Simple and robust DNA extraction method for the large-scale analysis of genotypes containing high polyphenolic content, such as landracesof Solanum tuberosum and Zea mays

Abstract

M. Barra, E. Salazar, M. Beltrán, and B. Sagredo. 2012. Simple and robust DNA extraction method for the large-scale analysis of genotypes containing high polyphenolic content, such as landraces of Solanum tuberosum and Zea mays. Cien Inv. Agr. 39(3): 593-601. The extraction of high-quality DNA is essential for studies conducted at the molecular level in species with an abundance of contaminants in their tissues, such as some landraces of potato and maize, in which it is difficult to extract good-quality genomic DNA. Compounds such as polyphenols interfere with the amplification of DNA during polymerase chain reaction (PCR), which makes it difficult to conduct PCR-based studies of molecular markers. This article describes a simple and robust protocol for DNA isolation that was applied to potato and maize landraces and resulted in a high DNA concentration with excellent purity and quality. This method is based on the method of Krizman et al. (2006) with some modifications and was tested on potato leaves and young maize leaves. The protocol was compared with one commercial DNA extraction kit and three methods that were previously described for plant DNA extraction: Keb-Llanes et al. (2002), for plants with high polyphenolic content; Fulton et al. (1995), which was developed for Solanaceae and other herbaceous plants; and the original Krizman et al. (2006) cetyltrimethylammonium bromide (CTAB)-activated charcoal method . The method proposed is suitable for isolating DNA from potato and maize genotypes with high polyphenol contents, yielding high-quality DNA that can be utilized for molecular techniques that involve PCR, cutting with restriction enzymes and other applications. The method is short, similar to a commercial kit, but cheaper. The amount of DNA extracted from maize using our method is superior compared with other published methods tested in this article.

An efficient and economical method for extraction of DNA amenable to biotechnological manipulations, from diverse soils and sediments

“…) and plant genomic DNA (Barra et al . ). Use of activated charcoal in combination with other purifying agents, such as anion exchange resins (Desai and Madamwar ) and PVPP (Verma and Satyanarayana ), has also been reported in isolation of pure metagenomic DNA from activated sludge samples and soil, respectively.…”

“…It has the ability to remove a number of nonbiodegradable organic substances including humic acid, lignin sulfonate, tannic acid, arabic gum along with several biodegradable substances from waste water (Seo and Ohgaki 2001). Activated charcoal has been used for obtaining pure plasmid (Kim et al 2010) and plant genomic DNA (Barra et al 2012). Use of activated charcoal in combination with other purifying agents, such as anion exchange resins (Desai and Madamwar 2006) and PVPP (Verma and Satyanarayana 2011), has also been reported in isolation of pure metagenomic DNA from activated sludge samples and soil, respectively.…”

Comparative analysis of different methods of Hedera helix DNA extraction and molecular evidence of the functionality in PCR

“…Polysacharides are difficult to separate from DNA during the extraction (Porebski et al, 1997;Lade et al, 2014) and in the subsequent PCR they interfere with polymerases. Moreover, phenol, alkaloids, polyphenols Rogers and Bendich (1994) and polysacharides were identified to inhibit enzymes in the PCR or restriction reactions (Barra et al, 2012). Therefore, the first step in the work-flow of plant DNA analyses is to choose the most appropriate extraction method.…”

Comparison of four DNA extraction methods in Heliconia (Heliconia L.)

“…Methods 2 and 4 included PVP in their extraction buffer while Method 3 did not. PVP has been reported to form insoluble complexes with lactones and phenolic compounds (Kim et al, 1997;Barra et al, 2012), and the presence of these compounds in Heliconia spp. have been detected in different studies (Estrada et al, 2008;Hernández-Meneses et al, 2013).…”

“…These compounds may undergo rapid oxidation, binding tightly to the DNA and subsequently, co-precipitate with the DNA preventing its use in downstream applications (Porebski et al, 1997;Sheperd et al, 2002;Shabnam and Saeed, 2016;Swati et al, 2016). Tropical plants for example, often have high levels of polysaccharides, polyphenols, proteins, lipids, among others, which make the isolation and purification of DNA difficult (Barra et al, 2012;Cavallari et al, 2014;Huang et al, 2014). In addition, the presence of these compounds in the isolated DNA inhibits the activity of various enzymes used in molecular biology experiments, such as DNA *Corresponding author.…”