Resumo
DNA extraction is usually the first step to perform molecular studies. This process can be nonviable due to genomic DNA (gDNA) extraction commercial kits prices. Furthermore, available DNA extraction protocols generally have high specificity, limiting their use to specific sources of biological material. In order to reduce costs, optimize time and laboratory logistics, besides to demonstrate a versatile protocol, the present study worked on an efficient DNA extraction protocol from somatic and non-somatic cells, using biological material from sheep as a model. For that, gDNA was extracted from whole blood, spermatozoa, and hair bulb cells, collected from three adult sheep, transported at 5ºC and stored at -20ºC until lab procedures. After extraction, gDNA concentration and purity were evaluated in a nano spectrophotometer. gDNA concentration from whole blood was greater (p < 0.05) than extracted from hair bulb cells, which in turn was superior (p < 0.05) than in spermatozoa. Also, gDNA from whole blood and, followed by, sperm showed greater (p < 0.05) purity when compared to gDNA of hair bulb cells. Adapting a gDNA extraction protocol, originally developed for bovine whole blood, enabled to obtain and isolate gDNA in different nucleated sheep cells.(AU)
Assuntos
Animais , Feminino , DNA/genética , Ovinos/genética , Biotecnologia/instrumentação , Melhoramento GenéticoResumo
The principle and the techniques applied in DNA extraction play a pivotal role in the obtention of a purified genetic material. The present study investigates the efficiency of eight protocols in the DNA extraction of Hypostomus commersoni, an essential component of South American freshwater ichthyofauna. The quality of samples was assessed through spectrophotometry, gel electrophoresis, and PCR-RAPD markers amplification. The efficiency of DNA extraction was influenced both by the method applied and the target-tissue of choice. Higher concentrations and yield of DNA were obtained from ocular tissue, with a positive spectrum of incubation in lysis buffer for up to 36 hours after sample collection, using fresh tissues and in the presence of a high concentration of Proteinase K (20 mg.ml-1). In these conditions, samples were successfully amplified. To date, there is no record of description for the parameters analyzed in this work, neither the description of RAPD markers for the species H. commersoni.(AU)
Os princípios e as técnicas aplicadas na extração de DNA desempenham um papel crucial na obtenção de material genético purificado. O presente estudo investiga a eficiência de oito protocolos na extração de DNA de Hypostomus commersoni, um importante componente ictiofaunístico de riachos da América do Sul. A qualidade das amostras foi avaliada por espectrofotometria, eletroforese em gel e amplificação por marcadores de PCR-RAPD. A eficiência da extração de DNA foi influenciada tanto pelo método aplicado quanto pelo tecido-alvo de escolha. Maiores concentrações e rendimento de DNA foram obtidos a partir do tecido ocular, com um espectro positivo de incubação em tampão de lise por até 36 horas após a coleta da amostra, utilizando tecidos frescos e na presença de alta concentração de proteinase K (20 mg.ml-1). Nestas condições, as amostras foram amplificadas com sucesso. Até o momento, não há registro de descrição para os parâmetros analisados neste trabalho, nem a descrição de marcadores RAPD para a espécie H. commersoni.(AU)
Assuntos
Animais , Peixes-Gato/classificação , Peixes-Gato/genética , Técnica de Amplificação ao Acaso de DNA Polimórfico/veterinária , Variação GenéticaResumo
Abstract The principle and the techniques applied in DNA extraction play a pivotal role in the obtention of a purified genetic material. The present study investigates the efficiency of eight protocols in the DNA extraction of Hypostomus commersoni, an essential component of South American freshwater ichthyofauna. The quality of samples was assessed through spectrophotometry, gel electrophoresis, and PCR-RAPD markers amplification. The efficiency of DNA extraction was influenced both by the method applied and the target-tissue of choice. Higher concentrations and yield of DNA were obtained from ocular tissue, with a positive spectrum of incubation in lysis buffer for up to 36 hours after sample collection, using fresh tissues and in the presence of a high concentration of Proteinase K (20 mg.ml-1). In these conditions, samples were successfully amplified. To date, there is no record of description for the parameters analyzed in this work, neither the description of RAPD markers for the species H. commersoni.
Resumo Os princípios e as técnicas aplicadas na extração de DNA desempenham um papel crucial na obtenção de material genético purificado. O presente estudo investiga a eficiência de oito protocolos na extração de DNA de Hypostomus commersoni, um importante componente ictiofaunístico de riachos da América do Sul. A qualidade das amostras foi avaliada por espectrofotometria, eletroforese em gel e amplificação por marcadores de PCR-RAPD. A eficiência da extração de DNA foi influenciada tanto pelo método aplicado quanto pelo tecido-alvo de escolha. Maiores concentrações e rendimento de DNA foram obtidos a partir do tecido ocular, com um espectro positivo de incubação em tampão de lise por até 36 horas após a coleta da amostra, utilizando tecidos frescos e na presença de alta concentração de proteinase K (20 mg.ml-1). Nestas condições, as amostras foram amplificadas com sucesso. Até o momento, não há registro de descrição para os parâmetros analisados neste trabalho, nem a descrição de marcadores RAPD para a espécie H. commersoni.
Resumo
Many tropical trees have high canopies and their leaves are not accessible. Thus, the use of tissue from a more accessible organ (cambium) for DNA extraction may be an alternative for molecular studies. We adapted a feasible methodology for extracting genomic DNA from cambium tissue harvested in the field for the assessment with PCR. We tested three storage conditions (two buffers and a silica gel) and four periods of time after harvest. We used previously described protocols and tested them on three species that occur in Amazonian forests and other biomes: Anadenanthera peregrina var. peregrina, Cedrela fissilis, and Ceiba speciosa. Our protocol obtained suitable PCR-grade genomic DNA for DNA sequencing and microsatellite genotyping. We recommend the use of silica for long-term storage and the buffer with ascorbic acid for short-term storage.(AU)
Muitas árvores tropicais possuem dossel alto e folhas não facilmente acessíveis. O uso de tecido de um órgão mais acessível (câmbio) para extração de DNA pode ser uma alternativa para estudos moleculares. Nós adaptamos uma metodologia viável para extrair DNA genômico de tecido cambial coletado no campo para avaliação com PCR. Testamos três condições de armazenamento (dois tampões e sílica gel) e quatro períodos após a coleta. Utilizamos protocolos descritos anteriormente e os testamos em três espécies encontradas em florestas amazônicas e outros biomas: Anadenanthera peregrina var. peregrina, Cedrela fissilis e Ceiba speciosa. Nosso protocolo foi eficaz na obtenção de DNA adequado para sequenciamento e genotipagem de microssatélites. Recomendamos o uso de sílica para armazenamento de longo prazo e o tampão com ácido ascórbico para curto prazo.(AU)
Assuntos
Floresta Úmida , Ácido Ascórbico , DNA/isolamento & purificaçãoResumo
Background: Toxocara vitulorum is a involved in the Ascaridoidea family and is a large roundworm with a semi translucent, soft body surface and pinkish color. Female worms measure 8-30cm in length, male worms 6-25cm. The major hosts of T.vitulorum are buffalo (Bubalus bubalis) and cattle (Bos species) in the humid tropics of Asia, Africa and South America. The diagnosis of T. vitulorum infections is usually made by observing characteristic eggs in routine fecal examination. Serological methods are also used to diagnose Toxocariasis. However, in recent years, PCR, a new generation molecular diagnostic method, has been used. The genetic structure of T. vitulorum is little known compared with data available from other parasites. The present sutudy was designed to determine the T. vitulorum isolates by the genetic characterization of the mitochondrial cytochrome c oxidase subunit I (cox1) gene.Materials, Methods & Results: Adult worms were collected from the feces of two calves (East Anatolian Red) during visits to the clinic at the Department of Internal Medicine of Van Yuzuncu Yil University, Faculty of Veterinary Medicine. Worms were washed thoroughly in 0.85 % saline to remove any debris and fixed into 70 % ethanol. After repeated and thoroughly washing the specimens, total genomic DNA of parasite extraction was performed be employing DNA extraction reagent kit (Thermo, GeneJET Genomic DNA Purification Kit) according to manufacturers recommendations. After DNA amplification, a 446 bp fragment of cox1 of T. vitulorum were obtained in all three isolates. All generated sequences were registered in GenBank database with accession numbers including MG905159, MG911729 and MG911730. The cox1 of T. vitulorum examined differed from another two isolates extracted from Germany beef cattle (KY313642.1) and Sri Lanka buffalo calf (FJ664617.1) at NCBI database.[...]
Assuntos
Análise de Sequência de DNA/veterinária , Genes Mitocondriais , Toxocara/isolamento & purificação , Reação em Cadeia da Polimerase/métodosResumo
Background: Toxocara vitulorum is a involved in the Ascaridoidea family and is a large roundworm with a semi translucent, soft body surface and pinkish color. Female worms measure 8-30cm in length, male worms 6-25cm. The major hosts of T.vitulorum are buffalo (Bubalus bubalis) and cattle (Bos species) in the humid tropics of Asia, Africa and South America. The diagnosis of T. vitulorum infections is usually made by observing characteristic eggs in routine fecal examination. Serological methods are also used to diagnose Toxocariasis. However, in recent years, PCR, a new generation molecular diagnostic method, has been used. The genetic structure of T. vitulorum is little known compared with data available from other parasites. The present sutudy was designed to determine the T. vitulorum isolates by the genetic characterization of the mitochondrial cytochrome c oxidase subunit I (cox1) gene.Materials, Methods & Results: Adult worms were collected from the feces of two calves (East Anatolian Red) during visits to the clinic at the Department of Internal Medicine of Van Yuzuncu Yil University, Faculty of Veterinary Medicine. Worms were washed thoroughly in 0.85 % saline to remove any debris and fixed into 70 % ethanol. After repeated and thoroughly washing the specimens, total genomic DNA of parasite extraction was performed be employing DNA extraction reagent kit (Thermo, GeneJET Genomic DNA Purification Kit) according to manufacturers recommendations. After DNA amplification, a 446 bp fragment of cox1 of T. vitulorum were obtained in all three isolates. All generated sequences were registered in GenBank database with accession numbers including MG905159, MG911729 and MG911730. The cox1 of T. vitulorum examined differed from another two isolates extracted from Germany beef cattle (KY313642.1) and Sri Lanka buffalo calf (FJ664617.1) at NCBI database.[...](AU)
Assuntos
Toxocara/isolamento & purificação , Análise de Sequência de DNA/veterinária , Genes Mitocondriais , Reação em Cadeia da Polimerase/métodosResumo
The bacterium Xylella fastidiosa, the causal agent of citrus variegated chlorosis (CVC), is dependent on vector insects for its spread and infection of citrus hosts. The insects, leafhoppers (Hemiptera: Cicadellidae) transmit the bacteria to healthy plants after feeding on infected plants. The objective of this study was to develop and compare methods for extracting genomic DNA from sharpshooters to detect X. fastidiosa. The DNA extraction from insects was performed according to a phenol-chloroform based DNA extraction in conjunction with two commercial kits, Dneasy® Plant Mini Kit and blood & tissue Dneasy® Handbook (Qiagen Inc., Valencia, CA, USA). The heads of the following species of sharpshooter were used: Dilobopterus costalimai, Acrogonia citrina, Oncometopia facialis, Bucephalogonia xanthophis, Macugonalia leucomelas and Homalodisca ignorata. Based on the numeric differences between independent samples, the results showed the use of the phenol:chloroform extraction method (36/50 positives) and the DNeasy® Plant Mini Kit (33/50) resulting in the most detections of X. fastidiosa from leafhopper samples. As these two methods detected Xylella in the greatest number of infected samples, they may be more efficient to use for detection purposes in leafhoppers.(AU)
A bactéria Xylella fastidiosa, agente causal da clorose variegada dos citros, é dependente da ação de insetos vetores para sua disseminação e infecção em plantas cítricas hospedeiras. Os insetos cigarrinhas (Hemiptera: Cicadellidae) transmitem a bactéria para plantas sadias depois de se alimentarem das plantas contaminadas. O objetivo deste trabalho foi desenvolver e comparar métodos de extração de DNA genômico de cigarrinhas a fim de detectar a bactéria X. fastidiosa. A extração de DNA foi realizada de acordo com protocolo à base de fenol-clorofórmio em conjunto com dois kits comerciais: Dneasy® Plant Mini Kit and Blood & Tissue Dneasy® Handbook (Qiagen Inc., Valencia, CA, USA). Utilizou-se a cabeça das seguintes espécies de cigarrinhas: D. costalimai, A. citrina, O. facialis, B. xanthophis, M. leucomelas e H. ignorata. Com base nas diferenças numéricas entre as amostras independentes, os resultados mostraram o uso do método de extração fenol-clorofórmio (36/50 positivos) e o kit comercial DNeasy® Plant Mini Kit (33/50) resultando nas maiores detecções de X. fastidiosa nas amostras de cigarrinhas. Como esses dois métodos detectaram a presença de X. fastidiosa no maior número de amostras infectadas, eles podem ser mais eficientes para o uso na detecção em cigarrinhas.(AU)
Assuntos
Reação em Cadeia da Polimerase/métodos , Citrus , Xylella , Pragas da Agricultura , HemípterosResumo
The bacterium Xylella fastidiosa, the causal agent of citrus variegated chlorosis (CVC), is dependent on vector insects for its spread and infection of citrus hosts. The insects, leafhoppers (Hemiptera: Cicadellidae) transmit the bacteria to healthy plants after feeding on infected plants. The objective of this study was to develop and compare methods for extracting genomic DNA from sharpshooters to detect X. fastidiosa. The DNA extraction from insects was performed according to a phenol-chloroform based DNA extraction in conjunction with two commercial kits, Dneasy® Plant Mini Kit and blood & tissue Dneasy® Handbook (Qiagen Inc., Valencia, CA, USA). The heads of the following species of sharpshooter were used: Dilobopterus costalimai, Acrogonia citrina, Oncometopia facialis, Bucephalogonia xanthophis, Macugonalia leucomelas and Homalodisca ignorata. Based on the numeric differences between independent samples, the results showed the use of the phenol:chloroform extraction method (36/50 positives) and the DNeasy® Plant Mini Kit (33/50) resulting in the most detections of X. fastidiosa from leafhopper samples. As these two methods detected Xylella in the greatest number of infected samples, they may be more efficient to use for detection purposes in leafhoppers.(AU)
A bactéria Xylella fastidiosa, agente causal da clorose variegada dos citros, é dependente da ação de insetos vetores para sua disseminação e infecção em plantas cítricas hospedeiras. Os insetos cigarrinhas (Hemiptera: Cicadellidae) transmitem a bactéria para plantas sadias depois de se alimentarem das plantas contaminadas. O objetivo deste trabalho foi desenvolver e comparar métodos de extração de DNA genômico de cigarrinhas a fim de detectar a bactéria X. fastidiosa. A extração de DNA foi realizada de acordo com protocolo à base de fenol-clorofórmio em conjunto com dois kits comerciais: Dneasy® Plant Mini Kit and Blood & Tissue Dneasy® Handbook (Qiagen Inc., Valencia, CA, USA). Utilizou-se a cabeça das seguintes espécies de cigarrinhas: D. costalimai, A. citrina, O. facialis, B. xanthophis, M. leucomelas e H. ignorata. Com base nas diferenças numéricas entre as amostras independentes, os resultados mostraram o uso do método de extração fenol-clorofórmio (36/50 positivos) e o kit comercial DNeasy® Plant Mini Kit (33/50) resultando nas maiores detecções de X. fastidiosa nas amostras de cigarrinhas. Como esses dois métodos detectaram a presença de X. fastidiosa no maior número de amostras infectadas, eles podem ser mais eficientes para o uso na detecção em cigarrinhas.(AU)
Assuntos
Reação em Cadeia da Polimerase/métodos , Citrus , Xylella , Pragas da Agricultura , HemípterosResumo
The objective of this study was to determine the genome association between markers of bovine LD BeadChip with dairy important traits. Information of breeding program of the Universidad Nacional de Colombia was used. BLUP-EBVs were used for dairy yield (DY), fat percentage (FP), proteinpercentage (PP) and somatic cell score (SCS). 150 animals were selected for blood or semen DNA extraction and genotyping with BovineLD BeadChip (Illumina). Autosomal information was retained and the editing information was performed using Plink v1.07 software. The effects of SNPs were determined by Bayes C with GS3 software. The minor allele frequency for most of the markers on the bead chip was high, which increases the probability of finding important loci segregating in the population. Estimations of fraction markers with an effect were close to zero in almost all cases. The most important markers were mapped by trait using ENSEMBL. A total of 6,510 autosomal SNPs were retained, out of which only a proportion with effect was taken from the mixed function of Bayes C. Important genes as ANKS1B, CLCN1, NMBR and CTSD, were found for each trait for AL, FP, PP and SCS respectively. Finally, Bayes C estimation allowed to identify specific SNPs possibly associated with QTLs.(AU)
O Objetivo deste estudo foi determinar a associação genômica entre os marcadores do chip bovino LD (LD BeadChip) com características importantes na produção de leite. Foram utilizadas informações do programa de melhoramento genético da Universidade Nacional de Colômbia. BLUPEBVs foram utilizados para a produção de leite (DY), porcentagem de gordura (FP), porcentagem deproteína (PP) e escore de células somáticas (SCS). 150 animais foram selecionados para extração de DNAdo sangue ou sêmen e genotipados com o chip BovineLD (Illumina). A informação autossômica foi mantidae a edição da informação foi executada usando o programa Plink v1.07. Os efeitos dos SNPs foram determinados por Bayes C com o programa GS3. A frequência do alelo menor para a maioria dos marcadores no chip foi alta, o que aumenta a probabilidade de encontrar locos importantes segregando na população. As estimativas da fração de marcadores, com efeito, foram próximas de zero em quase todas as situações. Os marcadores mais importantes foram mapeados com ENSEMBL. Um total de 6510 SNPs autossômicos foram preservados, dos quais apenas uma proporção foi tomada com efeito a partir da função mista de Bayes C. Para cada característica foram encontrados genes importantes, como ANKS1B, CLCN1, NMBR e CTSD, para AL, FP, PP e SCS, respectivamente. Finalmente, a estimativa de Bayes-C permitiu aidentificação de SNPs com possível associação com QTLs.(AU)
Assuntos
Animais , Feminino , Bovinos , Bovinos/genética , Bovinos/fisiologia , Teorema de Bayes , Genômica/classificação , Polimorfismo de Nucleotídeo Único , Indústria de LaticíniosResumo
The objective of this study was to determine the genome association between markers of bovine LD BeadChip with dairy important traits. Information of breeding program of the Universidad Nacional de Colombia was used. BLUP-EBVs were used for dairy yield (DY), fat percentage (FP), proteinpercentage (PP) and somatic cell score (SCS). 150 animals were selected for blood or semen DNA extraction and genotyping with BovineLD BeadChip (Illumina). Autosomal information was retained and the editing information was performed using Plink v1.07 software. The effects of SNPs were determined by Bayes C with GS3 software. The minor allele frequency for most of the markers on the bead chip was high, which increases the probability of finding important loci segregating in the population. Estimations of fraction markers with an effect were close to zero in almost all cases. The most important markers were mapped by trait using ENSEMBL. A total of 6,510 autosomal SNPs were retained, out of which only a proportion with effect was taken from the mixed function of Bayes C. Important genes as ANKS1B, CLCN1, NMBR and CTSD, were found for each trait for AL, FP, PP and SCS respectively. Finally, Bayes C estimation allowed to identify specific SNPs possibly associated with QTLs.
O Objetivo deste estudo foi determinar a associação genômica entre os marcadores do chip bovino LD (LD BeadChip) com características importantes na produção de leite. Foram utilizadas informações do programa de melhoramento genético da Universidade Nacional de Colômbia. BLUPEBVs foram utilizados para a produção de leite (DY), porcentagem de gordura (FP), porcentagem deproteína (PP) e escore de células somáticas (SCS). 150 animais foram selecionados para extração de DNAdo sangue ou sêmen e genotipados com o chip BovineLD (Illumina). A informação autossômica foi mantidae a edição da informação foi executada usando o programa Plink v1.07. Os efeitos dos SNPs foram determinados por Bayes C com o programa GS3. A frequência do alelo menor para a maioria dos marcadores no chip foi alta, o que aumenta a probabilidade de encontrar locos importantes segregando na população. As estimativas da fração de marcadores, com efeito, foram próximas de zero em quase todas as situações. Os marcadores mais importantes foram mapeados com ENSEMBL. Um total de 6510 SNPs autossômicos foram preservados, dos quais apenas uma proporção foi tomada com efeito a partir da função mista de Bayes C. Para cada característica foram encontrados genes importantes, como ANKS1B, CLCN1, NMBR e CTSD, para AL, FP, PP e SCS, respectivamente. Finalmente, a estimativa de Bayes-C permitiu aidentificação de SNPs com possível associação com QTLs.
Assuntos
Feminino , Animais , Bovinos , Bovinos/fisiologia , Bovinos/genética , Genômica/classificação , Teorema de Bayes , Indústria de Laticínios , Polimorfismo de Nucleotídeo ÚnicoResumo
Background: Control of oral lesions contributes directly to the survival, and welfare of captive animals, and studies show that the genus Ateles has a higher prevalence of widespread periodontal disease compared to other genera. Anaerobic microbial species, considered as periodontal pathogens, are part of the biofilm community that contributes to the development of periodontitis. The present study aimed to detect periodontopathogenos in the oral cavity of two captive white-cheeked spider monkeys (Ateles marginatus) submitted for assessment oral and subgingival curettage.Case: We evaluated one pair of captive white-cheeked spider monkeys, one male (A) and one female (B), of 15 years of age with an average weight of 7 kg. Animals were fed daily with rations for primates, including fruit, vegetables, and raw eggs. The animals underwent oral evaluation, and following the charting of odontogram and photographic documentation, both were classified with periodontal disease stage III, according to the AVDC (American College of Veterinary Dentistry). They presented with moderate periodontitis, characterized by a loss of 25 to 50% of periodontal insertion and exposure of furcation degree 2, measured through clinical survey. During intraoral review, animals underwent subgingival curettage with curette of Gracey on the surface of the canine vestibular (C) and four top bilateral premolars (4PM). Antibiotics were not used at the time of collection, for dealing with routine procedures of clinical evaluation. The animals showed an increase in the volume of hemorrhagic features in the vestibular region between C and the second pre molar (2PM) on the upper right. Incisional biopsy was collected immediately at the end of the assessment, for the purpose of histopathological analyses. The samples from subgingival collection were immediately deposited in microtubes containing 500 µL of 0.9% saline solution and kept at -18°C until the time of genomic DNA extraction.[...]
Assuntos
Masculino , Feminino , Animais , Atelinae/microbiologia , Doenças Periodontais/diagnóstico , Doenças Periodontais/veterinária , Infecções por Bactérias Gram-Negativas/veterinária , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/veterináriaResumo
Background: Control of oral lesions contributes directly to the survival, and welfare of captive animals, and studies show that the genus Ateles has a higher prevalence of widespread periodontal disease compared to other genera. Anaerobic microbial species, considered as periodontal pathogens, are part of the biofilm community that contributes to the development of periodontitis. The present study aimed to detect periodontopathogenos in the oral cavity of two captive white-cheeked spider monkeys (Ateles marginatus) submitted for assessment oral and subgingival curettage.Case: We evaluated one pair of captive white-cheeked spider monkeys, one male (A) and one female (B), of 15 years of age with an average weight of 7 kg. Animals were fed daily with rations for primates, including fruit, vegetables, and raw eggs. The animals underwent oral evaluation, and following the charting of odontogram and photographic documentation, both were classified with periodontal disease stage III, according to the AVDC (American College of Veterinary Dentistry). They presented with moderate periodontitis, characterized by a loss of 25 to 50% of periodontal insertion and exposure of furcation degree 2, measured through clinical survey. During intraoral review, animals underwent subgingival curettage with curette of Gracey on the surface of the canine vestibular (C) and four top bilateral premolars (4PM). Antibiotics were not used at the time of collection, for dealing with routine procedures of clinical evaluation. The animals showed an increase in the volume of hemorrhagic features in the vestibular region between C and the second pre molar (2PM) on the upper right. Incisional biopsy was collected immediately at the end of the assessment, for the purpose of histopathological analyses. The samples from subgingival collection were immediately deposited in microtubes containing 500 µL of 0.9% saline solution and kept at -18°C until the time of genomic DNA extraction.[...](AU)
Assuntos
Animais , Masculino , Feminino , Atelinae/microbiologia , Doenças Periodontais/diagnóstico , Doenças Periodontais/veterinária , Infecções por Bactérias Gram-Negativas/veterinária , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/veterináriaResumo
Abstract The principle and the techniques applied in DNA extraction play a pivotal role in the obtention of a purified genetic material. The present study investigates the efficiency of eight protocols in the DNA extraction of Hypostomus commersoni, an essential component of South American freshwater ichthyofauna. The quality of samples was assessed through spectrophotometry, gel electrophoresis, and PCR-RAPD markers amplification. The efficiency of DNA extraction was influenced both by the method applied and the target-tissue of choice. Higher concentrations and yield of DNA were obtained from ocular tissue, with a positive spectrum of incubation in lysis buffer for up to 36 hours after sample collection, using fresh tissues and in the presence of a high concentration of Proteinase K (20 mg.ml-1). In these conditions, samples were successfully amplified. To date, there is no record of description for the parameters analyzed in this work, neither the description of RAPD markers for the species H. commersoni.
Resumo Os princípios e as técnicas aplicadas na extração de DNA desempenham um papel crucial na obtenção de material genético purificado. O presente estudo investiga a eficiência de oito protocolos na extração de DNA de Hypostomus commersoni, um importante componente ictiofaunístico de riachos da América do Sul. A qualidade das amostras foi avaliada por espectrofotometria, eletroforese em gel e amplificação por marcadores de PCR-RAPD. A eficiência da extração de DNA foi influenciada tanto pelo método aplicado quanto pelo tecido-alvo de escolha. Maiores concentrações e rendimento de DNA foram obtidos a partir do tecido ocular, com um espectro positivo de incubação em tampão de lise por até 36 horas após a coleta da amostra, utilizando tecidos frescos e na presença de alta concentração de proteinase K (20 mg.ml-1). Nestas condições, as amostras foram amplificadas com sucesso. Até o momento, não há registro de descrição para os parâmetros analisados neste trabalho, nem a descrição de marcadores RAPD para a espécie H. commersoni.
Resumo
Background: In many parts of the Old World, domesticated camels (genus - Camelus) are an essential resource, providing food, labor, commodities, and sport to millions of people Of the three extent species, two have been domesticated (singlehumped dromedarius, Camelus dromedarius, and two humped Bactrian camels Camelus bactrianus) and one remains wild (two-humped wild Bactrian camels Camelus ferus). All three species possess a variety adaptations to harsh desert conditions, including mechanisms to tolerate of extreme temperatures, dehydration, and sandy terrain. People residing in harsh climate zones of the world are being benefitted by raising camels in terms of draft, milk, meat, hides and wool from centuries. There are different breeds of dromedary camels distributed in various parts of Pakistan; however there have been scarcity of research work on camels in Pakistan. Identification of novel link between Camel breeders with fatal neurodegenerative disorders is presence or not can be detect by a Prion gene and it was not carried out in Pakistan soil to date. Prion diseases which are a group of fatal neurodegenerative disorders affect both animals and humans. It is believed that the prions are infectious agents responsible for transmissible spongiform encephalopathies. In this study we report the first study on Prion protein gene in dromedary camels of Pakistan. Material, Methods & Results: Genes are the blueprint of life and determine the functional aspects of cellular mechanisms. Genomic DNA of the enrolled blood samples was extracted using the Nucleospin® DNA extraction kit. Genomic DNA was run on Agarose gel electrophoresis, checked the Genomic DNA quality and amplified using prion region specific primer pair. Prion protein gene was amplified (770 bp) in 35 individuals of seven dromedary camel breeds from the province...[...]
Assuntos
Animais , Camelus/genética , Filogenia , Paquistão , Polimorfismo Genético , Príons/análise , Príons/genética , Deleção de GenesResumo
Background: In many parts of the Old World, domesticated camels (genus - Camelus) are an essential resource, providing food, labor, commodities, and sport to millions of people Of the three extent species, two have been domesticated (singlehumped dromedarius, Camelus dromedarius, and two humped Bactrian camels Camelus bactrianus) and one remains wild (two-humped wild Bactrian camels Camelus ferus). All three species possess a variety adaptations to harsh desert conditions, including mechanisms to tolerate of extreme temperatures, dehydration, and sandy terrain. People residing in harsh climate zones of the world are being benefitted by raising camels in terms of draft, milk, meat, hides and wool from centuries. There are different breeds of dromedary camels distributed in various parts of Pakistan; however there have been scarcity of research work on camels in Pakistan. Identification of novel link between Camel breeders with fatal neurodegenerative disorders is presence or not can be detect by a Prion gene and it was not carried out in Pakistan soil to date. Prion diseases which are a group of fatal neurodegenerative disorders affect both animals and humans. It is believed that the prions are infectious agents responsible for transmissible spongiform encephalopathies. In this study we report the first study on Prion protein gene in dromedary camels of Pakistan. Material, Methods & Results: Genes are the blueprint of life and determine the functional aspects of cellular mechanisms. Genomic DNA of the enrolled blood samples was extracted using the Nucleospin® DNA extraction kit. Genomic DNA was run on Agarose gel electrophoresis, checked the Genomic DNA quality and amplified using prion region specific primer pair. Prion protein gene was amplified (770 bp) in 35 individuals of seven dromedary camel breeds from the province...[...](AU)
Assuntos
Animais , Camelus/genética , Príons/análise , Príons/genética , Paquistão , Polimorfismo Genético , Filogenia , Deleção de GenesResumo
With the objective of detecting the presence of caprine lentivirus (CLV) in ewe milk and in ram semen, ten matrixes and four reproducers experimentally infected with CLV were used. Samples of ewe milk were collected during the four months of lactation, five collections per animal, totaling 50 samples. Regarding the rams, eight semen collections were made per animal, during one year of experimentation, totaling 32 samples. The milk and semen samples were submitted to DNA extraction and the nested polymerase chain reaction test (nPCR) to detect CLV proviral DNA. Eight (16%) of the milk samples were positive in nPCR originating from two ewes. Only one (3.12%) semen sample was positive. The amplification products were sequenced, and were confirmed to be a CLV genomic sequence. Thus, the presence of CLV proviral DNA in sheep milk and semen was demonstrated, confirming the feasibility of infection between species, and alerting to the risk of spreading infections.(AU)
Com o objetivo de detectar a presença do lentivírus caprino (LVC) no leite de ovelhas e no sêmen de carneiros, utilizaram-se 10 matrizes e quatro reprodutores infectados experimentalmente com o LVC. Foram coletadas amostras de leite das ovelhas durante os quatro meses de lactação, ocorrendo cinco coletas por animal, totalizando 50 amostras. Quanto aos carneiros, realizaram-se oito coletas de sêmen por animal, durante um ano de experimentação, totalizando 32 amostras. As amostras de leite e de sêmen foram submetidas à extração de DNA e à prova de reação em cadeia da polimerase do tipo nested (nPCR) visando à detecção de DNA proviral do LVC. Oito (16%) amostras de leite foram positivas na nPCR oriundas de duas ovelhas. Apenas uma (3,12%) amostra de sêmen apresentou positividade. Produtos da amplificação foram sequenciados, confirmando-se tratar de sequência genômica do LVC. Dessa forma, demonstrou-se a presença do DNA proviral do LVC em leite e sêmen de ovinos, confirmando a viabilidade da infecção entre espécies e, assim, alertando sobre o risco de que a infecção seja disseminada.(AU)
Assuntos
Animais , Masculino , Feminino , Lentivirus/isolamento & purificação , Leite/virologia , Ruminantes/virologia , Sêmen/virologia , Transmissão de Doença Infecciosa/veterinária , Reação em Cadeia da Polimerase/veterináriaResumo
With the objective of detecting the presence of caprine lentivirus (CLV) in ewe milk and in ram semen, ten matrixes and four reproducers experimentally infected with CLV were used. Samples of ewe milk were collected during the four months of lactation, five collections per animal, totaling 50 samples. Regarding the rams, eight semen collections were made per animal, during one year of experimentation, totaling 32 samples. The milk and semen samples were submitted to DNA extraction and the nested polymerase chain reaction test (nPCR) to detect CLV proviral DNA. Eight (16%) of the milk samples were positive in nPCR originating from two ewes. Only one (3.12%) semen sample was positive. The amplification products were sequenced, and were confirmed to be a CLV genomic sequence. Thus, the presence of CLV proviral DNA in sheep milk and semen was demonstrated, confirming the feasibility of infection between species, and alerting to the risk of spreading infections.(AU)
Com o objetivo de detectar a presença do lentivírus caprino (LVC) no leite de ovelhas e no sêmen de carneiros, utilizaram-se 10 matrizes e quatro reprodutores infectados experimentalmente com o LVC. Foram coletadas amostras de leite das ovelhas durante os quatro meses de lactação, ocorrendo cinco coletas por animal, totalizando 50 amostras. Quanto aos carneiros, realizaram-se oito coletas de sêmen por animal, durante um ano de experimentação, totalizando 32 amostras. As amostras de leite e de sêmen foram submetidas à extração de DNA e à prova de reação em cadeia da polimerase do tipo nested (nPCR) visando à detecção de DNA proviral do LVC. Oito (16%) amostras de leite foram positivas na nPCR oriundas de duas ovelhas. Apenas uma (3,12%) amostra de sêmen apresentou positividade. Produtos da amplificação foram sequenciados, confirmando-se tratar de sequência genômica do LVC. Dessa forma, demonstrou-se a presença do DNA proviral do LVC em leite e sêmen de ovinos, confirmando a viabilidade da infecção entre espécies e, assim, alertando sobre o risco de que a infecção seja disseminada.(AU)
Assuntos
Animais , Masculino , Feminino , Lentivirus/isolamento & purificação , Leite/virologia , Sêmen/virologia , Ruminantes/virologia , Transmissão de Doença Infecciosa/veterinária , Reação em Cadeia da Polimerase/veterináriaResumo
The aim of the study was to establish a DNA isolation protocol Nectandra megapotamica (Spreng.) Mez., able to obtain samples of high yield and quality for use in genomic analysis. A commercial kit and four classical methods of DNA extraction were tested, including three cetyltrimethylammonium bromide (CTAB)-based and one sodium dodecyl sulfate (SDS)-based methods. Three drying methods for leaves samples were also evaluated including drying at room temperature (RT), in an oven at 40ºC (S40), and in a microwave oven (FMO). The DNA solutions obtained from different types of leaves samples using the five protocols were assessed in terms of cost, execution time, and quality and yield of extracted DNA. The commercial kit did not extract DNA with sufficient quantity or quality for successful PCR reactions. Among the classic methods, only the protocols of Dellaporta and of Khanuja yielded DNA extractions for all three types of foliar samples that resulted in successful PCR reactions and subsequent enzyme restriction assays. Based on the evaluated variables, the most appropriate DNA extraction method for Nectandra megapotamica (Spreng.) Mez. was that of Dellaporta, regardless of the method used to dry the samples. The selected method has a relatively low cost and total execution time. Moreover, the quality and quantity of DNA extracted using this method was sufficient for DNA sequence a...
O objetivo do estudo foi estabelecer um protocolo de isolamento de DNA de Nectandra megapotamica (Spreng.) Mez., capaz de obter amostras de alto rendimento e qualidade para emprego em análises genômicas. Foram testados um kit comercial e quatro métodos clássicos de extração de DNA, incluindo três métodos baseados em brometo de cetiltrimetilamónio (CTAB) e um baseado em dodecil sulfato de sódio (SDS). Três métodos de secagem de amostras de folhas foram também avaliados, incluindo a secagem à temperatura ambiente (RT), em estufa a 40ºC (S40), e em forno microondas (FMO). As soluções de DNA obtidas a partir de diferentes tipos de amostras foliares utilizando os cinco protocolos foram avaliadas em termos de custo, tempo de execução e qualidade e rendimento de DNA extraído. O kit comercial não extraiu DNA com quantidade ou qualidade suficiente para o sucesso das reações de PCR. Entre os métodos clássicos, apenas os protocolos Dellaporta e Khanuja proporcionaram extração de DNA para os três tipos de amostras foliares, que resultaram em reações de PCR e ensaios com enzimas de restrição bem sucedidos. Com base nas variáveis avaliadas, o método de extração de DNA mais apropriado para Nectandra megapotamica (Spreng.) Mez. foi o Dellaporta, independentemente do método utilizado para secar as amostras. O método selecionado apresenta custo e tempo de execução total relativamente baixo. Além...
Assuntos
DNA , Lauraceae/anatomia & histologia , Lauraceae/genéticaResumo
A algaroba, Prosopis juliflora (Sw) DC, é considerada uma alternativa na alimentação de animais em regiões semiáridas, apresentando importância econômica e social. A referida espécie tem recebido atenção por pesquisadores em estudos genéticos moleculares, embora, pouco se sabe sobre a sua diversidade e estrutura genética. Assim, objetivou-se gerar informações relativas à extração de ácidos nucleicos, uso de marcadores moleculares e estimativas de diversidade e estrutura genética que contribuam para subsidiar ações de manejo e melhoramento genético em P. juliflora (Sw.) DC. Para isto, foram testados sete protocolos de extração de DNA genômico disponíveis na literatura, utilizados em diferentes espécies vegetais. Os protocolos foram otimizados por meio da eliminação do uso de -mercaptoetanol, nitrogênio líquido, proteinase K e RNAses. Após as etapas de extração, foi analisada a qualidade e quantidade de DNA obtido e, por fim, realizado teste de amplificação com uso de marcador molecular Resistance Gene Analogs - RGA. Ao final, selecionou-se os três protocolos mais eficientes para a obtenção do DNA genômico: tampão SDS 10%, CTAB 5% e Sorbitol; sendo utilizado o SDS 10% para extração do DNA nos próximos estudos. Posteriormente, foram caracterizados e selecionados 17 combinações de marcadores moleculares RGA, em 20 amostras de DNA genômico da planta extraídas de espécimes coletadas no município de Itapetinga, Bahia. Com base nos perfis de amplificação os pares de iniciadores foram classificados como: Adequado; Razoável e Inadequado. Também foram realizadas análises descritivas associadas ao número de marcadores gerados, onde os percentuais de polimorfismo variou entre 60% e 100%, a média da Heterozigosidade Esperada (He) foi de 0,21 e, o conteúdo de informações polimórficas (PIC) médio foi de 0,17. Dos 17 pares de iniciadores analisados, 10 foram selecionados para estimar a diversidade e a estrutura genética de população referente à sete populações naturalizadas da mesorregião centro sul baiano, composta por 48 acessos e 144 acessos da coleção de germoplasma do Instituto Federal Baiano foram incluídas nas análises. Com base nos perfis de amplificação, realizou-se a genotipagem e a construção da tabela de dados binários. As análises descritivas, Análise de Variância Molecular (AMOVA) e Análise de Coordenadas Principais (PCoA) também foram realizadas. Observamos um total de 147 marcas, cuja média de polimorfismo foi de 51,7% , sendo 12 marcas raras e cinco exclusivas. A Heterozigosidade esperada apresentou média de 0,17, variando de 0,05 em Anagé e 0,32 em Caculé. O conteúdo de informação de polimorfismo variou de 0,04 a 0,25 com média de 0,14. Os acessos estudados apresentaram estruturação em dois pools gênicos e subestruturação em três pools gênicos. A partir dos atributos genéticos moleculares apresentados nesse estudo, a presente Tese busca subsidiar futuros planos de manejo das populações de Prosopis juliflora naturalizada na mesorregião sudoeste da Bahia.
The mesquite, Prosopis juliflora (Sw) DC, is considered an alternative for animal feeding in semiarid regions, presenting economic and social importance. The specie has received attention by researchers in molecular genetic studies, although, there are not much studies about its diversity and genetic structure. This study aimed to generate information related to the extraction of nucleic acids, use of molecular markers and estimation of diversity and genetic structure that contribute to support management and genetic improvement actions of P. juliflora (Sw.) DC. It was used seven genomic DNA extraction protocols available in the literature, used in different botanical species. The protocols were optimized by eliminating the use of -mercaptoethanol, liquid nitrogen, proteinase K and RNAses. After the extraction steps were done, the quality and quantity of DNA obtained was analyzed and an amplification test was performed using a molecular marker Resistance Gene Analogs - RGA. At the end, three most efficient protocols for obtaining genomic DNA were selected: 10% SDS buffer, 5% CTAB and Sorbitol; using 10% SDS for DNA extraction in future studies. Subsequently, 17 combinations of RGA molecular markers were characterized and selected on 20 samples of plant genomic DNA extracted from specimens collected in the county of Itapetinga, Bahia. Based on the amplification profiles the primer pairs were classified as: Adequate; Reasonable and Inappropriate. It was also performed the description of the analyzes associated with the number of generated markers, where the polymorphism percentages ranged between 60% and 100%, the average Expected Heterozygosity (H) was 0.21, and the average polymorphic information content (PIC) was of 0.17. From the 17 pairs of primers analyzed, 10 were selected to estimate the diversity and genetic structure of the population referring to seven naturalized populations from the central southern region of Bahia, comprising 48 accessions and 144 accessions from the germplasm collection of the Instituto Federal Baiano were included in the analyses. Based on the amplification profiles, genotyping and the construction of the binary data table were performed. Descriptive analyses, Molecular Variance Analysis (AMOVA) and Principal Coordinate Analysis (PCoA) were also performed. It was observed a total of 147 brands, whose average polymorphism was 51.7%, being 12 rare brands and five exclusive ones. Expected heterozygosity averaged 0.17, ranging from 0.05 in Anagé to 0.32 in Caculé. The polymorphism information content ranged from 0.04 to 0.25 with a mean of 0.14. The studied accessions presented structure in two gene pools and substructure in three gene pools. From the molecular genetic attributes presented in this study, this doctoral dissertation intends to support future management plans for populations of Prosopis juliflora naturalized in the southwestern mesoregion of Bahia.
Resumo
The aim of the study was to establish a DNA isolation protocol Nectandra megapotamica (Spreng.) Mez., able to obtain samples of high yield and quality for use in genomic analysis. A commercial kit and four classical methods of DNA extraction were tested, including three cetyltrimethylammonium bromide (CTAB)-based and one sodium dodecyl sulfate (SDS)-based methods. Three drying methods for leaves samples were also evaluated including drying at room temperature (RT), in an oven at 40ºC (S40), and in a microwave oven (FMO). The DNA solutions obtained from different types of leaves samples using the five protocols were assessed in terms of cost, execution time, and quality and yield of extracted DNA. The commercial kit did not extract DNA with sufficient quantity or quality for successful PCR reactions. Among the classic methods, only the protocols of Dellaporta and of Khanuja yielded DNA extractions for all three types of foliar samples that resulted in successful PCR reactions and subsequent enzyme restriction assays. Based on the evaluated variables, the most appropriate DNA extraction method for Nectandra megapotamica (Spreng.) Mez. was that of Dellaporta, regardless of the method used to dry the samples. The selected method has a relatively low cost and total execution time. Moreover, the quality and quantity of DNA extracted using this method was sufficient for DNA sequence a...(AU)
O objetivo do estudo foi estabelecer um protocolo de isolamento de DNA de Nectandra megapotamica (Spreng.) Mez., capaz de obter amostras de alto rendimento e qualidade para emprego em análises genômicas. Foram testados um kit comercial e quatro métodos clássicos de extração de DNA, incluindo três métodos baseados em brometo de cetiltrimetilamónio (CTAB) e um baseado em dodecil sulfato de sódio (SDS). Três métodos de secagem de amostras de folhas foram também avaliados, incluindo a secagem à temperatura ambiente (RT), em estufa a 40ºC (S40), e em forno microondas (FMO). As soluções de DNA obtidas a partir de diferentes tipos de amostras foliares utilizando os cinco protocolos foram avaliadas em termos de custo, tempo de execução e qualidade e rendimento de DNA extraído. O kit comercial não extraiu DNA com quantidade ou qualidade suficiente para o sucesso das reações de PCR. Entre os métodos clássicos, apenas os protocolos Dellaporta e Khanuja proporcionaram extração de DNA para os três tipos de amostras foliares, que resultaram em reações de PCR e ensaios com enzimas de restrição bem sucedidos. Com base nas variáveis avaliadas, o método de extração de DNA mais apropriado para Nectandra megapotamica (Spreng.) Mez. foi o Dellaporta, independentemente do método utilizado para secar as amostras. O método selecionado apresenta custo e tempo de execução total relativamente baixo. Além...(AU)