A proposal for standardization in forensic equine DNA typing: allele nomenclature for 17 equine‐specific STR loci

In this study, a proposal is presented for the allele nomenclature of 17 polymorphic STR loci (AHT4, AHT5, ASB2, ASB17, ASB23, CA425, HMS1, HMS2, HMS3, HMS6, HMS7, HTG4, HTG6, HTG7, HTG10, LEX3 and VHL20) for equine genotyping (Equus caballus). The nomenclature is based on sequence data of the polymorphic region of the STR loci as recommended by the DNA commission of the International Society for Forensic Genetics for human DNA typing. For each STR locus, several alleles were selected and animals homozygous for those alleles were subjected to sequence analysis. The alleles of the 17 STR loci consisted either of simple (10), compound (6) or complex repeat patterns (1). Only a limited number of alleles with the same fragment size showed different repeat structures. The allele designation described here was based on the number of repeats, including all variable regions within the amplified fragment.     

A proposal for standardization in forensic bovine DNA typing: allele nomenclature of 16 cattle-specific short tandem repeat loci

In this study, a proposal is presented for the allele nomenclature of 16 polymorphic short tandem repeat (STR) loci ( BM1824 , BM2113 , ETH10 , ETH225 , INRA023 , SPS115 , TGLA122 , TGLA126 , TGLA227 , ETH3 , TGLA53 , BM1818 , CSRM60 , CSSM66 , HAUT27 and ILSTS006 ) for bovine genotyping ( Bos taurus ). The nomenclature is based on sequence data of the polymorphic region(s) of the STR loci as recommended by the DNA commission of the International Society of Forensic Genetics for human DNA typing. To cover commonly and rarely occurring alleles, a selection of animals homozygous for the alleles at these STR loci were analysed and subjected to sequence studies. The alleles of the STR loci consisted either of simple or compound dinucleotide repeat patterns. Only a limited number of alleles with the same fragment size showed different repeat structures. The allele designation described here was based on the number of repeats including all variable regions within the amplified fragment. The set of 16 STR markers should be propagated for the use in all bovine applications including forensic analysis.     

Number of STR Repeats as a Potential New Quantitative Genetic Marker for Complex Diseases, Illustrated by Schizophrenia

It has proved difficult to find strong and replicable genetic linkages for complex diseases, since each susceptibility gene makes only a modest contribution to onset. This is partly because high-efficacy genetic markers are not usually available. The aim of this article is to explore the possibility that the total number of tandem repeats in one STR locus, rather than the frequencies of different alleles, is a higher efficacy quantitative genetic marker. DNA samples were collected from schizophrenic patients and from a control population. Alleles of the short tandem repeats (STR) loci D3S1358, vWA, and FGA were determined using the STR Profiler Plus PCR amplification kit. The two groups did not differ statistically in the frequencies of alleles at the D3S1358, vWA, or FGA loci. However, a significant difference was obtained in the vWA locus when the total number of core unit repeats was compared between the schizophrenia and control groups (33.28+/-2.61 vs. 32.35+/-2.58, P<0.05). It seems that the number of STR repeats may be a new, quantitative, and higher efficacy genetic marker for directly indicating genetic predisposition to complex hereditary diseases such as schizophrenia.     

Mutation and selection processes regulating short tandem repeats give rise to genetic and phenotypic diversity across species

Short tandem repeats (STRs) are units of 1–6 bp that repeat in a tandem fashion in DNA. Along with single nucleotide polymorphisms and large structural variations, they are among the major genomic variants underlying genetic, and likely phenotypic, divergence. STRs experience mutation rates that are orders of magnitude higher than other well-studied genotypic variants. Frequent copy number changes result in a wide range of alleles, and provide unique opportunities for modulating complex phenotypes through variation in repeat length. While classical studies have identified key roles of individual STR loci, the advent of improved sequencing technology, high-quality genome assemblies for diverse species, and bioinformatics methods for genome-wide STR analysis now enable more systematic study of STR variation across wide evolutionary ranges. In this review, we explore mutation and selection processes that affect STR copy number evolution, and how these processes give rise to varying STR patterns both within and across species. Finally, we review recent examples of functional and adaptive changes linked to STRs.     

A simple method for the detection of neurologic disorders associated with CAG repeat expansion using PCR-microtiter plate hybridization

A new screening method was developed for the detection of CAG expanded alleles in patients with hereditary ataxia using polymerase chain reaction-based microtiter plate hybridization (PCR-MPH). The system can be applied to detect pathologic alleles by hybridization with the immobilized (CAG)48 repeat probe derived from the unrelated gene 'ERDA1' except for the CAG repeats. We examined 10 individuals with SCA3, 10 with Huntington disease and 30 normal controls (31 controls for SCA3) using this method. The results showed that a clear discrimination was possible in all cases. We suggest that this system be made available for mass screening of patients with hereditary ataxia disorders. This report is the first to demonstrate that a PCR-MPH system can be successfully applied to DNA size differentiation in addition to base pair mismatches. Also, our design of the probe is unique in that the probe motif stem from the unrelated gene sequence and not from the synthetic oligonucleotides.     

A minisatellite and a microsatellite polymorphism within 1.5 kb at the human muscle glycogen phosphorylase (PYGM) locus can be amplified by PCR and have combined informativeness of PIC 0.95.

We sequenced a genomic clone (pMCMP1), previously reported to detect a VNTR polymorphism at the PYGM locus, and found a dinucleotide repeat segment (CA)14(GA)25 and a complex (AT)-repeat-rich segment containing 63 repeats spanning 160 bp. Resolution of PCR-amplified genomic DNA from the (CA)(GA) repeat region on DNA sequencing gels revealed a highly informative polymorphism with alleles differing by 2-bp intervals and ranging in size from 156 to 190 bp. Among three racial groups, a total of 18 alleles were observed. Fourteen alleles were observed in Caucasians (PIC 0.89), 12 alleles in American Blacks (PIC 0.89), and 9 alleles in Pima Indians (PIC 0.73). PCR amplification of the (AT) repeat region and resolution of the products on DNA sequencing gels revealed a complex variable length polymorphism with alleles distributed in size from 367 to 970 bp. Twenty-eight alleles were found in American Blacks (PIC 0.94), 6 alleles in Pima Indians (PIC 0.70), and 11 alleles in Caucasians (PIC 0.71). Comparison of the previously described VNTR RFLP alleles visualized by Southern hybridization to the PCR products described in this report demonstrated that the polymorphism described in both assays was identical. However, a larger number of alleles could be detected from the PCR-amplified products. Combined informativeness, PIC 0.95, for the two polymorphisms was determined from haplotype analysis of 100 Caucasian chromosomes. Therefore, for genotyping purposes, informativeness is maximized from using both polymorphisms.     

中国人诱导型一氧化氮合酶基因STR多态性研究

一氧化氮（nitric oxide,NO）作为一种可在细胞间自由扩散的信使分子在神经递质传递和血管舒张调节等生理与病理过程中起着重要作用。NO通过一氧化氮合酶（nitric oxide synthase,NOS）催化L-精氨酸的氧化反应而生成。目前在哺乳动物中已发现细胞来源、表达方式和活性调节不同的3种NOS同工酶,分别为神经原型NOS(meuronal NOS,nNOS）、诱导型NOS(inducible NOS,iNOS）和内皮细胞型NOS（endothelial NOS,eNOS）。3种NOS由位于不同染色体上的基因所编码。人iNOS基因位于第17号染色体长臂(17q11.2～17q12）,全长约37kb,含有26个外显子,iNOS可在多种类型的细胞中通过IL-1、IFN-a、INF-γ等细胞因子和其他介质的刺激作用而诱导表达。iNOS基因5‘-端调控区内存在一个CCTTT串联重复的多态性位点,这一多态性基因座已居北爱尔兰的糖尿病患者中证实与微血管病变有关。另有实验表明,CCTTT串联重复序列的变化对iNOS基因的转录将产生不同影响。目前在东方种族中有关iNOS基因CCTTT串联重复多态性尚未见报道。将303名中国汉族人基因组DNA用于iNOS基因CCTTT串联重复多态性分析,鉴定出了12种等位基因和49种基因型,其中重复17次、18次和19次的等位基因是在人类中首次发现的新等位基因。统计学检验和比较表明,中国汉族人iNOS基因CCTTT串联重复序列的6个等位基因频率与来自英格兰的高加索人差异显著,说明这一多态性位点的等位基因频率分布存在种族差异。在中国正常人群中获得的有关iNOS基因STR多态性的统计资料,对于进一步研究这一多态性基因座与心脑血管疾病的相关性将有重要应用价值。     

Human minisatellite alleles detectable only after PCR amplification.

We present evidence that a proportion of alleles at two human minisatellite loci is undetected by standard Southern blot hybridization. In each case the missing allele(s) can be identified after PCR amplification and correspond to tandem arrays too short to detect by hybridization. At one locus, there is only one undetected allele (population frequency 0.3), which contains just three repeat units. At the second locus, there are at least five undetected alleles (total population frequency 0.9) containing 60-120 repeats; they are not detected because these tandem repeats give very poor signals when used as a probe in standard Southern blot hybridization, and also cross-hybridize with other sequences in the genome. Under these circumstances only signals from the longest tandemly repeated alleles are detectable above the nonspecific background. The structures of these loci have been compared in human and primate DNA, and at one locus the short human allele containing three repeat units is shown to be an intermediate state in the expansion of a monomeric precursor allele in primates to high copy number in the longer human arrays. We discuss the implications of such loci for studies of human populations, minisatellite isolation by cloning, and the evolution of highly variable tandem arrays.     

Repeat instability at human minisatellites arising from meiotic recombination.

Little is known about the role of meiotic recombination processes such as unequal crossover in driving instability at tandem repeat DNA. Methods have therefore been developed to detect meiotic crossovers within two different GC-rich minisatellite repeat arrays in humans, both in families and in sperm DNA. Both loci normally mutate in the germline by complex conversion-like transfer of repeats between alleles. Analysis shows that inter-allelic unequal crossovers also occur at both loci, although at low frequency, to yield simple recombinant repeat arrays with exchange of flanking markers. Equal crossovers between aligned alleles, resulting in recombinant alleles but without change in repeat copy number, also occur in sperm at a similar frequency to unequal crossovers. Both crossover and conversion show polarity in the repeat array and are co-suppressed in an allele showing unusual germline stability. This provides evidence that minisatellite conversion and crossover arise by a common mechanism, perhaps by alternative processing of a meiotic recombination initiation complex, and implies that minisatellite instability is a by-product of meiotic recombination in repeat DNA. While minisatellite recombination is infrequent, crossover rates indicate that the unstable end of a human minisatellite can act as a recombination warm-spot, even between sequence-heterologous alleles.     

High-throughput sequencing of core STR loci for forensic genetic investigations using the Roche Genome Sequencer FLX platform

The analysis and profiling of short tandem repeat (STR) loci is routinely used in forensic genetics. Current methods to investigate STR loci, including PCR-based standard fragment analyses and capillary electrophoresis, only provide amplicon lengths that are used to estimate the number of STR repeat units. These methods do not allow for the full resolution of STR base composition that sequencing approaches could provide. Here we present an STR profiling method based on the use of the Roche Genome Sequencer (GS) FLX to simultaneously sequence multiple core STR loci. Using this method in combination with a bioinformatic tool designed specifically to analyze sequence lengths and frequencies, we found that GS FLX STR sequence data are comparable to conventional capillary electrophoresis-based STR typing. Furthermore, we found DNA base substitutions and repeat sequence variations that would not have been identified using conventional STR typing.     

An exhaustive DNA micro-satellite map of the human genome using high performance computing

The current pace of the generation of sequence data requires the development of software tools that can rapidly provide full annotation of the data. We have developed a new method for rapid sequence comparison using the exact match algorithm without repeat masking. As a demonstration, we have identified all perfect simple tandem repeats (STR) within the draft sequence of the human genome. The STR elements (chromosome, position, length and repeat subunit) have been placed into a relational database. Repeat flanking sequence is also publicly accessible at http://grid.abcc.ncifcrf.gov. To illustrate the utility of this complete set of STR elements, we documented the increased density of potentially polymorphic markers throughout the genome. The new STR markers may be useful in disease association studies because so many STR elements manifest multiallelic polymorphism. Also, because triplet repeat expansions are important for human disease etiology, we identified trinucleotide repeats that exist within exons of known genes. This resulted in a list that includes all 14 genes known to undergo polynucleotide expansion, and 48 additional candidates. Several of these are non-polyglutamine triplet repeats. Other examinations of the STR database demonstrated repeats spanning splice junctions and identified SNPs within repeat elements.     

Vntr Allele Frequency Distributions under the Stepwise Mutation Model: A Computer Simulation Approach

Variable numbers of tandem repeats (VNTRs) are a class of highly informative and widely dispersed genetic markers. Despite their wide application in biological science, little is known about their mutational mechanisms or population dynamics. The objective of this work was to investigate four summary measures of VNTR allele frequency distributions: number of alleles, number of modes, range in allele size and heterozygosity, using computer simulations of the one-step stepwise mutation model (SMM). We estimated these measures and their probability distributions for a wide range of mutation rates and compared the simulation results with predictions from analytical formulations of the one-step SMM. The average heterozygosity from the simulations agreed with the analytical expectation under the SMM. The average number of alleles, however, was larger in the simulations than the analytical expectation of the SMM. We then compared our simulation expectations with actual data reported in the literature. We used the sample size and observed heterozygosity to determine the expected value, 5th and 95th percentiles for the other three summary measures, allelic size range, number of modes and number of alleles. The loci analyzed were classified into three groups based on the size of the repeat unit: microsatellites (1-2 base pair (bp) repeat unit), short tandem repeats [(STR) 3-5 bp repeat unit], and minisatellites (15-70 bp repeat unit). In general, STR loci were most similar to the simulation results under the SMM for the three summary measures (number of alleles, number of modes and range in allele size), followed by the microsatellite loci and then by the minisatellite loci, which showed deviations in the direction of the infinite allele model (IAM). Based on these differences, we hypothesize that these three classes of loci are subject to different mutational forces.     

Microarray-based STR genotyping using RecA-mediated ligation

We describe a novel assay capable of accurately determining the length of short tandem repeat (STR) alleles. STR genotyping is achieved utilizing RecA-mediated ligation (RML), which combines the high fidelity of RecA-mediated homology searching with allele-specific ligation. RecA catalyzes the pairing of synthetic oligonucleotides with one strand of a double-stranded DNA target, in this case a PCR amplicon. Ligation occurs only when two adjacent oligonucleotides are base paired to the STR region without any overlap or gap. RecA activity is required to overcome the inherent difficulty of annealing repeated sequences in register. This assay is capable of determining STR genotypes of human samples, is easily adapted to high throughput or automated systems and can have widespread utility in diagnostic and forensic applications.     

SSR allelic variation in almond (Prunus dulcis Mill.)

Sixteen SSR markers including eight EST-SSR and eight genomic SSRs were used for genetic diversity analysis of 23 Chinese and 15 international almond cultivars. EST- and genomic SSR markers previously reported in species of Prunus, mainly peach, proved to be useful for almond genetic analysis. DNA sequences of 117 alleles of six of the 16 SSR loci were analysed to reveal sequence variation among the 38 almond accessions. For the four SSR loci with AG/CT repeats, no insertions or deletions were observed in the flanking regions of the 98 alleles sequenced. Allelic size variation of these loci resulted exclusively from differences in the structures of repeat motifs, which involved interruptions or occurrences of new motif repeats in addition to varying number of AG/CT repeats. Some alleles had a high number of uninterrupted repeat motifs, indicating that SSR mutational patterns differ among alleles at a given SSR locus within the almond species. Allelic homoplasy was observed in the SSR loci because of base substitutions, interruptions or compound repeat motifs. Substitutions in the repeat regions were found at two SSR loci, suggesting that point mutations operate on SSRs and hinder the further SSR expansion by introducing repeat interruptions to stabilize SSR loci. Furthermore, it was shown that some potential point mutations in the flanking regions are linked with new SSR repeat motif variation in almond and peach. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Features of the DNA fingerprinting probe pITZ1

Stringently controlled plasmids generate DNA fingerprint patterns in mammals when used at low hybridization temperatures. In order to develop a probe for use in paternity testing in cattle we screened a bovine, partial genomic plasmid library with the PCR-amplified ori region of plasmid P1. Of eight isolated clones one generated strong band patterns at high stringency in various mammalian species (data not shown). Sequence analysis revealed an imperfect, compound dinucleotide repeat region, which was PCR-amplified and cloned into the plasmid vector pUC19. Fingerprint results generated by this probe (termed pITZ1) in cattle are compared with the results generated by VNTR-probe pV47, which itself was developed by screening a human chromosome 16 library with tandem repeats of bacteriophage M13. Probe pITZ1 is useful in conjunction with other VNTR-probes for DNA fingerprinting in cattle and donkey populations. The dinucleotide repeat region responsible for the band patterns generated with pITZ1 is close to an Alu -like sequence, which may be involved in eukaryotic replication mechanisms.     

Responder (Rsp) alleles in the segregation distorter (SD) system of meiotic drive in Drosophila may represent a complex family of satellite repeat sequences

In D. melanogaster males carrying Segregation Distorter (SD) second chromosomes, sperm receiving sensitive alleles of the Responder (Rsp) locus are subject to high rates of dysfunction. The Rsp region is located in 2R immediately adjacent to the centromere in heterochromatic band 39, and covers roughly 600 kb of material, of which approximately 85 kb is comprised of several hundred copies of a 240-bp satellite DNA sequence. Cytological observations as well as molecular analysis of rearrangements which bisect h39 indicate that sensitivity of the Rsp target to SD action is also subdivisible, and sensitivities of the component pieces appear to be correlated with copy number of the 240 bp repeat. In an attempt to examine possible higher order sequence structure for these blocks, PCR using single primers derived from a canonical repeat was used to identify potential reversals of direction of tandem arrays; that is, head-to-head or tail-to-tail junctions. Surprisingly, for two different Rsp alleles, only a single such reversal product for each was identified, differing in size and sequence between alleles. Sequencing of PCR products identified diverged copies of the canonical repeats that would not have been found using the levels of DNA stringency employed in earlier studies. Examination of Southern digests and slot-blots for DNA quantification indicates that adding the estimated numbers of such diverged copies to the canonical repeat copies discovered earlier is potentially sufficient to account for the entire 600 kb Rsp region. This adds strength to the hypothesis that this extended family of repeats is in fact the target of SD-mediated sperm dysfunction. Implications of these results for understanding the evolution of repetitive DNA are also discussed.     

Genotyping HapSTR loci: phase determination from direct sequencing of PCR products

HapSTRs combine information from a microsatellite (or simple tandem repeat, STR) with one or more single nucleotide polymorphisms in the DNA sequence immediately flanking the STR. These loci may offer increased power for the estimation of demographic parameters, but also present some challenges for data collection and analysis. We describe a process for inferring HapSTR alleles, including the flanking haplotypes, STR alleles and their phase relative to each other, directly from DNA sequence electropherograms of PCR products from heterozygous individuals. Our approach eliminates the need for more costly and time-consuming processes, such as cloning or acrylamide gel electrophoresis to separate alleles prior to sequencing.     

Early-glycation of apolipoprotein E: effect on its binding to LDL receptor,scavenger receptor A and heparan sulfates

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) causes substantial morbidity afflicting approximately 10% of adult males. Treatment is often empirical and ineffective since the etiology is unknown. Other prostate and genitourinary diseases have genetic components suggesting that CP/CPPS may also be influenced by genetic predisposition. We recently reported a highly polymorphic short tandem repeat (STR) locus near the phosphoglycerate kinase gene within Xq11-13. Because this STR is in a region known to predispose towards other prostate diseases, we compared STR polymorphisms in 120 CP/CPPS patients and 300 control blood donors. Nine distinct allele sizes were detected, ranging from 8 to 15 repeats of the tetrameric STR plus a mutant allele (9.5) with a six base deletion in the flanking DNA sequence. The overall allele size distribution in the CP/CPPS patients differed from controls (Chi-square=19.252, df=8, P=0.0231). Frequencies of two specific alleles, 9.5 and 15, differed significantly in CP/CPPS vs. control subjects and allele 10 differed with marginal significance. Alleles 9.5 and 10 were both more common in CP/CPPS patients than controls while allele 15 was less common. These observations suggest that Xq11-13 may contain one or more genetic loci that predispose toward CP/CPPS. Further investigations involving family studies, larger patient populations, and other control groups may help elucidate this potential genetic predisposition in CP/CPPS.     

Novel Y-chromosome Short Tandem Repeat Variants Detected Through the Use of Massively Parallel Sequencing

Massively parallel sequencing(MPS) technology is capable of determining the sizes of short tandem repeat(STR) alleles as well as their individual nucleotide sequences. Thus, single nucleotide polymorphisms(SNPs) within the repeat regions of STRs and variations in the pattern of repeat units in a given repeat motif can be used to differentiate alleles of the same length. In this study, MPS was used to sequence 28 forensically-relevant Y-chromosome STRs in a set of 41 DNA samples from the 3 major U.S. population groups(African Americans, Caucasians, and Hispanics).The resulting sequence data, which were analyzed with STRait Razor v2.0, revealed 37 unique allele sequence variants that have not been previously reported. Of these, 19 sequences were variations of documented sequences resulting from the presence of intra-repeat SNPs or alternative repeat unit patterns. Despite a limited sampling, two of the most frequently-observed variants were found only in African American samples. The remaining 18 variants represented allele sequences for which there were no published data with which to compare. These findings illustrate the great potential of MPS with regard to increasing the resolving power of STR typing and emphasize the need for sample population characterization of STR alleles.