Methodology A rapid and inexpensive method for isolation of total DNA from Trichoderma spp (Hypocreaceae)

Vazquez-Angulo, J C; Méndez-Trujillo, Vianey; González-Mendoza, Daniel; Morales, Adriana; Grimaldo-Juarez, Onecimo; Cervantes-Díaz, Lourdes

doi:10.4238/2012.may.15.8

Cited by 10 publications

(6 citation statements)

References 4 publications

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“…In developing our new protocol, we aimed for a method that would (a) consistently produce good-quality DNA for biome characterization, (b) be scalable across samples of vastly different size, (c) be economically feasible (i.e., cheap), (d) require little dedicated equipment and (e) present no health hazards, thus excluding methods based on phenol extraction (California et al, 2012). To minimize the time spent on method development, we naturally based it on previously established protocols.…”

Section: A New Methodsmentioning

confidence: 99%

“…The large size of many fruiting bodies poses challenges for sample handling and makes standard DNA extraction methods ungainly. More important, the complex chemical properties of fungi, such as high amounts of chitin, trehalose and secondary metabolites, have impaired the PCR steps critical in molecular analyses (California et al, 2012;Cubero, Crespo, Fatehi, & Bridge, 1999;Haugland, Heckman, & Wymer, 1999). The solutions developed to date have generally been optimized for relatively small-sized samples.…”

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confidence: 99%

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Finding flies in the mushroom soup: Host specificity of fungus‐associated communities revisited with a novel molecular method

et al. 2018

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Fruiting bodies of fungi constitute an important resource for thousands of other taxa. The structure of these diverse assemblages has traditionally been studied with labour-intensive methods involving cultivation and morphology-based species identification, to which molecular information might offer convenient complements. To overcome challenges in DNA extraction and PCR associated with the complex chemical properties of fruiting bodies, we developed a pipeline applicable for extracting amplifiable total DNA from soft fungal samples of any size. Our protocol purifies DNA in two sequential steps: (a) initial salt-isopropanol extraction of all nucleic acids in the sample is followed by (b) an extra clean-up step using solid-phase reversible immobilization (SPRI) magnetic beads. The protocol proved highly efficient, with practically all of our samples-regardless of biomass or other properties-being successfully PCR-amplified using metabarcoding primers and subsequently sequenced. As a proof of concept, we apply our methods to address a topical ecological question: is host specificity a major characteristic of fungus-associated communities, that is, do different fungus species harbour different communities of associated organisms? Based on an analysis of 312 fungal fruiting bodies representing 10 species in five genera from three orders, we show that molecular methods are suitable for studying this rich natural microcosm. Comparing to previous knowledge based on rearing and morphology-based identifications, we find a species-rich assemblage characterized by a low degree of host specialization. Our method opens up new horizons for molecular analyses of fungus-associated interaction webs and communities. Fruiting bodies of fungi constitute an important resource for thousands of other taxa. The structure of these diverse assemblages has traditionally been studied with labour-intensive methods involving cultivation and morphology-based species identification, to which molecular information might offer convenient complements. To overcome challenges in DNA extraction and PCR associated with the complex chemical properties of fruiting bodies, we developed a pipeline applicable for extracting amplifiable total DNA from soft fungal samples of any size. Our protocol purifies DNA in two sequential steps: (a) initial salt-isopropanol extraction of all nucleic acids in the sample is followed by (b) an extra clean-up step using solid-phase reversible immobilization (SPRI) magnetic beads. The protocol proved highly efficient, with practically all of our samples-regardless of biomass or other properties-being successfully PCR-amplified using metabarcoding primers and subsequently sequenced. As a proof of concept, we apply our methods to address a topical ecological question: is host specificity a major characteristic of fungus-associated communities, that is, do different fungus species harbour different communities of associated organisms? Based on an analysis of 312 fungal fruiting bodies representing 10 species in five genera fro...

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Section: A New Methodsmentioning

confidence: 99%

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confidence: 99%

Finding flies in the mushroom soup: Host specificity of fungus‐associated communities revisited with a novel molecular method

et al. 2018

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“…The concentration of potassium, sodium, magnesium, calcium and phosphorous is also high [ 47 , 48 , 49 ]. Some of these compounds hinder nucleic acid extraction by either interacting with the buffer or precipitating with the DNA or by collating into the DNA strand [ 50 ]. Adding MES (2-(N-morpholino) ethanesulfonic acid), PEG (polyethylene glycol) and Na 2 SO 3 to the buffer is suggested to help eliminate these compounds [ 18 , 51 , 52 ].…”

Section: Resultsmentioning

confidence: 99%

A Comprehensive High-Quality DNA and RNA Extraction Protocol for a Range of Cultivars and Tissue Types of the Woody Crop Avocado

Nath

Fletcher

Hayward

et al. 2022

Plants

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High-quality DNA and RNA forms the basis of genomic and genetic investigations. The extraction of DNA and RNA from woody trees, like avocado (Persea americana Mill.), is challenging due to compounds which interact with nucleic acids and influence separation. Previously reported methods of DNA and RNA extraction from avocado have issues of low yield, quality and applicability across different cultivars and tissue types. In the current study, methods have been optimised for high-quality DNA extraction from 40 avocado cultivars and RNA extraction from multiple tissue types, including roots, stem, leaves, flowers and fruits. The method is based on the modification of the cetyltrimethylammonium bromide buffer, centred around the specific optimisation of chemicals, such as sodium dodecyl sulphate, polyvinylpyrrolidone, sodium sulphite, polyethylene glycol and β-mercaptoethanol. The DNA extraction method yielded high-molecular weight DNA from the leaf tissue of 40 avocado cultivars belonging to Mexican, Guatemalan and West Indian avocado horticultural groups. The method was further optimised for RNA extraction from different avocado plant parts, enabling extraction using amounts as low as ~10 mg of starting material. The DNA and RNA extracted was successfully used for long- and short-read sequencing and gene expression analysis. The methods developed may also be applicable to other recalcitrant plant species.

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“…However, DNA purity can be an important issue for PCR success, as shown for plants [ 27 ]. Polysaccharides and pigments impair DNA purity, and have been described as an important issue in PCR amplification of Trichoderma [ 28 ]. In these cases, DNA extraction and DNA purification are therefore essential steps for a successful PCR amplification.…”

Section: Discussionmentioning

confidence: 99%

Colony-PCR Is a Rapid Method for DNA Amplification of Hyphomycetes

Walch

Knapp

Rainer

et al. 2016

JoF

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Fungal pure cultures identified with both classical morphological methods and through barcoding sequences are a basic requirement for reliable reference sequences in public databases. Improved techniques for an accelerated DNA barcode reference library construction will result in considerably improved sequence databases covering a wider taxonomic range. Fast, cheap, and reliable methods for obtaining DNA sequences from fungal isolates are, therefore, a valuable tool for the scientific community. Direct colony PCR was already successfully established for yeasts, but has not been evaluated for a wide range of anamorphic soil fungi up to now, and a direct amplification protocol for hyphomycetes without tissue pre-treatment has not been published so far. Here, we present a colony PCR technique directly from fungal hyphae without previous DNA extraction or other prior manipulation. Seven hundred eighty-eight fungal strains from 48 genera were tested with a success rate of 86%. PCR success varied considerably: DNA of fungi belonging to the genera Cladosporium, Geomyces, Fusarium, and Mortierella could be amplified with high success. DNA of soil-borne yeasts was always successfully amplified. Absidia, Mucor, Trichoderma, and Penicillium isolates had noticeably lower PCR success.

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Methodology A rapid and inexpensive method for isolation of total DNA from Trichoderma spp (Hypocreaceae)

Cited by 10 publications

References 4 publications

Finding flies in the mushroom soup: Host specificity of fungus‐associated communities revisited with a novel molecular method

Finding flies in the mushroom soup: Host specificity of fungus‐associated communities revisited with a novel molecular method

A Comprehensive High-Quality DNA and RNA Extraction Protocol for a Range of Cultivars and Tissue Types of the Woody Crop Avocado

Colony-PCR Is a Rapid Method for DNA Amplification of Hyphomycetes

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