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.     

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.     

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.     

Escherichia coli DNA polymerase IV contributes to spontaneous mutagenesis at coding sequences but not microsatellite alleles

Slipped strand mispairing during DNA synthesis is one proposed mechanism for microsatellite or short tandem repeat (STR) mutation. However, the DNA polymerase(s) responsible for STR mutagenesis have not been determined. In this study, we investigated the effect of the Escherichia colidinB gene product (Pol IV) on mononucleotide and dinucleotide repeat stability, using an HSV-tk gene episomal reporter system for microsatellite mutations. For the control vector (HSV-tk gene only) we observed a statistically significant 3.5-fold lower median mutation frequency in dinB(-) than dinB(+) cells (p<0.001, Wilcoxon Mann Whitney Test). For vectors containing an in-frame mononucleotide allele ([G/C](10)) or either of two dinucleotide alleles ([GT/CA](10) and [TC/AG](11)) we observed no statistically significant difference in the overall HSV-tk mutation frequency observed between dinB(+) and dinB(-) strains. To determine if a mutational bias exists for mutations made by Pol IV, mutational spectra were generated for each STR vector and strain. No statistically significant differences between strains were observed for either the proportion of mutational events at the STR or STR specificity among the three vectors. However, the specificity of mutational events at the STR alleles in each strain varied in a statistically significant manner as a consequence of microsatellite sequence. Our results indicate that while Pol IV contributes to spontaneous mutations within the HSV-tk coding sequence, Pol IV does not play a significant role in spontaneous mutagenesis at [G/C](10), [GT/CA](10), or [TC/AG](11) microsatellite alleles. Our data demonstrate that in a wild type genetic background, the major factor influencing microsatellite mutagenesis is the allelic sequence composition.     

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.     

A hemizygous short tandem repeat polymorphism 3′ to the human phosphoglycerate kinase gene

The human phosphoglycerate kinase (PGK) gene is located within Xq11-Xq13, a region implicated in genitourinary diseases including: prostate cancer, androgen insensitivity, perineal hypospadias, and other genetic abnormalities. The PGK gene and the androgen receptor gene are in linkage disequilibrium. PGK has been mapped extensively for nuclease-sensitive sites, methylation sites, and flanking DNA sequences. A PGK-associated BstXI polymorphism has been used to determine clonality of neoplastic tissues. Using fluorescent PCR product analysis and DNA sequencing, we discovered that a short tandem repeat (STR) in the 3 flanking region of the PGK gene is polymorphic. Among 231 individuals, there were nine distinct alleles, including eight based on variations in the number of TATC repeats. The PGK STR demonstrated hemizygosity, consistent with its X-chromosomal location and with an absence of cross-hybridizing autosomal homologs. The polymorphic PGK STR shows promise for rapid investigation of neoplastic clonality, for personal identification, and for studies of inherited predisposition to urologic disorders.     

Mutational bias in penguin microsatellite DNA

Analysis of nucleotide sequence variation at a microsatellite DNA locus revealed extensive size homoplasy of alleles in Adélie penguins (Pygoscelis adeliae). Variation in the flanking regions at this locus allowed discrimination between mechanisms proposed for length changes in microsatellite DNA alleles. We further examined the structure of alleles for the same microsatellite DNA locus across 11 additional species of penguin (Spheniscidae) by mapping allele sequences onto an independent penguin phylogeny. Our analysis indicated that the repeat motifs appear to have evolved independently on several occasions. We observed sequence instability in the region bordering the repeat tract with a transversional bias predominating. We propose that this bias results from inaccurate DNA replication owing to the sequence context of this repeat tract. Because we show that regions flanking repeat sequences exhibit this mutational bias, this cautions against the use of such regions for phylogeny reconstruction.     

A study on the relationship between TCTA tetranucleotide polymorphism of the HPRT gene and primary hyperuricemia

We examined polymorphism of the TCTA tetranucleotide sequence in the 3rd intron of the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene in the Han population of Ningxia Province in China. We also looked for a possible relationship between STR polymorphism in the 3rd intron of the HPRT gene and primary hyperuricemia. We used Chelex-100 to extract DNA, then PCR, PAGE and silver staining for allele genotyping and DNA sequencing to obtain the distribution of the alleles. We found, for the first time, that there is high STR polymorphism in the 3rd intron of the HPRT gene. We detected 5 STR alleles in this intron in the Han population of Ningxia Province, with 15 genotypes in females; significant differences were observed in the distribution of alleles and genotypes between control and patient groups for both males and females. Alleles of the TCTA repeat in the 3rd intron of the HPRT gene were found to be associated with primary hyperuricemia; consequently, these alleles may be considered risk factors for primary hyperuricemia.     

Short tandem repeat polymorphism in the flanking region of the human phosphoglycerate kinase gene in a Japanese population

The human phosphoglycerate kinase (PGK1) gene is located within Xqll-Xql3 and is closely linked to the androgen receptor gene within a region implicated in a number of X-chromosome-linked urologic disorders. A polymorphism of a TATC short tandem repeat (STR) is present downstream from the PGK1 3' nuclease-sensitive site. We present the PGK1 flanking STR sequence and population genetic data for 190 Japanese males and 83 Japanese females. Ten STR alleles and 29 genotypes were identified in the population. Five alleles--*10, *11, *12, *13, and *14--were common in the Japanese with frequencies greater than 10%. No significant deviations from Hardy-Weinberg equilibrium were established. The power of discrimination was 0.993 for females and 0.819 for males; heterozygosity was 0.759 for females; and the polymorphic information content was 0.936. These data indicate that this STR locus shows a high degree of polymorphism in this Japanese population and may prove to be a useful genetic marker in forensic medicine, in determining the clonality of neoplasms, and potentially in studying predisposition to prostate cancer and other urologic diseases.     

The single strand conformational analysis of cattle and human single nucleotide polymorphisms may be biased towards specific sequence motifs that minimize local secondary structure of single strand DNA

Single strand conformational polymorphisms (SSCP) resulting from point mutations were found to be associated preferentially with two DNA sequence motifs. These motifs are (1) three or more of the same base but in which the polymorphism is not due to length variation and (2) a region of polypurine or polypyrimidine bases. These motifs were identified after SSCP alleles from cattle were sequenced. The sequence difference and flanking sequence for each single nucleotide polymorphism are shown. The motifs were also found in SSCP from humans chosen at random from the literature, in which the alleles had been sequenced. There is a low level of complementarity of adjacent bases in these motifs and they should represent regions of low secondary structure in the single stranded DNA. Regions of high secondary structure, such as palindromes, were found in the same sample to have allelic variation that was not detected by SSC analysis. These results give a rule of thumb for selecting the particular part of a DNA fragment to be selected for testing for polymorphisms, but this rule clashes with rules used to design primers to amplify sequences using the PCR, namely, minimise hydrogen bonding within and between primers and reduce self-complementarity.     

An Evaluation of Evolutionary Constraints on Microsatellite Loci Using Null Alleles

A test to evaluate constraints on the evolution of single microsatellite loci is described. The test assumes that microsatellite alleles that share the same flanking sequence constitute a series of alleles with a common descent that is distinct from alleles with a mutation in the flanking sequence. Thus two or more different series of alleles at a given locus represent the outcomes of different evolutionary processes. The higher rate of mutations within the repeat region (10(-3) or 10(-4)) compared with that of insertion/deletion or point mutations in adjacent flanking regions (10(-9)) or with that of recombination between the repeat and the point mutation (10(-6) for sequences 100 bp long) provides the rationale for this assumption. Using a two-phase, stepwise mutation model we simulated the evolution of a number of independent series of alleles and constructed the distributions of two similarity indices between pairs of these allele series. Applying this approach to empirical data from locus AG2H46 of Anopheles gambiae resulted in a significant excess of similarity between the main and the null series, indicating that constraints affect allele distribution in this locus. Practical considerations of the test are discussed.     

Comparative analysis of allele polymorphism of three short tandem repeats in the Russian, Uzbek and Georgian populations

The allele polymorphism of the AGC short tandem repeat (STR) of exon 1 of the androgen receptor (AR) gene located in Xq11-12, ATCT STR of intron 40 of the von Willebrand factor (vWF) gene located in chromosome 12p12, and AGAT STR of an anonymous DNA sequence (STRX1) from the short arm of the X chromosome was analyzed in the Georgian, Uzbek, and Russian populations. Polymerase chain reaction (PCR) with DNA of unrelated persons revealed 14 AR, 7 vWF, and 7 STRX1 alleles in Georgians; 14, 8, and 6 alleles, respectively, in Uzbeks; and 16, 8, and 9 alleles, respectively, in Russians. The heterozygosity at these STR was 0.61, 0.78, and 0.46 in Georgians; 0.60, 0.83, and 0.44 in Uzbeks; and 0.80, 0.70, and 0.58 in Russians. The correspondence of genotype frequencies to the Hardy-Weinberg equilibrium was observed with AR STR in Russians and Uzbeks, STRX1 STR in Georgians, and vWF in all three populations. A significant deviation from the equilibrium was found for STRX1 in Russians and Uzbeks and AR in Georgians. The potential of individualization was 0.05 for AR, 0.13 for vWF, and 0.18 for STRX1 in Georgians; 0.04, 0.09, and 0.13, respectively in Uzbeks; and 0.05, 0.14, and 0.07, respectively, in Russians. The allele and genotype frequency distributions of each STR were analyzed in all three populations. Allele frequencies in the populations were compared by the Kolmogorov-Smirnov test. The Russian population significantly differed in allele frequencies of the three STR from Uzbeks and in those of STRX1 and AR from Georgians. Georgians and Uzbeks significantly differed in vWF and STRX1 frequencies. The possibility of using the three STR in molecular diagnosis of the corresponding monogenic diseases, population genetic studies, and personal identification is discussed.     

STRBase: a short tandem repeat DNA database for the human identity testing community

The National Institute of Standards and Technology (NIST) has compiled and maintained a Short Tandem Repeat DNA Internet Database (http://www.cstl.nist.gov/biotech/++ +strbase/) since 1997 commonly referred to as STRBase. This database is an information resource for the forensic DNA typing community with details on commonly used short tandem repeat (STR) DNA markers. STRBase consolidates and organizes the abundant literature on this subject to facilitate on-going efforts in DNA typing. Observed alleles and annotated sequence for each STR locus are described along with a review of STR analysis technologies. Additionally, commercially available STR multiplex kits are described, published polymerase chain reaction (PCR) primer sequences are reported, and validation studies conducted by a number of forensic laboratories are listed. To supplement the technical information, addresses for scientists and hyperlinks to organizations working in this area are available, along with the comprehensive reference list of over 1300 publications on STRs used for DNA typing purposes.     

Isolation and characterization of two alleles of the chicken cytochrome c gene.

Analysis of total chicken DNA by genomic blot hybridization indicates that only one cytochrome c gene exists in the chicken genome. The two alleles of this single cytochrome c gene have been isolated from a Charon 4A-chicken genomic library. This isolation made use of the yeast CYC1 cytochrome c gene as a specific hybridization probe. The 2 chicken alleles, CC9 and CC10, have been sequenced. The amino acid sequence predicted by these 2 alleles is identical, and agrees with the published chicken cytochrome c protein sequence. The flanking regions of these 2 alleles exhibit approximately 1% divergence, indicating a very limited polymorphism. Comparative sequence analysis with the flanking regions of previously isolated cytochrome c genes (yeast and rat) indicate no significant regions of homology. The presence of only one cytochrome c-like sequence in the chicken genome is in striking contrast with mammalian genomes, which contain as many as 20-30 cytochrome c-like sequences.     

Effect of DNA flanking sequence on charge transport in short DNA duplexes

Several factors can influence charge transport (CT)-mediated DNA, such as sequence, distance, base stacking, base pair mismatch, conformation, tether length, etc. However, the DNA context effect or how flanking sequences influence redox active drugs in the DNA CT reaction and later in DNA enzymatic repair and synthesis is still not well understood. The set of seven DNA molecules in this study have been characterized well for the study of flanking sequence effects. These DNA duplexes are formed from self-complementary strands and contain the common central four-base sequence 5'-A-G-C-T-3', flanked on both sides by either (AT)(n) or (AA)(n) (n = 2, 3, or 4) or AA(AT)(2). UV-vis, fluorescence, UV melting, circular dichroism, and cyclic voltammetry experiments were used to study the flanking sequence effect on CT-mediated DNA by using daunomycin or adriamycin cross-linked with these seven DNA molecules. Our results showed that charge transport was related to the flanking sequence, DNA melting free energy, and ionic strength. For (AA)(n) or (AT)(n) species of the same length, (AA)(n) series were more stable and more efficient CT was observed through the (AA)(n) series. The same trend was observed for (AA)(n)() and (AT)(n) series at different ionic strengths, further supporting the idea that flanking sequence can result in different base stacking and modulate charge transport through these seven DNA molecules.     

Common benzothiazole and benzoxazole fluorescent DNA intercalators for studying Alzheimer Aβ1-42 and prion amyloid peptides

DNA-based methods for human identification principally rely upon genotyping of short tandem repeat (STR) loci. Electrophoretic-based techniques for variable-length classification of STRs are universally utilized, but are limited in that they have relatively low throughput and do not yield nucleotide sequence information. High-throughput sequencing technology may provide a more powerful instrument for human identification, but is not currently validated for forensic casework. Here, we present a systematic method to perform high-throughput genotyping analysis of the Combined DNA Index System (CODIS) STR loci using short-read (150 bp) massively parallel sequencing technology. Open source reference alignment tools were optimized to evaluate PCR-amplified STR loci using a custom designed STR genome reference. Evaluation of this approach demonstrated that the 13 CODIS STR loci and amelogenin (AMEL) locus could be accurately called from individual and mixture samples. Sensitivity analysis showed that as few as 18,500 reads, aligned to an in silico referenced genome, were required to genotype an individual (>99% confidence) for the CODIS loci. The power of this technology was further demonstrated by identification of variant alleles containing single nucleotide polymorphisms (SNPs) and the development of quantitative measurements (reads) for resolving mixed samples.     

A large population genetic study of 15 autosomal short tandem repeat loci for establishment of Korean DNA Profile Database

Genotyping of highly polymorphic short tandem repeat (STR) markers is widely used for the genetic identification of individuals in forensic DNA analyses and in paternity disputes. The National DNA Profile Databank recently established by the DNA Identification Act in Korea contains the computerized STR DNA profiles of individuals convicted of crimes. For the establishment of a large autosomal STR loci population database, 1805 samples were obtained at random from Korean individuals and 15 autosomal STR markers were analyzed using the AmpFlSTR Identifiler PCR Amplification kit. For the 15 autosomal STR markers, no deviations from the Hardy-Weinberg equilibrium were observed. The most informative locus in our data set was the D2S1338 with a discrimination power of 0.9699. The combined matching probability was 1.521 × 10⁻¹⁷. This large STR profile dataset including atypical alleles will be important for the establishment of the Korean DNA database and for forensic applications.     

Parentage analysis for three Romanian families by DNA-fingerprinting

In forensic medicine, DNA fingerprinting for human identification and paternity testing is becoming a necessary procedure. The genetic locus D1S80 (MCT118) with Hinf I polymorphism of its 5' flanking sequence, HUMTH01 and D21S11 have been successfully amplified from human genomic DNA isolated from blood (50 ng from each sample) by the polymerase chain reaction (PCR) using oligonucleotide primers complementary to the flanking sequences as primers for amplification. DNA bands were detected by ethidium bromide staining after electrophoresis on agarose gels or high-resolution SDS-PAGE. Analysis of these VNTR loci was thus achieved without the need for Southern blot or radioactive material. The small size of the DNA fragments produced in the PCR amplification permitted good resolution of individual alleles. The precise specification of the number of tandem repeats present in each allelic fragment was reproducible from one analysis to another. The aim of this study includes three paternity testing cases; they are the first three human DNA-fingerprints performed in Romania.     

Short tandem repeat typing technologies used in human identity testing

Short tandem repeat (STR) typing methods are widely used today for human identity testing applications including forensic DNA analysis. Following multiplex PCR amplification, DNA samples containing the length-variant STR alleles are typically separated by capillary electrophoresis and genotyped by comparison to an allelic ladder supplied with a commercial kit. This article offers a brief perspective on the technologies and issues involved in STR typing.