Genetic polymorphisms of 20 short tandem repeat loci from the Han population in Henan,China

The aim of this study was to investigate the genetic polymorphism of 20 short tandem repeat (STR) loci including D1S1656, D2S1338, D3S1358, D5S818, D6S1043, D7S820, D8S1179, D12S391, D13S317, D16S539, D18S51, D19S433, D21S11, CSF1PO, FGA, Penta D, Penta E, TH01, TPOX, and vWA in Han population of Henan, China and to assess its value in forensic science. Genomic DNA was extracted from 274 blood samples of unrelated healthy individuals in the Henan Han population. Alleles were amplified with PowerPlex® 21 system kit and PCR products were detected with ABI3130 genetic analyzer (Applied Biosystems) and the data were analyzed with modified PowerStats v1.2. A total of 229 alleles were observed in this Han population and the allelic frequencies ranged from 0.0020 to 0.5090 in the present study. Observed genotype distributions for each locus do not show deviations from Hardy–Weinberg equilibrium expectations (p < 0.05). The combined power of discrimination, combined power of exclusion, and combined matching probability of this 20 STR loci were 0.999999999, 0.999999994603, and 4.0433 × 10^?24, respectively. The 20 STR loci are highly polymorphic in the Han population of Henan, China and they may be of great value in forensic science and human population genetics.     

PCR-free digital minisatellite tandem repeat genotyping

We demonstrated a proof‐of‐concept for novel minisatellite tandem repeat typing, called PCR‐free digital VNTR (variable number tandem repeat) typing, which is composed of three steps: a ligation reaction instead of PCR thermal cycling, magnetic bead‐based solid‐phase capture for purification, and an elongated sample stacking microcapillary electrophoresis (μCE) for sensitive digital coding of repeat number. We designed a 16‐bp fluorescently labeled ligation probe which is complementary to a repeat unit of a biotinylated synthetic template mimicking the human D1S80 VNTR locus and is randomly hybridized with the minisatellite tandem repeats. A quick isothermal ligation reaction was followed to link the adjacent ligation probes on the DNA templates, and then the ligated products were purified by streptavidin‐coated magnetic beads. After a denaturing step, a large amount of ligated products whose size difference was equivalent to the repeat unit were released and recovered. Through the elongated sample stacking μCE separation on a microdevice, the fluorescence signal of the ligated products was generated in the electropherogram and the peak number was directly counted which was exactly matched with the repeat number of VNTR locus. We could successfully identify the minisatellite tandem repeat number with only 5 fmol of DNA template in 30 min.     

Slippage mutation rates in 15 autosomal short tandem repeat loci for forensic purposes in a Southeastern Brazilian population

Well‐defined estimates of mutation rates in highly polymorphic tetranucleotide STR loci are a prerequisite for human identification in genetics laboratory routines useful for civil and criminal investigations. Studying 15 autosomal STR loci of forensic interest (CSF1PO, D2S1338, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D19S433, D21S11, FGA, TH01, TPOX, and vWA), we detected 193 slippage mutations (189 one‐step and four two‐step mutations) in 148 875 parent‐child allelic transfers from 5171 paternity cases with true biological relationship (15 096 individuals; 4754 trios and 417 duos; 9925 meiosis) from the state of São Paulo, a very representative population of Brazil. The overall mutation rate was 1.3 × 10^?3 and the highest rates were observed at loci vWA (2.8 × 10^?3), FGA and D18S51 (2.7 × 10^?3 for both), while loci TH01 and TPOX did not present any mutations. The mean slippage mutation rate of paternal origin (1.8 × 10^?3) was six times higher than that observed for maternal origin (0.3 × 10^?3).     

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.     

AOTF-based multicolor fluorescence detection for short tandem repeat (STR) analysis in an electrophoretic microdevice

An acousto-optic tunable filter (AOTF) has been used to perform multicolor fluorescence detection for four and five-color short tandem repeat (STR) analysis on glass microchips. Matrix files were initially generated by collecting and comparing the laser-induced fluorescence emission of the labels specific to a particular STR kit, and raw data was processed to remove spectral overlap. The AmpFlSTR kits used in this work include Profiler Plus and COfiler, which are four-color kits used in tandem to address the core STR loci, as well as the five-color Identifiler kit, which contains each of the loci. In contrast to previous reports on multicolor detection for STR analysis on microchips, this detection system is characterized by a single filter and detector, and reports the first five-color genotyping application on-chip. This capability matches the portability and reduced scale of the microchip with the state-of-the-art in multicolor STR analysis kits.     

Mimicry of tandem repeat peptides against cell surface carbohydrates

Our approach to multivalent peptide construction relies on tentacle peptides, also known as a multiple antigenic peptides, which contain two and four repeats of a selected peptide. In this communication, we report the results of preliminary studies aimed at (1) the selection of short peptides against the carbohydrate, sLeX, (2) the synthesis of tentacle dimers and tetramers of the selected peptides, and (3) the determination of affinities and specificities of the peptides to several related carbohydrates by using the surface plasmon resonance (SPR) and the equilibrium dialysis techniques. Binding affinity studies, as well as assays of in vitro binding of the peptides to a sLeX-specific cell line, have shown that the tetrameric peptides bind to the cell surface sugars.     

Microdevice DNA forensics by the simple tandem repeat method

We review recent experiments on DNA forensics by the simple tandem repeat (STR) method using a 16-lane micromachined device as the active separation element. Separations by linear polyacrylamide matrices show very high data quality metrics when evaluated with statistically significant data sets. Full 16-locus multiplexes are verified on the multilane system. Multi-donor mixed samples are studied in the context of the limits of the laser-induced fluorescence detector and data-reduction software. The microdevice appears to be posed to outperform current capillary arrays in terms of stability and, through specialized sample loading, in the interpretation of complex mixtures.     

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 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.     

A novel nomenclature for the hypervariable short tandem repeat APOAI1

A novel nomenclature for the hypervariable microsatellite DNA, APOAI1 locus, is proposed. The complex nature of the repeat unit in this locus results in alleles separated by a single base. Polymerase chain reaction (PCR) products amplified from this locus were separated by single-strand conformation polymorphism (SSCP) electrophoresis. All the single-stranded DNA bands on the SSCP gel were removed from the gel and a second amplification performed. Homozygous DNA fragments amplified from single-stranded DNA were sequenced. From the 100 individuals studied, 30 alleles and 73 genotypes were found. A system of nomenclature for the APOAI1 locus is provided that is logical and in line with previous models. Using the primers described, the locus can be amplified and alleles designated on the basis of size. This system of nomenclature will assist in the exchange of data between laboratories for this locus.     

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.     

Validation of the HumDXS6807 short tandem repeat polymorphism for forensic application.

This paper presents sequence and population genetic data of the HumDXS6807 short tandem repeat (also known as CHLC.GATA52B03) which is a tetranucleotide repeat polymorphism representing seven alleles of 251-275 bp in length. HumDXS6807 is located at Xpter-Xp22.2, i.e., at a genetic distance of more than 87 and 151 cM from the well-known markers HumARA and HumHPRTB, respectively. Kinship tests in 157 family trios revealed a typical X-linked codominant inheritance; mutations were not found. Population genetic data were obtained by analyzing a Caucasian population sample comprising 308 females and 209 males: polymorphism information content (PIC) = 0.640; Heterozygosity (Het) = 0.668; Mean exclusion chance (MEC) = 0.414. The HumDXS6807 allele distribution met the Hardy-Weinberg expectations.     

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.     

Valence-controlled protein conjugation on nanoparticles via re-arrangeable multivalent interactions of tandem repeat protein chains

Precise control of the number of conjugated proteins on a nanoparticle surface has long been a highly challenging task. Here, we developed a one-pot, purification-free strategy for valency-controlled conjugation of tandem repeat protein chains on gold nanoparticles. Protein chains were designed to contain multiple, regularly spaced binding modules, which can multivalently interact with coating molecules on nanoparticle surfaces. We discovered that a slow increase of this interaction strength facilitates full participation of repeated binding modules on a protein chain for surface binding (as well as dynamic rearrangement) on a single nanoparticle, which resulted in stable protein chain wrapping around nanoparticles. By varying the protein chain length, a defined number of protein chains were conjugated on gold nanoparticles with difference sizes. Various high-order nanoparticle structures were accurately assembled with these valence-controlled protein–particle conjugates. The present strategy offers a highly dynamic but controlled protein coating approach on solid surfaces of diverse nanostructures. In addition, this work also provides a valuable clue to understand dynamic binding processes of multivalent repeat proteins.

Tandem repeat protein chains were wrapped around nanoparticles via re-arrangeable multivalent interactions for valence controlled protein conjugation.     

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.     

Temperature and pH studies of short tandem repeat systems using capillary electrophoresis at elevated pH

The DNA secondary structure can affect the migration time and precision of DNA separations in the physical gels used in capillary electrophoresis (CE). To counteract these effects, DNA typing is performed using elevated temperatures (60 degrees C) and high concentrations (7 M) of urea. These conditions affect the precision and lifetime of the analysis. To better understand the effects of these conditions on the reproducibility of DNA migration, we examined the effects of temperature and pH on short tandem repeat (STR) analysis using the PE/ABI 310 Genetic Analyzer. Separations were performed using the Profiler + multiplex system, a set of coamplified STRs with a 4-base repeat motif, labeled at the 5'-end using fluorescent dyes. The analytical separations were obtained using a commercial buffer at pH 8 and an experimental buffer consisting of 3% hydroxyethylcellulose at pH settings ranging from 8-12. Multichannel laser-induced fluorescence detection was used. Temperatures were examined from 30-70 degrees C. The results demonstrate the fact that highly efficient separations can be carried out at alkaline pH. In addition, improvements in temperature stability were seen when compared to results at lower pH. However, high concentrations of urea were found to be necessary to achieve optimal resolution.     

Strain discrimination of Yersinia pestis using a SERS-based electrochemically driven melting curve analysis of variable number tandem repeat sequences

Strain discrimination within genetically highly similar bacteria is critical for epidemiological studies and forensic applications. An electrochemically driven melting curve analysis monitored by SERS has been utilised to reliably discriminate strains of the bacterial pathogen Yersinia pestis, the causative agent of plague. DNA amplicons containing Variable Number Tandem Repeats (VNTRs) were generated from three strains of Y. pestis: CO92, Harbin 35 and Kim. These amplicons contained a 10 base pair VNTR repeated 6, 5, and 4 times in CO92, Harbin 35 and Kim respectively. The assay also included a blocker oligonucleotide comprising 3 repeats of the 10-mer VNTR sequence. The use of the blocker reduced the effective length of the target sequence available to bind to the surface bound probe and significantly improved the sensitivity of the discrimination. The results were consistent during three replicates that were carried out on different days, using different batches of PCR product and different SERS sphere segment void (SSV) substrate. This methodology which combines low cost, speed and sensitivity is a promising alternative to the time consuming current electrophoretic methods.     

Characterization of a highly variable short tandem repeat polymorphism at the D2S1242 locus

We report the evaluation of short tandem repeat (STR) locus D2S1242 (GDB ID G00-309-429) for forensic purposes, investigated by polymerase chain reaction (PCR) amplification and both native and denaturating polyacrylamide gel electrophoresis in 147 unrelated Austrians. No deviations from Hardy-Weinberg expectations were observed. The mean exclusion chance (MEC) was 0.669, the discriminating power (DP) was 0.947, and the observed heterozygosity rate was 0.856. An allelic ladder consisting of eight sequenced alleles (141-167 and 175 bp) was constructed. Sequence analysis revealed that the locus comprised two repeat motifs varying in number between alleles GAAA and GAAG. According to the number of tetranucleotide repeats the smallest allele was designated as 10 and the largest allele as 18.     

Optimal population analysis

An easy method of approximating the electron density of a molecule by expressing it as a sum of squares of atomic orbitals is described. The coefficients are determined by minimizing the error in the electric field. The optimal population analysis is an integration of this optimized density over the orbitals belonging to one atom. The Mulliken population analysis can be interpreted as the integral of a Mulliken density that is a similar expression with fixed coefficients. The two densities are compared in détail using calculations on the water molecule and, more briefly, HF, LiH, BH, and C₂H₄. The error in the densities is identified and examined. The optimal population analysis is strongly recommended as a practical improvement to the Mulliken populations.