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We describe a novel method for rapidly identifying and distinguishing between different DNA sequences using short tandem repeat (STR) analysis and DNA microarrays. The method can be used to deduce identity, length, and number of STRs of the target molecule. We refer to this technique as the "variable-length probe array" method for STR profiling (VLPA). The method involves hybridization of the unknown STR target sequence to a DNA microarray displaying complementary probes that vary in length to cover the range of possible STRs. A post-hybridization enzymatic digestion of the DNA hybrids is then used to selectively remove labeled single-stranded regions of DNA from the microarray surface. The number of repeats in the unknown target is then deduced based on the pattern of target DNA that remains hybridized to the array. This DNA profiling technique is useful for performing forensic analysis to uniquely identify individual humans or other species.

The Centre of Forensic Sciences, in both its Toronto and Northern Regional Laboratories (Sault Ste. Marie, Ontario), has completed internal validation of the Promega PowerPlex®16 DNA typing system in accordance with the revised SWGDAM validation guidelines (January 2004). The Promega PowerPlex®16 DNA typing system amplifies the 13 CODIS loci along with Penta D, Penta E, and Amelogenin in a single reaction, affording the laboratory greater efficiencies, discrimination power, and throughput. Methodologies, techniques and platforms already in place for current multiplex systems were employed. Detection of amplified product was achieved using 5% Long Ranger® Singel Polyacrylamide gels on ABI 377 Automated Sequencers. Robust performance of the PowerPlex®16 system was obtained using 1ng of target DNA in a 15 pL reaction amplified for a total of 30 PCR cycles. With these conditions, 0.5 pL of amplified product yielded average combined allelic peak heights in excess of 1000 rfu per locus. Sensitivity stud...

A population study on 10 tetrameric STR loci (D3S1358, VWA, D8S1179, D21S11, D18S51, D16S539, D2S1338, D19S433, THO1 and FGA) was performed on 104 unrelated individuals from Romania (Bucharest area), by mean of multiplex PCR (AmpFl STR SGM plus) and capillary electrophoresis. The allele frequencies and the usual forensic statistical parameters were defined. A brief comparison of the allele frequency distributions with other available data from European populations is presented. All the analyzed loci met Hardy–Weinberg equilibrium expectations. The high discrimination and exclusion power of the combined system showed the forensic efficiency of these 10 genetic markers. The study is considered to be very useful for paternal lineage testing and identification purposes in current forensic practice with Romanian individuals.

In this study the development of a 13-locus multiplex-PCR system fitting the updated demands for paternity testing in Germany is described. For this purpose an existing multiplex PCR system that allows the simultaneous amplification of eight different STR loci together with the sex-specific locus amelogenin ( genRESMPX-2, Serac, Germany) was extended. Whereas some of the primers were taken from the underlying multiplex system, suitable primer sequences were chosen for the STR loci D19S433, TPOX, TH01, D16S539, D5S818, D2S1338 and FGA. Primers of loci resulting in potentially overlapping fragment sizes were labelled with the fluorescent dyes 6-FAM, JOE and NED. Reaction conditions, such as annealing temperature, concentrations of primers and polymerase or buffer conditions were optimised to obtain a robust amplification and reproducible genotype analysis for various sample sources. Full DNA profiles from single source samples were reliably typed from template DNA amounts of as low as 120 pg, suggesting a potential use of this system also in forensic casework analysis. With a mean exclusion chance (MEC) of 99.9989% and a power of discrimination (P(D)) of about 1x10(14) (Caucasians), the new multiplex PCR system provides a significant and sensitive system for forensic DNA analysis. On the basis of these studies, a commercial kit system is now provided by Serac (Bad Homburg, Germany, genRESMPX-3).

Analysis of length polymorphism at short tandem repeat (STR) loci utilizing the polymerase chain reaction (PCR) process has proven to be an ideal assay for human identification purposes. The short length of STR loci coupled with the amplification of target sequence through PCR allows for a robust, sensitive, and specific assay for highly polymorphic markers. A multiplex containing fifteen STR loci plus the gender-determining locus Amelogenin was developed to provide a single amplification/detection of all CODIS (Combined DNA Index System) STR loci (CSF1PO, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, FGA, TH01, TPOX, and vWA) as well as two internationally-accepted STRs (D2S1338 and D19S433). By incorporating five-dye fragment analysis technology and non-nucleotide linkers, previously optimized AmpFlSTR kit primer sequences have been maintained. This kit has been developed in accordance with the standards of the forensic community as defined by the DNA Advisory Board. Validation studies were performed to include developmental validation, and the results support the use of the AmpFlSTR Identifiler PCR Amplification Kit for human identity and parentage testing.

The Y-PLEX 12 system, developed for use in human identification, enables simultaneous amplification of eleven polymorphic short tandem repeat (STR) loci, namely DYS392, DYS390, DYS385 a/b, DYS393, DYS389I, DYS391, DYS389II, DYS 19, DYS439 and DYS438, residing on the Y chromosome and Amelogenin. Amelogenin provides results for gender identification and serves as internal control for PCR. The validation studies were performed according to the DNA Advisory Board's (DAB) Quality Assurance Standards. The minimal sensitivity of the Y-PLEX 12 system was 0.1 ng of male DNA. The mean stutter values ranged between 3.76-15.72%. A full male profile was observed in mixture samples containing 0.5 ng of male DNA and up to 400 ng of female DNA. Amelogenin did not adversely affect the amplification of Y-STRs in mixture samples containing male and female DNA. The primers for the Y-STR loci present in Y-PLEX 12 are specific for human DNA and some higher primates. None of the primate samples tested provided a complete profile at all 11 Y-STR loci amplified with the Y-PLEX 12 system. Y-PLEX 12 is a sensitive, valid, reliable, and robust multiplex system for forensic analysis, and it can be used in human forensic and male lineage identification cases.

Human chromosome Y-specific short tandem repeat (Y-specific STR) markers have useful properties for forensic applications. However, there is a need to develop more Y-specific STR markers because the discriminating power of each STR locus is limited. In the present study, we describe our results on two new Y-specific STR markers, which were initially reported by White et al. [Genomics 57 (1999) 433] as Y-GATA-C4 and Y-GATA-A10. The distributions of alleles and haplotypes for both Y-specific STR loci were studied in a Chinese Han population sample. Allele determination was carried out by comparison with sequenced allelic ladders which were made in-house. Following the recommendations of the International Society of Forensic Genetics, the allele classification for both Y-specific STR loci was based on the number of repeat motifs. The results show that the haplotype diversity, the power of discrimination and the exclusion probability in the Chinese population for both Y-specific STR loci were 0.9090. The implication from this study is that Y-GATA-C4 and Y-GATA-A10 are useful Y-specific STR markers for forensic DNA typing in Chinese populations.

In order to get more available STR loci for forensic DNA analysis in Chinese populations, we investigated 10 tetranucleotide STR loci on chromosome 9 selected from GDB (D9S917, D9S254, D9S1121, D9S1118, D9S301, D9S1122, D9S922, D9S252, D9S2026, D9S934). Some of these were then chosen for further evaluation. 1039 EDTA-blood samples of unrelated individuals and some biological materials obtained from crime scene were used in this study. Amp-FLP techniques were employed for DNA typing. All alleles were cloned in plasmids and sequenced with an ABI 310. For each locus, the cloned alleles were amplified and the PCR products were mixed to make an allelic ladder for DNA typing. The software POPGENE was used to analyze the allele frequencies and heterozygosities of 10 STR loci in Chinese Han population. The results showed that compared with the other nine loci, allele frequencies of D9S1118 had a preferable distribution in Chinese Han population. The exclusion probability was 0.6405, while the discrimination power was 0.9424. Analyzing biological materials from crime scenes showed that typing D9S1118 had a higher success rate. All of these results suggest that D9S1118 as a marker could be used for forensic DNA analysis in Chinese populations.

DNA typing was performed on 508 unrelated Japanese volunteers using the 15 short tandem repeats (STR) loci, that is, D3S1358, TH01, D21S11, D18S51, Penta E, D5S818, D13S317, D7S820, D16S539, CSF1PO, Penta D, vWA, D8S1179, TPOX, and FGA, present in the PowerPlex™ 16 System. The allele frequencies, heterozygosity, power of discrimination, mean exclusion chance and polymorphism information content of all loci were calculated by statistical analysis. The combined matching probability and the combined mean exclusion chance in Japanese was 1 in 1.81×017 and 0.9999989, respectively. None of the 15 loci were found to deviate from Hardy–Weinberg equilibrium (HWE) according to the results of the exact test and the homozygosity test. There was also no evidence for correlation of alleles between loci. This study demonstrates that this multiplex system is a useful and convenient tool for forensic identification and parentage testing in Japan.

Short tandem repeats (STR) are useful to forensic science and are characterized by their small range of alleles, their high sensitivity and their suitability if the DNA is degraded. In our research, allele frequencies of 15 STR loci (CSF1PO, TPOX, TH01, D5S818, D7S820, D13S317, D16S539, D21S11, D18S51, FGA, vWA, D8S1179, D3S1358, PentaE, PentaD), together with the Amelogenin locus, were obtained in a sample of over 200 people from the Beijing-Han population, China. The results suggest that the 15 loci are excellent markers for identity and paternity testing. This is the first report on statistical data of all the 15 loci in the Beijing-Han population.

Pentanucleotide tandem repeat markers are of interest for forensic science because they may present less stutter in electrophoretic patterns. We focused on the analysis of the DNA sequence for each allele at two pentanucleotide STR loci, D6S957 and D10S2325, in order to understand their structures in the human genome and to construct allelic ladders, necessary for forensic DNA typing. In order to evaluate the forensic applicability of both pentanucleotide tandem repeat loci and to construct a preliminary database, the genotype distributions and allele frequencies in different ethnic groups were investigated. The population samples included Caucasians (Germans) and Asians (Chinese). The Amp-FLP technique was employed for DNA typing. An example of each allele and new alleles were sequenced. Allele determination for each pentanucleotide STR locus was carried out by comparison with a sequenced allelic ladder made in-house. Both pentanucleotide STR markers provided easily interpretable results. No evidence of deviation from Hardy–Weinberg equilibrium was observed. In 64 confirmed father/mother/child trios, no mutation event was observed. Using a maximum likelihood method, the mutation rate at both STR loci was indirectly estimated as 2.510 5 , suggesting both pentanucleotide STR markers would be useful for

Short tandem repeat systems on the X chromosome are the natural counterpart to the well-established Y-chromosomal STR loci. The X-linked systems are inherited as a single haplotype only in males, whereas in females, the X chromosomes recombine and exhibit the same characteristics as the autosomes. Nevertheless, X-linked systems may provide a useful tool in paternity cases with female offspring, in particular when the alleged father is not available for testing, or in forensic identification cases based on the comparison with firstor second-degree relatives. Only a small number of STR loci have been described on the X chromosome, and a number of these are not highly informative. Therefore, we have investigated the four X-chromosomal STR loci: DXS101, DXS8377, HPRTB and STRX-1 [1–5], to develop a rapid multiplex PCR typing system suitable for fluorescent detection and to perform population genetic studies among Germans. Whereas HPRTB and STRX-1 contain tetrameric repeats, DXS101 and DXS8377 have trimeric repeats. All four loci are highly polymorphic exhibiting up to 18 common alleles at each locus (Table 1).

Genotyping of 15 STR loci plus Amelogenin by use of the commercial kit PowerplexR 16 system Promega [1], on an ABI PRISMk 377 DNA Sequencer (Perkin Elmer), is generally carried out on 36-cm plates. We demonstrate that genotyping of samples amplified with Powerplex 16 can be accomplished efficiently on 12-cm plates [2]. To guarantee sufficient accuracy, different conditions were tested in over 300 runs in which 12-cm plates were used. There are two advantages of performing electrophoresis, genotyping 16 loci per lane, with 12-cm plates rather than 36-cm plates with the ABI PRISM 377 DNA Sequencer: firstly, gel casting is more practical; secondly and more importantly, the same gel can be used for up to three consecutive runs because the lower voltage of the run exposes the gel to lower stress, maintaining sensitivity, separation, and correct allele determination.

Short tandem repeat (STR) loci in human genome are widely used in human identity testing and forensic casework, and most commercial kits available are based on multiplex amplification of common 10–16 STR loci with each analysis range specifically established in some populations. Therefore, this system may miss unusually small or large repeats beyond predefined analysis range. Here, we report unusually large repeats of 25-, 26- and novel 30-repeat in PentaE locus, suggesting that it may not be so rare in a certain population and necessitating extensive validation process of multiplex system prior to introduction into the population concerned.

We performed multiplex polymerase chain reaction (PCR) for the TH01, TPOX, CSF1PO, and vWA loci using a newly designed pair of primers that yield smaller fragments than reported previously [Fujii et al., J Hum Genet 45 (2000) 303; Lederer et al., Int J Legal Med 114 (2000) 87]. These loci can be detected in the range of 74-143 bp amplifying products. This system required genomic DNA in a range of 80 pg to 2 ng, and proved to be a sensitive typing method. We compared our system against the GenePrint Fluorescent STR Multiplex Systems CTTv (Promega, Madison, WI, USA), using DNA extracted from old bloodstains left to stand for 17-26 years at room temperature. With our designed system, all allele-typing efforts were successful in the range of 1-5 ng DNA, while no signal peaks were detected, even with when using 10 ng of DNA GenePrint Fluorescent STR Multiplex Systems CTTv.

In this study, we describe a protocol of generating a large number of candidate SNPs and STR loci quickly and cost-efficiently for an endangered species, snub-nosed monkeys (Rhinopithecus roxellana), using next-generation sequencing technology. First, we built a reduced representation library of snub-nosed monkeys through digesting the DNA of four individuals using HaeIII and pooling the 350–500 bp fragments together. Second, we sequenced the DNA pool using pyrosequencing technology. Third, we analyzed sequencing reads using bioinformatic software. We detected 23 putative SNPs, 10 polymorphic STR loci, and 347 candidate STR loci (number of repeats greater than 9). The markers generated by this study can be used to address questions regarding genetic diversity, population structure, phylogeography, and conservation of this endangered primate.

It is well known that mutations in the primer binding site of STR loci can cause "null alleles". If a sample is typed with different sets of PCR primers (for example if different kits or kits of different manufacturers are used) discordant results might be observed, thus making the use of different STR kits for deviant genotypes necessary. Here we present another reason for using different kits: Insertion-/deletion polymorphism between the repeat region of the STR locus and the primer binding site can also cause genotypes that vary between different kits/primer sets.

Short tandem repeats (STRs) are highly mutable genetic elements that often reside in functional genomic regions. The cumulative evidence of genetic studies on individual STRs suggests that STR variation profoundly affects phenotype and contributes to trait heritability. Despite recent advances in sequencing technology, STR variation has remained largely inaccessible across many individuals compared to single nucleotide variation or copy number variation. STR genotyping with short-read sequence data is confounded by (1) the difficulty of uniquely mapping short, low-complexity reads and (2) the high rate of STR amplification stutter. Here, we present MIPSTR, a robust, scalable, and affordable method that addresses these challenges. MIPSTR uses targeted capture of STR loci by single-molecule Molecular Inversion Probes (smMIPs) and a unique mapping strategy. Targeted capture and mapping strategy resolve the first challenge; the use of single molecule information resolves the second challenge. Unlike previous methods, MIPSTR is capable of distinguishing technical error due to amplification stutter from somatic STR mutations. In proof-of-principle experiments, we use MIPSTR to determine germ-line STR genotypes for 102 STR loci with high accuracy across diverse populations of the plantA. thaliana. We show that putatively functional STRs may be identified by deviation from predicted STR variation and by association with quantitative phenotypes. Employing DNA mixing experiments and a mutant deficient in DNA repair, we demonstrate that MIPSTR can detect low-frequency somatic STR variants. MIPSTR is applicable to any organism with a high-quality reference genome and is scalable to genotyping many thousands of STR loci in thousands of individuals.

X-chromosome short tandem repeats (X-STR) analysis has been confirmed to be effective for kinship testing such as in deficiency paternity cases. The aim of this study was to develop a new multiplex polymerase chain reaction (PCR) system that can simultaneously amplify 9 X-STR loci (GATA172D05, DXS10159, DXS6797, HPRTB, DXS10079, DXS6789, DXS9895, DXS10146 and GATA31E08) in the same PCR reaction, and to obtain the database of the 9 X-STR loci in three ethnic populations in China. The genetic data of 815 (404 females and 411 males) unrelated Han Chinese from Hubei province, and Yi and Zhuang Chinese from Yunnan province were analyzed by using this multiplex system. The results showed that a total of 93 alleles for all these loci were found, and 7 to 20 alleles for each locus were observed. All of the analyzed loci were in agreement with Hardy-Weinberg equilibrium after Bonferroni correction in the three studied populations. The polymorphism information content (PIC) and power of discrimination (PD) in females were 0.6566–0.8531 and 0.8639–0.9684, respectively. Pairwise comparisons of allele frequency distribution showed significant differences in the most of these loci between different populations. The results indicate that this multiplex system is very useful for forensic analysis of different ethnic populations in China.

Short tandem repeats are among the most polymorphic loci in the human genome. These loci play a role in the etiology of a range of genetic diseases and have been frequently utilized in forensics, population genetics, and genetic genealogy. Despite this plethora of applications, little is known about the variation of most STRs in the human population. Here, we report the largest-scale analysis of human STR variation to date. We collected information for nearly 700,000 STR loci across more than 1000 individuals in Phase 1 of the 1000 Genomes Project. Extensive quality controls show that reliable allelic spectra can be obtained for close to 90% of the STR loci in the genome. We utilize this call set to analyze determinants of STR variation, assess the human reference genome’s representation of STR alleles, find STR loci with common loss-of-function alleles, and obtain initial estimates of the linkage disequilibrium between STRs and common SNPs. Overall, these analyses further elucidate the scale of genetic variation beyond classical point mutations., American Society for Engineering Education. National Defense Science and Engineering Graduate Fellowship

Mixed semen stains from multiple contributors are challenging samples in sexual assault casework, and it is crucial to obtain the DNA profiles of different donors to allow the evidence to play an important role in investigations and judicial proceedings. Current standard procedures, including preferential lysis, are incapable of separating single-source sperm from multiple male donors. Mixed profiles are often obtained and may not directly exclude or identify suspects. In this case, computational methods for mixture interpretation are often used, which rely on different types of calculation models. Here, we explored a new strategy for sperm cell isolation and detection from mixtures. It is a direct way to obtain genotypes of different sperm donors compared to computation-based mixture interpretation. Laser capture microdissection (LCM) and low volume-PCR (LV-PCR) were used for single sperm isolation and detection. The platform was sensitive; profiling of a single sperm cell generated a minimum of 13-16 loci in 73.1% of Y short tandem repeat (Y-STR) assays. A new Y-STR and autosomal STR multiplex system (YA-STR) were optimized by the combination of the Y-STR locus and 10 autosomal STR (auto STR) loci. The Y-STR locus acted as a tag to discriminate profile groups from different donors. Subsequently, consensus auto STR profiles of various persons could be received. The accuracy and availability of this method were evaluated on a three-donor semen mixture and found to be effective for the resolution of a multi-donor sperm mixture.

Regularly, STR results obtained with different PCR amplification kits are compared, for instance with old cases, when revisiting cold cases or when addressing cross-border crimes. It is known that differences in primer design for the same loci in different kits may give rise to null alleles or shifted alleles. In this study, the genotyping results of 2085 Dutch male samples were compared for six autosomal STR kits (Promega's PowerPlex (R) 16, ESX-16 and ESI-17 Systems, Qiagen's Investigator (R) ESSplex Kit and Applied Biosystems' AmpFlSTR (R) Identifiler and NGM PCR Amplification Kits). A total of 19 discordant autosomal genotyping results were obtained that were examined by sequence analysis using Roche-454 next generation sequencing and/or Sanger sequencing. A further 25 discordances were found and sequenced for the Amelogenin locus. The 24 samples showing the same primer binding site mutation at the Amelogenin locus were subjected to X-STR analysis in order to assess whether they could share a common origin, which appeared not to be the case. Based on the sequencing results, we set the final genotypes and determined the allele frequencies of 23 autosomal STRs for the Dutch reference database. (C) 2014 The Authors. Published by Elsevier Ireland Ltd. All rights reserved.

Tandem repeats are short DNA sequences that are repeated head-to-tail with a propensity to be variable. They constitute a significant proportion of the human genome, also occurring within coding and regulatory regions. Variation in these repeats can alter the function and/or expression of genes allowing organisms to swiftly adapt to novel environments. Importantly, some repeat expansions have also been linked to certain neurodegenerative diseases. Therefore, accurate sequencing of tandem repeats could contribute to our understanding of common phenotypic variability and might uncover missing genetic factors in idiopathic clinical conditions. However, despite long-standing evidence for the functional role of repeats, they are largely ignored because of technical limitations in sequencing, mapping and typing. Here, we report on a novel capture technique and data filtering protocol that allowed simultaneous sequencing of thousands of tandem repeats in the human genomes of a three generation family using GS-FLX-plus Titanium technology. Our results demonstrated that up to 7.6% of tandem repeats in this family (4% in coding sequences) differ from the reference sequence, and identified a de novo variation in the family tree. The method opens new routes to look at this underappreciated type of genetic variability, including the identification of novel disease-related repeats. ispartof: Nucleic Acids Research vol:42 issue:9 pages:5728-5741 ispartof: location:England status: published

A series of validation experiments was performed for a Y chromosome specific STR multiplex system following the suggestions made by the Technical Working Group DNA Analysis Methods (TWGDAM). The multiplex PCR products were detected on Perkin-Elmer 373 and 377 automated sequencers using two labeling colors. No problems regarding the stability, robustness and sensitivity of the Y STR multiplex were observed. Mixture studies revealed a cut off rate similar to autosomal STRs for mixtures of male DNAs and no interference of any female admixture. The comparison of the Y STR results to the autosomal typing results for 56 nonprobative semen stains and swabs, showed a slightly higher success rate in detecting the semen donor’s alleles for the Y STR multiplex. Two examples are shown to illustrate the usefulness of Y STR typing for DNA mixtures. In one case the Y STR results confirmed an isolated exclusion; in the other case, the interpretation of a mixture was clarified since the Y STR results proved the presence of DNA from at least two semen donors. Y STR typing is a valuable addition to the forensic DNA testing panel.

We present a dense STR/linkage disequilibrium(LD)/gene map between the RING3 and HLA-B loci, reference allelic sizes on the most prevalent HLA haplotypes and their allelic frequencies in pedigree founders. This resource will facilitate LD, evolution and gene mapping studies, including comparisons of HLA and STR haplotypes and identification of HLA recombinants. The map was constructed by testing novel and previously reported STRs using a panel of 885 individuals in 211 families and 60 DNA samples from cell lines and bone marrow donors homozygous in the HLA-A, -B and -DR loci selected from over 15 000 entries into the registry of Swedish bone marrow donors. We have also analysed the variability of STR alleles/haplotypes on the most prevalent HLA haplotypes to identify STRs useful for fine mapping of disease genes in the region previously implicated in susceptibility to many disorders. The analysis of 40 HLA-A*01, B*0801, DRB1*03011, DQB1*0201 haplotypes in homozygous donors showed a surprising stability in 23 STRs between the class II recombination hot spot and HLA-B, with the average of 1.9% (16/838) variant alleles. However, 40% variant alleles were found at the D6S2670 locus in intron 19 of the tenascin-X gene both in the families and homozygous donors. The nucleotide sequence analysis of this STR showed a complex polymorphism consisting of tetra- (CTTT)(8-18) and penta-nucleotide (CTTTT)(1-2) repeats, separated by an intervening non-polymorphic sequence of 42 bp. The HLA-A1, B*0801, DRB1*03011, DQB1*0201 haplotypes had five (CTTT)(14-18)/(CTTTT)(2) variants with a predominant (CTTT)(16) allele, implicating the tetranucleotide component as the source of this ancestral haplotype diversification, which may be due to the location of D6S2670 in the region of the highest GC content in the human MHC.

We devised a multiplex polymerase chain reaction (PCR) amplification and loading system for the convenient typing of 168 short tandem repeat (STR) polymorphic markers in a commercially available screening primer set for human linkage analysis. We genotyped all these 168 STR loci with 32 healthy unrelated Japanese, calculated allele frequencies at each STR locus, and performed three kinds of tests for Hardy-Weinberg equilibrium (HWE). Significant deviations from HWE in all three tests were observed at only three loci, and the average heterozygosity in the Japanese (0.733) was slightly lower than that in Caucasians (0.773). We also examined 32 Caucasians at some selected loci, to be compared with Japanese. Some markers showed greatly different heterozygosities or allelic distributions in Japanese and Caucasian populations. In two groups of STRs, those with and without irregular alleles (or inter-alleles), the former had a higher proportion of bimodal allelic distribution and possessed more alleles per locus than the latter. However, no significant differences in the observed and expected heterozygosities, or in the powers of discrimination, were found between the two groups. The present basic study of allele frequency databases of these STRs will contribute to further applications in forensic science and human genetics.

Human cytomegalovirus (HCMV) strains display genetic polymorphisms, and these polymorphisms can be analyzed to study viral transmission and pathogenesis. Recently, short tandem repeat (STR) length polymorphisms have been identified in the HCMV genome. We assessed the utility of STRs in characterizing HCMV strains and found that a multiplexed PCR assay using primers based upon these STRs accurately maps HCMV strains. Using primers for 10 microsatellite regions, the STR profiles of 44 wild-type and 2 laboratory strains of HCMV were characterized. The results of STR analysis were compared with those for strain characterization using nucleotide sequencing and restriction fragment length polymorphism analysis. In each instance, STR analysis accurately and specifically identified strains that were indistinguishable or distinct by conventional molecular analysis. Analysis of short tandem repeats also detected polymorphisms that supported simultaneous excretion of two HCMV strains. These results indicate that STR analysis allows rapid, precise molecular characterization of HCMV strains.

Human chromosome Y-specific short tandem repeat (Y-specific STR) markers have useful properties for forensic applications. However, there is a need to develop more Y-specific STR markers, because the discriminating power of each STR locus is limited. In the present study, we describe our results on six new Y-specific STR markers that were initially located using sequence database information by Ayub et al. and were named DYS434, DYS435, DYS436, DYS437, DYS438 and DYS439. Our studies focused on the analysis of the DNA sequence for each allele at all six Y-specific STR loci in order to understand their structures in the human genome and to construct human allelic ladders, which are necessary for forensic DNA typing. In addition, the haplotype distribution for all six analyzed loci was studied in a Chinese Han population sample. The results indicate that DYS434, DYS435, DYS436, DYS437, DYS438 and DYS439 are useful Y-specific STR markers for forensic sciences.

The allele distributions at the 12 short tandem repeat (STR) loci D3S1358, HUMvWA, HUMFIBRA/FGA, HUMTHO1, HUMTPOX, HUMCSF1P0, D5S818, D13S317, D7S820, D8S1179, D21S11 and D18S51 have been determined for 284 unrelated Chinese in Hong Kong. The combined probability of identity for the 12 STR loci was about 4.1 x 10(-14) and the overall probability of excluding paternity 0.999978. None of the 12 loci were found to deviate from Hardy-Weinberg expectations according to the results of the exact test. There was also little evidence for association of alleles between loci. The results demonstrate that the loci are useful for forensic human identification and parentage testing for the Chinese population in Hong Kong.

The advent of PCR technology and use of short tandem repeat (STR) loci improves throughput and reduces costs whilst a high level of discrimination can be achieved. A new system, comprising seven STRs, was developed to compliment the existing systems. This paper describes the preparation of allelic ladders of the most commonly observed alleles of a new STR multiplex system (third generation; TGM multiplex); all alleles have been sequenced. Meioses studies estimated a mutation rate of 0‐0.4% across loci. Statistical independence was investigated by employing exact tests; w 2 -tests and excess homozygosity tests. The results demonstrated that the allele proportions do not differ from those expected and that there was no consequential dependence between loci. The discriminating power of the system was examined using 295 Caucasian, 140 Afro-Caribbean and 212 Asian unrelated samples, and was found to be approximately 1 in 50 million, 1 in 85 million and 1 in 20 million for each of these groups, respectively. # 2001 Elsevier Science Ireland Ltd. All rights reserved.

Conventional karyotyping for antenatal diagnosis is time consuming and hence there has been a growing interest in more rapid techniques for detection of chromosomal aneuploidies. Around 95% of Down syndrome cases are due to free trisomy 21.The aims of this study were to demonstrate sensitivity of DNA diagnosis of Down syndrome using polymerase chain reaction (PCR) and short tandem repeat (STR) markers, and to determine the parental origin of the nondisjoined chromosome.DNA polymorphism was studied using two tetranucleotide STR markers, D21S2055 situated at 21q22.2 and D21S11 situated at 21q21.1. PCR conditions for both markers were standardized. PCR products were analyzed in 15% poly-acrylamide gels. The results obtained by STR analysis were verified with the chromosomal analysis and quantitative fluorescent (QF)-PCR.These two STR markers were able to detect 86.7% cases of trisomy 21. Parental origin of extra chromosome was assigned to 77% of detected cases.The PCR-based DNA diagnostic method was found to be sensitive, reproducible, and efficient, not only for diagnosis of trisomy 21, but also for tracing allelic transmission from parents to the offspring.

Allele frequencies and haplotypes of 11 Y-chromosome STR loci, DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385 ab, DYS438, DYS439 and DYS437 were determined in 320 unrelated Yunnan Han Chinese males. A total of 293 haplotypes were identified, of which 268 were unique, 23 were shared in two individuals, and 2 were shared in three individuals. The allele diversity values for each locus ranged from 0.4087 (DYS438) to 0.9701 (DYS385). The allele observed haplotypes diversity value was 0.9994. The combined Y-chromosome STR polymorphisms provide a powerful discrimination tool for routine forensic applications.

On human chromosomes, a short sequence of DNA is known to repeat a number of times. These repeats are called variable number of tandem repeat (VNTR) or short tandem repeat (STR) which has a short repeat core. VNTR and STR are used in the field of forensic science, evolution, and anthropology. In this work, we examined allele frequencies of one VNTR (YNZ22) and three STRs (NeuR, D21S11, Humth01) in a Korean population sample by polymerase chain reaction (PCR) followed by high-resolution polyacrylamide gel electrophoresis (PAGE) with silver stain. Subsequently, the polymorphism information content (PIC) was calculated: the highest PIC was observed in the NeuR locus (0.95680) and lowest in the Humth01 locus (0.75809).

For two short tandem repeats at the D22S683 and D6S477 loci, 30 and 22 selected alleles, respectively were sequenced. A total of 20 different alleles were found for the D22S683 locus and 12 alleles for the D6S477 locus. In both systems the alleles were designated according to the total number of repeats. D22S683 is a hypervariable STR consisting of blocks of (TATC) repeats with a basic sequence structure (TATATC)n (TATC)n (ATC)0-1 (TATC)n. The D6S477 locus consists of blocks of (TCTA) repeats with a basic sequence structure (TCTA)n (TA)1 (TCTA)0-2 (TA)0-1 (TCTA)n. Population data showed a heterozygosity of 0.89 for D22S683 and 0.75 for D6S477. These STRs are promising markers for forensic genetics as they are robust and can be easily included in multiplexes.

Short tandem repeat (STR) markers are currently used to define loss of heterozygosity (LOH) of genes and chromosomes in tumors. Chromosome 6 and chromosome 15 STR markers are applied to define loss of HLA and related genes (e.g. TAP and beta(2)m) The number of STR identified in the HLA region is still increasing. In this study, seven representative STR markers covering the 6p/6q arms of chromosome 6 including the HLA region and two for chromosome 15 flanking the beta(2)m gene, were selected as minimally required for reliable LOH studies. A multiplex polymerase chain reaction (PCR) strategy is proposed when small number of cells are available in microdissected tumor samples.

A novel DNA forensics platform has been developed that is based on automated high throughput electrospray ionization mass spectrometry (ESI-MS). The approach uses ESI-MS to "weigh" DNA markers with enough accuracy to yield an unambiguous base composition (A w , G x , C y , T z ) which are then used to derive a DNA profile for an individual. A number of blinded studies have been performed to evaluate the platform for both STR and mtDNA typing. The same platform is used for both analyses and offers advantages over the conventional typing approaches. This approach facilitates the analysis of mtDNA samples containing length heteroplasmy in the HV1/HV2 regions in situations that are not amenable to sequencing. Based on the base compositions profiles from a 24-primer pair assay which covers CRS 15924–16428 and 31–576, this approach is more resolving than traditional sequencing approaches which cover 16024–16365 and 73–340 and facilitates mixture analysis. STR markers can be analyzed in a similar fashion. The high mass accuracy of the measurements affords the ability to detect SNPs within the STR markers. A survey of samples from different population groups suggests that the frequency of SNPs in several of the CODIS loci is in excess of 30%; SNPs were identified in 10 of the 13 core loci.

Three multiplex PCRs were developed for the analysis of 14 single-copy and 4 multi-copy Y chromosome Short Tandem Repeat (STR) loci routinely used by several public genealogical databases. These assays were used in addition to PowerPlex ® Y for the analysis of 245 DNA samples from a genealogical project. In total 244 different haplotypes composed of 37–40 alleles were identified with one haplotype identical between two males with the same surname. The multi-copy loci DYS464 and DYS724 were the most polymorphic with a gene diversity of at least 0.964. The use of DYS454 and DYS455 can be questioned as these loci had the lowest gene diversity (0.039 and 0.269, respectively).

DNA degradation may cause the loss of the longer short tandem repeat (STR) markers, resulting in DNA profiles with lower discrimination power. We compared standard STR profiling with DNA repair enzyme incubation, and genotyping with mini-STRs or (tri-allelic) single nucleotide polymorphisms (SNPs) in progressively degraded, UV-irradiated DNA samples. In highly degraded DNA samples, most of the standard STR markers fail to amplify, while mini-STRs and especially (tri-allelic) SNPs still provide valuable information.

STR multiplex is a practical and simple method to obtain large amounts of important information in forensic and population genetic studies. The present work describes a new multiplex system that allows the simultaneous analysis of 4 X-STR markers, namely DXS9902, DXS6800, DXS6799 and DXS7132, as the tool of approach for X-STR studies. In addition, this work presents the genotyping results obtained for a sample 400 individuals (200 males and 200 females) from Beijing Han ethnic group in China.

Allele and genotype frequencies for eight tetrameric short tandem repeat (STR) loci were established from 200 unrelated Thai individuals using one multiplex polymerase chain reaction (PCR) and five single-locus PCRs, followed by electrophoresis of the PCR products in denaturing polyacrylamide gels and subsequent detection by silver staining. The loci were CSF1PO, TPOX, TH01, F13A01, FESFPS, F13B, LPL, and vWA. All loci met Hardy–Weinberg expectations ( P >0.05). The range of observed heterozygosity was between 0.42 and 0.86. The discrimination power and the average power of exclusion from the combination of the 8 STR loci was 0.9999995 and 0.9911, respectively. Hence, the combination of these useful systems provides powerful DNA typing and can be used as a battery for person identification and paternity testing.

A population study on two new short tandem repeat (STR) loci D6S477 and D19S433 was performed on 214 unrelated Italian Caucasians. The DNA was amplified by PCR and separation and detection of the amplified STR fragments were carried out by use of a PE/ABD PRISM 377 DNA sequencer 377 automated system (Applied Biosystems Division/Perkin Elmer). Both loci meet Hardy-Weinberg expectations. There is no evidence for departures from expectations between the two loci. The combined probability of discrimination and probability of exclusion for the two STR loci are 0.997161 and 0.883183, respectively. The results demonstrate that these loci can be useful for human identification in forensic cases in Italy.

A multiplex PCR was developed for the analysis of the sex-determining gene Amelogenin, four conventional STR (short tandem repeat; THO1, D18S51, D21S11 and FGA) loci with a reduced amplicon size and four miniSTR loci (D1S1677, D2S441, D10S1248 and D22S1045). A concordance study in a population of 198 Belgians revealed no differences for the conventional STR loci while a sensitivity study showed a reproducible DNA profile with as low as 30pg of input DNA.

Insertion–deletion (InDel) markers, which combine the advantages of both short tandem repeats (STR) and single nucleotide polymorphisms (SNP), are suitable for personal identification and parentage testing. To overcome the limitation of poor biostatistical power for diallelic InDels, higher polymorphic multi-InDel markers with more than 3 allele markers have been successfully designed and developed. In this study, suspected one-step mutations at the Penta E locus (18 to 19, 15 to 16) were observed in two paternity cases tested with the AGCU Expressmarker kit (AGCU, China) including 22 STRs. A multiplex consisting of 20 multi-InDel loci was applied to these two cases. Full profiles of 20 multi-InDel markers were obtained and no mutations were found. The paternity index (PI) increased from 618 to 71,226 and from 2475 to 335,775 by combining both STR and multi-Indel markers, respectively. So the multi-Indels provided efficient genetic information for forensic paternity studies. The results underline the importance of the multi-InDel multiplex as a supplementary test to STRs in special cases.

Population data of 15 short tandem repeat (STR) loci were determined from 500 unrelated Latvian individuals. The samples were amplified using the AmpFlSTR® NGMTM PCR Amplification Kit and the PowerPlex® ESX 16 System. No significant deviation from the Hardy–Weinberg equilibrium was detected, except for locus D3S1358. Calculated forensically relevant population statistics show that the investigated loci are very discriminating in the Latvian population, with a combined discrimination power of 0.9999999999999999998. A comparison with previously published allele frequency data from other populations is presented.

DNA separations which traditionally have been performed by slab gel or capillary electrophoresis, may now be conducted via time-of-flight mass spectrometry (TOF-MS). The advantages of using a mass spectrometry approach for short tandem repeat (STR) characterization include a dramatic increase in both the speed of analysis and the accuracy of mass measurements. We report here typing of the STR loci TH01, TPOX, and CSF1PO as well as the sex-typing marker amelogenin using TOF-MS. Allelic ladders, which are typically used with electrophoretic separation systems to correct for mobility differences of DNA fragments under various conditions, are not needed for accurate genotyping with TOF-MS. A mass precision of 0.1% RSD, which corresponds to approximately 0.1 nucleotide, was routinely observed. Mass accuracies were better than a fraction of a single nucleotide when a daily mass calibration was used. STR microvariants, such as the TH01 allele 9.3, could be detected and resolved from alleles which differ by as little as a single base. In addition, the smaller PCR product sizes (55– 125 bp) examined in this study have the potential advantage of being more successful when amplifying forensic samples with degraded DNA.

This paper reports the composition of a new reference allelic ladder mixture for use with a multiplex DNA profiling system consisting of six short tandem repeat loci. The loci included in this mixture are HUMTH01, D21S11, D18S51, D8S1179, HUMVWAF31/A, HUMFIBRA/FGA and an amelogenin sex test. Sequence analysis of individual ladder alleles was carried out and allelic designations made in accordance with the recommendations of the International Society of Forensic Haemogenetics (1992; 1994). A series of rare alleles which increase the range of alleles previously reported were identified. By including some of the rare alleles into the ladder marker system, we have significantly improved the ability to identify new alleles in unknown samples.

A single copy fragment (FD14-ca 1) cantaining 22 CA repetitive units was isolated with (CA)15 oligonucleotide probe from a human chromosome 14q24.3 probe pool generated by microdissection, and proved to be a new short tandem repeat (STR) sequence through querying in GenBank of NCBI. The STR has 11 alleles in Chinese. and its polymorphic information content (PIC) is 0.85. Mendelian segregation was shown in 2 Chinese pedigrees with two generations; This STR has been accurately relocalized on chromosome 14q24.3 by fluorescencein situ hybridization (FISH). The accession numbers of this STR in GDB and Gnknk database are D14S1435 and G31413 respectively. This STR would be able to be regarded as a novel genetic marker which can increase the genetic map accuracy in this chromosome region and improve the gene diagnosis on some genetic diseases located in chromosome 14q24.3 band.

Nowadays Short Tandem Repeats Polymorphisms are the most utilized genetic markers in forensic biology. A new kind of STRs profiling system is based on the amplification of shorter fragments compared to the conventional STR multiplexes; because of their length, these “Mini” STR amplicons could be obtained even from extremely degraded DNA and/or from few copies of template DNA. Mini STR kits for Human Identification have been already validated and placed on the market; moreover they are related to those loci which are commonly detected by traditional STRs kits, allowing comparison between data yielded with both methods. In this paper we report a homicide casework occurred in the South of Italy: a young guy was killed and then burnt. Nothing inside could be collected apart a burnt stub which was subjected to a comparative genotype analysis between STRs and MiniSTRs in order to verify efficiency and discrimination power of the latter ones. Our results show that MiniSTRs permit a reliable determination of 9 human allelic loci whereas traditional STRs typing protocols offer only a partial resulting genotyping profile. MiniSTRs turned out to be more robust and sensitive markers than the traditional ones. In the next future we aim to apply this method to several kinds of degraded samples in order to confirm the above mentioned observations.

Since relatively few genital injuries are discovered at the medical examination, the detection of other elements such as the presence of semen is important to confirm the presumption of sexual assaults. For many years, cytology and the detection of acid phosphatase were considered to provide forensic evidence. In the past 2 years commercial Y-STR kits were developed to enable forensic practitioners to use these multiplex systems in male specific identification. DNA male profile was obtained from evidence samples, using Y-STR, when only autosomal STR profile from victims was detected.