A novel,integrated forensic microdevice on a rotation‐driven platform: Buccal swab to STR product in less than 2 h

This work describes the development of a novel microdevice for forensic DNA processing of reference swabs. This microdevice incorporates an enzyme‐based assay for DNA preparation, which allows for faster processing times and reduced sample handling. Infrared‐mediated PCR (IR‐PCR) is used for STR amplification using a custom reaction mixture, allowing for amplification of STR loci in 45 min while circumventing the limitations of traditional block thermocyclers. Uniquely positioned valves coupled with a simple rotational platform are used to exert fluidic control, eliminating the need for bulky external equipment. All microdevices were fabricated using laser ablation and thermal bonding of PMMA layers. Using this microdevice, the enzyme‐mediated DNA liberation module produced DNA yields similar to or higher than those produced using the traditional (tube‐based) protocol. Initial microdevice IR‐PCR experiments to test the amplification module and reaction (using Phusion Flash/SpeedSTAR) generated near‐full profiles that suffered from interlocus peak imbalance and poor adenylation (significant ?A). However, subsequent attempts using KAPA 2G and Pfu Ultra polymerases generated full STR profiles with improved interlocus balance and the expected adenylated product. A fully integrated run designed to test microfluidic control successfully generated CE‐ready STR amplicons in less than 2 h (<1 h of hands‐on time). Using this approach, high‐quality STR profiles were developed that were consistent with those produced using conventional DNA purification and STR amplification methods. This method is a smaller, more elegant solution than current microdevice methods and offers a cheaper, hands‐free, closed‐system alternative to traditional forensic methods.     

A valveless microfluidic device for integrated solid phase extraction and polymerase chain reaction for short tandem repeat (STR) analysis

A valveless microdevice has been developed for the integration of solid phase extraction (SPE) and polymerase chain reaction (PCR) on a single chip for the short tandem repeat (STR) analysis of DNA from a biological sample. The device consists of two domains--a SPE domain filled with silica beads as a solid phase and a PCR domain with an ~500 nL reaction chamber. DNA from buccal swabs was purified and amplified using the integrated device and a full STR profile (16 loci) resulted. The 16 loci Identifiler? multiplex amplification was performed using a non-contact infrared (IR)-mediated PCR system built in-house, after syringe-driven SPE, providing an ~80-fold and 2.2-fold reduction in sample and reagent volumes consumed, respectively, as well as an ~5-fold reduction in the overall analysis time in comparison to conventional analysis. Results indicate that the SPE-PCR system can be used for many applications requiring genetic analysis, and the future addition of microchip electrophoresis (ME) to the system would allow for the complete processing of biological samples for forensic STR analysis on a single microdevice.     

Integrated DNA purification, PCR, sample cleanup, and capillary electrophoresis microchip for forensic human identification

A fully integrated microdevice and process for forensic short tandem repeat (STR) analysis has been developed that includes sequence-specific DNA template purification, polymerase chain reaction (PCR), post-PCR cleanup and inline injection, and capillary electrophoresis (CE). Fragmented genomic DNA is hybridized with biotin-labeled capture oligos and pumped through a fluidized bed of magnetically immobilized streptavidin-coated beads in microchannels where the target DNA is bound to the beads. The bead-DNA conjugates are then transferred into a 250 nL PCR reactor for autosomal STR amplification using one biotin and one fluorescence-labeled primer. The resulting biotin-labeled PCR products are electrophoretically injected through a streptavidin-modified capture gel where they are captured to form a concentrated and purified injection plug. The thermally released sample plug is injected into a 14 cm long CE column for fragment separation and detection. The DNA template capture efficiency provided by the on-chip sequence-specific template purification is determined to be 5.4% using K562 standard DNA. This system can produce full 9-plex STR profiles from 2.5 ng input standard DNA and obtain STR profiles from oral swabs in about 3 hours. This fully integrated microsystem with sample-in-answer-out capability is a significant advance in the development of rapid, sensitive, and reliable micro-total analysis systems for on-site human identification.     

Sample stacking capillary electrophoretic microdevice for highly sensitive mini Y short tandem repeat genotyping

Lab‐on‐a‐chip provides an ideal platform for short tandem repeat (STR) genotyping due to its intrinsic low sample consumption, rapid analysis, and high‐throughput capability. One of the challenges, however, in the forensic human identification on the microdevice is the detection sensitivity derived from the nanoliter volume sample handling. To overcome such a sensitivity issue, here we developed a sample stacking CE microdevice for mini Y STR genotyping. The mini Y STR includes redesigned primer sequences to generate smaller‐sized PCR amplicons to enhance the PCR efficiency and the success rate for a low copy number and degraded DNA. The mini Y STR amplicons occupied in the 5‐ and 10‐mm stacking microchannels are preconcentrated efficiently in a defined narrow region through the optimized sample stacking CE scheme, resulting in more than tenfold improved fluorescence peak intensities compared with that of a conventional cross‐injection microcapillary electrophoresis method. Such signal enhancement allows us to successfully analyze the Y STR typing with only 25 pg of male genomic DNA, with high background of female genomic DNA, and with highly degraded male genomic DNA. The combination of the mini Y STR system with the novel sample stacking CE microdevice provides the highly sensitive Y STR typing on a chip, making it promising to perform high‐performance on‐site forensic human identification.     

Differential pre‐amplification of STR loci for fragmented forensic DNA profiling

DNA profiling of short tandem repeats (STR) has been successfully used for the identification of individuals in forensic samples, accidents and natural disasters. However, STR profiling of DNA isolated from old crime scenes and damaged biological samples is difficult due to DNA degradation and fragmentation. Here, we show that pre‐amplification of STR loci using biotinylated primers for the STR loci is an efficient strategy to obtain STR profiling results from fragmented forensic samples. Analysis of STR loci with longer amplicon sizes is generally hampered, since these relatively long loci are vulnerable to DNA fragmentation. This problem was overcome by using reduced or increased primer concentrations for loci with shorter or longer amplicon sizes, respectively, in our pre‐amplification strategy. In addition, pre‐amplification of STR loci into two groups of short or long amplicon size increases the efficiency of STR profiling from highly fragmented forensic DNA samples. Therefore, differential pre‐amplification of STR loci is an effective way to obtain DNA profiling results from fragmented forensic samples.     

Improved STR analysis of degraded DNA from human skeletal remains through in-house MPS-STR panel

DNA analysis of degraded samples and low-copy number DNA derived from skeletal remains, one of the most challenging forensic tasks, is common in disaster victim identification and genetic analysis of historical materials. Massively parallel sequencing (MPS) is a useful technique for STR analysis that enables the sequencing of smaller amplicons compared with conventional capillary electrophoresis (CE), which is valuable for the analysis of degraded DNA. In this study, 92 samples of human skeletal remains (70+ years postmortem) were tested using an in-house MPS-STR system designed for the analysis of degraded DNA. Multiple intrinsic factors of DNA from skeletal remains that affect STR typing were assessed. The recovery of STR alleles was influenced more by DNA input amount for amplification rather than DNA degradation, which may be attributed from the high quantity and quality of libraries prepared for MPS run. In addition, the higher success rate of STR typing was achieved using the MPS-STR system compared with a commercial CE-STR system by providing smaller sized fragments for amplification. The results can provide constructive information for the analysis of degraded sample, and this MPS-STR system will contribute in forensic application with regard to skeletal remain sample investigation.     

Short tandem repeat analysis in Japanese population

Short tandem repeats (STRs), known as microsatellites, are one of the most informative genetic markers for characterizing biological materials. Because of the relatively small size of STR alleles (generally 100-350 nucleotides), amplification by polymerase chain reaction (PCR) is relatively easy, affording a high sensitivity of detection. In addition, STR loci can be amplified simultaneously in a multiplex PCR. Thus, substantial information can be obtained in a single analysis with the benefits of using less template DNA, reducing labor, and reducing the contamination. We investigated 14 STR loci in a Japanese population living in Sendai by three multiplex PCR kits, GenePrint PowerPlex 1.1 and 2.2. Fluorescent STR System (Promega, Madison, WI, USA) and AmpF/STR Profiler (Perkin-Elmer, Norwalk, CT, USA). Genomic DNA was extracted using sodium dodecyl sulfate (SDS) proteinase K or Chelex 100 treatment followed by the phenol/chloroform extraction. PCR was performed according to the manufacturer's protocols. Electrophoresis was carried out on an ABI 377 sequencer and the alleles were determined by GeneScan 2.0.2 software (Perkin-Elmer). In 14 STRs loci, statistical parameters indicated a relatively high rate, and no significant deviation from Hardy-Weinberg equilibrium was detected. We apply this STR system to paternity testing and forensic casework, e.g., personal identification in rape cases. This system is an effective tool in the forensic sciences to obtain information on individual identification.     

High-speed analysis of multiplexed short tandem repeats with an electrophoretic microdevice

We report the development of a robust and effective method for multiplexed short tandem repeat (STR) analysis within a chip-based microdevice. The method uses a laser-induced fluorescence detection system and simultaneously detects three- and four-color multiplexed polymerase chain reaction (PCR) samples. Analyses of the eight combined DNA index system (CODIS) STR loci were performed in 20 min with single-base-pair resolution ranging from 0.75 to 1. A simultaneous analysis of fifteen loci-ladders and a gender marker Amelogenin based on the PowerPlexTM 16 System was achieved in less than 35 min. The system is capable of repetitive operation and may be extended to high-throughput multilane devices that could be readily interfaced to an automated sample loading system.     

Platinum nanoparticle-facilitated reflective surfaces for non-contact temperature control in microfluidic devices for PCR amplification

The polymerase chain reaction (PCR) is critical for amplification of target sequences of DNA or RNA that have clinical, biological or forensic relevance. While extrinsic Fabry-Perot interferometry (EFPI) has been shown to be adequate for non-contact temperature sensing, the difficulty in defining a reflective surface that is semi-reflective, non-reactive for PCR compatibility and adherent for thermal bonding has limited its exploitation. Through the incorporation of a reflective surface fabricated using a thermally driven self-assembly of a platinum nanoparticle monolayer on the surface of the microfluidic chamber, an enhanced EFPI signal results, allowing for non-contact microfluidic temperature control instrumentation that uses infrared-mediated heating, convective forced-air cooling, and interferometic temperature sensing. The interferometer is originally calibrated with a miniature copper-constantan thermocouple in the PCR chamber resulting in temperature sensitivities of -22.0 to -32.8 nm·°C(-1), depending on the chamber depth. This universal calibration enables accurate temperature control in any device with arbitrary dimensions, thereby allowing versatility in various applications. Uniquely, this non-contact temperature control for PCR thermocycling is applied to the amplification of STR loci for human genetic profiling, where nine STR loci are successfully amplified for human identification using the EFPI-based non-contact thermocycling.     

Improved STR typing of telogen hair root and hair shaft DNA

Today the STR typing of telogen hair and hair shafts is regarded as a challenge. The small DNA quantity in the hair is highly degraded. Another problem are PCR inhibitors in the hair. In particular hair pigments, the melanins, are known to inhibit PCR. Hairs are exposed to sunlight and partly to chemical oxidation processes, which make them even more difficult to analyze. To increase the chances of a correct typing of hair, the small amount of DNA must be successfully isolated and the inhibitors have to be removed or neutralized. Furthermore, miniSTR typing improves the analysis of stains with degraded DNA like it is the case with hair. We introduce a nonorganic extraction method and in addition a miniSTR concept which is promising in typing stains with little and degraded DNA, especially hairs. The miniSTR concept including five database STRs (SE33, VWA, TH01, FGA, D3S1358) and the gender typing system Amelogenin was optimized for the amplification of hair DNA. Compared to commercial STR kits, this approach resulted in considerably higher success rates.     

Development of a novel forensic STR multiplex for ancestry analysis and extended identity testing

There is growing interest in developing additional DNA typing techniques to provide better investigative leads in forensic analysis. These include inference of genetic ancestry and prediction of common physical characteristics of DNA donors. To date, forensic ancestry analysis has centered on population‐divergent SNPs but these binary loci cannot reliably detect DNA mixtures, common in forensic samples. Furthermore, STR genotypes, forming the principal DNA profiling system, are not routinely combined with forensic SNPs to strengthen frequency data available for ancestry inference. We report development of a 12‐STR multiplex composed of ancestry informative marker STRs (AIM‐STRs) selected from 434 tetranucleotide repeat loci. We adapted our online Bayesian classifier for AIM‐SNPs: Snipper, to handle multiallele STR data using frequency‐based training sets. We assessed the ability of the 12‐plex AIM‐STRs to differentiate CEPH Human Genome Diversity Panel populations, plus their informativeness combined with established forensic STRs and AIM‐SNPs. We found combining STRs and SNPs improves the success rate of ancestry assignments while providing a reliable mixture detection system lacking from SNP analysis alone. As the 12 STRs generally show a broad range of alleles in all populations, they provide highly informative supplementary STRs for extended relationship testing and identification of missing persons with incomplete reference pedigrees. Lastly, mixed marker approaches (combining STRs with binary loci) for simple ancestry inference tests beyond forensic analysis bring advantages and we discuss the genotyping options available.     

Construction of two fluorescence‐labeled non‐combined DNA index system miniSTR multiplex systems to analyze degraded DNA samples in the Chinese Han Population

MiniSTR loci have been demonstrated to be an effective approach in recovering genetic information from degraded specimens, because of the reduced PCR amplicon sizes which improved the PCR efficiency. Eight non‐combined DNA index system miniSTR loci suitable for the Chinese Han Population were analyzed in 300 unrelated Chinese Han individuals using two novel five fluorescence‐labeled miniSTR multiplex systems(multiplex I: D10S1248, D2S441, D1S1677 and D9S2157; multiplex II: D9S1122, D10S1435, D12ATA63, D2S1776 and Amelogenin). The allele frequency distribution and forensic parameters in the Chinese Han Population were reported in this article. The Exact Test demonstrated that all loci surveyed here were found to be no deviation from Hardy–Weinberg equilibrium. The accumulated power of discrimination and power of exclusion for the eight loci were 0.999999992 and 0.98, respectively. The highly degraded DNA from artificially degraded samples and the degraded forensic case work samples was assessed with the two miniSTR multiplex systems, and the results showed that the systems were quite effective.     

An automated instrument for human STR identification: design, characterization, and experimental validation

The microfluidic integration of an entire DNA analysis workflow on a fully integrated miniaturized instrument is reported using lab‐on‐a‐chip automation to perform DNA fingerprinting compatible with CODIS standard relevant to the forensic community. The instrument aims to improve the cost, duration, and ease of use to perform a “sample‐to‐profile” analysis with no need for human intervention. The present publication describes the operation of the three major components of the system: the electronic control components, the microfluidic cartridge and CE microchip, and the optical excitation/detection module. Experimental details are given to characterize the level of performance, stability, reliability, accuracy, and sensitivity of the prototype system. A typical temperature profile from a PCR amplification process and an electropherogram of a commercial size standard (GeneScan 500?, Applied Biosystems) separation are shown to assess the relevance of the instrument to forensic applications. Finally, we present a profile from an automated integrated run where lysed cells from a buccal swab were introduced in the system and no further human intervention was required to complete the analysis.     

Evaluation of microfluidics-based droplet PCR combined with multiplex STR system in forensic science

Because of its excellent monodispersity, high throughput, and low volume, microfluidics-based droplet PCR has become the core technology of digital PCR, next-generation sequencing, and other technology platforms. This study constructed a microfluidic water-in-oil droplet PCR system and amplified a commercially available forensic 22-plex short tandem repeat detection system. We analyzed the sensitivity, concordance, amplification efficiency of the droplet PCR, and influence factors of the above aspects. The droplet PCR showed high concordance with conventional bulk PCR and had high sensitivity as 0.125 ng. Furthermore, we observed the performance of droplet PCR in high-order mixed DNA. As the mixture ratios from 10:1 to 30:1, droplet PCR presented more mixture proportion (Mx) increased loci from 11 (57.89%) to 17 (89.47%). In the mixture ratios 20:1, 25:1, and 30:1, significant Mx differences between droplet PCR and bulk PCR were observed (p < 0.05). The results showed that the droplet PCR could improve the identification of the minor contributor's DNA in a two-person mixture and alleviate the imbalanced amplification problem. This study provides a reference and basis for the wide application of droplet PCR in forensic science.     

Microchip free-flow electrophoresis on glass substrate using laser-printing toner as structural material

In this work, a microfluidic free-flow electrophoresis device, obtained by thermal toner transferring on glass substrate, is presented. A microdevice can be manufactured in only 1 h. The layout of the microdevice was designed in order to improve the fluidic and electrical characteristics. The separation channel is 8 microm deep and presents an internal volume of 1.42 microL. The deleterious electrolysis effects were overcome by using a system that isolates the electrolysis products from the separation channel. The Joule heating dissipation in the separation channel was found to be very efficient up to a current density of 8.83 mA/mm(2) that corresponds to a power dissipation per unit volume of running electrolyte of 172 mW/microL. Promising results were obtained in the evaluation of the microdevices for the separation of ionic dyes. The microfluidic device can be used for a continuous sample pretreatment step for micro total analysis system.     

A microfluidic device integrated with multichamber polymerase chain reaction and multichannel separation for genetic analysis

This work describes a microfluidic device integrated with multichamber polymerase chain reaction (PCR) and multichannel separation for parallel genetic analysis. The microdevice consists of three functional units: temperature control, multiple PCR (four chambers PCR), and multiple channel separation (four separation channels, each channel connected to a PCR chamber). Platinum (Pt)/titanium (Ti) microheater was used to ensure homogeneous temperature field, and Pt-chip sensor was used for temperature monitoring. The interface between chip-PCR and chip separation was simplified by connecting the PCR chamber with separation channel directly. After chip-PCR, PCR products were introduced into parallel separation channels for subsequent separation/detection by applying an electric field automatically. This microdevice was successfully applied for detection of pathogens including hepatitis B virus (HBV) and Mycobacterium tuberculosis (MTB), and genotyping of human leucocyte antigen (HLA)-B27 as well, demonstrating the feasibility of the integrated microdevice for parallel genetic analysis.     

Suspension bead array branch migration displacement assay for rapid STR analysis

STR analysis is commonly used in forensic and genetic studies. STRs are currently discriminated based on size, primarily by gel- and column-based approaches. Hybridization-based approaches have the potential to allow high-throughput analysis of STRs; however, development of such approaches has been limited by the difficulty in discriminating between STRs of similar length. We have recently described several innovations to enable STR analysis using an array-based hybridization approach for high- throughput STR analysis. Here we extend that approach by incorporating the array into microspheres and adding a discriminatory branch migration displacement step. This microsphere-based platform uses Luminex xMAP technology and improves the sensitivity, selectivity, and speed of the assay. We demonstrate the feasibility, speed, and reliability of the assay for STR detection by correctly analyzing two STR loci in 20 forensic DNA samples of known STR type. The multiplex, bead-based approach provides a high-throughput and more portable STR analysis.     

An integrated passive micromixer-magnetic separation-capillary electrophoresis microdevice for rapid and multiplex pathogen detection at the single-cell level

Here we report an integrated microdevice consisting of an efficient passive mixer, a magnetic separation chamber, and a capillary electrophoretic microchannel in which DNA barcode assay, target pathogen separation, and barcode DNA capillary electrophoretic analysis were performed sequentially within 30 min for multiplex pathogen detection at the single-cell level. The intestine-shaped serpentine 3D micromixer provides a high mixing rate to generate magnetic particle-pathogenic bacteria-DNA barcode labelled AuNP complexes quantitatively. After magnetic separation and purification of those complexes, the barcode DNA strands were released and analyzed by the microfluidic capillary electrophoresis within 5 min. The size of the barcode DNA strand was controlled depending on the target bacteria (Staphylococcus aureus, Escherichia coli O157:H7, and Salmonella typhimurium), and the different elution time of the barcode DNA peak in the electropherogram allows us to recognize the target pathogen with ease in the monoplex as well as in the multiplex analysis. In addition, the quantity of the DNA barcode strand (～10(4)) per AuNP is enough to be observed in the laser-induced confocal fluorescence detector, thereby making single-cell analysis possible. This novel integrated microdevice enables us to perform rapid, sensitive, and multiplex pathogen detection with sample-in-answer-out capability to be applied for biosafety testing, environmental screening, and clinical trials.     

Developmental validation of the Microreader™ 20A ID system

The Microreader? 20A ID system is designed for forensic applications such as personal identification, parentage testing, and research. It includes 13 combined DNA index system (CODIS) short tandem repeat (STR) loci (CSF1PO, FGA, TH01, TPOX, vWA, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, and D21S11), three expanded CODIS STR loci (D12S391, D19S433, and D2S1338), three non‐CODIS STR loci (D6S1043, Penta D, and Penta E), and the amelogenin locus in one reaction with a six‐dye fluorescent (FAM, HEX, TAMAR, ROX, PUR, and QD550) analysis system. In this study, the Microreader? 20A ID system was validated according to the Scientific Working Group on DNA Analysis Methods validation guidelines for forensic DNA Analysis methods and Chinese national standard, including PCR‐based studies, sensitivity study, precision, and accuracy evaluation, stutter calculation, inhibitor tests, species specificity, and DNA mixture studies. Our results suggest that the Microreader? 20A ID system is a useful tool for personal identification and parentage testing.