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Evaluation of DNA extraction protocols for Brucella abortus PCR detection in aborted fetuses or calves born from cows experimentally infected with strain 2308; Avaliação de diferentes protocolos de extração de DNA para detecção de Brucella abortus a partir de abortos ou de bezerros nascidos de vacas experimentalmente infectadas com estirpe 2308

MATRONE, Marianna; KEID, Lara Borges; ROCHA, Vivianne Cambuí Mesquita; VEJARANO, M. P; IKUTA, Cássia Yumi; ROSALES RODRIGUEZ, Cesar Alejandro; FERREIRA, Fernando; DIAS, Ricardo Augusto; FERREIRA NETO, José Soares
Fonte: Sociedade Brasileira de Microbiologia; São Paulo Publicador: Sociedade Brasileira de Microbiologia; São Paulo
Tipo: Artigo de Revista Científica
ENG
Relevância na Pesquisa
66.23%
O objetivo do presente estudo foi aperfeiçoar a detecção de Brucella abortus pela PCR em homogeneizados de órgãos de fetos abortados por vacas infectadas, importante mecanismo para descobrir focos da doença na fase de erradicação. Assim, foram comparados diferentes protocolos de extração de DNA, visando à detecção de B. abortus pela PCR em amostras clínicas colhidas de abortos ou de bezerros oriundos de vacas desafiadas com a estirpe 2308 de B. abortus. Para tanto, foram construídos dois grupos padrão ouro com base na bacteriologia clássica, constituídos por: 32 pulmões (17 positivos), 26 baços (11 positivos), 23 fígados (8 positivos) e 22 linfonodos bronquiais (7 positivos). Todas essas amostras foram submetidas a três protocolos de extração de DNA, seguidos do mesmo processo de amplificação com os primers B4 e B5. Nos resultados acumulados por órgão, a proporção de positivos nos pulmões foi maior que a encontrada nos fígados (p=0,04) e nos linfonodos bronquiais (p=0,004), e igual a verificada nos baços (p=0,18). Nos resultados acumulados por método de extração de DNA, a proporção de positivos para o protocolo de Boom foi maior que a verificada para o PK (p<0,0001) e GT (p=0,0004). Não houve diferença entre os protocolos PK e GT (p=0...

Evaluation of DNA extraction protocols for Brucella abortus pcr detection in aborted fetuses or calves born from cows experimentally infected with strain 2308; Avaliação de diferentes protocolos de extração de DNA para detecção de Brucella abortus a partir de abortos ou de bezerros nascidos de vacas experimentalmente infectadas com estirpe 2308

MATRONE, M.; KEID, L.B.; ROCHA, V.C.M.; VEJARANO, M.P.; IKUTA, C.Y.; RODRIGUEZ, C.A.R.; FERREIRA, F.; DIAS, R.A.; FERREIRA NETO, J.S
Fonte: Sociedade Brasileira de Microbiologia Publicador: Sociedade Brasileira de Microbiologia
Tipo: Artigo de Revista Científica
ENG
Relevância na Pesquisa
66.23%
The objective of the present study was to improve the detection of B. abortus by PCR in organs of aborted fetuses from infected cows, an important mechanism to find infected herds on the eradication phase of the program. So, different DNA extraction protocols were compared, focusing the PCR detection of B. abortus in clinical samples collected from aborted fetuses or calves born from cows challenged with the 2308 B. abortus strain. Therefore, two gold standard groups were built based on classical bacteriology, formed from: 32 lungs (17 positives), 26 spleens (11 positives), 23 livers (8 positives) and 22 bronchial lymph nodes (7 positives). All samples were submitted to three DNA extraction protocols, followed by the same amplification process with the primers B4 and B5. From the accumulated results for organ, the proportion of positives for the lungs was higher than the livers (p=0.04) or bronchial lymph nodes (p=0.004) and equal to the spleens (p=0.18). From the accumulated results for DNA extraction protocol, the proportion of positives for the Boom protocol was bigger than the PK (p<0.0001) and GT (p=0.0004). There was no difference between the PK and GT protocols (p=0.5). Some positive samples from the classical bacteriology were negative to the PCR and viceversa. Therefore...

Comparação de protocolos de extração de DNA para detecção de Mycobacterium bovis através da PCR em homogeneizados de órgãos bovinos; DNA extraction protocols comparison for detecting Mycobacterium bovis by PCR in bovine organs homogenates

Ribeiro, Daniella Carvalho
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Dissertação de Mestrado Formato: application/pdf
Publicado em 18/12/2006 PT
Relevância na Pesquisa
66.13%
A tuberculose bovina, cujo agente etiológico é o Mycobacterium bovis, é uma zoonose de caráter crônico que representa uma das principais enfermidades em rebanhos bovinos. Devido às perdas consideráveis de bacilos viáveis nos processos de descontaminação, e também pelo consumo de várias semanas entre o isolamento primário e a identificação final da espécie, métodos moleculares funcionam como metodologia diagnóstica auxiliar à bacteriologia clássica. Estes métodos têm ampla aplicação no diagnóstico e tipificação da tuberculose bovina, no entanto, a baixa sensibilidade quando aplicados diretamente sobre homogeneizados de tecidos tem limitado sua difusão como método diagnóstico de rotina. A solução do problema, ao que tudo indica, passa por métodos de extração mais eficientes e menos vulneráveis aos fatores inibidores da amplificação. Assim, foram selecionadas 60 lesões granulomatosas de 60 bovinos condenados em abatedouro, 30 positivas ao isolamento de Mycobacterium bovis (padrão ouro positivo) e 30 negativas (padrão ouro negativo). Dessas lesões foram obtidos homogeneizados que foram submetidos a diferentes protocolos de extração de DNA, que por sua vez foram todos submetidos ao mesmo protocolo de amplificação. Os resultados mostraram que o protocolo de BOOM et al. (1990) modificado apresentou sensibilidade superior ao isolamento de M. bovis pelo método clássico.; The bovine tuberculosis...

Avaliação de métodos de extração de DNA de Cryptosporidium spp. em amostras fecais e comparação de nested PCR com o médodo coproparasitológico de centrífugo-flutuação em sacarose; Evaluation of DNA extraction methods of Cryptosporidium spp. in feces and comparison of nested PCR with sucrose centrifugal flotation method

Funada, Mikaela Renata
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Dissertação de Mestrado Formato: application/pdf
Publicado em 19/02/2009 PT
Relevância na Pesquisa
66.22%
O desempenho de seis métodos de extração de DNA de oocistos de Cryptosporidium spp. em fezes foram avaliados pela nested PCR do gene SSU rRNA. Os métodos consistem na combinação com pequenas variações de duas técnicas para a liberação de esporozoítos (indução de excistamento/E ou choque térmico/C) e três técnicas de purificação de DNA (fenol-clorofórmio/F, GuSCN-sílica/S ou kit QIAmp DNA Stool Mini/K). Para a avaliação da sensibilidade analítica, os testes foram realizados a partir de diluições seriadas de oocistos purificados, na ausência e na presença de 100 μl de fezes bovinas. A sensibilidade diagnóstica foi avaliada pela comparação com o método microscópico de centrífugo-flutuação em sacarose (padrão-ouro) em 15 amostras fecais de diferentes hospedeiros naturalmente infectados. Na ausência de fezes, foram avaliados apenas os métodos EF, ES, CF e CS, sendo que EF apresentou sensibilidade analítica superior, possibilitando a detecção de até 1 oocisto. Na presença de fezes, os métodos EF, EK e CK apresentaram desempenhos equivalentes, com sensibilidade de 104 oocistos. As maiores sensibilidades diagnósticas foram obtidas pelos métodos EK e CK, que possibilitaram a detecção de 13 (86...

Avaliação de diferentes protocolos de extração de DNA para detecção de Brucella abortus a partir de diferentes tecidos de vacas infectadas experimentalmente com a cepa 2308; Evaluation of different protocols of DNA extraction for Brucella abortus detection from different tissues from experimentally infected cows with 2308 strain

Ruibal, Maria Del Pilar Vejarano
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Dissertação de Mestrado Formato: application/pdf
Publicado em 19/06/2009 PT
Relevância na Pesquisa
66.23%
Este estudo comparou o desempenho de quatro protocolos de extração de DNA a partir de homogeneizados de diferentes órgãos provenientes de vacas infectadas experimentalmente com a B. abortus 2308. Os protocolos de extração comparados foram o método de GT (lise com isotiocianato de guanidina), Boom (lise com GT e tratamento com suspensão carreadora Diatomaceous earth), PK (lise com proteinase K) e Santos (lise por fervura e congelamento com nitrogênio líquido). Foram constituídos os grupos padrão ouro positivo e negativo baseados na bacteriologia clássica e compostos por: 54 cotilédones (27 pos. e 27 neg.), 39 linfonodos supra mamários (12 pos. e 27 neg.), 44 pré-escapulares (17 pos. e 27 neg.), 33 fígados (6 pos. e 27 neg.), 37 baços (10 pos. e 27 neg.), e 34 úberes (7 pos. e 27 neg.). Todas as amostras foram submetidas aos quatro protocolos de extração e a um mesmo processo de amplificação com os primers B4 e B5. Nos resultados consolidados por órgãos, a proporção de positivos no cotilédone foi maior do que a encontrada no linfonodo supramamário (p=0,0001), linfonodo pré-escapular (p<0,0001), fígado (p=0,0006), baço (p<0,0001) ou úbere (p=0,0019). Os resultados acumulados para os métodos de extração mostraram que o protocolo de Santos teve maior sensibilidade relativa do que o método de Boom (p=0...

Aperfeiçoamento de uma técnica para extrair DNA de água do mar e comparação de métodos de extração de DNA na caracterização de uma comunidade bacteriana marinha.; The improvement of a technique for seawater DNA extracting and the comparison of DNA extraction methods for the characterization of a marine bacterial community.

Passini, Maicon Ricardo Zieberg
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Dissertação de Mestrado Formato: application/pdf
Publicado em 21/05/2015 PT
Relevância na Pesquisa
66.27%
Com as limitações para cultivar os micro-organismos, tornou-se necessário caracterizar molecularmente os recursos genéticos microbianos. Entretanto, como estes estudos são realizados através do DNA, podemos encontrar distintos resultados dependendo da metodologia de extração de DNA utilizada. Assim, essa pesquisa buscou aperfeiçoar uma técnica para extrair DNA de água do e para demonstrar a importância dos métodos de extração de DNA no estudo dos micro-organismos por métodos independentes de cultivo, foi caracterizada, através da técnica de DGGE, a diversidade de uma comunidade bacteriana marinha usando diferentes métodos de extração de DNA. O aperfeiçoamento da metodologia de extração de DNA de água do mar resultou em um protocolo com a qualidade do DNA similar à do protocolo original, com rendimento melhor e aproximadamente quatro vezes mais rápido. Em relação à caracterização, verificamos, pelo DGGE, distintos perfis da comunidade microbiana marinha, tanto em relação à presença e ausência das bandas de DNA, como em relação à sua intensidade.; With the limitations of microorganisms culture techniques, it has become necessary to use molecular techniques to characterize microbial genetic resources. However...

Comparison of three DNA extraction methods for Mycobacterium bovis, Mycobacterium tuberculosis and Mycobacterium avium subsp. avium

Amaro, Ana; Duarte, Elsa; Amado, Alice; Ferronha, Helena; Botelho, Ana
Fonte: Universidade de Évora Publicador: Universidade de Évora
Tipo: Artigo de Revista Científica Formato: 109804 bytes; application/pdf
ENG
Relevância na Pesquisa
66.26%
Aims: To compare three methods for DNA extraction from Mycobacterium bovis, Mycobacterium tuberculosis and Mycobacterium avium subsp. avium. Methods and Results: The DNA was extracted from mycobacterial cultures using enzymatic extraction, combined bead beating and enzymatic extraction and cetyltrimethylammonium bromide (CTAB) extraction. The yield and quality of DNA were compared by spectrophotometry, agarose gel electrophoresis, restriction endonuclease analysis and PCR. The combined bead beating and enzymatic extraction method yielded more DNA. However, that method produced some sheared DNA, visible either by agarose gel electrophoresis or by restriction endonuclease analysis. All methods were appropriate for PCR amplification of a 123 bp fragment of IS6110 in M. bovis and M. tuberculosis, and of a 1700 bp fragment of FR300 region in M. avium avium. Conclusions: Combined bead beating and enzymatic extraction method was the most efficient and easy method for extracting DNA from bacteria of the M. tuberculosis complex. Significance and Impact of the Study: The results reveal important differences among the DNA extraction methods for mycobacteria, which are relevant for the success of further downstream molecular analysis.

Isolation of marine meiofauna from sandy sediments From decanting to DNA extraction

Fonseca, Vera; Packer, Margaret; Carvalho, Gary; Power, Deborah; Lambshead, John; Creer, Simon
Fonte: Nature Publishing Group Publicador: Nature Publishing Group
Tipo: Artigo de Revista Científica
Publicado em //2011 ENG
Relevância na Pesquisa
66.13%
This protocol describes the separation of marine meiofauna from sediment and subsequent environmental DNA extraction. In this study meiofauna samples were taken with a 45 mm core from the upper 5 to 10 cm of sediment layer. Separation from sediment was achieved using a decantation process followed by isolation from fine silt using repetitive centrifugation steps with a 1.16 specific gravity (sg) LUDOX-TM solution. Meiofauna were deliberately separated from macrofauna by using a 1 mm sieve on top of a bottle-top sterile 45 !M sieve. High quality DNA was subsequently obtained using the QIAamp DNA Blood Maxi Kit (Qiagen) with minor adjustments to the manufacturer’s protocol. This procedure allowed efficient isolation of meiofaunal representatives from marine sediments and also extraction of high quality environmental DNA that can be used for downstream metagenetic analysis.

Evaluation of three different DNA extraction methods from blood samples collected in dried filter paper in Plasmodium subpatent infections from the Amazon region in Brazil

Miguel,Renata Bortolasse; Coura,José Rodrigues; Samudio,Franklyn; Suárez-Mutis,Martha Cecília
Fonte: Instituto de Medicina Tropical Publicador: Instituto de Medicina Tropical
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/06/2013 EN
Relevância na Pesquisa
66.16%
Asymptomatic Plasmodium infection is a new challenge for public health in the American region. The polymerase chain reaction (PCR) is the best method for diagnosing subpatent parasitemias. In endemic areas, blood collection is hampered by geographical distances and deficient transport and storage conditions of the samples. Because DNA extraction from blood collected on filter paper is an efficient method for molecular studies in high parasitemic individuals, we investigated whether the technique could be an alternative for Plasmodium diagnosis among asymptomatic and pauciparasitemic subjects. In this report we compared three different methods (Chelex®-saponin, methanol and TRIS-EDTA) of DNA extraction from blood collected on filter paper from asymptomatic Plasmodium-infected individuals. Polymerase chain reaction assays for detection of Plasmodium species showed the best results when the Chelex®-saponin method was used. Even though the sensitivity of detection was approximately 66% and 31% for P. falciparum and P. vivax, respectively, this method did not show the effectiveness in DNA extraction required for molecular diagnosis of Plasmodium. The development of better methods for extracting DNA from blood collected on filter paper is important for the diagnosis of subpatent malarial infections in remote areas and would contribute to establishing the epidemiology of this form of infection.

Cheap, rapid and efficient DNA extraction method to perform multilocus microsatellite genotyping on all Schistosoma mansoni stages

Beltran,S; Galinier,R; Allienne,JF; Boissier,J
Fonte: Instituto Oswaldo Cruz, Ministério da Saúde Publicador: Instituto Oswaldo Cruz, Ministério da Saúde
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/08/2008 EN
Relevância na Pesquisa
66.09%
Schistosomes are endoparasites causing a serious human disease called schistosomiasis. The quantification of parasite genetic diversity is an essential component to understand the schistosomiasis epidemiology and disease transmission patterns. In this paper, we propose a novel assay for a rapid, low costly and efficient DNA extraction method of egg, larval and adult stages of Schistosoma mansoni. One euro makes possible to perform 60,000 DNA extraction reactions at top speed (only 15 min of incubation and 5 handling steps).

Direct RAPD evaluation of bacteria without conventional DNA extraction

Araújo,Welington Luiz; Angellis,Derlene Attili de; Azevedo,João Lúcio
Fonte: Instituto de Tecnologia do Paraná - Tecpar Publicador: Instituto de Tecnologia do Paraná - Tecpar
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/07/2004 EN
Relevância na Pesquisa
66.13%
The present work reports successful DNA amplification of Pantoea agglomerans and Bacillus pumilus through Random Amplified Polymorphic DNA (RAPD). For this, template DNA was obtained without conventional DNA extraction. The procedure was as follows: cultures grown for 20 hours in 5 mL LB medium were centrifuged and the resulting preparation was suspended in TE buffer. After boiling, the cell suspension was diluted and 2.0 µl were used in reactions of 15 µl. The results showed no significant differences among the RAPD profile of the PCR reactions derived from the boiling and phenol extraction methods, suggesting the utilization of this method for genetic population analysis.

Rapid yeast DNA extraction by boiling and freeze-thawing without using chemical reagents and DNA purification

Silva,Gildo Almeida da; Bernardi,Taís Letícia; Schaker,Patrícia Dayane Carvalho; Menegotto,Morgana; Valente,Patricia
Fonte: Instituto de Tecnologia do Paraná - Tecpar Publicador: Instituto de Tecnologia do Paraná - Tecpar
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/04/2012 EN
Relevância na Pesquisa
66.12%
The purpose of this work was to study a rapid yeast DNA extraction by boiling and freeze-thawing processes without using chemical reagents or any purification procedures, to obtain a high grade PCR-product. A specific DNA fragment of the 18S region of Dekkera bruxellensis and Saccharomyces cerevisiae was chosen. The described boiling and freeze-thawing protocols generated the PCR-grade product preparations and could be used to process many samples. The amplification of the fragments could be observed after 30 and 35 cycles. These processes of extraction without using any kind of chemical reagents, especial water, and purification procedures proved to be efficient, reproducible, simple, fast, and inexpensive.

Evaluation of DNA extraction protocols for Brucella abortus pcr detection in aborted fetuses or calves born from cows experimentally infected with strain 2308

Matrone,M.; Keid,L.B.; Rocha,V.C.M.; Vejarano,M.P.; Ikuta,C.Y.; Rodriguez,C.A.R.; Ferreira,F.; Dias,R.A.; Ferreira Neto,J.S
Fonte: Sociedade Brasileira de Microbiologia Publicador: Sociedade Brasileira de Microbiologia
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/09/2009 EN
Relevância na Pesquisa
66.19%
The objective of the present study was to improve the detection of B. abortus by PCR in organs of aborted fetuses from infected cows, an important mechanism to find infected herds on the eradication phase of the program. So, different DNA extraction protocols were compared, focusing the PCR detection of B. abortus in clinical samples collected from aborted fetuses or calves born from cows challenged with the 2308 B. abortus strain. Therefore, two gold standard groups were built based on classical bacteriology, formed from: 32 lungs (17 positives), 26 spleens (11 positives), 23 livers (8 positives) and 22 bronchial lymph nodes (7 positives). All samples were submitted to three DNA extraction protocols, followed by the same amplification process with the primers B4 and B5. From the accumulated results for organ, the proportion of positives for the lungs was higher than the livers (p=0.04) or bronchial lymph nodes (p=0.004) and equal to the spleens (p=0.18). From the accumulated results for DNA extraction protocol, the proportion of positives for the Boom protocol was bigger than the PK (p<0.0001) and GT (p=0.0004). There was no difference between the PK and GT protocols (p=0.5). Some positive samples from the classical bacteriology were negative to the PCR and viceversa. Therefore...

Comparison of DNA extraction protocols for microbial communities from soil treated with biochar

Leite,D.C.A.; Balieiro,F.C.; Pires,C.A.; Madari,B.E.; Rosado,A.S.; Coutinho,H.L.C.; Peixoto,R.S.
Fonte: Sociedade Brasileira de Microbiologia Publicador: Sociedade Brasileira de Microbiologia
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/01/2014 EN
Relevância na Pesquisa
66.21%
Many studies have evaluated the effects of biochar application on soil structure and plant growth. However, there are very few studies describing the effect of biochar on native soil microbial communities. Microbial analysis of environmental samples requires accurate and reproducible methods for the extraction of DNA from samples. Because of the variety among microbial species and the strong adsorption of the phosphate backbone of the DNA molecule to biochar, extracting and purifying high quality microbial DNA from biochar-amended soil is not a trivial process and can be considerably more difficult than the extraction of DNA from other environmental samples. The aim of this study was to compare the relative efficacies of three commercial DNA extraction kits, the FastDNA® SPIN Kit for Soil (FD kit), the PowerSoil® DNA Isolation Kit (PS kit) and the ZR Soil Microbe DNA Kit MiniprepTM (ZR kit), for extracting microbial genomic DNA from sand treated with different types of biochar. The methods were evaluated by comparing the DNA yields and purity and by analysing the bacterial and fungal community profiles generated by PCR-DGGE. Our results showed that the PCR-DGGE profiles for bacterial and fungal communities were highly affected by the purity and yield of the different DNA extracts. Among the tested kits...

DNA extraction protocols cause differences in 16S rRNA amplicon sequencing efficiency but not in community profile composition or structure

Rubin, Benjamin E R; Sanders, Jon G; Hampton-Marcell, Jarrad; Owens, Sarah M; Gilbert, Jack A; Moreau, Corrie S
Fonte: BlackWell Publishing Ltd Publicador: BlackWell Publishing Ltd
Tipo: Artigo de Revista Científica
EN_US
Relevância na Pesquisa
66.14%
The recent development of methods applying next-generation sequencing to microbial community characterization has led to the proliferation of these studies in a wide variety of sample types. Yet, variation in the physical properties of environmental samples demands that optimal DNA extraction techniques be explored for each new environment. The microbiota associated with many species of insects offer an extraction challenge as they are frequently surrounded by an armored exoskeleton, inhibiting disruption of the tissues within. In this study, we examine the efficacy of several commonly used protocols for extracting bacterial DNA from ants. While bacterial community composition recovered using Illumina 16S rRNA amplicon sequencing was not detectably biased by any method, the quantity of bacterial DNA varied drastically, reducing the number of samples that could be amplified and sequenced. These results indicate that the concentration necessary for dependable sequencing is around 10,000 copies of target DNA per microliter. Exoskeletal pulverization and tissue digestion increased the reliability of extractions, suggesting that these steps should be included in any study of insect-associated microorganisms that relies on obtaining microbial DNA from intact body segments. Although laboratory and analysis techniques should be standardized across diverse sample types as much as possible...

An assessment of the efficiency of fungal DNA extraction methods for maximizing the detection of medically important fungi using PCR

Karakousis, A.; Tan, L.W.; Ellis, D.; Alexiou, H.; Wormald, P.J.
Fonte: Elsevier Science BV Publicador: Elsevier Science BV
Tipo: Artigo de Revista Científica
Publicado em //2006 EN
Relevância na Pesquisa
66.24%
To date, no single reported DNA extraction method is suitable for the efficient extraction of DNA from all fungal species. The efficiency of extraction is of particular importance in PCR-based medical diagnostic applications where the quantity of fungus in a tissue biopsy may be limited. We subjected 16 medically relevant fungi to physical, chemical and enzymatic cell wall disruption methods which constitutes the first step in extracting DNA. Examination by light microscopy showed that grinding with mortar and pestle was the most efficient means of disrupting the rigid fungal cell walls of hyphae and conidia. We then trialled several published DNA isolation protocols to ascertain the most efficient method of extraction. Optimal extraction was achieved by incorporating a lyticase and proteinase K enzymatic digestion step and adapting a DNA extraction procedure from a commercial kit (MO BIO) to generate high yields of high quality DNA from all 16 species. DNA quality was confirmed by the successful PCR amplification of the conserved region of the fungal 18S small-subunit rRNA multicopy gene.; http://www.elsevier.com/wps/find/journaldescription.cws_home/506034/description#description; A. Karakousis, L. Tan, D. Ellis, H. Alexiou and P.J. Wormald

Residual soil DNA extraction increases the discriminatory power between samples

Young, J.M.; Weyrich, L.S.; Clarke, L.J.; Cooper, A.
Fonte: Springer Publicador: Springer
Tipo: Artigo de Revista Científica
Publicado em //2015 EN
Relevância na Pesquisa
66.22%
Forensic soil analysis relies on capturing an accurate and reproducible representation of the diversity from limited quantities of soil; however, inefficient DNA extraction can markedly alter the taxonomic abundance. The performance of a standard commercial DNA extraction kit (MOBIO PowerSoil DNA Isolation kit) and three modified protocols of this kit: soil pellet re-extraction (RE); an additional 24-h lysis incubation step at room temperature (RT); and 24-h lysis incubation step at 55°C (55) were compared using high-throughput sequencing of the internal transcribed spacer I ribosomal DNA. DNA yield was not correlated with fungal diversity and the four DNA extraction methods displayed distinct fungal community profiles for individual samples, with some phyla detected exclusively using the modified methods. Application of a 24 h lysis step will provide a more complete inventory of fungal biodiversity, and re-extraction of the residual soil pellet offers a novel tool for increasing discriminatory power between forensic soil samples.; Jennifer M. Young, Laura S. Weyrich, Laurence J. Clarke, Alan Cooper

DNA extraction from paraff in-em bedded tissues using a salting-out procedure, a reliable method for PCR amplification of archiva1 material

Howe, J.R.; Klimstra, D.S.; Cordon-Cardo, C.
Fonte: Murcia : F. Hernández Publicador: Murcia : F. Hernández
Tipo: Artigo de Revista Científica Formato: application/pdf
ENG
Relevância na Pesquisa
66.18%
Many techniques have been described for the extraction of DNA from paraffin-embedded tissues. Numerous efforts have been directed at simplification of these methods for rapid analysis using PCR. One disadvantage to some of the simpler procedures is inefficient PCR amplification, and for more involved ones using phenol/chloroform extraction, reduction in the yield of DNA. In the present study we report the use of a novel salting-out procedure that was utilized to extract DNA from 259 separate microdissection specimens of formalin-fixed, paraffin-embedded tissue sections. These sections were derived from 97 patients with tumors of the ampulla of Vater resected between 1965 and 1995 at our institution. The mean DNA yield was 22.75 pg (median 13.2130.25) and the mean 2601280 absorbance ratio was 1.68 (median 1.7020.25). Al1 specimens (2591259) were successfully used to amplify K-ras exon 1 by a nested PCR technique. These results indicate that this DNA extraction method produces good yields of quality DNA, even from specimens several decades old.

DNA extraction from hair shafts of wild Brazilian felids and canids

ALBERTS, C. C.; RIBEIRO-PAES, J. T.; ARANDA-SELVERIO, G.; CURSINO-SANTOS, J. R.; MORENO-COTULIO, V. R.; OLIVEIRA, A. L. D.; PORCHIA, B. F. M. M.; SANTOS, W. F.; SOUZA, E. B.
Fonte: FUNPEC-EDITORA Publicador: FUNPEC-EDITORA
Tipo: Artigo de Revista Científica
ENG
Relevância na Pesquisa
66.18%
Wild felids and canids are usually the main predators in the food chains where they dwell and are almost invisible to behavior and ecology researchers. Due to their grooming behavior, they tend to swallow shed hair, which shows up in the feces. DNA found in hair shafts can be used in molecular studies that can unravel, for instance, genetic variability, reproductive mode and family structure, and in some species, it is even possible to estimate migration and dispersion rates in given populations. First, however, DNA must be extracted from hair. We extracted successfully and dependably hair shaft DNA from eight wild Brazilian felids, ocelot, margay, oncilla, Geoffroy's cat, pampas cat, jaguarundi, puma, and jaguar, as well as the domestic cat and from three wild Brazilian canids, maned wolf, crab-eating fox, and hoary fox, as well as the domestic dog. Hair samples came mostly from feces collected at the Sao Paulo Zoo and were also gathered from non-sedated pet or from recently dead wild animals and were also collected from museum specimens. Fractions of hair samples were stained before DNA extraction, while most samples were not. Our extraction protocol is based on a feather DNA extraction technique, based in the phenol: chloroform: isoamyl alcohol general method...

Comparison of different methodologies for DNA extraction from Aegla longirostri

Bitencourt,João Vitor Trindade; Roratto,Paula Angélica; Bartholomei-Santos,Marlise Ladvocat; Santos,Sandro
Fonte: Instituto de Tecnologia do Paraná - Tecpar Publicador: Instituto de Tecnologia do Paraná - Tecpar
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/11/2007 EN
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The aim of this study was to compare some DNA extraction methodologies for Aegla longirostri. The protocols were based on the traditional phenol-chloroform DNA extraction methodology and using a commercial kit for DNA extraction. They differed in tissues used, the addition - or not - of beta-mercaptoethanol to the lysis buffer, times and methods for the animal's conservation (frozen, in ethanol or fresh). Individuals stored at -20°C for a long time supplied lower molecular weight DNA than those stored for a short time. The best yield for the specimens preserved in ethanol was obtained for 15 days storage in 95% ethanol. The kit resulted in a low quantity of high molecular weight DNA. The best protocol for DNA extraction from Aeglidae, and probably for other crustaceans should, therefore, utilize fresh specimens, with addition of beta-mercaptoethanol to the lysis buffer.