Exploring Rapid and Efficient Protocol for Isolation of Fungal DNA

Tripathy, Swapan K.; Maharana, Manasmita; Ithape, Dinesh Manohar; Lenka, Devraj; Mishra, Dayanidhi; Prusti, Arjun; Mohanty, Digbijaya Swain Ranjan; Raj, K.R. Reshmi

doi:10.20546/ijcmas.2017.603.113

Cited by 11 publications

(10 citation statements)

References 29 publications

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“…The combination of components in this buffer were extremely efficient in the lysis of cell wall and cell membrane of G. zonatum. Due to the anionic and cationic conditions of SDS and CTAB, these buffers work well with each other in solubilising proteins and lipids (Tripathy et al, 2017). Generally, Ganoderma species contains β-Glucans as the major active polysaccharides (Obodai et al, 2017) and are rich in phenolic compounds from fruiting bodies and mycelia (Mishra et al, 2018).…”

Section: Resultsmentioning

confidence: 99%

A RAPID AND EFFICIENT DNA EXTRACTION PROTOCOL FOR Ganoderma zonatum, A BASAL AND UPPER STEM ROT PATHOGEN OF OIL PALM IN MALAYSIA

Nagappan

Kadri

Chin

et al. 2020

JOPR

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Ganoderma zonatum (G. zonatum) is associated with both the basal and upper stem rot diseases in oil palm. Despite the severity of these diseases, there is only limited information on the molecular characteristics of this oil palm pathogen. Most of the studies on G. zonatum related to oil palm are focused on the epidemiology and genetic diversity of the organism. In other palm species, G. zonatum has also been identified as the causal agent of bud rot disease. To further characterize the organism using molecular techniques, the ability to isolate good quality DNA samples is important. In this study, seven DNA extraction protocols were evaluated and the best protocol, Boehm protocol, had the highest yield of good quality DNA. The protocol was able to yield 208.95 ± 4.52 µg DNA per gram of sample with purities above 1.80 for A 260/280 and 2.0 for A 260/230. This extraction protocol is a rapid and efficient protocol that employs cetyl trimethylammonium bromide (CTAB), sodium dodecyl sulphate (SDS), β-mercaptoethanol and Proteinase K in the lysis buffer. The Boehm protocol was further tested on three other Ganoderma species found in the oil palm plantations and a medicinal fungus, G. lucidum. It was noted that the protocol was efficient, with high yields for G. zonatum when compared to the other four species. This is probably due to the fact that extraction protocols for each organism requires specific optimization to obtain optimal yield and purity. In conclusion, the Boehm protocol was best suited for genomic DNA extraction of G. zonatum and found suitable for downstream applications such as PacBio sequencing.

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Section: Resultsmentioning

confidence: 99%

A RAPID AND EFFICIENT DNA EXTRACTION PROTOCOL FOR Ganoderma zonatum, A BASAL AND UPPER STEM ROT PATHOGEN OF OIL PALM IN MALAYSIA

Nagappan

Kadri

Chin

et al. 2020

JOPR

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“…To streamline the DNA extraction process, a tissue homogeniser was utilized as a closed system to crush the sample and avoid contamination. Other methods to pulverize and crush samples or to maximise absorption can, however, also be applied, such as freeze drying (Griffin et al, 2002), liquid nitrogen (Tripathy et al, 2017;Wu et al, 2001), magnetic beads (Faggi et al, 2005) or using mini pestles and grinding with sea sand (Yee et al, 2018). This method can be used for large scale DNA extraction because at any given time, samples can be processed in within 70 minutes.…”

Section: Discussionmentioning

confidence: 99%

“…mortar and pestle or freeze drying (Al-Samarrai and Schmid, 2000;Yang et al, 2016a)), while chemical lysis is done by treating cells with a lysis buffer (Dairawan and Shetty, 2020). Popular lysis buffers include Cetyltrimethylammonium Bromide (CTAB), sodium dodecyl sulfate (SDS), and ethylenediaminetetraacetic acid (EDTA) (Dairawan and Shetty, 2020;Tripathy et al, 2017). DNA is typically purified through phenol/chloroform extraction and precipitated using ethanol (Aamir et al, 2015;Butler, 2012;Dairawan and Shetty, 2020).…”

Section: Introductionmentioning

confidence: 99%

Genomic DNA extraction from minimal amount of dried mushroom samples

Strauss

Ghosh

Murray

et al. 2021

MBJ

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Aims: To develop a simple and rapid genomic DNA extraction technique for dried (≈ 1 year old) mushroom fruiting bodies that yields high-quality DNA, suitable for use by post-graduate institutions. Method: Small amounts (0.04 g) of pulverized dried mushroom sample were incubated in a Tris /EDTA/SDS lysis buffer (100mM:10mM: 2%) at 65°C to lyse the chitinous fungal cell walls. Genomic DNA purification was performed using chloroform isoamyl alcohol (24:1), and DNA was precipitated using 100% ethanol. Results: Genomic DNA was successfully extracted under 70 minutes from 16 samples morphologically identified as Panaeolus, Copelandia, Gymnopilus, Pluteus and Favolus species. DNA concentrations were on average of 696.9ng/µL. PCR successfully amplified the ITS-5.8S region. The protocol has been successfully used by numerous post-graduate students in our research programme. Conclusion:The rapid and easy protocol produced high-quality genomic DNA void of any inhibitors that is suitable for downstream molecular implications across multiple mushroom genera. Noticeably, this method requires only minute quantities (0.04g) of starting material and is ideal for student training in higher academic institutions.

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“…DNA quantification was conducted using nano drops AE-Nano200 Nucleic Acid Analyzer version 2.0, while, the purity value was determined by calculating the absorbance ratio Å260/Å280. Meanwhile, DNA qualitative analysis was determined using 0.8% agarose gel electrophoresis (Tripathy et al, 2017).…”

Section: Methodsmentioning

confidence: 99%

Identification endophytic fungi of Nutgrass (Cyperus rotundus L.) as solubilizing phosphate and indole-3-acetic acid producers

Kusmiyati¹,

Wicaksono²,

Maknuna³

2021

bio

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Low phosphate content in the soil can cause insufficient plant needs. Besides, the endophytic fungi of nutgrass have the potential as a phosphate solvent and can produce IAA (Indole-3-Acetic Acid). This study aimed to determine the levels of solubilizing phosphate and production of IAA (Indole-3-Acetic Acid) by the endophytic fungi of nutgrass and to identify the isolates based on rDNA-ITS sequences. The methods used were the isolation of endophytic fungi, analysis of solubilizing levels of phosphate and IAA production, and molecular analysis with rDNA-ITS sequences. Results of isolation and purification, found five isolates coded URT1, URT2, URT3, URT4, and URT5. The endophytic fungi of nutgrass were able to solubilizing phosphate levels around 54.03 - 87.83 ppm, with the highest levels produced by URT4 isolate. IAA levels around 5.58 - 45.50 ppm, with the highest levels produced by URT1 isolate. Based on molecular analysis with rDNA-ITS sequences, it showed that URT4 had 97.42% similarity to Aspergillus tereus species, while UTR1 had 100% similarity to Fusarium oxyporum species. To conclude, the endophytic fungi of nutgrass from A. tereus and F. oxyporum species have high levels of solubilizing phosphate and IAA production so that they are potential candidates for biofertilizer.

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Exploring Rapid and Efficient Protocol for Isolation of Fungal DNA

Cited by 11 publications

References 29 publications

A RAPID AND EFFICIENT DNA EXTRACTION PROTOCOL FOR Ganoderma zonatum, A BASAL AND UPPER STEM ROT PATHOGEN OF OIL PALM IN MALAYSIA

A RAPID AND EFFICIENT DNA EXTRACTION PROTOCOL FOR Ganoderma zonatum, A BASAL AND UPPER STEM ROT PATHOGEN OF OIL PALM IN MALAYSIA

Genomic DNA extraction from minimal amount of dried mushroom samples

Identification endophytic fungi of Nutgrass (Cyperus rotundus L.) as solubilizing phosphate and indole-3-acetic acid producers

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