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The isolation of DNA from placental tissue suspected of infection is an important tool for identifying microorganisms such as bacteria, fungi, and viruses associated with complications during and after pregnancy. While experts primarily process placental tissue, the preservation methods employed pose challenges to extracting complete DNA. Therefore, selecting the appropriate protocol is paramount to achieving greater efficiency in obtaining genetic material.

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Molecular markers are important tools in the characterization of plant genetic diversity and can provide support for conservation strategies for endangered populations. The different molecular techniques involve the evaluation of many individuals; therefore, it is crucial to have fast, efficient, and inexpensive methods for DNA extraction. Given the importance of the Aroeira (Myracrodruon urundeuva Fr. All.) it is pertinent to optimize a protocol that allows the obtainment of intact and pure DNA, aiming to assist conservation strategies for this species that is threatened with extinction. Thus, this study aimed to compare five DNA extraction methods: Dellaporta et al. (1983), Doyle and Doyle (1987) modified, Ferreira and Grattapaglia (1995), Romano and Brasileiro (2015), and Khanuja et al. (1999) and optimize the most efficient protocol for M. urundeuva. The modified DNA extraction protocol proposed by Doyle and Doyle (1987), using 100 mg of leaf tissue and 6 µl of ß-mercaptoethanol was the protocol that presented the sharpest bands after DNA electrophoresis and after the reactions of amplification employing Polymerase Chain Reaction (PCR). Therefore, it is suggested to use the protocol described by Doyle and Doyle (1987) modified for the extraction of DNA from young M. urundeuva leaves to carry out techniques involving molecular markers.

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An optimized high-quality DNA isolation protocol was developed using body segment tissue from the Fall Armyworm (Spodoptera frugiperda), that will allow documenting genetic variability based on biotypes, facilitating studies on the appearance, distribution and population dynamics of the fall armyworm at the molecular level. The resulting protocol is an easy-to-use, timesaving method that can rapidly achieve high quality, high-yielding total genomic DNA, using chemicals and everyday consumables available in a molecular laboratory. This new method of DNA extraction avoids the contamination of polysaccharides, salts, phenols, proteins and other cellular by-products that can interfere with subsequent reactions. DNA purity estimates reveal A260: A280 ratios greater than 1.9, which were evidenced by quality test on agarose gel, observing complete integrity and high purity of the resulting samples, and yielded 30-99 µg/g of total DNA. Therefore, the quality of the DNA produced from this extraction is suitable for subsequent molecular applications: (i) next generation whole genome sequencing, (ii) conventional polymerase chain reaction for genotyping, (iii) barcodes and (iv) gene cloning. In addition, to become an anticipating diagnostic tool for invasive lepidopteran larval stages:•The resulting protocol is an easy-to-use time-saving method.•This new extraction method prevents contamination from polysaccharides, salts, phenols, proteins, and other cellular sub-products.•DNA purity estimations reveal A260:A280 ratios above 1.9.

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"Sucupira branca" is a plant found in the Brazilian Cerrado and is adapted to low fertility soils, and its fruit extract has anti-inflammatory, healing, antiulcerogenic, antimicrobial, cercaricidal, leishmanicidal and antioxidant activities. Furthermore, it provides protection against oxidative stress, is a natural biocontrol agent of Aedes aegypti, has very resistant wood, is a melliferous plant and has been used in reforestation programs. The development of conservation strategies is important for maintaining diversity in natural populations of "sucupira branca" since these populations are in the process of genetic erosion. Molecular biology techniques, which are important for characterizing the genetic diversity of plants to develop conservation strategies, require sufficient high-quality genomic deoxyribonucleic acid (DNA). This study aimed to compare five methods to extract DNA from "sucupira branca". The quality and concentration of DNA were revealed by agarose gel electrophoresis, and only the protocols of Dellaporta, Wood and Hicks et al. (1983) and Khanuja et al. (1999) did not result in satisfactory quantities of DNA. When PCR (Polymerase Chain Reaction) was performed with three inter-simple sequence repeat (ISSR) primers, DNA was successfully amplified from extractions performed with the protocols proposed by Doyle and Doyle (1987), Romano and Brasileiro (1998) and Ferreira and Grattapaglia (1995), which are less expensive than commercial purification kits. These protocols resulted in DNA of sufficient quality and quantity after the amplification reactions were performed.

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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)

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The objective of this study was to test the efficiency of preservation and maceration methods for Euterpe precatoria leaflet tissue to obtain genomic DNA for molecular studies. The leaflets of E. precatoria were collected in an experimental field at Embrapa Acre, Brazil. The study was conducted in a completely randomized design with 10 replicates, in a 12 × 2 factorial structure, with 12 storage treatments (fresh; lyophiliser 3 days; refrigerator 3, 5, and 7 days; silica gel 7, 10, 20, and 30 days; and transport buffer 3, 5, and 7 days) and two leaf tissue maceration methods (liquid nitrogen and the TissueLyser®). Statistically significant differences in the obtained DNA concentration were found between the maceration and storage treatments. The TissueLyser® macerator produced higher DNA concentrations when compared to liquid nitrogen. For the storage treatments, five groups were formed based on DNA concentration when macerated with the TissueLyser® and two groups when macerated with liquid nitrogen. The DNA concentrations ranged from 285.00 ng/µ L (7 days in transport buffer) to 702.00 ng/µ L (30 days in silica gel) when the leaflets were macerated with liquid nitrogen, and from 572.73 ng/µ L (30 days in silica gel) to 2,850.00 ng/µ L (3 days in lyophiliser) using the TissueLyser® macerator. The DNA purity (A260/A280 nm) varied from 1.30 to 1.70 when the leaflets were macerated with liquid nitrogen and from 1.30 to 1.90 with the TissueLyser® macerator. Despite the variations in leaf tissue preservation and DNA concentration, all treatments were effective for DNA isolation and it was possible to amplify genomic regions of microsatellite markers by PCR. It was concluded that leaflets of E. precatoria stored in a lyophiliser and processed with an automatic macerator resulted in satisfactory DNA for molecular studies.

O objetivo deste estudo foi testar a eficiência de métodos de preservação e maceração de tecidos de folíolos de Euterpe precatoria para obter DNA genômico para estudos moleculares. Os folíolos de E. precatoria foram coletados no campo experimental da Embrapa Acre, Brasil. O estudo foi conduzido em delineamento inteiramente casualizado com 10 repetições, em esquema fatorial 12 × 2, com 12 tratamentos de armazenamento (fresco; liofilizado 3 dias; geladeira 3, 5, e 7; sílica gel 7, 10, 20 e 30 dias e tampão de transporte 3, 5 e 7 dias) e dois tipos de maceração do tecido foliar (nitrogênio líquido e TissueLyser®). Para a variável concentração de DNA houve diferença estatística entre os tipos maceração e de armazenamento. O macerador TissueLyser® apresentou maiores concentrações de DNA quando comparado ao nitrogênio líquido.Para os tipos de armazenamento verificou-se formação de cinco grupos quando macerados TissueLyser® e dois grupos quando macerados com nitrogênio líquido. As concentrações de DNA variaram de 285,00 ng/µ L (7 dias em tampão de transporte) a 702,00 ng/µ L (30 dias em sílica gel) quando maceradas com nitrogênio líquido. Quando maceradas com macerador TissueLyser® variaram de 572,73 ng/µ L (30 dias em sílica gel) a 2.850,00 ng/µ L (3 dias em liofilizador). A pureza do DNA (A260/A280 nm) variou de 1,30 a 1,70 quando os folíolos foram macerados com nitrogênio líquido e de 1,30 a 1,90 quando macerados em macerador TissueLyser®. Apesar das variações na conservação e concentração dos tecidos foliares, todos os tratamentos foram eficazes para o isolamento do DNA e amplificaram regiões de marcadores microssatélites. Concluiu-se que folíolos de E. precatoria armazenados em liofilizador e macerados com macerador automático resultaram em DNA satisfatório para estudos moleculares.

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Next-generation genome mapping through nanochannels (Bionano optical mapping) of plant genomes brings genome assemblies to the 'nearly-finished' level for reliable and detailed gene annotations and assessment of structural variations. Despite the recent progress in its development, researchers face the technical challenges of obtaining sufficient high molecular weight (HMW) nuclear DNA due to cell walls which are difficult to disrupt and to the presence of cytoplasmic polyphenols and polysaccharides that co-precipitate or are covalently bound to DNA and might cause oxidation and/or affect the access of nicking enzymes to DNA, preventing downstream applications. Here we describe important improvements for obtaining HMW DNA that we tested on Solanum crops and wild relatives. The methods that we further elaborated and refined focus on •Improving flexibility of using different tissues as source materials, like fast-growing root tips and young leaves from seedlings or in vitro plantlets.•Obtaining nuclei suspensions through either lab homogenizers or by chopping.•Increasing flow sorting efficiency using DAPI (4',6-diamidino-2-phenylindole) and PI (propidium iodide) DNA stains, with different lasers (UV or 488 nm) and sorting platforms such as the FACSAria and FACSVantage flow sorters, thus making it appropriate for more laboratories working on plant genomics. The obtained nuclei are embedded into agarose plugs for processing and isolating uncontaminated HMW DNA, which is a prerequisite for nanochannel-based next-generation optical mapping strategies.

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The Anacardiaceae is an important and worldwide distributed family of ecological and socio-economic relevance. Notwithstanding that, molecular studies in this family are scarce and problematic because of the particularly high concentration of secondary metabolites-i.e. tannins and oleoresins-that are present in almost all tissues of the many members of the group, which complicate the purification and amplification of the DNA. The objective of this work was to improve an available DNA isolation method for Schinopsis spp. and other related Anacardiaceae, as well as the PCR protocols for DNA amplification of the chloroplast trnL-F, rps16 and ndhF and nuclear ITS-ETS fragments. The modifications proposed allowed the extraction of 70-120 µg of non-degraded genomic DNA per gram of dry tissue that resulted useful for PCR amplification. PCR reactions produced the expected fragments that could be directly sequenced. Sequence analyses of amplicons showed similarity with the corresponding Schinopsis accessions available at GenBank. The methodology presented here can be routinely applied for molecular studies of the group aimed to clarify not only aspects on the molecular biology but also the taxonomy and phylogeny of this fascinating group of vascular plants.

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Stool is chemically complex and the extraction of DNA from stool samples is extremely difficult. Haemoglobin breakdown products, such as bilirubin, bile acids and mineral ions, that are present in the stool samples, can inhibit DNA amplification and cause molecular assays to produce false-negative results. Therefore, stool storage conditions are highly important for the diagnosis of intestinal parasites and other microorganisms through molecular approaches. In the current study, stool samples that were positive for Giardia intestinalis were collected from five different patients. Each sample was stored using one out of six different storage conditions [room temperature (RT), +4ºC, -20ºC, 70% alcohol, 10% formaldehyde or 2.5% potassium dichromate] for DNA extraction procedures at one, two, three and four weeks. A modified QIAamp Stool Mini Kit procedure was used to isolate the DNA from stored samples. After DNA isolation, polymerase chain reaction (PCR) amplification was performed using primers that target the β-giardin gene. A G. intestinalis-specific 384 bp band was obtained from all of the cyst-containing stool samples that were stored at RT, +4ºC and -20ºC and in 70% alcohol and 2.5% potassium dichromate; however, this band was not produced by samples that had been stored in 10% formaldehyde. Moreover, for the stool samples containing trophozoites, the same G. intestinalis-specific band was only obtained from the samples that were stored in 2.5% potassium dichromate for up to one month. As a result, it appears evident that the most suitable storage condition for stool samples to permit the isolation of G. intestinalis DNA is in 2.5% potassium dichromate; under these conditions, stool samples may be stored for one month.

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In this work, genomic DNA of Streptococcus pyogenes, S. mutans and S. sobrinus was isolated using two methods: either using the detergent cetyltrimethylammonium bromide (CTAB) at 65ºC; or by applying ultrasound to a mixture of silica and celite in CTAB. The composite method that used ultrasound was the more efficient, allowing the straightforward extraction of genomic DNA from Gram-positive bacteria with good quality and reproducibility.

O gênero Streptococcus encontra-se amplamente distribuído na natureza e algumas espécies constituem a microbiota humana da cavidade bucal, como Streptococcus pyogenes, que pode estar associado a importantes doenças humanas, Streptococcus mutans e Streptococcus sobrinus, relacionados à cárie dental. O DNA genômico destas três espécies foi isolado utilizando-se dois métodos, o primeiro utilizando o detergente brometo de cetiltrimetilamônio (CTAB) à 65ºC e outro associando ultra-som a uma mistura de sílica e celite em CTAB. O método que possibilitou a extração do DNA genômico das bactérias Gram positivas, com qualidade, boa reprodutibilidade fácil execução foi aquele que utilizou ultra-som associado à sílica e celite em CTAB.

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Microsatellite markers have proved to be useful in genetic diversity assessments of sweetpotato (Ipomoea batatas) but practical DNA extraction methods to ensure good quality and quantity DNA for these studies are yet to be established. This study compares the efficiency of three modified methodologies for DNA extraction of six sweetpotato landraces using the CTAB extraction buffer in regard to quantity and purity of DNA quantification and microsatellite band patterns. All methodologies yielded satisfactory results, but the method based in leaf tissue macerated in liquid nitrogen was deemed more adequate because of its simplicity and lower cost. However, the method based in dry leaf tissue was considered more advantageous, first because elicits practicability in the plant acquisition and drying process, especially when the collection is performed in situ, and also because its simplicity makes possible the cold storage of the dry, ground samples for future DNA extractions.

Os marcadores microssatélites são úteis para a análise da diversidade genética de variedades tradicionais de batata-doce (Ipomoea batatas). Para estes estudos, métodos práticos de extração de DNA precisam ser estabelecidos para assegurar uma boa qualidade e quantidade de DNA extraído. Assim, foi comparada a eficiência de três metodologias para extração de DNA usando o tampão de extração CTAB, todas com modificações. Para verificar a quantidade e pureza na quantificação de DNA, bem como o padrão de bandas de microssatélites para as três metodologias utilizaram-se seis etnovariedades de batata-doce. Os testes mostraram que as três metodologias apresentaram resultados satisfatórios. Uma das metodologias baseada em tecido foliar macerado em nitrogênio líquido mostrou-se a mais adequada devido à simplicidade e menor custo. Entretanto, o método baseado em tecido foliar seco foi o mais vantajoso devido à praticidade na aquisição da planta e no processo de secagem, principalmente quando a coleção encontra-se em condições in situ, e pela possibilidade do armazenamento refrigerado das amostras secas e maceradas para futuras extrações de DNA.

RESUMO

Microsatellite markers have proved to be useful in genetic diversity assessments of sweetpotato (Ipomoea batatas) but practical DNA extraction methods to ensure good quality and quantity DNA for these studies are yet to be established. This study compares the efficiency of three modified methodologies for DNA extraction of six sweetpotato landraces using the CTAB extraction buffer in regard to quantity and purity of DNA quantification and microsatellite band patterns. All methodologies yielded satisfactory results, but the method based in leaf tissue macerated in liquid nitrogen was deemed more adequate because of its simplicity and lower cost. However, the method based in dry leaf tissue was considered more advantageous, first because elicits practicability in the plant acquisition and drying process, especially when the collection is performed in situ, and also because its simplicity makes possible the cold storage of the dry, ground samples for future DNA extractions.

Os marcadores microssatélites são úteis para a análise da diversidade genética de variedades tradicionais de batata-doce (Ipomoea batatas). Para estes estudos, métodos práticos de extração de DNA precisam ser estabelecidos para assegurar uma boa qualidade e quantidade de DNA extraído. Assim, foi comparada a eficiência de três metodologias para extração de DNA usando o tampão de extração CTAB, todas com modificações. Para verificar a quantidade e pureza na quantificação de DNA, bem como o padrão de bandas de microssatélites para as três metodologias utilizaram-se seis etnovariedades de batata-doce. Os testes mostraram que as três metodologias apresentaram resultados satisfatórios. Uma das metodologias baseada em tecido foliar macerado em nitrogênio líquido mostrou-se a mais adequada devido à simplicidade e menor custo. Entretanto, o método baseado em tecido foliar seco foi o mais vantajoso devido à praticidade na aquisição da planta e no processo de secagem, principalmente quando a coleção encontra-se em condições in situ, e pela possibilidade do armazenamento refrigerado das amostras secas e maceradas para futuras extrações de DNA.

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This study describes a rapid procedure for the isolation of genomic DNA from Staphylococcus aureus that yielded a good amount of high quality DNA for the amplification of staphylococcal enterotoxins genes (A, B, C, D, and E) and the TSST-1 gene as well as enzymatic restriction (HaeIII) from environmental isolates. With this method, it was possible to detect these genes in a sample containing as little as 10(5) cells with positive PCR reactions obtained from approximately 10pg of DNA in a final reaction volume of 25µl.(AU)

Descreve-se um procedimento rápido para extração de DNA genômico de isolados de Staphylococcus aureus capaz de produzir DNA estafilocócico em qualidade e quantidade suficiente para a amplificação de genes que codificam enterotoxinas estafilocócicas (A - E) e para TSST-1 e restrição enzimática (HaeIII) de isolados ambientais. O método proposto foi capaz de detectar esses genes em um produto de extração contendo tanto quanto 10(5) células, e reações positivas de PCR foram obtidas de aproximadamente 10pg de DNA.(AU)

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This study describes a rapid procedure for the isolation of genomic DNA from Staphylococcus aureus that yielded a good amount of high quality DNA for the amplification of staphylococcal enterotoxins genes (A, B, C, D, and E) and the TSST-1 gene as well as enzymatic restriction (HaeIII) from environmental isolates. With this method, it was possible to detect these genes in a sample containing as little as 10(5) cells with positive PCR reactions obtained from approximately 10pg of DNA in a final reaction volume of 25µl.

Descreve-se um procedimento rápido para extração de DNA genômico de isolados de Staphylococcus aureus capaz de produzir DNA estafilocócico em qualidade e quantidade suficiente para a amplificação de genes que codificam enterotoxinas estafilocócicas (A - E) e para TSST-1 e restrição enzimática (HaeIII) de isolados ambientais. O método proposto foi capaz de detectar esses genes em um produto de extração contendo tanto quanto 10(5) células, e reações positivas de PCR foram obtidas de aproximadamente 10pg de DNA.

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