A modular microfluidic system for deoxyribonucleic acid identification by short tandem repeat analysis

Microfluidic technology has been utilized in the development of a modular system for DNA identification through STR (short tandem repeat) analysis, reducing the total analysis time from the ∼6 h required with conventional approaches to less than 3 h. Results demonstrate the utilization of microfluidic devices for the purification, amplification, separation and detection of 9 loci associated with a commercially-available miniSTR amplification kit commonly used in the forensic community. First, DNA from buccal swabs purified in a microdevice was proven amplifiable for the 9 miniSTR loci via infrared (IR)-mediated PCR (polymerase chain reaction) on a microdevice. Microchip electrophoresis (ME) was then demonstrated as an effective method for the separation and detection of the chip-purified and chip-amplified DNA with results equivalent to those obtained using conventional separation methods on an ABI 310 Genetic Analyzer. The 3-chip system presented here demonstrates development of a modular, microfluidic system for STR analysis, allowing for user-discretion as to how to proceed after each process during the analysis of forensic casework samples.     

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.     

"Paterniplex", a highly discriminative decaplex STR multiplex tailored for investigating special problems in paternity testing

The goal of the study was to develop a STR multiplex ("Paterniplex") that is--as supplement to commercially available multiplex kits like the Identifiler kit (Applied Biosystems, Foster City, CA)--suitable for solving complex paternity cases such as deficiency cases or cases with mutations. The Paterniplex comprises the nine highly polymorphic STRs D8S1132, D7S1517, D10S2325, D12S391, Se33, D17S976, Penta E, Penta D and FGA in addition to Amelogenin as sex determination marker. The loci were selected because of their high degree of polymorphism (higher than that of the widely used TH01 marker). Only one locus, FGA, is shared with the Identifiler kit to avoid sample mix up. The study further gives details on the population genetics of the loci in a German Caucasian population (allelic distribution, Hardy-Weinberg Equilibrium and forensic efficiency markers such as the Discriminating Power) and three examples for cases that could not be solved using commercially available kits alone, but using the Paterniplex in addition to a commercial kit.     

A convenient guideline to determine if two Y‐STR profiles are from the same lineage

Y chromosome STR loci are used in forensics primarily for identification purposes by determining the male lineages. The Henan province in China has established a large Y‐STR (>200 000 profiles) database for criminal investigations. A large proportion of the Y‐STR profiles in the database were generated using either the Applied Biosystems Yfiler^? or Yfiler^? Plus PCR Amplification kits. The additional loci in the Yfiler Plus kit as compared to the Yfiler kit results in a concomitant cumulative mutation rate increase across the loci. Therefore, in those cases when two profiles have one to a few mismatched loci, it is difficult to determine if they are from the same lineage. In this study, 7405 unrelated male profiles were manually selected from the database. Analysis showed higher power of discrimination than the corresponding Yfiler haplotypes. Further, the distributions of the number of mismatched loci and the mismatched steps were generated for father‐son, grandfather‐grandson, uncle‐nephew, and cousins (i.e. one, two, three, and four meioses, respectively) by exhaustive pairwise comparison of the unrelated profiles using a dynamic programming approach. The same distributions were generated for unrelated pairs with mutation rates of the loci. With the distributions, the false negative and false positive rates were determined. Two Yfiler profiles with ≤2 mismatched loci or ≤2 steps are more likely from the same lineage than unrelated lineages, and two Yfiler Plus profiles with ≤4 mismatched loci or ≤5 mismatched steps are more likely from the same lineage.     

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.     

A powerful,novel, multiplex typing system for six short tandem repeat loci and the allele frequency distributions in two Japanese regional populations

A new multiplex system for six tetranucleotide short tandem repeat (STR) loci was devised based on multicolor dye technology. Six loci (D20S480, D6S2439, D6S1056, D9S1118, D4S2639, and D17S1290), each with high discriminating power (each unbiased estimates of expected heterozygosity, Exp. Hz, > 0.80 in a preliminary test), were selected from more than 100 tetranucleotide STRs included in a commercially available primer set. These loci were also selected so as not to link with general STRs in commercially released kits including the combined DNA index system (CODIS) 13 core STRs. The primers were newly designed in the present study, in which each amplicon size had a range of less than 200 base pairs (bp), in order to genotype from highly degraded template DNA. Using this system, we genotyped 270 Honshu (mainland)-Japanese and 187 Okinawa-Japanese. From these allele frequencies, we performed three tests, a homozygosity test, a likelihood ratio test and an exact test for Hardy-Weinberg equilibrium (HWE), and no significant deviations (p < 0.05) from HWE were observed. We also compared the allele distributions at six STRs between both populations, and they were significantly different (p < 0.05) at three loci (D6S2439, D9S1118 and D4S2639). Furthermore, the Exp. Hz and the power of discrimination (PD) at all loci in the Honshu-Japanese population were higher than 0.80 and 0.93, respectively. These statistical values for discriminating power in the Honshu-Japanese were almost the same as in the Okinawa-Japanese. This novel, multiplex polymerase chain reaction (PCR) amplification and typing system for six STR loci thus promises to be a convenient and informative new DNA profiling system in the forensic field.     

Fluorescence energy transfer-labeled primers for high-performance forensic DNA profiling

A fluorescence energy transfer (ET) dye-labeled STR typing system (ET 16-plex) is developed for the markers used in the commercial STR typing kit PowerPlex 16, and its performance assessed using a 96-lane microfabricated capillary array electrophoresis (muCAE) system. The ET 16-plex amplicons displayed 1.6-9-fold higher fluorescence intensities compared to those produced using the single-dye (SD)-labeled multiplex kits. The ET multiplex delivered full STR profiles from 62.5 pg of DNA; half the input required for the SD kits while maintaining a similar heterozygote allele balance. This increased sensitivity should improve typing of poor-quality DNA samples by making minor or imbalanced alleles more readily detectable at the low copy number (LCN) threshold. The ET 16-plex also generated complete profiles with only 28 PCR cycles; this capability should improve LCN typing by reducing the amplification time and drop-in allele incidence. To confirm the practical advantages of ET-labeled primers, six previously problematic casework samples were tested and only the ET 16-plex kit was able to capture additional allele data. The successful development and demonstration of ET primers for higher sensitivity STR typing offers a simple solution to improving current commercial multiplex typing capability. The superior spectral properties and universal compatibility with any primer sequence provided by ET cassettes will make future multiplex construction more facile and straightforward. The pairing of ET cassette technology with the muCAE system illustrates not only an enhanced STR typing platform, but a significant step toward a higher-efficiency forensic laboratory enabled by better chemistry and microfluidics.     

Genetic profile characterization and population study of 21 autosomal STR in Chinese Kazak ethnic minority group

Short tandem repeat loci have been recognized as useful tools in the routine forensic application and in recent decades, more and more new short tandem repeat (STR) loci have been constantly discovered, studied, and applied in forensic caseworks. In this study, we investigated the genetic polymorphisms of 21 STR loci in the Kazak ethnic minority as well as the genetic relationships between the Kazak ethnic minority and other populations. Allelic frequencies of 21 STR loci were obtained from 114 unrelated healthy Kazak individuals in the Ili Kazak Autonomous Prefecture, Xinjiang Uigur Autonomous Region of China. We observed a total of 159 alleles in the group with the allelic diversity values ranging from 0.0044 to 0.5088. The highest polymorphism was found at D19S433 locus and the lowest was found at D1S1627. Statistical analysis of the generated data indicated no deviation from Hardy–Weinberg equilibriums at all 21 STR loci. In order to estimate the population differentiation, allelic frequencies of all STR loci of the Kazak were compared with those of other neighboring populations using analysis of molecular variance method. Statistically significant differences were found between the studied population and other populations at 2–7 STR loci. A neighbor‐joining tree was constructed based on allelic frequencies of the 21 STR loci and phylogenetic analysis indicates that the Kazak has a close genetic relationship with the Uigur ethnic group. The present results may provide useful information for forensic sciences and population genetics studies, and can also increase our understanding of the genetic background of this group. The present findings showed that all the 21 STR loci are highly genetically polymorphic in the Kazak group, which provided valuable population genetic data for the genetic information study, forensic human individual identification, and paternity tests.     

Who touched the document?: A new overall strategy for collection and identification of DNA from the questioned documents as a supportive evidence

The questions on which judges/prosecutors apply for expertise are mostly about by whom a document was drafted/signed. In this study, a new collective strategy was constructed including a collection method, a modified-silica-based DNA isolation method, and a novel purification method on four contact traces formed on four different paper surface during writing, using PCR with AmpFlSTR®GlobalFiler™ STR kit (after experimental comparison between three different kits) and identification using CE. This collective analysis approach is more sensitive and superior to its equivalents on questioned documents in literature because quantifiable amounts of touch DNA and profiles with high loci percentages (100% on day 1, 72.72% after 1 week) were obtained up to 1 week even after the most challenging conditions of sample forming that a forensic scientist can meet; as washing hands just before drafting and using a very low pressure in a shorter time (simulating a simple contact real conditions while drafting), using no visualizing technique that damages the document. Using the strategy, four most commonly used paper types were compared, to see in which of them DNA could be recovered better. The success of this strategy was shown on the 1-day to 10-year-old real samples from a diary and some archive documents from a law office (including the mix-DNA and different ballpoint pens). Thus, it became possible to show if a person had touched the document, in high success rates up to 1 week as a secondary evidence, when primary evidences are insufficient for the detection of document fraud offenses.     

Evaluation of extraction buffers using the current approach of detecting multiple allergenic and nonallergenic proteins in food

The detection of food allergens has been a challenge because of the increasing need to ensure the absence of undeclared allergens in foods. The current trend in the detection of some food allergens, like peanuts, is based on the detection of multiple allergenic and nonallergenic proteins, and this is the approach that kit manufacturers have adopted. Because commercial kits differ in their ability to detect allergens, regulatory agencies, the food industry, and kit manufacturers are working together to standardize the detection methods. Three kits for the detection of peanuts have been evaluated for performance by the AOAC Research Institute. For this evaluation, a peanut butter suspension was used as a reference material. Several kit components contribute to between-kit analytical variation, even when the same sample is used. One component of commercial kits, which may be contributing to this variability, is the sample extraction buffer. In this study, differences in extractability of 3 allergenic foods were evaluated by using 4 different extraction buffers. The conclusion is that optimum allergen extractability was buffer-dependent, and no single buffer is appropriate for use as a universal extraction solution for all allergenic foods. Therefore, a thorough evaluation of sample preparation buffers needs to be performed for every individual allergenic food. In light of the results obtained, the current approach used for detection of peanut allergens based on the detection of multiple allergenic and nonallergenic proteins is being analyzed.     

17 to 23: A novel complementary mini Y‐STR panel to extend the Y‐STR databases from 17 to 23 markers for forensic purposes

A Y‐STR multiplex system has been developed with the purpose of complementing the widely used 17 Y‐STR haplotyping (AmpFlSTR Y Filer® PCR Amplification kit) routinely employed in forensic and population genetic studies. This new multiplex system includes six additional STR loci (DYS576, DYS481, DYS549, DYS533, DYS570, and DYS643) to reach the 23 Y‐STR of the PowerPlex® Y23 System. In addition, this kit includes the DYS456 and DYS385 loci for traceability purposes. Male samples from 625 individuals from ten worldwide populations were genotyped, including three sample sets from populations previously published with the 17 Y‐STR system to expand their current data. Validation studies demonstrated good performance of the panel set in terms of concordance, sensitivity, and stability in the presence of inhibitors and artificially degraded DNA. The results obtained for haplotype diversity and discrimination capacity with this multiplex system were considerably high, providing further evidences of the suitability of this novel Y‐STR system for forensic purposes. Thus, the use of this multiplex for samples previously genotyped with 17 Y‐STRs will be an efficient and low‐cost alternative to complete the set of 23 Y‐STRs and improve allele databases for population and forensic purposes.     

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.     

Sizing short tandem repeat alleles in capillary array gel electrophoresis instruments.

A 96-capillary array gel electrophoresis Applied Biosystems 3700 instrument has been used to analyse AMPF/STR SGM Plus short tandem repeat (STR) loci for forensic applications. This multiplex consists of ten STR loci plus the Amelogenin locus and currently forms the basis of the UK National DNA database that currently holds more than 1 million profiles. Of particular interest is the accuracy of allele designation that is determined by comparison with standard control allelic ladder markers. Some loci have higher standard deviations than others. In particular the high-molecular-weight HUMFIBRA alleles have high standard deviations of the order of 0.15 and it is these alleles that are most likely to be misdesignated. However, this risk is minimised by the analysis of at least five different allelic ladders across the array to estimate the mean size of each allele. In conjunction with this, a series of guidelines that can be programmed into expert systems are used to minimise risks of misdesignation. The efficacy of the procedures utilised are tested by computer simulation and demonstrated to be robust.     

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.     

Development of a new 25plex STRs typing system for forensic application

We have developed a novel STR 25‐plex florescence multiplex‐STR kit (DNATyper25) to genotype 23 autosomal and two sex‐linked loci for forensic applications and paternity analysis. Of the 23 autosomal loci, 20 are non‐CODIS. The sex‐linked markers include a Y‐STR locus (DYS391) and the Amelogenin gene. We present developmental validation studies to show that the DNATyper25 kit is reproducible, accurate, sensitive, and robust. Sensitivity testing showed that full profiles were achieved with as low as 125 pg of human DNA. Specificity testing demonstrated a lack of cross reactivity with a variety of commonly encountered non‐human DNA contaminants. Stability testing showed that full profiles were obtained with humic acid concentration ≤60 ng/μL and hematin concentration <400 μM. For forensic evaluation, the 23 autosomal STRs followed the Hardy–Weinberg equilibrium. In an analysis of 509 Chinese (CN) Hans, we detected a combined total of 181 alleles at the 23 autosomal STR loci. Since these autosomal STRs are independent from one another, PM was 8.4528 × 10^?22, TDP was 0.999 999 999 999 999 999 999, CEP was 0.999 999 8395. The forensic efficiency parameters demonstrated that these autosomal STRs are highly polymorphic and informative in the Han population of China. We performed population comparisons and showed that the Northern CN Han has a close genetic relationship with the Luzhou Han, Tujia, and Bai populations. We propose that the DNATyper25 kit will be useful for cases where paternity analysis is difficult and for situations where DNA samples are limited in quantity and low in quality.     

Allele frequency of 19 autosomal STR loci in the Bai population from the southwestern region of mainland China

The aim of this study was to investigate a 19 STR loci database using the Bai population from China. This multiplex amplification kit included 13 CODIS STR markers and six plus STR markers (D19S433, Penta E, D2S1338, Penta D, D6S1043, and D12S391) that were successfully analyzed by using 1158 DNA samples from the Bai population from the southwestern part of mainland China. These results indicate that this multiplex amplification kit may provide significant polymorphic information for kinship testing and relationship investigations.     

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.     

Detection of hazelnuts and almonds using commercial ELISA test kits

Three commercial sandwich enzyme-linked immunosorbent assay (ELISA) test kits for the detection of hazelnuts and almonds were evaluated. Limits of detection and dynamic ranges were determined for hazelnuts and almonds spiked into cooked oatmeal, dipping chocolate, and muffins (baked). The limit of detection values varied from 1 to 38 μg/g, depending on the food matrix and ELISA test kit. Percent recoveries based on the standards supplied with the test kits varied from 10% to 170%. It is impossible to ascertain whether the percent recoveries reflect the performance of the ELISAs or differences between the protein content of the nuts used to spike the samples and the test kit standards. Unfortunately, reference materials do not exist that can be used to compare the results from different test kits and standardize the test kit standards. Also, insufficient knowledge regarding the epitope specificity of the antibodies used in the ELISAs further hinders interpretation of the results generated by the different test kits.     

Locus-specific brackets for reliable typing of Y-chromosome short tandem repeat markers

Short tandem repeat (STR) loci, widely used as genetic markers in disease diagnostic studies and human identity applications, are traditionally genotyped through comparison of allele sizes to a sequenced allelic ladder. Allelic ladders permit a floating bin allele calling method to be utilized, which enables reliable allele calling across laboratories, instrument platforms, and electrophoretic conditions. Precise sizing methods for STR allele calling involving fixed bins can also be used when a high degree of precision has been demonstrated within an instrument platform and a set of electrophoretic conditions. An alternative method for reliable genotyping of STR markers, locus-specific brackets (LSBs), is introduced here. LSBs are artificial alleles created through molecular biology manipulations to be shorter or longer than alleles commonly seen in populations under investigation. The size and repeat number of measured alleles are interpolated between the two LSB products that are mixed with the polymerase chain reaction-amplified STR alleles. The advantages and limitations of the LSB approach are described along with a concordance study between the LSB typing approach and other STR typing methods. Complete agreement was observed with 162 samples studied at 5 Y-chromosome loci.     

Grazing-exit and micro X-ray fluorescence analyses for chemical microchips.

Grazing-exit x-ray fluorescence (GE-XRF) and micro x-ray fluorescence (micro-XRF) methods were applied to chemical microchips as a detection method. Since an energy-dispersive x-ray detector was used, the simultaneous detection of multiple elements was possible. An analyzing region was especially designed on the microchip so that a sample solution could be dried and concentrated in a suitable area corresponding to the size of the primary x-ray beam. Finally, it was confirmed that both analytical methods could be combined well for use with a microchip. In GE-XRF, the background intensity in the XRF spectrum was reduced at grazing-exit angles. In addition, a good relationship between the x-ray fluorescence intensities and the concentrations of standard solutions that were introduced into the microchip was obtained. This indicates that the GE-XRF method is feasible for trace elemental analysis in chemical microchip systems. In micro-XRF, an attempt was made to concentrate and dry the analyte within a small analyzing region. The preliminary results indicated that the micro-XRF method could be applied for the analysis of microchips.