CTAB methods for DNA extraction of sweetpotato for microsatellite analysis (original) (raw)
Related papers
Métodos CTAB de extração de DNA para a análise de microssatélites em batata-doce
2009
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.
Several extraction methods of genomic DNA for identification and characterization of genetic diversity in different plant species are routinely applied during molecular analysis. However, the presence of undesirable compounds such as polyphenols and polysaccharides is one of the biggest problems faced during the isolation and purification of high quality DNA in plants. Therefore, achievement of fast and accurate methods for DNA extraction is crucial in order to produce pure samples. Leaves of strawberry genotypes (Fragaria ananassa) have high contents of polysaccharides and polyphenols which increase the sample viscosity and decrease the DNA quality, interfering with the PCR performance. Thereby, in this study we evaluated the quality and amount of genomic DNA extracted from young leaves of strawberry after tissue lyophilization and maceration in presence of polivinilpirrolidone (PVP). The CTAB method was used as reference procedure and it was modified to improve the DNA extraction. The modifications consisted of tissue lyophilization overnight until it was completely freeze-dried and addition of PVP during the tissue maceration in liquid nitrogen. The results showed the efficiency and reliability of the modified method compared to the unmodified method, indicating that combination of lyophilization and PVP improve the quality and amount of the DNA extracted from strawberry leaves. RESUMO Vários métodos de extração de DNA genômico para a identificação e caracterização da diversidade genética em diferentes espécies de plantas são rotineiramente aplicados durante a análise molecular. Entretanto, a presença de compostos indesejáveis, tais como polifenóis e polissacarídeos, é um dos maiores problemas que ocorrem durante o isolamento e purificação de DNA de alta qualidade em plantas. Dessa forma, o sucesso no desenvolvimento de métodos de extração de DNA rápidos e acurados é crucial para produzir amostras puras. Folhas de genótipos de morangueiro (Fragaria ananassa) têm elevado conteúdo de polissacarídeos e polifenóis que aumentam a viscosidade da amostra e reduzem a qualidade do DNA, interferindo no desempenho da PCR. Neste estudo, avaliamos a qualidade e a quantidade de DNA genômico extraído de folhas jovens de morangueiro após a liofilização do tecido e a maceração na presença de polivinilpirrolidona (PVP). O método CTAB foi utilizado como procedimento de referência e foi modificado para melhorar a extração do DNA. As modificações consistiram na liofilização do tecido a baixa temperatura até que ele tivesse sido desidratado completamente, associada à adição de PVP durante a maceração do tecido no nitrogênio líquido. Os resultados demonstraram a eficiência e a confiabilidade do método modificado comparado ao método não modificado, indicando que a combinação da liofilização com PVP melhora a qualidade e quantidade do DNA extraído de folhas de morangueiro.
Optimización de un protocolo para aislamiento de DNA de hojas de Saccharum officinarum
Revista Mexicana de Ciencias Agrícolas, 2017
La extracción de DNA libre de polisacáridos, polifenoles, proteínas y RNA de plantas constituye el paso inicial para diversos estudios en Biología molecular. Existen diversos métodos de extracción de DNA específicos para caña de azúcar (Saccharum officinarum), sin embargo, consumen mucho tiempo y utilizan reactivos y equipos costosos. El objetivo del presente estudio fue optimizar un protocolo rápido, eficiente y de bajo costo para extraer DNA de hojas de S. officinarum. Las variables evaluadas fueron rendimiento, pureza, integridad y funcionalidad del DNA purificado de hojas de caña de azúcar de 6 meses de edad. Se utilizaron técnicas de espectrofotometría, electroforesis en geles de agarosa y marcadores moleculares para evaluar las variables anteriores. Las variables rendimiento (mg/g) y pureza (A260:280 y A260:230) del DNA extraído mostraron diferencias altamente significativas (p˃ 0.05) con el protocolo I. Los mejores resultados se obtuvieron con la interacción del protocolo I +...
Extraction of high-quality DNA from ethanol-preserved tropical plant tissues
BMC Research Notes, 2014
Background: Proper conservation of plant samples, especially during remote field collection, is essential to assure quality of extracted DNA. Tropical plant species contain considerable amounts of secondary compounds, such as polysaccharides, phenols, and latex, which affect DNA quality during extraction. The suitability of ethanol (96% v/v) as a preservative solution prior to DNA extraction was evaluated using leaves of Jatropha curcas and other tropical species.
A rapid and simple method for small-scale DNA extraction inAgavaceae and other tropical plants
Plant Molecular Biology Reporter, 2002
Abstract. This protocol permits the simultaneous extraction of clean DNA from many samples with little reagent waste, thus decreasing the cost of analysis per sample. The procedure is rapid, permitting the processing of 80-100 samples per day. Using this protocol, we analyzed naturally propagated and micropropagated populations of henequen and other Agavaceae species using amplified fragment length polymorphism (AFLP). Agaves have succulent leaves with a content that is high in fiber and chemical compounds. Therefore, this protocol should work for other tropical and subtropical plant species. The protocol involves precipitation and resuspension of DNA 3 times at the end of the preparation; this increases DNA digestibility and the sharpness of AFLP bands. Full text † : This manuscript, in detail, is available only in the electronic version of the Plant Molecular Biology Reporter.
Extraction of DNA from milligram amounts of fresh, herbarium and mummified plant tissues
Plant molecular biology, 1985
We have developed a DNA extraction procedure for milligram amounts of plant tissue. Yields ranged from 0.3-200 nanograms of DNA per milligram of tissue. The factors affecting yield are discussed. Fresh tissue, as well as herbarium specimens (22-118 years old) and mummified seeds and embryos (500 to greater than 44 600 years old) were used. All tissues attempted (57 types from 29 species) yielded measurable amounts of DNA. In no case tested was inhibition observed for restriction enzymes BamHI or EcoRI.
DNA extraction from silica gel-preserved common bean (Phaseolus vulgaris L.) leaves
African Journal of Biotechnology
Extraction of non-degraded and contaminant-free DNA from field specimen requires collection under liquid nitrogen which is not readily available in resource constrained laboratories in low and middle income countries (LMICs). A method of extracting DNA from silica gel-preserved common bean (Proteus vulgaris L.) leaves is presented. The method, which does not involve the use of phenol, chloroform or isoamyl alcohol also obviates the need for low temperature incubation during the DNA extraction steps and the grinding of desiccated leaf tissue in liquid nitrogen. It relies on inactivating proteins using SDS and proteinase K along with precipitation of polysaccharides using a high salt solution (0.8 M NaCl). DNA is further purified by exploiting its insolubility in aqueous media. High quality pure DNA (mean concentration 2.84 ± 0.013 µg/g of dry leaf tissue) with mean DNA purity values of 2.1 ± 0.1 was extracted. The DNA was also found to be free of protein and polysaccharide contamination. This method enables DNA amplification using molecular markers routinely used in molecular biology laboratories like random amplified polymorphic (RAPD) markers, inter simple sequence repeat (ISSR) markers, sequence-characterized amplified region (SCAR) markers and simple sequence repeat (SSR) markers. The findings of this study show that it is possible to obtain high quality DNA from leaf tissue preserved in silica gel. The method used in this research will be invaluable to resource constrained laboratories in low and middle income countries (LMICs) that cannot afford to buy or access liquid nitrogen in order to extract high quality DNA and for research groups undertaking field surveys that require several days or weeks off station without laboratory freezers to maintain the integrity of the tissues which is crucial for obtaining high quality DNA.
DNA Isolation Protocol for Plants
A simple and efficient protocol for isolating genomic DNA from fresh and dry leaves of Vigna aconitifolia and V. trilobata was developed. It involves a modified CTAB procedure using 3% CTAB, 4% -mercaptoethanol, 2 M NaCl and 5% PVP. The extraction was carried out at 70°C. A slight increase in the concentrations of these chemical components and temperature helped in the removal of secondary metabolites and polysaccharides from the DNA preparation. The quantity and purity of isolated DNA was higher when compared with DNA extracted by the methods of and Doyle and Doyle (1990) [9, 13]. The DNA yield ranged from 45 -55 µg per g of leave samples and it was 1.25 times greater in dried than fresh samples. The DNA samples were found suitable for analysis with restriction enzyme digestion and randomamplification of polymorphic DNA (RAPD).
A b s t r a c t DNA based markers have extensively been used in genome mapping. Restriction Fragment Length Polymorphism (RFLP) markers were the first to be used for this purpose. With the advent of Taq DNA Polymerase, PCR-based markers have become popular since they require less time, effort and expense for molecular mapping. PCR based studies such as in construction of a linkage map, estimating genetic diversity in a germplasm etc. involves a limited number of samples (50-200). Such studies require isolation of sufficiently pure DNA, which is suitable both for the purpose as well as for preserving the same for a considerable period of time. Although the DNA needed per reaction in PCR based markers is very low, the number of PCR reactions to be performed is large and hence a good amount of DNA would be needed for such studies. We have developed a simple method for isolation of DNA from plant tissue (leaf or seed). The method is suitable for isolating DNA from a small to medium number of plant samples. The DNA can be stored for a longer duration. The method involves extraction of DNA using a buffer (pH 8.0) containing Tris (100 mM), EDTA (20 mM), 7 M urea, 0.5 M NaCl and 0.1% β-mercaptoethanol, followed by purification of DNA with phenol, chloroform and Isoamlyalcohol and finally precipitation of DNA by sodium acetate and isopropanol. The protocol is simple and fast as it does not involve time consuming steps such as incubation at higher temperatures, does not require expensive chemicals such as proteinase K, liquid nitrogen etc., and no special equipment is needed.