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PubMed1999

Analysis and interpretation of short tandem repeat microvariants and three-banded allele patterns using multiple allele detection systems.

The Palm Beach County Sheriffs Office (PBSO) Crime Laboratory and the Alabama Department of Forensic Sciences (ADFS) have validated and implemented analysis of short tandem repeat (STR) sequences on casework using silver staining kit and SYBR Green I detection systems and are presently validating fluorescently tagged STR alleles using the Hitachi FMBIO 100 instrument. Concurrently, the Broward County Sheriff's Office (BSO) Crime Laboratory is validating the ABI Prism310 Genetic Analyzer capillary electrophoresis STR detection system (ABI CE310) from Perkin Elmer Applied BioSystems. During the course of analyzing over 10,000 individuals for the STR loci CSF1PO, TPOX and THO1 (CTT) using silver staining for allele detection, 42 samples demonstrated alleles that were "off ladder," contained three-banded patterns at a single locus, or exhibited an apparent THO1 "9.3,10" allele pattern. PBSO, ADFS and BSO Crime Laboratories have collaborated on the verification of the allele patterns observed in these 42 samples using the following allele detection systems: (1) manual silver staining, (2) SYBR Green I staining, and/or (3) fluorescently tagged amplified products separated by polyacrylamide gel electrophoresis or capillary electrophoresis followed by laser detection. Regardless of the CTT allele detection system utilized, concordant results were obtained for 41 of the 42 samples. The only exception was a sample in which a wide band within the THO1 locus was identified as a THO1 "9.3, 10" genotype by silver staining kit and SYBR Green I staining but was verified to be a THO1 "9.3" homozygote by all other allele detection systems. Manual allele detection could readily identify microvariants, as a visual assessment of stained gels clearly shows that alleles do not migrate coincident with well-characterized allele size standards. As would be predicted, however, the manual detection systems did not provide adequate resolution to approximate the basepair size for off-ladder variants. All fluorescent software program systems were consistent in designating alleles "not in range" or "off ladder," thereby indicating true microvariants. All single-locus three-banded patterns were detected using all of the STR multiplex systems. In addition, individual locus-specific primers verified multiplexed amplified products were specific for the locus in question.

Alleles↗
PubMed2001

New polymorphic short tandem repeats for PCR-based Charcot-Marie-Tooth disease type 1A duplication diagnosis.

BACKGROUND: Charcot-Marie-Tooth disease type 1A (CMT1A) accounts for 70-90% of cases of CMT1 and is most frequently caused by the tandem duplication of a 1.4-Mb genomic fragment on chromosome 17p12. Molecular diagnosis of CMT1A has been based primarily on pulsed-field electrophoresis, fluorescence in situ hybridization, polymorphic allele dosage analysis, and quantitative PCR. We sought to improve the fidelity and applicability of PCR-based diagnosis by developing a panel of novel, highly polymorphic short tandem repeats (STRs) from within the CMT1A duplicated region. METHODS: We used a recently available genomic sequence to identify potentially polymorphic simple repeats. We then amplified these sequences in a multiethnic cohort of unaffected individuals and assessed the heterozygosity and number of alleles for each STR. Highly informative markers were then tested in a set of previously diagnosed CMT1A duplication patients, and the ability to identify the genomic duplication through the presence of three bands was assessed. RESULTS: We identified 34 polymorphic markers, 15 of which were suitable for CMT1A diagnosis on the basis of high heterozygosity in different ethnic groups, peak uniformity, and a large number of alleles. On the basis of the fluorescent dye and allele range of each marker, we developed two panels, each of which could be analyzed concurrently. Panel 1, which comprised 10 markers, detected 37 of 39 duplications, whereas panel 2, which comprised the remaining 5 markers, identified 21 of 39 duplications. Through the combination of both panels, we identified 39 of 39 duplications in previously diagnosed CMT1A patients. CONCLUSIONS: The newly developed 15-marker set has the capability of detecting > 99% of duplications and thus is a powerful and versatile diagnostic tool.

Asian People↗
PubMed2004

[Genetic polymorphism of short tandem repeat loci D1S549, D3S1754 and D12S375 in Qingdao Han population].

OBJECTIVE: To know the genotype and allele frequency distribution of D1S549, D3S1754 and D12S375 in Chinese Han population in the Qingdao area and to study the three short tandem repeat(STR) loci for genetic application. METHODS: ACD-blood specimens were collected from the unrelated individuals in Qingdao. The DNA samples were extracted with the use of Chelex method and were amplified by polymerase chain reaction (PCR) technique. The PCR products were analyzed by polyacrylamide gel electrophoresis and were visualized by silver staining. RESULTS: Eight alleles were found at D1S549 locus, eight alleles at D3S1754 locus and five alleles at D12S375 locus, and 22, 19 and 14 genotypes were identified respectively. No deviation from Hardy-Weinberg equilibrium was observed in the three loci. The heterozygosities expected of them were 0.7988, 0.7087 and 0.75 respectively. The exclusion probability was calculated as 0.6592 for D1S549, and 0.5605 for D3S1754, and 0.5864 for D12S375. The discriminating power of the three loci were 0.9143, 0.8382 and 0.8861. Comparison of the allelic frequencies in Qingdao area with those in Hans of Chengdu area by chi-square test showed a difference statistically significant at D1S549 locus but no difference at D3S1754 and D12S375 loci. CONCLUSION: This study reveals the structure of the three loci and the obtained data are beneficial to understanding the population genetics in Chinese Han population. All of the three loci have higher chance of exclusion and higher discriminating power, and they will be useful markers for individual identification, paternity test and genetics purposes.

China↗
PubMed2004

Short tandem repeat analysis to monitor chimerism in macaca fascicularis.

Chimerism assessment following bone marrow transplantation (BMT) in cynomolgus monkeys (cynos) has been hampered by the lack of good engraftment markers. In human BMT, such markers have been provided by short tandem repeat (STR) loci. We tested the idea that techniques effective for detecting human STR could be readily adapted to cynos. Genomic DNA was extracted from cyno unseparated blood or peripheral cell subsets. With only slight modifications, reagents for detecting human STR alleles were used to amplify and detect cyno STRs and to quantitate allelic mixtures on an automated sequencer. Of the 15 STR loci tested, only CSF1PO, D18S51, and FGA successfully amplified, with seven, seven and two alleles, respectively. CSF1PO and D18S51 heterozygosity (80% and 55%, respectively) allowed use of these two loci for chimerism quantitation after BMT. The successful adaptation of human STR reagents to monitor chimerism in transplanted cynos will facilitate the use of this species in preclinical tolerance studies.

Animals↗
PubMed2006

Quantification of chimerism within peripheral blood, bone marrow and purified leukocyte subsets: comparison of singleplex and multiplex PCR amplification of short tandem repeat (STR) loci.

OBJECTIVE AND METHODS: Chimerism analysis has become a routine diagnostic procedure after haematopoietic allogeneic stem cell transplantation for early detection of relapse of disease or graft failure. Whereas some centres developed individual in-house short tandem repeat (STR) systems, others prefer commercial multiplex PCR systems. However, little is known about inter-assay variation, which could have a significant impact on treatment decision. We therefore compared two commercial multiplex PCR kits with our in-house STR system using different sample sources, such as peripheral blood (PB), bone marrow (BM) and specific leukocyte subsets. RESULTS: Fifty samples of eighteen paediatric patients were analysed. For neither material, PB, BM and leukocyte subtypes, a significant difference between the STR systems tested was observed. Chimerism analyses of each single STR primer, which is component of both the in-house and the commercial STR system, did not reveal significant differences. CONCLUSION: Our analysis demonstrates that similar results can be obtained with both assays, even when using various sample sources. Further evaluation of different test systems will help to increase interlaboratory standardisation of chimerism analyses for early clinical intervention.

Adolescent↗
PubMed2001

Standardisation of multiplex fluorescent short tandem repeat analysis for chimerism testing.

To evaluate the origin of cells after allogeneic haematopoietic stem cell transplantation we optimised and evaluated two commercially available systems (AmpFlSTR Profiler Plus and GenePrint Powerplex-16) which are based on multiplex fluorescent short tandem repeat (STR) analysis. A standard procedure for interpretation of electropherographs was found essential to obtain reproducible results. On the basis of the relative length of donor and recipient alleles, TYPE-I (no shared alleles are used to calculate chimerism), TYPE-II (one shared and one unshared allele is used to calculate chimerism) or TYPE-III (not informative) allelic distribution types were distinguished. Also, stutter peaks were recognised as an important criterion to exclude a marker for analysis. Intralaboratory and multicentre evaluation of the AmpFlSTR Profiler Plus system showed that mixed blood samples could be determined with an absolute deviation of less than 2%. A sensitivity threshold was set at 5% for TYPE-I and 10% for TYPE-II markers since relative imprecision increases at low chimerism values. No significant difference of calculated chimerism values was observed between STR markers shared between both systems. By monitoring 26 allogeneic peripheral blood stem cell transplants, the applicability of the proposed method was demonstrated.

Alleles↗
PubMed2005

Multiplex fluorescent analysis of four short tandem repeats for rapid haemophilia A molecular diagnosis.

Indirect molecular diagnosis of hemophiliaA (HA) is carried out by analyzing intragenic polymorphic markers described along the coagulation factorVIII (FVIII) gene. Several studies have demonstrated that the two commonly used intronic short tandem repeats (STR13 and STR22) located in the FVIII gene are highly informative for this task. Two extragenic markers closely linked to FVIII (DXS1073 and DXS1108) have also been described as valuable tools for gene tracking. The objective of the present work was to develop a rapid, single-tube automated method to simultaneously analyze these four STRs. Consistent amplification was achieved by quadruplex fluorescent PCR and the products were analyzed by capillary electrophoresis. Validation of the method included DNA analysis of 88 individuals from a control population, 45 HA patients and 32 individuals from 10 HA-affected families. Statistical study showed that the STR13, STR22 and DXS1108 loci were in significant linkage disequilibrium, whereas DXS1073 was not. Nevertheless, the combination of the four markers offered a high heterozygosity rate (>90%) that improved tracing of FVIII gene inheritance. Optimal results with application to single cells in a HA preimplantation genetic diagnosis (PGD) protocol demonstrated the sensitivity of the technique. In conclusion, the automated fluorescent method described is an extremely rapid, simple and highly informative one that is easy to standardize and allows direct comparison of results among different groups working with genetic counseling, prenatal diagnosis and PGD in HA-affected families.

Factor VIII↗
PubMed1998

Short tandem repeat (STR) locus HUMD8S306 in a large population sample from Germany.

Applied DNA typing in medico-legal investigations, in criminalistic practice, and in paternity cases often relies on high inclusion and exclusion probabilities. For that reason, the short autosomal tandem repeat locus D8D306 was validated for forensic use and incorporated into a nonoverlapping multiplex reaction with HUMDHFRP2 and HUMCD4: The allele frequencies of D8S306 in four different regions of Germany (n = 1220 alleles) were determined for use in a population database; the allele distributions did not significantly deviate from each other. The hererozygosity of D8S306 is 83%, expected exclusion chance in stain cases is 96% (paternity cases: 69%), the lowest amount of successfully amplified DNA was 30 pg. The alleles are in Hardy-Weinberg equilibrium.

Alleles↗
PubMed2000

Short tandem repeat methodology for genotypic identification of single-person versus multi-person use of syringes.

OBJECTIVE: To develop laboratory methods to differentiate between single- versus multi-person use of syringes by injection drug users. METHODS: Forensic short tandem repeat (STR) genetic analysis was undertaken to cross-validate a test panel of trace blood contents from syringes representing single- versus multi-person syringe use. Laboratory-simulated scenarios of needle sharing generated 34 syringe washes that were blinded for evaluation. Polymerase chain reaction was used to amplify the polymorphic STR locus D6S502 from blood trace contents in used syringes. Alleles were sized and quantified using a commercial gene sequencer. A statistical algorithm was developed to determine the number of alleles present in the amplified DNA fragments. Syringes with more than two expected alleles were considered to represent multi-person syringe use. Sensitivity, specificity and the kappa coefficient were calculated. RESULTS: Allelic matrix-based analysis of alleles from the single STR successfully characterized single-use (n = 12) and multiple-use (n = 22) syringes with 68% sensitivity and 100% specificity upon re-analysis. The extent of agreement over and above chance (kappa = 0.6; P < 0.0001) indicated good agreement for differentiating single- versus multi-person syringe use. CONCLUSIONS: These findings suggest that improved genotypic STR analysis of syringe material could be an adjunct to methods for validating self-reported needle sharing, conducting behavioral surveillance of needle-sharing behaviors, and evaluating interventions such as needle-exchange programs. Assays based on multiple STR loci will undoubtedly improve upon the promising results obtained from laboratory simulations of needle sharing.

Alleles↗
PubMed1999

Two-color multiplexed analysis of eight short tandem repeat loci with an electrophoretic microdevice.

Single-channel microfabricated electrophoretic devices equipped with a dual-wavelength laser-induced fluorescence detection system were used for the fast analysis of an eight-loci, two-color multiplex short tandem repeat (STR) system for human identification. Routine analyses of the eight loci (CSF1PO, TPOX, TH01, vWA and D16S539, D7S820, D13S317, D5S818), requiring four-base resolution, were performed in only 2 min. Specific analyses for a microvariant allele (allele 9.3 of the TH01 locus) demanded single-base resolution and was performed in less than 10 min. The high accuracy of the microdevice for real-world STR sample analyses was demonstrated by comparison with conventional slab-gel electrophoresis. Our results show that a fast multiwavelength multichannel electrophoretic microsystem will be capable of routinely processing thousands of complex STR samples per day.

Color↗
PubMed2005

The DUB/USP17 deubiquitinating enzymes, a multigene family within a tandemly repeated sequence.

Here we report the identification of 10 human, 1 murine, and 2 rat ORFs, all of which represent additional members of the DUB/USP17 family of deubiquitinating enzymes. In addition, we demonstrate that this family constitutes part of a tandemly repeated sequence conserved throughout humans, mice, and rats. Furthermore, upon examination of the known family members we have found that the multiple genes observed, in contrast to other gene families, have arisen due to the independent expansion of an ancestral sequence within each species. This premise is further strengthened by the observation that the murine and rat genes span two exons while their human counterparts have one. These observations, in conjunction with previous work demonstrating that the DUB/USP17's are cytokine inducible and that they regulate both cell growth and survival, suggest that the DUB/USP17's are a large highly conserved family of genes that may play an important role in controlling cell fate.

Amino Acid Sequence↗
PubMed2000

Tandemly repeated sequences in mtDNA control region of whitefish, Coregonus lavaretus.

Length variation of the mitochondrial DNA control region was observed with PCR amplification of a sample of 138 whitefish (Coregonus lavaretus). Nucleotide sequences of representative PCR products showed that the variation was due to the presence of an approximately 100-bp motif tandemly repeated two, three, or five times in the region between the conserved sequence block-3 (CSB-3) and the gene for phenylalanine tRNA. This is the first report on the tandem array composed of long repeat units in mitochondrial DNA of salmonids.

Animals↗
PubMed2006

Specific interactions of divalent metal ions with a DNA duplex containing the d(CA)n/(GT)n tandem repeat.

Divalent metal ions are essential for maintaining functional states of the DNA molecule. Their participation in DNA structure is modulated by the base sequence and varies depending on the nature of the ion. The present investigation addresses the interaction of Ca2+ ions with a tandem repeat of two CA dinucleotides, (CA)2/(TG)2. The binding of Ca2+ to the repeat is monitored by nuclear magnetic resonance (NMR) spectroscopy using chemical shift mapping. Parallel experiments monitor binding of Mg2+ ions to the repeat as well as binding of each ion to a DNA duplex in which the (CA)2/(TG)2 repeat is eliminated. The results reveal that the direction and the magnitude of chemical shift changes induced by Ca2+ ions in the NMR spectra of the repeat are different from those induced by Mg2+ ions. The differences between the two cations are significantly diminished by the elimination of the (CA)2/(TG)2 repeat. These findings suggest a specific interaction of Ca2+ ions with the (CA)2/(TG)2 motif. The specificity of the interaction resides in the two A-T base pairs of the repeat, and it involves the major groove of the first A-T base pair and both grooves of the second A-T base pair.

Calcium↗
PubMed2003

Tandemly repeated DNA sequences and centromeric chromosomal regions of Arabidopsis species.

Despite their common function, centromeric DNA sequences are not conserved between organisms. Most centromeres of animals and plants so far investigated have now been shown to consist of large blocks of tandemly repeated satellite sequences that are embedded in recombination-deficient heterochromatic regions. This central domain of satellite sequences that is postulated to mediate spindle attachment is surrounded by pericentromeric sequences incorporating various classes of repetitive sequences often including retroelements. The centromeric satellite DNA sequences are amongst the most rapidly evolving sequences and pose some fundamental problems of maintaining function. In this overview, we will discuss work on centromeric repetitive sequences in Arabidopsis thaliana and its relatives, and highlight some of the common features that are emerging when analysing closely related species.

Arabidopsis↗
PubMed2005

Characterization of rDNAs and tandem repeats in the heterochromatin of Brassica rapa.

We describe the morphology and molecular organization of heterochromatin domains in the interphase nuclei, and mitotic and meiotic chromosomes, of Brassica rapa, using DAPI staining and fluorescence in situ hybridization (FISH) of rDNA and pericentromere tandem repeats. We have developed a simple method to distinguish the centromeric regions of mitotic metaphase chromosomes by prolonged irradiation with UV light at the DAPI excitation wavelength. Application of this bleached DAPI band (BDB) karyotyping method to the 45S and 5S rDNAs and 176 bp centromere satellite repeats distinguished the 10 B. rapa chromosomes. We further characterized the centromeric repeat sequences in BAC end sequences. These fell into two classes, CentBr1 and CentBr2, occupying the centromeres of eight and two chromosomes, respectively. The centromere satellites encompassed about 30% of the total chromosomes, particularly in the core centromere blocks of all the chromosomes. Interestingly, centromere length was inversely correlated with chromosome length. The morphology and molecular organization of heterochromatin domains in interphase nuclei, and in mitotic and meiotic chromosomes, were further characterized by DAPI staining and FISH of rDNA and CentBr. The DAPI fluorescence of interphase nuclei revealed ten to twenty conspicuous chromocenters, each composed of the heterochromatin of up to four chromosomes and/or nucleolar organizing regions.

Arabidopsis↗
PubMed2000

[Variable number of tandem repeats as an evidence of engraftment status after allogeneic peripheral blood stem cell transplantation].

OBJECTIVE: To evaluate engraftment status of patients after allogeneic peripheral blood stem cell transplantation (allo-PBSCT) based on red blood cell type, karyotype and variable number of tandem repeats (VNTR). METHODS: Amplification of 3 loci of VNTR in vitro by polymerase chain reaction (PCR) was preformed to detect the polymorphism of 15 patients post-PBSCT. Furthermore, analysis of karyotype in 7 sex-mismatched patients and red blood cell antigen switching in 3 ABO blood type mismatched patients were also employed. RESULTS: VNTR analysis in 15 patients revealed complete chimerism in 11 patients and mixed chimerism in 4. In the 7 sex-mismatched patients, karyotype analysis showed types of their donors. As for blood type analysis, it was after 2 months that the first patient transformed to his donor's type. One patient showed mixed status 7 months after transplantation. CONCLUSION: Amplification of VNTR loci by PCR for documenting engraftment status after allo-PBSCT is simple and sensitive. This technology is especially suitable for patients who have received stem cells with the same sex, same blood type and identical HLA type. Examination with several different VNTR loci will improve the power of discrimination.

Adolescent↗
PubMed2003

Multiplex amplification and fluorimetric detection of short tandem repeats for mixed chimerism after bone marrow transplant.

We present the clinical application of a simple method for mixed chimerism analysis after allogeneic bone marrow (BM) or peripheral blood stem cell (PBSC) transplant. A commercial kit which enables multiplex amplification of 9 highly polymorphic short tandem repeats in a single reaction tube was used. Molecular size and relative quantities of each amplified fragment are subsequently measured using an automated fluorimeter. The sensitivity and linearity were tested using whole blood or genomic DNA from two subjects, mixed in various proportions from 0.25% to 99.75%. In 70 donor-recipient pairs the median number of informative alleles for the assessment of relapse was 5.9 (range 3-11). Results showed that the linearity between the measured and expected relative quantities of amplified DNA showed a regression coefficient of 0.99 in the interval 10-90%. The mean sensitivity was 1.5% (range 0.5-2.5), greater than previously reported. A total of 70 cases of allogeneic transplant (49 with family and 21 with unrelated donors) were monitored before transplant and after 1, 2, 4, 6 or 12 months thereafter and then at 6 months intervals (range 6-36). In 18 patients mixed chimerism was observed 1 month from transplant, with donor allele percentage ranging from 1 to 6%. Fragment dimension reproducibility (CV 0.34, range 0.52-0.66) was confirmed by an internal DNA control and by amelogenin fragment length repeatability on all patients. In conclusion, the proposed method is sufficiently simple, rapid, sensible, specific and cost effective for the evaluation of mixed chimerism after BM or PBSC transplant in a clinical setting.

Bone Marrow Cells↗