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PubMed1976

[Influence of mutation in the gene-suppressor su-str on the virulence of Salmonella enteritidis].

Virulent and avirulent cells resistant to streptomycin were revealed in the population of avirulent streptomycin-resistant mutant (obtained from the supressor revertant str-d S. enteritidis mutant) in seeding on a medium with streptomycin. Transduction analysis of the isolated avirulent and virulent strains demonstrated that mutation in the gene-suppressor su-str+ led to the loss of virulence. In connection with the presence in the genom of suppressor revertants of the streptomycin-dependent mutants two mutations (su-su+ and str-d), which irrespective of one another led to the loss of virulence, a conclusion was drawn on the stability of avirulent properties of such strains and on future prospects for their use in the capacity of living vaccines.

Drug Resistance, Microbial↗
PubMed1996

STR typing of buccal swabs for paternity testing with reference to Japanese population data on the D20S85, D14S118, and D14S543 loci.

To simplify short tandem repeat (STR) typing of genomic DNA, we used buccal cells instead of blood cells. Buccal swabs taken from paternity-test trios were washed with 1 ml of distilled water by centrifugation, and the pellets were suspended in 20 microliters of distilled water. The suspensions were frozen at -80 degrees C and then thawed. Using a 1/5 volume of each suspension, we were able to type STRs at the TH01, D20S85, D14S118, and D14S543 loci by triplex PCR amplification of the first three markers and by simplex amplification of the other. In addition, we studied the polymorphisms at the D20S85, D14S118, and D14S543 loci in a Japanese population of 320 individuals. The four loci have a combined average exclusion power of 99.37%. In 24 cases of disputed paternity, the results of STR typing of the four loci agreed with those of conventional marker typing. In addition to being simple and rapid, STR typing of buccal swabs involves no painful procedure. It is therefore preferable for paternity tests on infants and small children.

Alleles↗
PubMed1996

STR typing of plasma DNA in a deficiency case of disputed maternity against a patient dying in the hospital.

In this deficiency duo case, the plasma that had been left over after clinical diagnostic tests was the only blood sample available from the deceased alleged mother. Since her blood had been ABO grouped during hospitalization, we tested the plaintiff for the ABO system first. Then, to draw as much genetic information as possible from their plasma, we phenotyped 13 plasma protein systems and, three and a half years later, analyzed three short tandem repeat (STR) loci. The conventional phenotyping and STR genotyping gave probabilities of maternity of 0.974 and 0.967, respectively, bringing the total probability to 0.999. The present study highlights the utility of plasma STR typing in case of need.

ABO Blood-Group System↗
PubMed1998

Subtyping of D11S488 STR alleles by single-strand conformation polymorphism (SSCP) analysis in two cases of disputed parentage.

In two normal trio cases of disputed parentage, we analyzed four short tandem repeat (STR) loci (FGA, TH01, D20S85, and D11S488). In one case, subtyping of alleles at the complex D11S488 locus by single-strand conformation polymorphism (SSCP) analysis increased the combined probability of paternity. In the other case, subtyping increased the number of loci by which the paternity was excluded, from two to three. The present study suggests that inclusion of complex STR loci in an STR typing program coupled with the use of SSCP analysis is a practical way of parentage testing because of a saving of time and work without sacrifice of reliability.

Alleles↗
PubMed1998

Development and population study of an eight-locus short tandem repeat (STR) multiplex system.

Amplification of short tandem repeat (STR) loci has become a useful tool for human identification applications. To improve throughput and efficiency for such uses, the polymorphic STR loci CSF1PO, TPOX, TH01, vWA, D16S539, D7S820, D13S317, D5S818, F13A01, FESFPS, F13B, and LPL have been evaluated, developed, and configured into fluorescently labeled multiplex systems. Eight of these STR loci were combined to generate the PowerPlex System, a two-color multiplex system that supports rapid, accurate, reliable analysis and designation of alleles. The remaining four loci comprise the FFFL System, a one-color multiplex system. The PowerPlex System may be evaluated alternatively as two one-color, four-locus multiplex systems, CTTv Multiplex and GammaSTR Multiplex. The products of multiplex amplification may be analyzed with a variety of fluorescence detection instruments. Determination of genotypes of over 200 individuals from each of three different population/ethnic groups revealed independence of inheritance of the loci and allowed calculation of matching probability, typical paternity index, and power of exclusion for each multiplex.

Alleles↗
PubMed2000

Additional highly polymorphic microsatellite (STR) loci for estimating kinship in rhesus macaques (Macaca mulatta).

Thirty-four short tandem repeat (STR) loci, not previously studied in rhesus macaques, were amplified by PCR. About one third of these were found to clearly and reliably amplify and exhibit high levels of genetic heterogeneity even in relatively inbred populations. These loci, together with 11 loci previously studied, were sufficiently informative to discretely differentiate between related and unrelated pairs and, in most cases, between parent/offspring and other relative pairs. An even greater number of hypervariable STR loci might be required to distinguish between half-sib and full-sib pairs in most rhesus populations.

Alleles↗
PubMed1995

Automated short tandem repeat (STR) analysis in forensic casework--a strategy for the future.

Short tandem repeat (STR) loci are routinely analysed for forensic purposes in the UK. Because small regions of DNa are amplified, successful results are more likely to be obtained from highly degraded material where the DNA fragment length may be < 500 bp. The method is superceeding conventional analysis with single locus probes (SLPs). Dimeric STR loci display stutter artefacts, hence STRs used in casework are restricted to tri or tetrameric loci. Some STRs are complex repeats and have more alleles than simple repeats - for example the locus D21S11 has 21 alleles which differ in size by 2 bp because of the presence/absence of a hexanucleotide within the block of tetrameric repeats. These loci are of great potential interest because they combine increased discriminating power with reduced potential to stutter. Multiplexing 4 different loci with different dye labelled primers (i.e. carrying out polymerase chain reaction of 4 loci simultaneously) using the ABD 373A automated sequencer enables a large numbers of samples to be processed. In addition data aquisition and manipulation is automated so that minimum postelectrophoresis operator input is required. It is our aim to develop a system equivalent in power to that of 4 single locus probes. To achieve this we have developed an octoplex system consisting of 7 loci and a sex test (amelogenin locus) which has a probability of chance of association of 10(-9); the power of this system is equivalent to that achieved by 4 conventional SLPs.

Alleles↗
PubMed1997

Variation at three short tandem repeat (STR) loci in Australians: forensic and ethnic considerations.

We report the results of typing three short tandem repeat (STR) polymorphisms by a multiplex system in three forensically relevant populations of Australia; (i) a general Australian, (ii) a Caucasian, and (iii) an Asian sample. The STRs are tetranucleotide repeats in the following autosomal genes; CSF1PO, TPOX and THO1, and collectively comprise the CTT multiplex (Promega). In all three population samples, each of the loci exhibited genotype frequencies consistent with those expected under Hardy-Weinberg equilibrium, and, further, there was no significant deviation from random association of alleles across separate loci. These two characteristics, plus the high discrimination power values, demonstrate that the CTT multiplex system is a powerful tool for DNA profiling in Australian jurisdictions. Australian Asians and Caucasians exhibit frequencies of alleles at these three STR loci very similar to those in other Asian and Caucasian samples. While most of the variation exists within any single population, there are still considerable differences between Asians and Caucasians. This difference between the two groups is minimal with respect to TPOX and greatest for THO1 alleles.

Alleles↗
PubMed1998

Analysis of multiplexed short tandem repeat (STR) systems using capillary array electrophoresis.

The profiling of polymorphic short tandem repeat (STR) markers is being applied to human identification, parentage testing and genetic mapping. Reliable genotyping of these markers is facilitated by polymerase chain reaction (PCR) amplification and high-resolution electrophoretic separation. Capillary array electrophoresis (CAE) offers very rapid, high-resolution separation of the amplified DNA and potential for automated sample processing not realized employing conventional slab-gel electrophoresis. The use of CAE to type DNA samples amplified at 11 genetic loci in multiplex profiles is presented. Two sets totaling 208 samples were amplified in a multiplex fashion using AmpFlSTR-Blue or AmpFlSTR-Green I and analyzed in a blind study using CAE. With the exception of one sample, the CAE genotyping results were in complete agreement with results obtained using a single-capillary system or two slab-gel electrophoresis systems. The sample, genotype TH01 7/10, migrated similar to TH01 6.3/9.3 allele sizes, which suggested a potential band migration shift. The recommended approach to such an observation is to analyze the sample again. The sample was rerun and correct genotype verified. Allelic ladder samples were analyzed multiple times by CAE to determine sizing accuracy and precision. The sizing of over 240 allelic ladder samples yielded an average within-run precision of +/- 0.13 bp and between-run precision of +/- 0.21 bp for fragments up to 350 bp. The CAE protocols permit processing of up to 96 multiplex STR samples in under 70 min.

Alleles↗
PubMed2004

Introduction of the QF-PCR analysis for the purposes of prenatal diagnosis in Bulgaria--estimation of applicability of 6 STR markers on chromosomes 21 and 18.

OBJECTIVE: The aim of our study was to estimate the observed heterozygosity and informativeness of 6 STR markers on chromosomes 18 and 21 in the Bulgarian population. We have evaluated the applicability of these markers used from other investigators for QF-PCR prenatal diagnosis of the most common autosomal aneuploidies in Bulgaria. METHODS: DNA samples (n = 486) were extracted from different fetal tissues (amniotic fluid cells, chorionic villus samples, and fetal tissue after abortions). PCR amplifications of 4 STR markers located on chromosome 21 (D21S11, D21S1411, D21S1270, and D21S1440) and 2 on chromosome 18 (D18S535 and D18S51) were performed. They were analysed on an automated sequencer, and the allele dosage ratios were calculated. RESULTS: The results indicate the selected markers as highly informative for our population and suitable for QF-PCR prenatal diagnosis in Bulgaria. All samples with trisomy 21 (n = 8), trisomy 18 (n = 4) and triploidy (n = 1) were correctly detected by our analysis. Thus, no false-negative results were observed. CONCLUSION: QF-PCR analysis could be an applicable alternative in prenatal and postnatal diagnosis in cases with a strong suspicion for particular autosomal aneuploidies (including chromosomes 21, 18, and 13) in small countries with limited resources like Bulgaria.

Aneuploidy↗
PubMed1990

The cyanelle str operon from Cyanophora paradoxa: sequence analysis and phylogenetic implications.

The str operon containing the genes for the ribosomal proteins S12 (rps12) and S7 (rps7) and for the elongation factors G (fus) and Tu (tufA) has been characterized for some cyanobacteria and chloroplasts from algae and higher plants. In the case of plastids a stepwise reduction by one and two genes, respectively, has been observed due to gene transfer to the nuclear genome. The nucleotide sequence of the str operon on the cyanelle genome from Cyanophora paradoxa was determined as a first example for a chlorophyll b-less plastid. It comprises rps12, rps7 and tufA which are closely linked and not interrupted by introns. Transcript analysis revealed cotranscription of the two ribosomal protein genes whereas tufA gave rise to a monocistronic mRNA. Phylogenetic studies using these three different traits allowed an assessment of the position of Cyanophora paradoxa among oxygenic photoautotrophs.

Amino Acid Sequence↗
PubMed1994

Synechocystis sp. PCC6803 fusB gene, located outside of the str operon, encodes a polypeptide related to protein synthesis factor EF-G.

Synechocystis sp. PCC6803, a cyanobacterium, possesses an unusual gene (fusB) which encodes a protein with strong homology to protein synthesis elongation factor G (EF-G), although it is not linked to the classical str operon. The fusB gene is redundant, since a Synechocystis gene similar to str operon-encoded fusA genes of other bacteria is also present (based on PCR and hybridization results). There is no evidence for the presence of a fusB homologue in other bacteria. The Synechocystis fusB gene encodes unusual amino acids at some positions that are highly conserved in fusA genes of other prokaryotes.

Amino Acid Sequence↗
PubMed1989

Characterization of the str operon genes from Spirulina platensis and their evolutionary relationship to those of other prokaryotes.

A 5.3 kb DNA segment containing the str operon (ca. 4.5 kb) of the cyanobacterium Spirulina platensis has been sequenced. The str operon includes the structural genes rpsL (ribosomal protein S12), rpsG (ribosomal protein S7), fus (translation elongation factor EF-G) and tuf (translation elongation factor EF-Tu). From the nucleotide sequence of this operon, the primary structures of the four gene products have been derived and compared with the available corresponding structures from eubacteria, archaebacteria and chloroplasts. Extensive homologies were found in almost all cases and in the order S12 greater than EF-Tu greater than EF-G greater than S7; the largest homologies were generally found between the cyanobacterial proteins and the corresponding chloroplast gene products. Overall codon usage in S. platensis was found to be rather unbiased.

Amino Acid Sequence↗
PubMed1994

Evaluation of an automated DNA profiling system employing multiplex amplification of four tetrameric STR loci.

We have examined the performance and reproducibility of an automated DNA profiling system which is based on the multiplex amplification of 4 tetrameric STR loci-HUMVWFA31/A. HUMTH01, HUMF13A1 and HUMFES/FPS. The system was able to type 100 pg of purified, undegraded, genomic DNA. At lower concentrations of DNA (below 100 pg), allelec drop-out occurred due to stochastic differences in allele copy number. Minor variation of individual PCR reagent concentrations or cycling temperatures did not result in a significant effect on the efficiency of amplification of any of the 4 loci in the quadruplex system. More substantial variation of reagent concentrations or cycling temperatures outside the optimum range of the system resulted in a reduction or complete loss of signal for one or more loci. This was also observed at high ionic strength or extreme pH. However, under all reagent concentrations and conditions studied, no artefact bands that could potentially result in the mistyping of a sample were apparent within the read region (130-240 bases) of the gel. Evaluation of both native and denaturing polyacrylamide gels revealed that, although native gels displayed faster run times, the sizing precision of such gels for certain STR loci was lower than that of denaturing gels. Also, artefact bands may be present within the read region of native gels. In conclusion the quadruplex amplification system described, coupled with automated fluorescence-based detection on denaturing polyacrylamide gels, appeared to be a robust and reliable system for individual identification.

Alleles↗
PubMed1996

The validation of a 7-locus multiplex STR test for use in forensic casework. (I). Mixtures, ageing, degradation and species studies.

We have evaluated a multiplex STR system for routine forensic use, which co-amplifies six short tandem repeat (STR) loci; HUMTH01, D21S11, D18S51, D8S1179, HUMVWF31/A and HUMFIBRA (FGA), in conjunction with the X-Y homologous gene Amelogenin. Analysis of PCR products employs denaturing polyacrylamide gels coupled with fluorescent labelled primers and detection is undertaken on ABD 373A automated sequencers. The technique was shown to be robust and reproducible when samples were analysed under conditions consistent with those encountered in a forensic environment. The system was demonstrated to be human specific and is suitable for use with both aged and degraded material. Somatic stability was proven with a wide range of tissue types and we were able to detect mixtures at ratios between 1:10 and 10:1. During this study no incidence of sample mis-typing due to allelic or locus drop-out was observed. Furthermore, although additional artefact bands were occasionally encountered these did not interfere with the interpretation of results. The performance of the system with poor quality samples demonstrated its suitability as a powerful tool in forensic investigation.

Adult↗
PubMed1996

The validation of a 7-locus multiplex STR test for use in forensic casework. (II), Artefacts, casework studies and success rates.

PCR-based DNA typing of biological evidence is now widely used in forensic analyses due to the obvious advantages of enhanced sensitivity, the ability to distinguish discrete alleles and efficacy with degraded samples. A multiplex short tandem repeat (STR) system has been previously developed which successfully co-amplifies six STR loci HUMTH01, D21S11, D18S51, D8S1179, HUMVWF31/A and HUMFIBRA (FGA) in conjunction with the X-Y homologous gene Amelogenin. This is known as the second generation multiplex system (SGM). Detection of the PCR products is undertaken on ABD 373A or 377 automated sequencers using denaturing polyacrylamide gels coupled with fluorescent-based technology. We have evaluated this system for routine forensic use and demonstrated that the technique is robust and reproducible under conditions consistent with those encountered in a forensic environment. A total of 132 stains from simulated and actual casework were analysed, together with relevant control areas and reference samples. The success rate was high with 76% of stains giving full profiles; we were also able to successfully detect and interpret mixtures. No mistyping was observed. A detailed examination of each of these profiles has assisted in the development of guidelines for casework interpretation. Although artefacts, stutter peaks and undenatured DNA were occasionally observed, these did not interfere with the accuracy of interpretation. In addition 38 samples, previously examined using the quadruplex system, were analysed with the SGM to enable a direct comparison to be made between the systems. The performance of the system with poor quality samples demonstrated its use as a rapid and powerful technique for individual identification.

Adult↗
PubMed1995

Swiss population data and forensic efficiency values on 3 tetrameric short tandem repeat loci-HUMTH01, TPOX, and CSF1PO-derived using a STR multiplex system.

Allele and genotype frequencies for 3 tetrameric short tandem repeat loci were determined in a Swiss population sample (n = 100) using the GenePrint STR Multiplex System, electrophoresis of the PCR products in DNA sequencing gels and subsequent detection of allelic fragments by silver staining. The loci are HUMTH01, TPOX, and CSF1PO. The observed heterozygosities are 83.0%, 60.0%, and 72.0%, respectively. The discrimination power determined for the individual loci is 0.914, 0.780, and 0.860, respectively, and the combined discrimination power for the triplex is 0.997. All loci meet Hardy-Weinberg expectations and after Bonferroni correction there was no evidence that the population sample deviates from expectations of independence. Moreover, independence of alleles at these STR loci with other PCR-based loci derived from the same Swiss population sample, previously reported, were considered. These loci were DQA1, LDLR, GYPA, HBGG, D7S8, GC and D1S80. Again, after Bonferroni correction there was no evidence that the population sample deviates from expectations of independence among alleles at the 10 different PCR-based loci. Thus, the allelic frequency data can be used in human identity testing to estimate the frequency of a multiple PCR-based DNA profile in the Swiss population.

Alleles↗
PubMed1995

PCR analysis of the short tandem repeat (STR) system HUMVWA31. Allele and genotype frequencies in an Italian population sample.

A population study in a sample of 211 unrelated individuals from 2 cities in North and Central Italy was carried out to investigate the short tandem repeat (STR) system HUMVWA31. Separation of PCR- amplified DNA fragments was performed by high-resolution horizontal denaturing polyacrylamide gel electrophoresis (PAGE) followed by silver staining. The 7 common alleles were found, together with a new smaller allele. Distribution of the observed genotypes did not deviate from Hardy-Weinberg (H-W) equilibrium. The power of discrimination for this locus was 0.93 and the chance of exclusion was 0.61. Good agreement was found between the allele frequencies in 2 Italian population samples and previous studies on Caucasians. The results of this study suggest that this STR system may be a useful tool in forensic investigations.

Alleles↗