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.     

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.     

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.     

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.     

Design and validation of an STR hexaplex assay for DNA profiling of grapevine cultivars

Although the analysis of length polymorphism at STR loci has become a method of choice for grape cultivar identification, the standardization of methods for this purpose lags behind that of methods for DNA profiling in human and animal forensic genetics. The aim of this study was thus to design and validate a grapevine STR protocol with a practically useful level of multiplexing. Using free bioinformatics tools, published primer sequences, and nucleotide databases, we constructed and optimized a primer set for the simultaneous analysis of six STR loci (VVIi51, scu08vv, scu05vv, VVMD17, VrZAG47, and VrZAG83) by multiplex PCR and CE with laser‐induced fluorescence, and tested it on 90 grape cultivars. The new protocol requires subnanogram quantities of the DNA template and enables automated, high‐throughput genetic analysis with reasonable discriminatory power. As such, it represents a step toward further standardization of grape DNA profiling.     

Exploring STR signal in the single‐ and multicopy number regimes: Deductions from an in silico model of the entire DNA laboratory process

Short tandem repeat (STR) profiling from DNA samples has long been the bedrock of human identification. The laboratory process is composed of multiple procedures that include quantification, sample dilution, PCR, electrophoresis, and fragment analysis. The end product is a short tandem repeat electropherogram comprised of signal from allele, artifacts, and instrument noise. In order to optimize or alter laboratory protocols, a large number of validation samples must be created at significant expense. As a tool to support that process and to enable the exploration of complex scenarios without costly sample creation, a mechanistic stochastic model that incorporates each of the aforementioned processing features is described herein. The model allows rapid in silico simulation of electropherograms from multicontributor samples and enables detailed investigations of involved scenarios. An implementation of the model that is parameterized by extensive laboratory data is publically available. To illustrate its utility, the model was employed in order to evaluate the effects of sample dilutions, injection time, and cycle number on peak height, and the nature of stutter ratios at low template. We verify the model's findings by comparison with experimentally generated data.     

CE analysis and molecular characterisation of depurinated DNA samples

A DNA sample was partially degraded by scalar heat-acid treatments to study the extent of apurinic-apyrimidinic (A-P) lesions produced along the molecule. A CE-UV method allowed us to measure the rate of depurination at pH 5.0 and 70°C which was calculated to be 5.41×10(-6) s(-1) for adenine and 6.27×10(-6) s(-1) for guanine. CE identified depurination on treated samples when it occurred with a loss of >4% of the basic moieties. The molecular features of the A-P enriched samples were investigated by using molecular assays (agarose gel electrophoresis, UV spectrophotometry and quantitative PCR) and the consistency of the results of the STR typing were compared with the degree of depurination of the PCR template. The treated DNA samples showed molecular features such as fragmentation, altered OD(260) /OD(280) ratios and decreased ability of the quantitative PCR to synthesise the human target, related to the severity of depurination. A satisfactory correlation between the degree of damage and the amount of residual PCR-sensitive target sequences was also demonstrated (r(2) =0.9717). The conventional and mini-STR typing of the samples showed that the genetic outcome was influenced by a depurination damage that exceeded 4% when locus drop-outs and artefactual PCR results were evident. As the success of STR typing depends on the integrity of the DNA recovered from the samples, the CE-UV, physical and molecular assays described here are proposed as a set of useful methods in the analysis of certain forensic and clinical samples, for a critical evaluation of the outcome of the genetic testing.     

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.     

Pyrosequencing‐based strategy for a successful SNP detection in two hypervariable regions: HV‐I/HV‐II of the human mitochondrial displacement loop

Forensic analysis of mitochondrial displacement loop (D‐loop) sequences using Sanger sequencing or SNP detection by minisequencing is well established. Pyrosequencing has become an important alternative because it enables high‐throughput analysis and the quantification of individual mitochondrial DNAs (mtDNAs) in samples originating from more than one individual. DNA typing of the mitochondrial D‐loop region is usually the method of choice if STR analysis fails because of trace amounts of DNA and/or extensive degradation. The main aim of the present work was to optimize the efficiency of pyrosequencing. To do this, 31 SNPs within the hypervariable regions I and II of the D‐loop of human mtDNA were simultaneously analyzed. As a novel approach, we applied two sets of amplification primers for the multiplexing assay. These went in combination with four sequencing primers for pyrosequencing. This method was compared with conventional sequencing of mtDNA from blood and biological trace materials.     

Forensic DNA typing by capillary electrophoresis using the ABI Prism 310 and 3100 genetic analyzers for STR analysis

DNA typing with short tandem repeat (STR) markers is now widely used for a variety of applications including human identification. Capillary electrophoresis (CE) instruments, such as the ABI Prism 310 and ABI 3100 Genetic Analyzers, are the method of choice for many laboratories performing STR analysis. This review discusses issues surrounding sample preparation, injection, separation, detection, and interpretation of STR results using CE systems. Requirements for accurate typing of STR alleles are considered in the context of what future analysis platforms will need to increase sample throughput and ease of use.     

Highly sensitive polymerase chain reaction-free quantum dot-based quantification of forensic genomic DNA

Forensic DNA samples can degrade easily due to exposure to light and moisture at the crime scene. In addition, the amount of DNA acquired at a criminal site is inherently limited. This limited amount of human DNA has to be quantified accurately after the process of DNA extraction. The accurately quantified extracted genomic DNA is then used as a DNA template in polymerase chain reaction (PCR) amplification for short tandem repeat (STR) human identification. Accordingly, highly sensitive and human-specific quantification of forensic DNA samples is an essential issue in forensic study. In this work, a quantum dot (Qdot)-labeled Alu sequence was developed as a probe to simultaneously satisfy both the high sensitivity and human genome selectivity for quantification of forensic DNA samples. This probe provided PCR-free determination of human genomic DNA and had a 2.5-femtogram detection limit due to the strong emission and photostability of the Qdot. The Qdot-labeled Alu sequence has been used successfully to assess 18 different forensic DNA samples for STR human identification.     

Ancient DNA profiling by megaplex amplications

Simultaneous amplification of nine human short tandem repeat (STR) DNA sequences and the amelogenin locus allows reducing to an absolute minimum the amount of sample material that is necessary for genetic identification or kinship analysis. Valuable remains can be studied this way without any visible damage, as is demonstrated by typing the DNA of a tooth root from the Saxon warrior Widukind, who died about 1200 years ago. The broad applicability of the megaplex approach is shown by typing bone and teeth specimens ranging from a few months to 3000 years of age employing AmpFlSTR Profiler Plus. Additionally, megaplex STR typing is the method of choice for proving the authenticity of molecular results derived from ancient degraded DNA.     

Cover Picture: Electrophoresis 21'2010

Issue no. 21 is a regular issue with "Emphasis on Nucleic Acids" and comprises 17 contributions distributed over 4 distinct parts. Part I is on nucleic acids and has 8 research articles on various aspects of nucleic acid research including an automated instrument for human STR identification, a high resolution chip‐CE assay for rapid detection of gene mutations and amplifications, a fluorescence‐based sequence‐specific primer PCR, pulsed field CE for RNA separation, methylated DNA immunoprecipitation sequencing, PCR‐SSCP, DNA methylation, and analysis of SNPs. Part II has 4 research articles dealing with studies on protein and proteomics analysis, e.g., microchip IEF, CE analysis of microheterogeneous glutelin subunits in rice, protein precipitation methods and prefractionation of plasma for proteomic analysis. Part III has 2 contributions that describe the separation of the enantiomers of DL‐isocitric acid by CE and the analysis of trace degradation products of S‐adenosylmethionine, which are decarboxylated diastereomers, by CE. The last 3 articles in this issue (Part IV) are on novel methodologies for facile interfacing of CE with MS, electrokinetic chromatography for the determination of contaminants and impurities of heparin samples, and particle electrophoresis. As such, issue no. 21 is packed with the latest developments and innovations in the field. Featured articles include: An automated instrument for Human STR identification: design, characterization, and experimental validation (doi: 10.1002/elps.201000305 )) Miniature flowing atmospheric‐pressure afterglow ion source for facile interfacing of capillary electrophoresis with mass spectrometry (doi: 10.1002/elps.201000350 )) Capillary electrophoresis for analysis of microheterogeneous glutelin subunits in rice (Oryza sativa L.). (doi: 10.1002/elps.201000333 ))     

Quantification of phosphorus in DNA using capillary electrophoresis hyphenated with inductively coupled plasma mass spectrometry

We have analyzed phosphorus in an enzymatically digested DNA molecule using capillary electrophoresis (CE) hyphenated with inductively coupled plasma mass spectrometry (ICP-MS). The DNA concentration was quantified by the phosphorus value obtained in the CE-ICP-MS analysis. The CE-ICP-MS measurement, for which the interface device AIF-01 equipped three layered nebulizer was adopted, was achieved with limited μL/min nebulizing without loss of sample in the vaporizing chamber. The samples of nucleotides and free phosphate were separated well in the CE-ICP-MS measurement, and the calibration curve (0.1-10μg/mL) of the phosphorus showed a linear (R(2)=0.999) increase in intensity. After digestion of the 100-bp double-strand DNA sample to deoxyribonucleotide-5'-monophosphates (dNMPs) by phosphodiesterase-I, phosphorus was detected by CE-ICP-MS without further purification steps. In this study, we applied two calculation schemes of DNA analysis using a dNMP concentration obtained from CE-ICP-MS. Comparative CE-ICP-MS analysis with DNA digested to dNMPs showed that the assay gave an equal value obtained from the total DNA quantification using fluorescence detection. The detection limits of the DNA sample obtained from these species and phosphorus in nucleotides using CE-ICP-MS were 3.1-26ng/mL. These LOD values were equal to the conventional fluorescence determination of DNA.     

A framework for the development of STR genotyping in domestic animal species: Characterization and population study of 12 canine X‐chromosome loci

This study reports the methodology used to search, select and characterize STR loci on the canine X chromosome using publicly available genome resources and following the current guidelines for human and non‐human forensic testing. After several rounds of selection, 12 X‐STR markers were optimized for simultaneous co‐amplification in a single PCR, and genetic profiles were determined in a sample of 103 unrelated dogs. Mendelian inheritance was verified and mutation rates were assessed using family groups. Alleles that varied in size were sequenced to create a standardized nomenclature proposal based on the number of repeats. All loci conformed to Hardy–Weinberg expectations. The resulting panel showed high forensic efficiency, presenting high values of power of discrimination (in males and females) and mean exclusion chance, both in trios involving female offspring and in duos composed of dam and male offspring. Its use may complement the information obtained by autosomal STR analysis and contribute to the resolution of complex cases of kinship in dogs. The presented methodology for the de novo construction of an STR multiplex may also provide a helpful framework for analogous work in other animal species. As an increasing number of reference genomes become available, convenient tools for individual identification and parentage testing based on STR loci selected from autosomes or sex chromosomes' sequences may be created following this strategy.     

Quantification of DNA in forensic samples

Quantification of DNA in a forensic sample is of major importance for proper DNA amplification and STR profiling. Several methods have been developed to quantify DNA, from basic UV spectrometry, through gel-based techniques, to dye staining, blotting techniques, and, very recently, DNA amplification methods (polymerase chain reaction, PCR). Early techniques simply measured total DNA, but newer techniques can specifically measure human DNA while excluding non-human DNA (foodstuff, animal, or bacterial contamination). These newer assays can be faster and less expensive than traditional methods, making them ideal for the busy forensic laboratory. This paper reviews classic and newer quantification techniques and presents methods recently developed by the authors on the basis of PCR of Alu sequences.     

Optimization of DNA extraction from dental remains

Efficient DNA extraction procedures is a critical step involved in the process of successful DNA analysis of such samples. Various protocols have been devised for the genomic DNA extraction from human tissues and forensic stains, such as dental tissue that is the skeletal part that better preserves DNA over time. However DNA recovery is low and protocols require labor‐intensive and time‐consuming step prior to isolating genetic material. Herein, we describe an extremely fast procedure of DNA extraction from teeth compared to classical method. Sixteen teeth of 100‐year‐old human remains were divided into two groups of 8 teeth and we compared DNA yield, in term of quantity and quality, starting from two different sample preparation steps. Specifically, teeth of group 1 were treated with a classic technique based on several steps of pulverization and decalcification, while teeth of group 2 were processed following a new procedure to withdraw dental pulp. In the next phase, the samples of both group underwent the same procedure of extraction, quantification and DNA profile analysis. Our findings provide an alternative protocol to obtain a higher amount of good quality DNA in a fast time procedure, helpful for forensic and anthropological studies.     

Interaction of humic acids with human DNA: proposed mechanisms and kinetics

Human DNA quantification by quantitative real-time PCR (QRT-PCR) has gained great importance in forensic DNA and ancient DNA studies. However, in such samples, DNA quantification is impaired by the frequently present humic acid (HA). We have previously shown that the addition of synthetic HA inhibits QRT-PCR. In this study we investigated the possible mechanisms of HA interaction with human DNA, and kinetics of QRT-PCR inhibition. In QRT-PCR with pure human DNA and no HA added, VMAX was 40. With DNA sample containing 4 microg/mL of HA, VMAX was 30.30 while the addition of extra Taq polymerase to the same sample changed VMAX into 38.91, amplifying between 80 and 90% of input DNA. The KM/VMAX ratio in all the samples remained constant, indicating that the mechanism of HA inhibition of QRT-PCR is uncompetitive by nature. Moreover, HA shifts the human DNA melting temperature point (Tm) from 75 to 87 degrees C and inhibits DNase I-mediated DNA cleavage, most probably affecting the enzyme's activity.     

A new autosomal STR nineplex for canine identification and parentage testing

A single multiplex PCR assay capable of simultaneously amplifying nine canine‐specific autosomal STR markers (FH3210, FH3241, FH2004, FH2658, FH4012, REN214L11, FH2010, FH2361 and the newly described C38) was developed for individual identification and parentage testing in domestic dogs. In order to increase genotyping efficiency, amplicon sizes were optimized for a 90–350 bp range, with fluorescently labelled primers for use in Applied Biosystems, Inc., platforms. The performance of this new multiplex system was tested in 113 individuals from a case‐study population and 12 random dogs from mixed‐breed origin. Co‐dominant inheritance of STR alleles was investigated in 101 father, mother and son trios. Expected heterozygosity values vary between 0.5648 for REN214L11 and 0.9050 for C38. The high level of genetic diversity observed for most markers provides this multiplex with a very high discriminating power (matching probability=1.63/10¹⁰ and matching probability among siblings=4.9/10³). Allele sequences and a proposal for standardized nomenclature are also herein presented, aiming at implementing the use of this system in forensic DNA typing and population genetic studies. This approach resulted in an optimized and well‐characterized canine DNA genotyping system that is highly performing and straightforward to integrate and employ routinely. Although this STR multiplex was developed for use and tested in a case‐study population, the Portuguese breed Cão de Gado Transmontano, it proved to be useful for general identification purposes or parentage testing.     

Inorganic oxidizer detection from propellants,pyrotechnics, and homemade explosive powders using gradient elution moving boundary electrophoresis

Advancement in rapid targeted chemical analysis of homemade and improvised explosive devices is critical for the identification of explosives-based hazards and threats. Gradient elution moving boundary electrophoresis (GEMBE), a robust electrokinetic separation technique, was employed for the separation and detection of common inorganic oxidizers from frequently encountered fuel-oxidizer mixtures. The GEMBE system incorporated sample and run buffer reservoirs, a short capillary (5 cm), an applied electric field, and a pressure-driven counterflow. GEMBE provided a separation format that allowed for continuous injection of sample, selectivity of analytes, and no sample cleanup or filtration prior to analysis. Nitrate, chlorate, and perchlorate oxidizers were successfully detected from low explosive propellants (e.g., black powders and black powder substitutes), pyrotechnics (e.g., flash powder), and tertiary explosive mixtures (e.g., ammonium nitrate- and potassium chlorate-based fuel-oxidizer mixtures). Separation of these mixtures exhibited detection without interference from a plethora of additional organic and inorganic fuels, enabled single particle analysis, and demonstrated semiquantitative capabilities. The bulk counterflow successfully excluded difficult components from fouling the capillary, yielding estimated limits of detection down to approximately 10 μmol/L. Finally, nitrate was separated and detected from postblast debris collected and directly analyzed from two nitrate-based charges.