RESUMO
The morphological identification of mites entails great challenges. Characteristics such as dorsal setae and aedeagus are widely used, but they show variations between populations, and the technique is time consuming and demands specialized taxonomic expertise that is difficult to access. A successful alternative has been to exploit a region of the mitochondrial cytochrome oxidase I (COI) gene to classify specimens to the species level. We analyzed the COI sequences of four mite species associated with cassava and classified them definitively by detailed morphological examinations. We then developed an identification kit based on the restriction fragment length polymorphism-polymerase chain reaction of subunit I of the COI gene focused on the three restriction enzymes AseI, MboII, and ApoI. This set of enzymes permitted the simple, accurate identification of Mononychellus caribbeanae, M. tanajoa, M. mcgregori, and Tetranychus urticae, rapidly and with few resources. This kit could be a vital tool for the surveillance and monitoring of mite pests in cassava crop protection programs in Africa, Asia, and Latin America.
Assuntos
Manihot/parasitologia , Reação em Cadeia da Polimerase/métodos , Tetranychidae/genética , Animais , Proteção de Cultivos/métodos , Enzimas de Restrição do DNA/genética , DNA Mitocondrial/genética , Complexo IV da Cadeia de Transporte de Elétrons/química , Complexo IV da Cadeia de Transporte de Elétrons/genética , Filogenia , Polimorfismo de Fragmento de Restrição , Alinhamento de Sequência , Especificidade da Espécie , Tetranychidae/anatomia & histologia , Tetranychidae/enzimologia , Fatores de TempoRESUMO
Several studies suggest the relation of DNA methylation to diseases in humans and important phenotypes in plants drawing attention to this epigenetic mark as an important source of variability. In the last decades, several methodologies were developed to assess the methylation state of a genome. However, there is still a lack of affordable and precise methods for genome wide analysis in large sample size studies. Methyl sensitive double digestion MS-DArT sequencing method emerges as a promising alternative for methylation profiling. We developed a computational pipeline for the identification of DNA methylation using MS-DArT-seq data and carried out a pilot study using the Eucalyptus grandis tree sequenced for the species reference genome. Using a statistic framework as in differential expression analysis, 72,515 genomic sites were investigated and 5,846 methylated sites identified, several tissue specific, distributed along the species 11 chromosomes. We highlight a bias towards identification of DNA methylation in genic regions and the identification of 2,783 genes and 842 transposons containing methylated sites. Comparison with WGBS, DNA sequencing after treatment with bisulfite, data demonstrated a precision rate higher than 95% for our approach. The availability of a reference genome is useful for determining the genomic context of methylated sites but not imperative, making this approach suitable for any species. Our approach provides a cost effective, broad and reliable examination of DNA methylation profile on MspI/HpaII restriction sites, is fully reproducible and the source code is available on GitHub (https://github.com/wendelljpereira/ms-dart-seq).
Assuntos
Análise Custo-Benefício , Metilação de DNA/genética , Eucalyptus/genética , Técnicas de Genotipagem/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Folhas de Planta/genética , Análise de Sequência de DNA/métodos , Árvores/genética , Cromossomos de Plantas/genética , Enzimas de Restrição do DNA/genética , Elementos de DNA Transponíveis/genética , Genes de Plantas/genética , Técnicas de Genotipagem/economia , Sequenciamento de Nucleotídeos em Larga Escala/economia , Projetos Piloto , Reprodutibilidade dos Testes , Mapeamento por Restrição , Análise de Sequência de DNA/economia , Sulfitos/farmacologiaRESUMO
Recombinant protein expression is widely used to produce large quantities of protein for diverse uses including functional characterization of selected sequences and vaccination trials. In the postgenomic era, high-throughput techniques that allow us to manipulate several sequences are needed. Cloning by in vivo recombination is a technique that consists in the insertion of a linear DNA into a linearized plasmid DNA by in vivo recombination using a recA+ E. coli strain. This methodology provides high-throughput cloning with high efficiency without the need for restriction enzyme digestion. In this chapter, we describe two protocols for DNA cloning: one using in vivo recombination and the other by using restriction enzymes. We also describe the application of different conditions to produce functional proteins that needs the incorporation of the amino acid selenocysteine (Sec), like thioredoxin-glutathione reductase enzyme.
Assuntos
Códon/genética , Escherichia coli/genética , Proteínas Recombinantes/genética , Clonagem Molecular/métodos , DNA/genética , Enzimas de Restrição do DNA/genética , Plasmídeos/genética , Selenocisteína/genéticaRESUMO
The advent of new DNA sequencing technologies leads to a dramatic increase in the number of available genome sequences and therefore of target genes with potential for functional analysis. The insertion of these sequences into proper expression vectors requires a simple an efficient cloning method. In addition, when expressing a target protein, quite often it is necessary to evaluate different DNA constructs to achieve a soluble and homogeneous expression of the target with satisfactory yields. The development of new molecular methods made possible the cloning of a huge number of DNA sequences in a high-throughput manner, necessary for meeting the increasing demands for soluble protein expression and characterization. In this chapter several molecular methods suitable for high-throughput cloning are reviewed.
Assuntos
Reação em Cadeia da Polimerase/métodos , Clonagem Molecular , Enzimas de Restrição do DNA/genéticaRESUMO
Exposure to ultraviolet (UV) radiation blocks DNA replication and arrests cellular division in Escherichia coli. Restoration of chromosome replication involves nucleoid reorganization, which involves the participation of the recombination-catalyzing proteins RecA, RecO, RecR and RecN. In this work, we evaluated the influence of recN, uvrA and recJ gene mutations on post-irradiation nucleoid reorganization. We used isogenic E. coli strains that are defective for these genes to study post-irradiation kinetics of the nucleoid shape fractions using fluorescence microscopy. The results showed that in the wild-type strain, post-irradiation nucleoid reorganization occurs, which restores the nucleoid shape fractions in the cells to those observed prior to irradiation. First, the nucleoid condenses into the central area of the irradiated cell. Second, the nucleoid disperses along the cell. Third, the cell enters the chromosome replicative phase and cytokinesis. Escherichia coli cells with a recN mutation did not exhibit increased nucleoid condensation, but chromosome replication and cytokinesis occurred. In the uvrA and recJ strains, the condensation step was delayed compared to the wild-type strain, and chromosome replication and cytokinesis did not occur. The results are discussed with an emphasis on the functions of RecN, UvrA and RecJ in nucleoid reorganization in UV-irradiated E. coli cells.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/metabolismo , Cromossomos Bacterianos/metabolismo , Enzimas de Restrição do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/efeitos da radiação , Exodesoxirribonucleases/metabolismo , Mutação , Raios Ultravioleta , Adenosina Trifosfatases/genética , Proteínas de Bactérias/genética , Enzimas de Restrição do DNA/genética , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/genética , Escherichia coli/enzimologia , Proteínas de Escherichia coli/genética , Exodesoxirribonucleases/genéticaRESUMO
Molecular markers can increase both the efficiency and speed of breeding programs. Functional markers that detect the functional mutations causing phenotypic changes offer a precise method for genetic identification. In this study, we used newly derived cleaved amplified polymorphic sequence markers to detect the functional mutations of tms5, which is a male sterile gene that is widely used in rice production in China. In addition, restriction cutting sites were designed to specifically digest amplicons of tms5 but not wild type (TMS5), in order to avoid the risk of false positive results. By optimizing the condition of the polymerase chain reaction amplifications and restriction enzyme digestions, the newly designed markers could accurately distinguish between tms5 and TMS5. These markers can be applied in marker-assisted selection for breeding novel thermo-sensitive genic male sterile (TGMS) lines, as well as to rapidly identify the TGMS hybrid seed purity.
Assuntos
Quimera/genética , Genes de Plantas , Marcadores Genéticos , Oryza/genética , Melhoramento Vegetal , Infertilidade das Plantas/genética , Mapeamento Cromossômico , Cruzamentos Genéticos , Primers do DNA/síntese química , Enzimas de Restrição do DNA/genética , Técnicas de Amplificação de Ácido Nucleico , Sementes/genéticaRESUMO
Microsatellite markers (also known as SSRs, Simple Sequence Repeats) are widely used in plant science and are among the most informative molecular markers for population genetic investigations, but the development of such markers presents substantial challenges. In this report, we discuss how next generation sequencing can replace the cloning, Sanger sequencing, identification of polymorphic loci, and testing cross-amplification that were previously required to develop microsatellites. We report the development of a large set of microsatellite markers for five species of the Neotropical cactus genus Pilosocereus using a restriction-site-associated DNA sequencing (RAD-seq) on a Roche 454 platform. We identified an average of 165 microsatellites per individual, with the absolute numbers across individuals proportional to the sequence reads obtained per individual. Frequency distribution of the repeat units was similar in the five species, with shorter motifs such as di- and trinucleotide being the most abundant repeats. In addition, we provide 72 microsatellites that could be potentially amplified in the sampled species and 22 polymorphic microsatellites validated in two populations of the species Pilosocereus machrisii. Although low coverage sequencing among individuals was observed for most of the loci, which we suggest to be more related to the nature of the microsatellite markers and the possible bias inserted by the restriction enzymes than to the genome size, our work demonstrates that an NGS approach is an efficient method to isolate multispecies microsatellites even in non-model organisms.
Assuntos
Cactaceae/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Repetições de Microssatélites/genética , Biologia Computacional , Análise Custo-Benefício , Primers do DNA , Enzimas de Restrição do DNA/genética , DNA de Plantas/genética , Marcadores Genéticos/genética , Variação Genética , Genótipo , Desequilíbrio de Ligação , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Polimorfismo Genético , Análise de Sequência de DNA , Especificidade da EspécieRESUMO
The aim of this study was to determine whether vitamin D receptor (VDR) genetic polymorphisms are associated with the susceptibility to pulmonary tuberculosis (PTB). MEDLINE and Embase databases and manual literature searches were used. A meta-analysis was conducted on the associations between the VDR FokI, TaqI, BsmI, and ApaI polymorphisms and PTB susceptibility. A total of 16 studies comprising 3231 patients and 3670 controls met the study inclusion criteria, consisting of 14 studies on the VDR FokI polymorphism, 13 on the VDR TaqI polymorphism, 8 on the VDR BsmI polymorphism, and 5 on the VDR ApaI polymorphism. Meta-analysis of the VDR FokI polymorphism showed no association between PTB and the f allele of the VDR FokI polymorphism (long variant) in all subjects (OR = 1.070, 95%CI = 0.979-1.169, P = 0.134). In contrast, after stratification by ethnicity, meta-analysis indicated that the VDR FokI F allele (short variant) was associated with PTB risk in an East Asian population (OR = 1.507, 95%CI = 1.192-1.906, P = 0.001). Meta-analysis revealed no association between PTB susceptibility and the VDR TaqI t allele in all study subjects (OR = 0.986, 95%CI = 0.839-1.159, P = 0.866) or in individual ethnic populations. Furthermore, a risk of PTB was not associated with the BsmI and ApaI polymorphisms. This meta-analysis suggested that the VDR FokI polymorphism is associated with a susceptibility to PTB in East Asians.
Assuntos
Estudos de Associação Genética , Predisposição Genética para Doença , Receptores de Calcitriol/genética , Tuberculose Pulmonar/genética , Alelos , Povo Asiático/genética , Enzimas de Restrição do DNA/genética , Genótipo , Humanos , Polimorfismo Genético , Fatores de Risco , Tuberculose Pulmonar/patologiaRESUMO
Real-time PCR based on the recN and gyrB genes was developed to detect four Streptococcus bovis/Streptococcus equinus complex (SBEC) subspecies from rectal swab specimens. The overall prevalence was 35.2%: Streptococcus gallolyticus subsp. gallolyticus (11.1%), S. gallolyticus subsp. pasteurianus (13%), Streptococcus infantarius subsp. coli (20.4%), and S. infantarius subsp. infantarius (11.1%). To conclude, these real-time PCR assays provide a reliable molecular method to detect SBEC pathogenic subspecies from rectal swab specimens.
Assuntos
Técnicas Bacteriológicas/métodos , Portador Sadio/diagnóstico , Fezes/microbiologia , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Infecções Estreptocócicas/diagnóstico , Streptococcus/isolamento & purificação , Adulto , Idoso , Idoso de 80 Anos ou mais , Proteínas de Bactérias/genética , Portador Sadio/epidemiologia , Portador Sadio/microbiologia , DNA Girase/genética , Enzimas de Restrição do DNA/genética , DNA Bacteriano/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Sondas de Oligonucleotídeos/genética , Prevalência , Infecções Estreptocócicas/epidemiologia , Infecções Estreptocócicas/microbiologia , Streptococcus/classificação , Streptococcus/genéticaRESUMO
Members of the genus Colletotrichum include some of the most economically important fungal pathogens in the world. Accurate diagnosis is critical to devising disease management strategies. Two species, Colletotrichum gloeosporioides and C. truncatum, are responsible for anthracnose disease in papaya (Carica papaya L.) and bell pepper (Capsicum annuum L.) in Trinidad. The ITS1-5.8S-ITS2 region of 48 Colletotrichum isolates was sequenced, and the ITS PCR products were analyzed by PCR-RFLP analysis. Restriction site polymorphisms generated from 11 restriction enzymes enabled the identification of specific enzymes that were successful in distinguishing between C. gloeosporioides and C. truncatum isolates. Species-specific restriction fragment length polymorphisms generated by the enzymes AluI, HaeIII, PvuII, RsaI, and Sau3A were used to consistently resolve C. gloeosporioides and C. truncatum isolates from papaya. AluI, ApaI, PvuII, RsaI, and SmaI reliably separated isolates of C. gloeosporioides and C. truncatum from bell pepper. PvuII, RsaI, and Sau3A were also capable of distinguishing among the C. gloeosporioides isolates from papaya based on the different restriction patterns that were obtained as a result of intra-specific variation in restriction enzyme recognition sites in the ITS1-5.8S-ITS2 rDNA region. Of all the isolates tested, C. gloeosporioides from papaya also had the highest number of PCR-RFLP haplotypes. Cluster analysis of sequence and PCR-RFLP data demonstrated that all C. gloeosporioides and C. truncatum isolates clustered separately into species-specific clades regardless of host species. Phylograms also revealed consistent topologies which suggested that the genetic distances for PCR-RFLP-generated data were comparable to that of ITS sequence data. ITS PCR-RFLP fingerprinting is a rapid and reliable method to identify and differentiate between Colletotrichum species.