A microwave-based method for nucleic acid isolation from environmental samples

Orsini, Massimiliano; Romano-Spica, Vincenzo

doi:10.1046/j.1472-765x.2001.00938.x

Cited by 85 publications

(64 citation statements)

References 19 publications

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“…Other methods, which use mortar mill grinding [64], grinding under liquid nitrogen [73,77], ultrasonication [48,51] and microwave thermal shock [47] have also been reported. Frostegard et al [17] observed that drying soil before grinding greatly improved the lysis efficiency.…”

Section: Cell Lysismentioning

confidence: 99%

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Extraction of DNA from soil

Robe¹,

Nalin²,

Capellano³

et al. 2003

European Journal of Soil Biology

254

164

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Section: Cell Lysismentioning

confidence: 99%

“…Another denaturing agent, guanidine isothiocyanate has been used for mRNA extraction from seeded soils [47,71] but has [71] not proven valuable when extracting DNA from soil samples [37,42].…”

Section: Cell Lysismentioning

confidence: 99%

Extraction of DNA from soil

Robe¹,

Nalin²,

Capellano³

et al. 2003

European Journal of Soil Biology

254

164

View full text Add to dashboard Cite

“…The extraction procedure described by Orsini and Romano-Spica avoids the co-purification of humic substances by applying a microwave thermal shock and the addition of polyvinylpyrrolidone at high concentrations [14]. As can be seen in table 1 Many workers have attempted to increase genomic DNA purity and yield from environmental samples by using various kinds of treatments.…”

Section: Resultsmentioning

confidence: 99%

“In‐gel patch electrophoresis:” A new method for environmental DNA purification

et al. 2005

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Most of the microorganism species are largely untapped and could represent an interesting reservoir of genes useful for biotechnological applications. Unfortunately, a major difficulty associated with the methods used to isolate environmental DNA is related to the contamination of the extracted material with humic substances. These polyphenolic compounds inhibit the DNA processing reactions and severely impede cloning procedures. In this work, we describe a rapid, simple and efficient method for the purification of genomic DNA from environmental samples: we added a chromatography step directly embedded into an agarose gel electrophoresis. This strategy enabled the DNA extraction from various environmental samples and it appeared that the purity grade was compatible with digestion by restriction enzymes and PCR amplifications.2

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“…The genomic DNA of Thermoactinomyces sp. CDF was extracted according to the method of Orsini & Romano-Spica (2001), except that the cells were disrupted by grinding with a mortar and pestle, rather than microwave treatment. A fragment of the gene (cdf) encoding protease CDF was amplified from the genomic DNA with Pfu DNA polymerase by PCR employing two consensus-degenerate hybrid oligonucleotide primers (CODEHOPs), F and R (Table 1), designed on the basis of two highly conserved amino acid sequences around the catalytic residues His and Ser of subtilases (Wu et al, 2004) ( Supplementary Fig.…”

Section: Methodsmentioning

confidence: 99%

Identification and characterization of a novel spore-associated subtilase from Thermoactinomyces sp. CDF

et al. 2009

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A gene encoding a spore-associated subtilase, designated protease CDF, was cloned from Thermoactinomyces sp. CDF and expressed in Escherichia coli. The enzyme gene is translated as a proform consisting of a 94 aa propeptide and a 283 aa mature protease domain. Phylogenetic analysis revealed that this enzyme belonged to the subtilisin family, but could not be grouped into any of its six known subfamilies. The mature protease CDF has an unusually high content of charged residues, which are mainly distributed on the enzyme surface. The recombinant proform of protease CDF formed inclusion bodies, but could be efficiently converted to the mature enzyme when the inclusion bodies were dissolved in alkaline buffers. The proform underwent a two-step maturation process, wherein the N-terminal part (85 residues) of the propeptide was autoprocessed intramolecularly, and the remaining 9-residue peptide was further processed intermolecularly. Protease CDF exhibited optimal proteolytic activity at 50-55 6C and pH 10.5-11.0. The enzyme was stable under high-pH conditions (pH 11.0-12.0), and NaCl could stabilize the enzyme at lower pH values. In addition, the enzyme was not dependent on calcium for either maturation or stability. By immunoblot analysis, protease CDF was found to be associated with spores, and could be extracted from the spores with 2 M KCl and alkaline buffers without damaging the coat layer, demonstrating that the protease CDF is located on the surface of the spore coat.

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A microwave-based method for nucleic acid isolation from environmental samples

Cited by 85 publications

References 19 publications

Extraction of DNA from soil

Extraction of DNA from soil

“In‐gel patch electrophoresis:” A new method for environmental DNA purification

Identification and characterization of a novel spore-associated subtilase from Thermoactinomyces sp. CDF

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