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PubMed2003

[Four Y-STR multiplex system by silver staining].

A multiplex PCR system has been developed to amplify 4 Y-chromosome specific short tandem repeats (STR), DYS391, GATA-A4, GATA-A10 and GATA-H4, simultaneously. PCR products were separated by polyacrylamide gels electrophoresis followed by silver stain. When 311 unrelated males from the Han population in Guangdong were detected by the multiplex system, DYS391, GATA-A4, GATA-A10 and GATA-H4 showed 5, 7, 6 and 5 alleles respectively. Total 98 haplotypes could be identified. Gene diversity value for the 4 STR was 0.4623, 0.6972, 0.7173 and 0.6015 respectively. The gene diversity value for the haplotypes of the 4 Y-STR reached 0.9755. The four Y-STR multiplex system will be very powerful for establishing Y-STR database, exploring human origin, paternity testing and personal identification.

English Abstract↗

PubMed2005

[A multiplex for simultaneous amplification of three Y-STR loci using modified primers].

OBJECTIVE: To develop a multiplex method for simultaneous detection of Y-STR loci. METHODS: The primers of Y-STR loci were devised for use in the simultaneous amplification of three Y-STR loci (DYS390, DYS391, DYS393). RESULTS: Simultaneous amplification of the three Y-STR loci was successfully performed using the modified primers in which the fragment length of DYS391 was shortened from 279-287 bp to 142-150 bp, and at the same time, the virile specificity of DYS393 was enhanced. CONCLUSION: The use of new-devised primers of Y-STR loci is a valid approach to rapid detection of multiple Y-STRs loci; it is worthy to be recommended in the field of forensic science.

Chromosomes, Human, Y↗

PubMed2005

[Distribution of haplotypes for four Y-sTR loci and validation in forensic science by using a double-fluorescent multiplex PCR system].

OBJECTIVE: We focus on developing a multiplex PCR system for Y-STR loci that can be detected by double fluorescent system and assessing their usefulness in forensic mixture samples. METHODS: The primers of four Y-STR loci (DYS-GATA-A10, DYS531, DYS557 and DYS448) amplified by multiplex PCR technique were labeled with fluorescence, then the PCR products of these Y-STRs loci were detecting and typing by ABI PRISM310 Genetic Analyzer. RESULTS: When 120 unrelated individuals from the Han population in Chengdu were detected by the system, Y-GATA-A10, DYS531, DYS557 and DYS448 showed 5, 5, 8, 7 alleles, respectively. A total of 78 different haplotypes was identified and the genetic diversity reached 0.9881. To the three cases of mixture stains failed by using conventional autosomal STR analysis, our multiplex system drew conforming conclusion comparing to the suspect's Y-STRs genotypes. CONCLUSION: Our results show that the multiplex system of four Y-STR will be very powerful for Y-STR database establishing, the paternity testing and mixture stains identifying.

Alleles↗

PubMed2005

[Study of Five STR Loci in A Chinese Han Population.].

To understanding the allele structure and genetic polymorphisms at five STR loci in Chinese Han population, and construct a preliminary database, EDTA-blood specimens were collected from unrelated individuals. DNA samples were extracted with Chelex method and were amplified by PCR technique. The PCR products were analyzed using both PAGE horizontal electrophoresis with discontinuous buffer system and automated fluorescence detection approach. As a result, three STRs consist of simple repeat motifs, while two STR contain a complex repeat structure. The STR polymorphisms at all of the five loci have been observed in Chinese Han population. In a word, the obtained data are beneficial to understanding the population genetics of the five STR loci in Chinese Han population. As a simple approach, the PAGE horizontal electrophoresis can be employed for typing the five STR markers.

Alleles↗

PubMed2005

Evaluation of sieving polymers for fast, reproducible electrophoretic analysis of short tandem repeats (STR) in capillaries.

Efficient capillary electrophoretic STR analysis requires rapid, reproducible and robust separation of DNA fragments with reasonable capillary longevity--this is currently accomplished using proprietary commercial polymeric sieving matrices specifically developed for this separation. These matrices, while effective, are costly and do not provide adequate resolution of STR DNA fragments in capillaries with shorter effective separation lengths, increasing the time required to accomplish the separation and minimizing the potential extrapolation to other miniaturized platforms. As the forensic community looks toward next generation microchip technology as a means of processing casework more rapidly, new sieving polymers need to be evaluated for utilization in this platform. The research presented here describes the assessment of commercially-available polymeric sieving matrices for STR analysis, with consideration given to feasibility of incorporation into a microdevice. Polymer composition, molecular weight, and concentration were evaluated, along with an assessment of the effects of buffer composition, separation temperature, and capillary length. These variables were evaluated individually or collectively on the ability to resolve STR DNA fragments and the reproducibility of the separations and the results compared to a proprietary commercial product. A 600,000 Da MW poly(ethylene oxide) (PEO) solution at a 3% (w/v) concentration was determined to be the most suitable matrix for these separations. This polymer, in coated capillaries, provided highly robust and reproducible separations, with near baseline resolution of fragments having single base differences. Reductions in the temperature of the separation, from 60 degrees C to 40 degrees C, and the urea concentration of the buffer, from 7 M to 3.5 M, provided increased longevity of the PEO polymer for repeated separations. Comparison of this polymer with currently specified commercial products used for STR analysis showed that the optimized PEO matrix provided superior separations under all conditions tested. In addition, PEO could be utilized in shorter capillary systems, with a concurrent decrease in analysis time, highlighting its potential for use in shortened capillary or microdevice systems.

Buffers↗

PubMed2002

[The polymorphism distributions of nine STR loci in Shanghai Han population].

By multiplex amplification and four fluorescent technique,the polymorphism distributions of nine STR loci, D3S1358, vWA,FGA, D8S1179, D21S11, D18S51, D5S818, D13S317 and D7S820 were investigated in Shanghai Han population.Gene frequency (Pi),power of discrimination (DP),polymorphism information content (PIC) expected heterozygosity (H) and probability of paternity exclusion (PE) were calculated. All loci meet Hardy-Weinberg equilibrium. DP of FGA locus,H of D8S1179 locus,PIC of D18S51 locus and PE of D18S51 locus are the biggest among nine STR loci. Cumulate DP (CDP) of nine STR loci is 0.9999996, Cumulate PE (CPE) of nine STR loci is 0.99991. Nine STR loci could be used as the genetic markers of Chinese population in the studies of anthropology, linkage analysis of genetic disease genes, individual identification and paternity test in forensic medicine.

English Abstract↗

PubMed2006

[Constructing of STR slippage model by optimizing some factors].

OBJECTIVE: To construct STR slippage model and study factors involved in this procedure. METHODS: DNA samples were amplified with the technology of Degenerate oligonucleotide- primed PCR, then their products were taken as later DNA template and their STR genotype were analyzed by optimizing several factors. RESULTS: STR slippage model was constructed. CONCLUSION: Several factors were involved in the produce of STR slippage, such as amount of modulate DNA, concentration of MgCl2, property of DNA polymerase, motif sequence of STR loci, sample, etc.

DNA↗

PubMed1998

Genotyping of forensic short tandem repeat (STR) systems based on sizing precision in a capillary electrophoresis instrument.

Automated fluorescence analysis of polymerase chain reaction (PCR)-amplified short tandem repeat (STR) systems by capillary electrophoresis (CE) is becoming an established tool both in forensic casework and in the implementation of both state and national convicted offender DNA databases. A new capillary electrophoresis instrument, the ABI Prism 310 Genetic Analyzer, along with the Performance Optimized Polymer 4 (POP-4) provides an automated and precise method for simultaneously analyzing ten fluorescently labeled STR loci from a single PCR amplification kit, which provides a power of discrimination of approximately one in five billion from a single PCR amplification. Data are presented on sizing precision, sizing accuracy, and resolution for the STR loci in the AmpFlSTR Profiler kit. Sizing accuracy is highly dependent on the electrophoresis system, and therefore the reporting of alleles based on the nucleotide size obtained from an electrophoresis system is not recommended for forensic work. The precision of the 310 capillary electrophoresis system, coupled with software developed for automated genotyping of alleles based on the use of an allelic ladder, allows for accurate genotyping of STR loci. Sizing precision of < or = 0.16 nucleotide standard deviation was obtained with this system, thus allowing for accurate genotyping of length variants that differ in length by a single nucleotide.

Alleles↗

PubMed2004

Forensic DNA typing by capillary electrophoresis using the ABI Prism 310 and 3100 genetic analyzers for STR analysis.

DNA typing with short tandem repeat (STR) markers is now widely used for a variety of applications including human identification. Capillary electrophoresis (CE) instruments, such as the ABI Prism 310 and ABI 3100 Genetic Analyzers, are the method of choice for many laboratories performing STR analysis. This review discusses issues surrounding sample preparation, injection, separation, detection, and interpretation of STR results using CE systems. Requirements for accurate typing of STR alleles are considered in the context of what future analysis platforms will need to increase sample throughput and ease of use.

Electrophoresis, Capillary↗

PubMed2006

Evaluation of whole-genome amplification of low-copy-number DNA in chimerism analysis after allogeneic stem cell transplantation using STR marker typing.

Whole-genome DNA amplification (WGA) is a promising method that generates large amounts of DNA from samples of limited quantity. We investigated the accuracy of a multiplex PCR approach to WGA over STR loci. The amplification bias within a locus and over all analyzed loci was investigated in relation to the amount of template in the WGA reaction, the specific STR locus, and allele length. We observed reproducible error-free STR profiles with 10 ng down to 1 ng of DNA template. The amplification deviation at a locus and between loci was within the intra-method reproducibility. WGA is the method of choice for amplifying nanogram amounts of genomic DNA for different applications. We detected unbalanced STR amplifications at one locus and between loci, allelic drop-outs, and additional alleles after WGA of low-copy-number DNA. We found that the high number of drop-outs and drop-ins could be eradicated using pooled DNA from separate WGA reactions even with as little as 100 pg of starting template. Nevertheless, the quality of the results was still not sufficient for use in routine chimerism analysis of limited specific cell populations after allogeneic stem cell transplantation.

Chimerism↗

PubMed1997

Detection of microsatellites by ethidium bromide staining. The analysis of an STR system in the human phenylalanine hydroxylase gene.

The analysis of short tandem repeat (STR) systems usually relies on polyacrylamide gel electrophoresis analysis followed by visualization with silver staining or autoradiography. Both these techniques may not be suitable for clinical laboratories. We developed a simple procedure based on the visualization of STR alleles by ethidium bromide staining. The 4-bp STR system analysed is located in the human phenylalanine hydroxylase gene. Alleles differing by 4 bp are clearly separated independently of the size of the amplified fragments and homozygous samples are easily identified by comparison of the relative intensity of the electrophoretic bands. This method could be applied to the analysis of other STR systems located in different genetic loci by carefully changing the electrophoretic conditions.

Alleles↗

PubMed1996

A highly variable STR at the D12S391 locus.

A total of 103 fragments in the STR D12S391 locus were sequenced. 24 different alleles were found which can be grouped into 12 allelic classes based on the total number of repeats. The structure of this compound STR consists of blocks of (AGAT) and (AGAC) repeats with a consensus structure (AGAT)8-17 (AGAC)6-10 (AGAT)0-1. Whereas shorter alleles only have (AGAT) repeats, > 225 bp alleles are more complex, having two motifs (AGAT) and (AGAC). Population data showed that this to be a highly polymorphic STR with a heterozygosity of 0.9. This fact together with its simple structure make this STR very suitable for forensic and genetic purposes.

Alleles↗

PubMed2003

The development of an 18-locus Y-STR system for forensic casework.

The aim of the present work was to improve the discriminatory potential, and hence the probative value, of Y-STR-based testing by extending the set of Y chromosome STR loci available for forensic casework. In accordance with the requirements of a Y chromosome multiplex analytical system developed specifically for forensic casework use, we have sought to maximize the number of loci able to be co-amplified, ensure appropriate assay sensitivity (1-2 ng of input genomic DNA), balance inter-locus signals and minimize confounding female DNA artifacts. Two Y chromosome STR systems, multiplex I (MPI) and multiplex II (MPII), have been developed which permit the robust co-amplification of 18 Y-STRs. The loci include DYS19, DYS385(a) and (b), DYS388, DYS389I and II, DYS390, DYS391, DYS392, DYS393, DYS425, DYS434, DYS437, DYS438, DYS439, Y-GATA-C4, Y-GATA-A7.1 (DYS460) and Y-GATA-H4. The two multiplex systems are robust over a wide range of primer, magnesium, and DNA polymerase concentrations and perform well under a variety of cycling conditions. Complete male haplotypes can be obtained with as little as 100-250 pg of template DNA. Although a limited number of female DNA artifacts are observed in mixed stains in which the male DNA comprises 1/100 of the total, the male profile is easily discernible. Slightly modified versions of MPI and MPII demonstrate a significant reduction in female artifacts. Thus, it may not be necessary to employ a differential extraction strategy to obtain a male haplotype (or haplotypes in the case of multiple male donors) in cases of sexual assault. The potential utility of MPI and MPII for forensic casework is exemplified by their ability to dissect out the male haplotype in post-coital vaginal swabs and to determine the number of male donors in mixed semen stains. This study has emphasized the need for novel Y-STR multiplexes developed for forensic use to undergo a series of validation exercises that go beyond simply optimizing the PCR reaction conditions. Specifically, stringent performance checks on their efficacy need to be carried out using casework-type specimens in order to determine potential confounding effects from female DNA.

Alleles↗

PubMed2004

A proposed nomenclature for 15 canine-specific polymorphic STR loci for forensic purposes.

We performed a population study on 15 polymorphic STR loci (FH2010, FH2079, PEZ2, VWF.X, FH2054, FH2087Ub, FH2611, WILMS-TF, PEZ12, PEZ15, PEZ6, FH2087Ua, ZUBECA4, ZUBECA6, FH2132) on 131 randomly selected dogs. Alleles were identified and grouped according to their estimated fragment length using fixed allelic bins encompassing one base-pair. The allele assignment was confirmed by sequence analysis of homozygote and cloned heterozygote alleles. In order to develop a uniform repeat-based nomenclature, extensive sequence analysis was performed on a selection of alleles from each STR locus. The proposed nomenclature refers to the internationally recognised recommendations for human-specific STR loci in forensic applications. The 15 canine-specific STR loci were grouped into 3 classes (simple STRs, compound STRs and complex/hypervariable STRs) according to their complexity and variability within the repeat structure. Finally, we evaluated the precision of fragment size estimation on a capillary electrophoresis platform and demonstrated reproducibility of fragment length estimation for single base-pair intermediate alleles.

Alleles↗

PubMed2004

Canine-specific STR typing of saliva traces on dog bite wounds.

Forensic investigations in dog attacks usually involve the examination of bite marks and toothprints, the dog's stomach and pathological methods. For identification of the offending dog we evaluated canine STR typing of saliva traces on dog bite marks. The specificity of 15 canine-specific STRs was tested on human-canine DNA mixtures prior to an applied study in which 52 cases of dog bites were investigated. The first-aid wound bandages as well as swab samples from the surrounding area of the wound were used for DNA analyses. Generally, it was possible to obtain a canine-specific STR profile from the dog's saliva left on the wound area, even when high background of human DNA was present (blood). Interestingly, we found canine STR typing to be more successful when the bandages and swabs showed high amounts of human blood, i.e. when the dog bite was severe. Canine saliva was then sometimes visible as white-coloured secretion on the human blood surface. Less severe bite cases, which did not result in bleeding wounds, showed less success in obtaining useful STR results, probably due to the fact that the surface of the wounds may have been treated before the victims consulted medical aid which therefore removed the canine cells.

Adolescent↗

PubMed2004

Y-STR analysis for detection and objective confirmation of child sexual abuse.

We evaluated 26 child sexual assault cases for the incorporation of Y-STR screening in the routine detection and objective confirmation of sexual contact between the child victim and the perpetrator. Various samples, e.g. vaginal or anal swabs from patients aged 2-17 years old (25 females, 1 male), were collected 6-72 h after the incident. Due to the limited amounts of DNA in these samples, total DNA was extracted using a one-step procedure and screened with autosomal STRs to detect signs of a victim-assailant DNA mixture and with Y-STRs for assailant DNA. Autosomal STRs failed to give signs of victim-assailant DNA mixtures while Y-STRs were detected in 24 of the 26 cases corresponding to a success rate of 92.3%. With the possible presence of both male sperm and/or male epithelial cells in forensic evidence, Y-STR DNA markers were detected regardless of external ejaculation, microscopic detection of sperm and with post-coital intervals of up to 72 h. While only partial profiles were generated owing to low quantities of male DNA present, Y-STR screening results can serve as objective evidence of sexual contact in child sexual abuse cases involving victims who do not have any previous sexual history. This type of evidence can corroborate child victim testimony and spare the child victim from further trauma caused by prolonged forensic investigations and court proceedings. Alternatively, Y-STR screening can provide objective proof of non-involvement of an accused with the victim.

Adolescent↗

PubMed2005

Y-chromosomal STR haplotypes in a Northeast Italian population sample using 17plex loci PCR assay.

One hundred fifty-five unrelated, autochthonous healthy males from Northeast Italy were typed for the 17 Y-chromosome short tandem repeat (STR) (Y-STR) loci DYS456, DYS389I, DYS390, DYS389II, DYS458, DYS19, DYS385, DYS393, DYS391, DYS439, DYS635, DYS392, Y GATA H4, DYS437, DYS438, DYS448 using the AmpFLSTR Yfiler polymerase chain reaction amplification kit. A total of 153 different haplotypes were observed, and among these, 151 were unique, while 2 were found two times. The overall haplotype diversity was 0.9997. Furthermore, 50 father-son pairs, previously confirmed by autosomal STR analysis, were typed using the same set of 17 Y-STR loci, and, among 850 allele transfers, three mutation events were identified, giving an average mutation rate of 3.53 x 10(-3) per locus per generation (95% confidence interval 0.73-1.03).

Chromosomes, Human, Y↗

PubMed1978

DNA sequences of promoter regions for the str and spc ribosomal protein operons in E. coli.

The DNA sequences have been determined for promoter regions of two ribosomal protein operons in E. coli, the str operon and the spc operon. The site of in vitro transcription initiation within each of these promoter regions has been determined. The start site of the str operon occurs 69 bases upstream from the initiation codon of the S12 gene. The start site of the spc operon occurs 72 bases upstream from the L14 gene, and only 91 bases downstream from the termination codon of the S17 gene (which is in the preceding S10 operon). Both promoters are similar to other sequenced promoters in that they each have an identifiable "Pribnow box" sequence 5 bases upstream from the transcription start site. The spc promoter has a long sequence of 2 fold symmetry centered within the Pribnow box; the str promoter has a shorter but similar symmetry. At positions -69 through -40 in the spc operon, another long region of symmetry is present which may be the termination signal of the preceding S10 operon. Extensive sequence similarity between the str and spc promoter regions is found downstream from the Pribnow box-that is, in a transcribed region preceding the translation start sites.

Bacterial Proteins↗