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1.
Leg Med (Tokyo) ; 47: 101776, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32795931

RESUMO

The RapidHIT™ ID system produces GlobalFiler™ analysis results after a short operating time. This device is effective because it automatically extracts DNA from oral mucosal cells or from blood stains and saliva collected at a crime scene, with subsequent polymerase chain reaction performed to produce a DNA profile. Two types of dedicated cartridges are available for RapidHIT™ ID: the RapidHIT™ ID ACE GlobalFiler Express sample cartridge for oral cells and other samples and the RapidINTEL™ sample cartridge for minute samples, such as blood stains. Previously validated specimens include oral mucosa cells and blood stains left at crime scenes. There have been no reports of blood and nail clipping samples collected from the postmortem bodies at the time of death. This report summarizes the results of using the RapidHIT™ ID system by collecting a variety of actual forensic samples from postmortem bodies at different stages of decomposition, which were subsequently analyzed using these cartridges.


Assuntos
Autopsia/métodos , Impressões Digitais de DNA/instrumentação , DNA/genética , DNA/isolamento & purificação , Genética Forense , Manchas de Sangue , Crime , Impressões Digitais de DNA/métodos , Humanos , Mucosa Bucal , Reação em Cadeia da Polimerase , Saliva
2.
Sci Rep ; 10(1): 12225, 2020 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-32699278

RESUMO

Massively parallel sequencing (MPS) has rapidly become a promising method for forensic DNA typing, due to its ability to detect a large number of markers and samples simultaneously in a single reaction, and sequence information can be obtained directly. In the present study, two kinds of forensic genetic markers, short tandem repeat (STR) and identity-informative single nucleotide polymorphism (iiSNP) were analyzed simultaneously using ForenSeq DNA Signature Prep Kit, a commercially available kit on MPS platform. A total of 152 DNA markers, including 27 autosomal STR (A-STR) loci, 24 Y chromosomal STR (Y-STR) loci, 7 X chromosomal STR (X-STR) loci and 94 iiSNP loci were genotyped for 107 Tibetan individuals (53 males and 54 females). Compared with length-based STR typing methods, 112 more A-STR alleles, 41 more Y-STR alleles, and 24 more X-STR alleles were observed at 17 A-STRs, 9 Y-STRs, and 5 X-STRs using sequence-based approaches. Thirty-nine novel sequence variations were observed at 20 STR loci. When the flanking regions were also analyzed in addition to target SNPs at the 94 iiSNPs, 38 more alleles were identified. Our study provided an adequate genotype and frequencies data of the two types of genetic markers for forensic practice. Moreover, we also proved that this panel is highly polymorphic and informative in Tibetan population, and should be efficient in forensic kinship testing and personal identification cases.


Assuntos
Repetições de Microssatélites/genética , Polimorfismo de Nucleotídeo Único/genética , Alelos , Impressões Digitais de DNA/métodos , Feminino , Frequência do Gene/genética , Marcadores Genéticos/genética , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Masculino , Análise de Sequência de DNA/métodos , Tibet
3.
Leg Med (Tokyo) ; 47: 101758, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32702606

RESUMO

We investigated whether bloodstain examination and DNA typing can be performed on washed bloodstains on clothes. Blood was dropped onto T-shirts made from 100% cotton or 100% polyester. After drying, the T-shirts were hand-washed with handwashing soap, dishwashing detergent, laundry detergent, soap, or just water until the bloodstains could not be seen. After drying the T-shirts, DNA and RNA were extracted simultaneously from the bloodstained areas using commercial kits. RNA was reverse-transcribed to DNA, and then the detection of the mRNAs for HBB, ACTB, and 18S rRNA was examined. DNA was quantified via real-time PCR, and then STR typing was performed with a commercial kit. The luminol and leucomalachite green tests were used as preliminary bloodstain tests, and an immuno-chromatography kit was used to identify human bloodstains. DNA could be extracted from all washed bloodstains, but more DNA was extracted from cotton T-shirts than from polyester T-shirts. STR typing was successful for all bloodstains without issues such as PCR inhibition. In the human bloodstain identification test using mRNA, almost all bloodstains produced a Ct value for HBB and all bloodstains produced a Ct value for 18S rRNA, whereas few bloodstains produced a Ct value for ACTB. All bloodstains reacted positively to luminol, but some were negative for leucomalachite green. Most of the bloodstains did not react positively in the human bloodstain identification test using the immuno-chromatography kit. The results suggest that human bloodstain identification and DNA typing can still be performed after clothes with bloodstains are washed.


Assuntos
Manchas de Sangue , Vestuário , Impressões Digitais de DNA/métodos , DNA/isolamento & purificação , Medicina Legal/métodos , Detergentes , Humanos , Temperatura , Água
4.
Leg Med (Tokyo) ; 47: 101727, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32562959

RESUMO

DNA profiling can identify an individual from a sample of biological material but it does not reveal what body fluid or tissue source the DNA profile originated from. In many cases it is important to know from what body fluid or tissue the DNA profile originated in order to provide crucial information necessary to the investigation, especially in cases where the victims are not able to give information about the dynamics of the event. For this purpose messenger RNA (mRNA) analysis has been shown to be a suitable method for the identification of body fluids, resulting in a trend to overcome the conventional approaches. Here we present the first report about case regarding a three-year-old child supposedly victim of a sexual assault with digital penetration. Thanks to the use of the combined DNA profiling and RNA analysis it was possible to demonstrate the sexual assault suffered by the victim.


Assuntos
Abuso Sexual na Infância , Vítimas de Crime , Impressões Digitais de DNA/métodos , Genética Forense/métodos , RNA Mensageiro/análise , Pré-Escolar , Feminino , Humanos , Unhas
6.
Gene ; 753: 144794, 2020 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-32464245

RESUMO

Novel microsatellite markers were developed to investigate the genetic diversity and DNA fingerprinting of bougainvillea cultivars. Total of 175 SSRs were designed from over 50,000 SSRs identified in the whole genome sequence data, 33 highly polymorphic markers were identified. These selected SSRs produced a total of 165 alleles with 2 (BOUG-3 and BOUG-50) to 9 (BOUG-69) alleles per loci with an average of 5 alleles per locus. The overall size of the amplified products ranged from 90 bp (BOUG-51 and BOUG-81) to 320 bp (BOUG-162). The gene diversity per locus ranged from 0.13 to 0.91 with a mean of 0.71. Primer BOUG-73 and BOUG-124 exhibited highest gene diversity with greater number of alleles. The mean Nei's genetic diversity index was 0.678 with range of 0.134 (BOUG-77) to 0.958 (BOUG-69). The UPGMA based dendrogram divided the cultivars into seven major clusters. Clustering pattern was more distinct for bract types and variegated cultivars which were also confirmed by PCA scatter plot diagram. The pair-wise genetic distance estimates ranged from 0.089 to 0.86 with an average of 0.56. Each of the 125 cultivar profiled had unique marker profile indicating that the SSR markers identified are useful for identification and differentiation of bougainvillea cultivars. These informative markers identified from the study will be of great utility to assess the genetic diversity, understanding the population structure and in marker assisted breeding for improvement of bougainvillea.


Assuntos
Repetições de Microssatélites/genética , Nyctaginaceae/genética , Alelos , Impressões Digitais de DNA/métodos , Marcadores Genéticos/genética , Variação Genética , Genótipo , Filogenia , Melhoramento Vegetal/métodos , Polimorfismo Genético
7.
Leg Med (Tokyo) ; 46: 101713, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32442862

RESUMO

An evaluation of a Rapid DNA system was performed using buccal swab samples and mock Disaster Victim Identification (DVI) samples collected postmortem. The allelic ladder success rate was 90% and samples analyzed simultaneously with this allelic ladder were used for further analysis. Sample success rate of the Rapid DNA system for buccal swab samples, and blood and muscle DVI samples were calculated. Success rates of buccal swab samples were 100% and 75% using cassettes preloaded with all reagents suitable for high- and low-DNA content samples, respectively. Success rates of fresh DVI samples were 80% to 100%. Success rates of putrefied DVI samples varied widely between 0% and 20% and 50% to 80% depending on cassette and sample types. Conventional DNA analysis was performed for comparison with the results of the Rapid DNA system. DNA quantity and degradation of human DNA were measured using quantitative polymerase chain reaction. DVI samples that yielded more than 1 ng/µL of DNA when extracted with conventional protocols were suitable for analysis using cassettes for both high- and low-DNA content samples. DVI samples with less than 0.1 ng/µL of DNA were suitable only for analysis using cassettes for low-DNA content samples. All alleles called and exported by the Expert system software implemented in the Rapid DNA system were concordant with allele calls made by conventional capillary electrophoresis DNA analysis.


Assuntos
Impressões Digitais de DNA/métodos , Vítimas de Desastres , Medicina Legal/métodos , Mucosa Bucal , DNA/análise , Humanos , Manejo de Espécimes
8.
Sci Rep ; 10(1): 5623, 2020 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-32221398

RESUMO

To facilitate the utility of SNP-based genotyping, we developed a new method called target SNP-seq which combines the advantages of multiplex PCR amplification and high throughput sequencing. Compared with KASP, Microarrays, GBS and other SNP genotyping methods, target SNP-seq is flexible both in SNPs and samples, yields high accuracy, especially when genotyping genome wide perfect SNPs with high polymorphism and conserved flanking sequences, and is cost-effective, requiring 3 days and $7 for per DNA sample to genotype hundreds of SNP loci. The present study established a DNA fingerprint of 261 cucumber varieties by target SNP-seq with 163 perfect SNPs from 4,612,350 SNPs based on 182 cucumber resequencing datasets. Four distinct subpopulations were found in 261 Chinese cucumber varieties: the north China type, the south China type, the Europe type, and the Xishuangbanna type. The north China type and Xishuangbanna type harbored lower genetic diversity, indicating greater risk of genetic erosion in these two subpopulations. Furthermore, a core set of 24 SNPs was able to distinguish 99% of the 261 cucumber varieties. 29 core cucumber backbone varieties in China were identified. Therefore, target SNP-seq provides a new way to screen out core SNP loci from the whole genome for DNA fingerprinting of crop varieties. The high efficiency and low cost of target SNP-seq is more competitive than the current SNP genotyping methods, and it has excellent application prospects in genetic research, as well as in promoting plant breeding processes in the near future.


Assuntos
Cucumis sativus/genética , Polimorfismo de Nucleotídeo Único/genética , Cruzamento/métodos , China , Impressões Digitais de DNA/métodos , Europa (Continente) , Testes Genéticos/métodos , Genoma de Planta/genética , Genótipo , Técnicas de Genotipagem/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos
9.
PLoS One ; 15(3): e0230390, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32176736

RESUMO

The aim of the study was to detect and genetically characterize Arcobacter butzleri in pet red-footed tortoises suspected for Campylobacter spp., using molecular techniques. A written consent from tortoise owners was obtained, after explaining the advantages of the research to tortoise owners of Grenada. Fecal samples were collected from 114 tortoises from five parishes of the country and cultured for Campylobacter spp. using selective culture techniques. A. butzleri was isolated from 4.39% of pet tortoises. Total thirteen isolates were obtained; all identified as A. butzleri by a universal and a species-specific Polymerase Chain Reaction (PCR) and direct sequencing. Genetic characterization of these isolates was performed based on Enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) that generated eight different genetic fingerprints with a discriminatory power of 0.91. Campylobacter species were not detected molecularly in any of the culture-positive samples. This is the first report of infection of pet tortoises in Grenada, West Indies with A. butzleri. This study emphasizes on the risk of zoonotic transmission of A. butzleri by exotic pets, which is a serious concern for public health.


Assuntos
Arcobacter/genética , Campylobacter/genética , Sequências Repetitivas de Ácido Nucleico/genética , Tartarugas/microbiologia , Animais , Campylobacter/isolamento & purificação , Impressões Digitais de DNA/métodos , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Fezes/microbiologia , Tartarugas/genética
10.
Sci Rep ; 10(1): 1945, 2020 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-32029845

RESUMO

Microhaplotypes are an emerging type of forensic genetic marker that are expected to support multiple forensic applications. Here, we developed a 124-plex panel for microhaplotype genotyping based on next-generation sequencing (NGS). The panel yielded intralocus and interlocus balanced sequencing data with a high percentage of effective reads. A full genotype was determined with as little as 0.1 ng of input DNA. Parallel mixture experiments and in-depth comparative analyses were performed with capillary-electrophoresis-based short tandem repeat (STR) and NGS-based microhaplotype genotyping, and demonstrated that microhaplotypes are far superior to STRs for mixture deconvolution. DNA from Han Chinese individuals (n = 256) was sequenced with the 124-plex panel. In total, 514 alleles were observed, and the forensic genetic parameters were calculated. A comparison of the forensic parameters for the 20 microhaplotypes with the top Ae values in the 124-plex panel and 20 commonly used forensic STRs showed that these microhaplotypes were as effective as STRs in identifying individuals. A linkage disequilibrium analysis showed that 106 of the 124 microhaplotypes were independently hereditary, and the combined match probability for these 106 microhaplotypes was 5.23 × 10-66. We conclude that this 124-plex microhaplotype panel is a powerful tool for forensic applications.


Assuntos
Genética Forense , Haplótipos/genética , Polimorfismo de Nucleotídeo Único/genética , Alelos , Grupo com Ancestrais do Continente Asiático/genética , DNA/genética , Impressões Digitais de DNA/métodos , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Desequilíbrio de Ligação/genética , Repetições de Microssatélites/genética , Probabilidade , Análise de Sequência de DNA/métodos
11.
Leg Med (Tokyo) ; 43: 101658, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31954956

RESUMO

Crimes committed with assault rifles are becoming increasingly prevalent in the United States. In the absence of other evidence, DNA analysis can often provide informative leads. Unfortunately, any DNA transferred to rifle components left behind at a crime scene is likely to be low in quantity and/or quality. Furthermore, collected evidence is unlikely to be processed immediately and may require storage. Long-term storage can subject DNA to damage and degradation, which ultimately affects DNA profile interpretation and may prevent the identification of potential suspects. This study assessed the ability of a new swab storage device, the SwabSaver®, to preserve "touch" DNA from AR-15 magazine rifles using three different collection devices. Three volunteers loaded bullet cartridges into plastic polymer and aluminum AR-15 magazines. DNA was collected with traditional cotton swabs, layered cotton paper swabs, or nylon-flocked swabs. Collection devices were then stored at room-temperature for up to two months in either the SwabSaver® device or an empty centrifuge tube. The results suggest that substrate and swab type had less of an effect on profile completeness than storage type. Furthermore, SwabSaver® storage yielded DNA quantities comparable to "touch" DNA extracted after 24 h.


Assuntos
Crime , Impressões Digitais de DNA/métodos , Manejo de Espécimes/métodos , Temperatura , Fatores de Tempo , Humanos
12.
Sci Justice ; 60(1): 1-8, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31924284

RESUMO

Human biological samples with multiple contributors remain one of the most challenging aspects of DNA typing within a forensic science context. With the increasing sensitivity of commercially available kits allowing detection of low template DNA, complex mixtures are now a standard component of forensic DNA evidence. Over the years, various methods and techniques have been developed to try to resolve the issue of mixed profiles. However, forensic DNA analysis has relied on the same markers to generate DNA profiles for the past 30 years causing considerable challenges in the deconvolution of complex mixed samples. The future of resolving complicated DNA mixtures may rely on utilising markers that have been previously applied to gene typing of non-forensic relevance. With Massively Parallel Sequencing (MPS), techniques becoming more popular and accessible even epigenetic markers have become a source of interest for forensic scientists. The aim of this review is to consider the potential of alleles from the Human Leukocyte Antigen (HLA) complex as effective forensic markers. While Massively Parallel Sequencing of HLA is routinely used in clinical laboratories in fields such as transplantation, pharmacology or population studies, there have not been any studies testing its suitability for forensic casework samples.


Assuntos
Alelos , Impressões Digitais de DNA/métodos , Genética Forense/métodos , Marcadores Genéticos , Antígenos HLA/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Repetições de Microssatélites , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNA
13.
Int J Legal Med ; 134(2): 461-471, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31897668

RESUMO

Bones are often found in mass grave crime scene. To increase DNA identification success rates, a highly efficient DNA extraction method should be selected. Several DNA extraction methods for human bones have been published yet never been systematically compared, and some are time-consuming or complex. As such, a quick and highly efficient DNA extraction method was developed and compared with three published methods (Hi-Flow silica-based, total demineralization (TD) and PrepFiler BTA) using 70 fresh and 22 casework bones from different body parts. The highest median DNA concentrations were obtained from developed method (135.85 ng/µL and 0.224 ng/µL for fresh and casework bones, respectively). For residual PCR inhibitors, the threshold cycle (Ct) of the internal positive control (IPC) showed that developed method and PrepFiler BTA removed most PCR inhibitors. Similarly, 95.45% of casework STR profiles obtained using the developed protocol meet the standard requirements for Australian National Criminal Investigative DNA Database (NCIDD) entry, followed by 86.35% using TD, 81.82% using PrepFiler BTA, and 45.45% using Hi-Flow. Additionally, DNA extracts from seven different bones revealed that the 1st distal phalange of the hand contained the highest DNA concentration of 338.43 ng/µL, which was three times higher than the tibia and femur. Our findings suggest that developed method was highly efficient for casework bone analysis. It significantly reduced the extraction processing time down to 4 h and is two to four times cheaper compared with other methods. In practice, both the extraction method and the bone sampling must be considered by a forensic DNA analyst to increase the chances of successful identification.


Assuntos
Osso e Ossos/química , Impressões Digitais de DNA/métodos , DNA/isolamento & purificação , Genética Forense/métodos , Repetições de Microssatélites , Densidade Óssea , Fêmur/química , Falanges dos Dedos da Mão/química , Humanos , Tíbia/química
14.
Leg Med (Tokyo) ; 43: 101660, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31911187

RESUMO

Buccal swabs from 200 unrelated Zimbabwean males were collected from voluntary participants located in Harare province. The 5-dye SureID® 27Y Human STR Identification Kit was used to perform multiplex polymerase chain reactions (PCR) and generate Y-chromosomal DNA profiles. This kit targets markers DYS456, DYS576, DYS570, DYS481, DYF387S1, DYS627, DYS393, DYS391, DYS390, DYS635, DYS449, DYS533, DYS438, DYS389I, DYS448, DYS389II, DYS19, GATA_H4, DYS518, DYS458, DYS460, DYS437, DYS439, DYS392, and DYS385, similar to the Yfiler® Plus Amplification Kit. A total of 161 haplotypes were generated with the PowerPlex® Y system, whereas 159 complete haplotypes were generated for the Yfiler® Plus system. Haplotype Discrimination Capacity (DC) with the Yfiler® Plus system was determined to be 0.9686, while the Genetic Diversity (GD) of the targeted loci ranged from 0.03748 at DYS392 to 0.867239 at DYS449. One haplotype contained the triallelic pattern 37, 38, and 39 at DYS387S1. In addition, marker DYS387S1 and marker DYS385 had 13 counts of microvariant alleles overall, while 9 null allele counts were noted at marker DYS448. Genetic distances between our population data and 22 other data sets from African countries and people of African descent were estimated and results showed significant genetic variation.


Assuntos
Cromossomos Humanos Y/genética , Impressões Digitais de DNA/métodos , Grupos Étnicos/genética , Repetições de Microssatélites/genética , Alelos , Variação Genética/genética , Haplótipos , Humanos , Zimbábue/etnologia
15.
J Forensic Sci ; 65(1): 209-213, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31433497

RESUMO

When using non-FTA cards in commercial multiplex STR kits for direct PCR, pretreatment steps with specific buffers are recommended. Here, we designed a rapid direct PCR method utilizing a non-FTA card, Oral Cell Sampling Kit, by omitting the pretreatment step involving Prep-n-Go™ Buffer, and it showed compatibility with the GlobalFiler™ Express PCR Amplification Kit, GlobalFiler™ PCR Amplification Kit, and PowerPlex® Fusion system. To optimize the PCR conditions, we tested the method with different final PCR volumes and cycles. Finally, we conducted a performance test using 50 Korean buccal samples and confirmed the high performance of the method, detecting more than 90% of the samples with full profiles when using GlobalFiler™ PCR Amplification Kit and PowerPlex® Fusion system at 29 cycles in a 10 µL final PCR volume. Thus, we report a simple direct PCR set-up to analyze reference samples collected using a non-FTA card manufactured in Korea.


Assuntos
Reação em Cadeia da Polimerase/métodos , Manejo de Espécimes/instrumentação , Impressões Digitais de DNA/métodos , Feminino , Humanos , Repetições de Microssatélites , Mucosa Bucal/química , República da Coreia , Manejo de Espécimes/métodos
16.
Int J Legal Med ; 134(1): 79-91, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31414202

RESUMO

Human dental remains encountered in criminal casework evidence, missing person cases, or mass disaster tragedies provide a valuable sample source for DNA typing when suitable soft tissue is unavailable. Using traditional methods, teeth samples can be challenging to process, resulting in low-quantity and/or quality nuclear DNA and insufficient profiles for comparisons. This study examines the performance of a three-part nuclear DNA analysis workflow for teeth samples based on (1) improved dental tissue recovery using the Dental Forensic Kit (DFKMR) (Universidad de los Andes) and DNA extraction with QuickExtract™ FFPE DNA Extraction Kit (Lucigen®), (2) quantification with InnoQuant® HY (InnoGenomics Technologies) for sensitive assessment of total human and male DNA quantity/quality, and (3) massively parallel sequencing for simultaneous genotyping of 231 short tandem repeat (STR) and single-nucleotide polymorphism (SNP) markers with the ForenSeq® DNA Signature Prep Kit (Verogen, Inc.). Initial evaluation of artificially degraded blood samples (n = 10) achieved highly sensitive and informative quantification results with InnoQuant® HY, enabling successful first pass genotyping with the MiSeq FGx® System. Twenty-three STR alleles (out of 85) and 70 identity informative SNP loci (out of 94) were recovered from two pg total long target DNA input (0.86 ng total short target input) and an InnoQuant degradation index (DI) of 460 (severely degraded). The three-part workflow was subsequently applied to teeth samples (dental pulp, root cement tissues; n = 13) with postmortem intervals (PMI) of the teeth ranging from 8 days to approximately 6 months. Informative SNP and STR DNA profiles were obtained, to include 78 STR alleles and 85 identity informative SNP loci typed (of 94 total SNP targets) in a 1 month, four-day PMI root cement sample with one pg total long target DNA input and a DI of 76. These data indicate successful performance of the proposed workflow from degraded DNA from teeth samples.


Assuntos
Impressões Digitais de DNA/métodos , DNA/isolamento & purificação , Odontologia Legal , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de DNA , Dente , Adolescente , Adulto , Alelos , Criança , Cemento Dentário , Polpa Dentária , Feminino , Marcadores Genéticos , Genótipo , Humanos , Masculino , Repetições de Microssatélites , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Reação em Cadeia da Polimerase em Tempo Real
17.
Methods Mol Biol ; 2057: 119-143, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31595476

RESUMO

Symbiotic nitrogen fixation (SNF) is a characteristic feature of nodulating legumes. The wild legumes are comparatively less explored for their SNF ability; hence, it is essential to study nodulation and identify the microsymbiont diversity associated with them. This chapter aims to describe the methodology for nodule hunting; trapping, isolation, and characterization of root nodule bacteria (RNB) at phenotypic, genotypic, and symbiotic levels. The documentation of nodulating native legume species and the rhizobial diversity associated with them in various parts of world has gained attention as this symbiotic association provides fixed nitrogen, improves productivity of plants in an ecofriendly manner. Before field-based applications the symbiotic bacteria need to be assessed for their N fixing ability as well as characterized at molecular level. The phylogeny based on symbiosis-essential genes supplemented with the host-range studies helps in better understanding of the symbiotaxonomy of rhizobia. More efficient symbiotic couples need to be screened by cross-nodulation studies for their application in agricultural practices.


Assuntos
Bactérias Fixadoras de Nitrogênio/isolamento & purificação , Rhizobium/isolamento & purificação , Nódulos Radiculares de Plantas/microbiologia , Simbiose/genética , Impressões Digitais de DNA/métodos , Fabaceae , Genes Essenciais , Nitrogênio/metabolismo , Fixação de Nitrogênio , Bactérias Fixadoras de Nitrogênio/genética , Bactérias Fixadoras de Nitrogênio/metabolismo , Filogenia , Rhizobium/genética , Rhizobium/metabolismo , Rhizobium/fisiologia , Simbiose/fisiologia
18.
J Forensic Sci ; 65(2): 380-398, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31580496

RESUMO

Most DNA evidence is a mixture of two or more people. Cybergenetics TrueAllele® system uses Bayesian computing to separate genotypes from mixture data and compare genotypes to calculate likelihood ratio (LR) match statistics. This validation study examined the reliability of TrueAllele computing on laboratory-generated DNA mixtures containing up to ten unknown contributors. Using log(LR) match information, the study measured sensitivity, specificity, and reproducibility. These reliability metrics were assessed under different conditions, including varying the number of assumed contributors, statistical sampling duration, and setting known genotypes. The main determiner of match information and variability was how much DNA a person contributed to a mixture. Observed contributor number based on data peaks gave better results than the number known from experimental design. The study found that TrueAllele is a reliable method for analyzing DNA mixtures containing up to ten unknown contributors.


Assuntos
Impressões Digitais de DNA/métodos , DNA/genética , Funções Verossimilhança , Modelos Genéticos , Software , Alelos , Genótipo , Humanos , Repetições de Microssatélites , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
19.
J Forensic Sci ; 65(1): 295-303, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30859587

RESUMO

A set of historic murders, known as the "Jack the Ripper murders," started in London in August 1888. The killer's identity has remained a mystery to date. Here, we describe the investigation of, to our knowledge, the only remaining physical evidence linked to these murders, recovered from one of the victims at the scene of the crime. We applied novel, minimally destructive techniques for sample recovery from forensically relevant stains on the evidence and separated single cells linked to the suspect, followed by phenotypic analysis. The mtDNA profiles of both the victim and the suspect matched the corresponding reference samples, fortifying the link of the evidence to the crime scene. Genomic DNA from single cells recovered from the evidence was amplified, and the phenotypic information acquired matched the only witness statement regarded as reliable. To our knowledge, this is the most advanced study to date regarding this case.


Assuntos
Vestuário , Impressões Digitais de DNA/métodos , Genética Forense/métodos , Homicídio/história , Manchas de Sangue , Vestuário/história , Vítimas de Crime , Criminosos , DNA Mitocondrial/genética , DNA Mitocondrial/isolamento & purificação , Fluorescência , História do Século XIX , Humanos , Raios Infravermelhos , Microdissecção e Captura a Laser , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNA , Análise de Célula Única , Reino Unido , Sequenciamento Completo do Genoma
20.
Int J Legal Med ; 134(2): 491-499, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30915532

RESUMO

The Y chromosome short tandem repeat (Y-STR) haplotyping method has been widely used in forensic applications. However, the existing Y-STR panels are not the ideal tools for criminal investigation and database applications because of their relatively low discriminatory capacity (DC) or high mutation rates. In the present study, the multiplex PCR assay (AGCU Y30) for simultaneous amplification of 30 slowly and moderately mutated Y-STR loci labeled by 6-dye fluorescence was developed and validated. The AGCU Y30 assay was capable of amplification purified DNA from casework and database samples on FTA™ cards in direct amplification module with a 10 µL reaction volume. Furthermore, the genetic diversities and forensic parameters of AGCU Y30 were performed using 719 unrelated male samples, demonstrating its high level of genetic polymorphisms and DC in Nantong Han population. This validation study demonstrated good sensitivity, mixture samples, inhibitor tolerance, precision, and concordance for the AGCU Y30, which is suitable for forensic investigation and database construction.


Assuntos
Cromossomos Humanos Y , Impressões Digitais de DNA/métodos , Repetições de Microssatélites , Reação em Cadeia da Polimerase Multiplex/métodos , Taxa de Mutação , Polimorfismo Genético , Animais , China , Corantes Fluorescentes , Marcadores Genéticos , Genética Populacional , Humanos , Masculino , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Especificidade da Espécie
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