

Comparative Analysis of ANDE 6C Rapid DNA Analysis System and Traditional Methods.

Rapid DNA analysis is an ultrafast and fully automated DNA-typing system, which can produce interpretable genetic profiles from biological samples within 90 minutes. This "swab in-profile out" method comprises DNA extraction, amplification by PCR multiplex, separation and detection of DNA fragments by capillary electrophoresis. The aim of study was the validation of the Accelerated Nuclear DNA Equipment (ANDE) 6C system as a typing method for reference samples according to the ISO/IEC 17025 standard. Here, we report the evaluation of the validity and reproducibility of results by the comparison of the genetic profiles generated by the ANDE 6C System with those generated by standard technologies. A quantity of 104 buccal swabs were analyzed both through the ANDE 6C technology and the traditional method (DNA extraction and quantification, amplification and separation by capillary electrophoresis). Positive typing was observed in 97% of cases for ANDE 6C technology with only three buccal swabs failing to reveal interpretable signals. Concordance was determined by comparing the allele calls generated by ANDE 6C and conventional technology. Comparison of 2800 genotypes revealed a concordance rate of 99.96%. These results met the ISO/IEC 17025 requirements, enabling us to receive the accreditation for this method. Finally, rapid technology has certainly reached a level of reliability which has made its use in laboratories of forensic genetics a reality.

Internal validation of the DNAscan/ANDE™ Rapid DNA Analysis™ platform and its associated PowerPlex® 16 high content DNA biochip cassette for use as an expert system with reference buccal swabs.

Rapid DNA platforms are fully integrated systems capable of producing and analyzing short tandem repeat (STR) profiles from reference sample buccal swabs in less than two hours. The technology requires minimal user interaction and experience making it possible for high quality profiles to be generated outside an accredited laboratory. The automated production of point of collection reference STR profiles could eliminate the time delay for shipment and analysis of arrestee samples at centralized laboratories. Furthermore, point of collection analysis would allow searching against profiles from unsolved crimes during the normal booking process once the infrastructure to immediately search the Combined DNA Index System (CODIS) database from the booking station is established. The DNAscan/ANDE™ Rapid DNA Analysis™ System developed by Network Biosystems was evaluated for robustness and reliability in the production of high quality reference STR profiles for database enrollment and searching applications. A total of 193 reference samples were assessed for concordance of the CODIS 13 loci. Studies to evaluate contamination, reproducibility, precision, stutter, peak height ratio, noise and sensitivity were also performed. The system proved to be robust, consistent and dependable. Results indicated an overall success rate of 75% for the 13 CODIS core loci and more importantly no incorrect calls were identified. The DNAscan/ANDE™ could be confidently used without human interaction in both laboratory and non-laboratory settings to generate reference profiles.

Developmental validation of the DNAscan™ Rapid DNA Analysis™ instrument and expert system for reference sample processing.

Since the implementation of forensic DNA typing in labs more than 20 years ago, the analysis procedures and data interpretation have always been conducted in a laboratory by highly trained and qualified scientific personnel. Rapid DNA technology has the potential to expand testing capabilities within forensic laboratories and to allow forensic STR analysis to be performed outside the physical boundaries of the traditional laboratory. The developmental validation of the DNAscan/ANDE Rapid DNA Analysis System was completed using a BioChipSet™ Cassette consumable designed for high DNA content samples, such as single source buccal swabs. A total of eight laboratories participated in the testing which totaled over 2300 swabs, and included nearly 1400 unique individuals. The goal of this extensive study was to obtain, document, analyze, and assess DNAscan and its internal Expert System to reliably genotype reference samples in a manner compliant with the FBI's Quality Assurance Standards (QAS) and the NDIS Operational Procedures. The DNAscan System provided high quality, concordant results for reference buccal swabs, including automated data analysis with an integrated Expert System. Seven external laboratories and NetBio, the developer of the technology, participated in the validation testing demonstrating the reproducibility and reliability of the system and its successful use in a variety of settings by numerous operators. The DNAscan System demonstrated limited cross reactivity with other species, was resilient in the presence of numerous inhibitors, and provided reproducible results for both buccal and purified DNA samples with sensitivity at a level appropriate for buccal swabs. The precision and resolution of the system met industry standards for detection of micro-variants and displayed single base resolution. PCR-based studies provided confidence that the system was robust and that the amplification reaction had been optimized to provide high quality results. The DNAscan integrated Expert System was examined as part of the Developmental Validation and successfully interpreted the over 2000 samples tested with over 99.998% concordant alleles. The system appropriately flagged samples for human review and failed both mixed samples and samples with insufficient genetic information. These results demonstrated the integrated Expert System makes correct allele calls without human intervention.

Rapid DNA analysis for automated processing and interpretation of low DNA content samples.

BACKGROUND: Short tandem repeat (STR) analysis of casework samples with low DNA content include those resulting from the transfer of epithelial cells from the skin to an object (e.g., cells on a water bottle, or brim of a cap), blood spatter stains, and small bone and tissue fragments. Low DNA content (LDC) samples are important in a wide range of settings, including disaster response teams to assist in victim identification and family reunification, military operations to identify friend or foe, criminal forensics to identify suspects and exonerate the innocent, and medical examiner and coroner offices to identify missing persons. Processing LDC samples requires experienced laboratory personnel, isolated workstations, and sophisticated equipment, requires transport time, and involves complex procedures. We present a rapid DNA analysis system designed specifically to generate STR profiles from LDC samples in field-forward settings by non-technical operators. By performing STR in the field, close to the site of collection, rapid DNA analysis has the potential to increase throughput and to provide actionable information in real time. RESULTS: A Low DNA Content BioChipSet (LDC BCS) was developed and manufactured by injection molding. It was designed to function in the fully integrated Accelerated Nuclear DNA Equipment (ANDE) instrument previously designed for analysis of buccal swab and other high DNA content samples (Investigative Genet. 4(1):1-15, 2013). The LDC BCS performs efficient DNA purification followed by microfluidic ultrafiltration of the purified DNA, maximizing the quantity of DNA available for subsequent amplification and electrophoretic separation and detection of amplified fragments. The system demonstrates accuracy, precision, resolution, signal strength, and peak height ratios appropriate for casework analysis. CONCLUSIONS: The LDC rapid DNA analysis system is effective for the generation of STR profiles from a wide range of sample types. The technology broadens the range of sample types that can be processed and minimizes the time between sample collection, sample processing and analysis, and generation of actionable intelligence. The fully integrated Expert System is capable of interpreting a wide range or sample types and input DNA quantities, allowing samples to be processed and interpreted without a technical operator.

Evaluation of the RapidHIT™ 200 and RapidHIT GlobalFiler(®) Express kit for fully automated STR genotyping.

The RapidHIT™ 200 Human Identification System and RapidHIT GlobalFiler(®) Express kit were evaluated and validated for use with single-source reference samples. It was of primary interest to evaluate the system for its efficacy as an expert system and to estimate a first pass success rate, as well as to identify the technical variables impacting that result. While results indicated that this instrument/kit combination can be used to accurately type single-source buccal samples, substantial variability in sensitivity and intra-color balance were observed, as were multiple artifacts, requiring extensive manual editing of the profiles. Artifacts included dye "blobs" and spectral overlap (pull-up) peaks that often originated from relatively low intensity allele peaks. Reduced intra-color balance, in combination with low sensitivity, occasionally resulted in instances of allelic dropout. Overall, 50% of the buccal samples analyzed in this study would have been successfully typed to give full GlobalFiler(®) profiles without the need for manual review and editing.