Catalogo Articoli (Spogli Riviste)

OPAC HELP

Titolo:
Evaluation and optimization of DNA extraction and purification procedures for soil and sediment samples
Autore:
Miller, DN; Bryant, JE; Madsen, EL; Ghiorse, WC;
Indirizzi:
Cornell Univ, Div Biol Sci, Microbiol Sect, Ithaca, NY 14853 USA Cornell Univ Ithaca NY USA 14853 ci, Microbiol Sect, Ithaca, NY 14853 USA
Titolo Testata:
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
fascicolo: 11, volume: 65, anno: 1999,
pagine: 4715 - 4724
SICI:
0099-2240(199911)65:11<4715:EAOODE>2.0.ZU;2-N
Fonte:
ISI
Lingua:
ENG
Soggetto:
POLYMERASE CHAIN-REACTION; MICROBIAL DNA; RAPID METHOD; HUMIC SUBSTANCES; BACTERIAL-DNA; RNA; MICROORGANISMS; AMPLIFICATION; SEPARATION; GENE;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Agriculture,Biology & Environmental Sciences
Life Sciences
Citazioni:
45
Recensione:
Indirizzi per estratti:
Indirizzo: Miller, DN USDA ARS, Meat Anim Res Ctr, Box 166, Clay Ctr, NE 68933 USA USDA ARS Box 166 Clay Ctr NE USA 68933 , Clay Ctr, NE 68933 USA
Citazione:
D.N. Miller et al., "Evaluation and optimization of DNA extraction and purification procedures for soil and sediment samples", APPL ENVIR, 65(11), 1999, pp. 4715-4724

Abstract

We compared and statistically evaluated the effectiveness of nine DNA extraction procedures by using frozen and dried samples of two silt loam soils and a silt loam wetland sediment with different organic matter contents. The effects of different chemical extractants (sodium dodecyl sulfate [SDS], chloroform, phenol, Chelex 100, and guanadinium isothiocyanate), different physical disruption methods (bead mill homogenization and freeze-thaw lysis), and lysozyme digestion were evaluated based on the yield and molecular size of the recovered DNA. Pairwise comparisons of the nine extraction procedures revealed that bead mill homogenization with SDS combined with either chloroform or phenol optimized both the amount of DNA extracted and the molecular size of the DNA (maximum size, 16 to 20 kb). Neither lysozyme digestion before SDS treatment nor guanidine isothiocyanate treatment nor addition of Chelex 100 resin improved the DNA yields. Bead mill homogenization in a lysis mixture containing chloroform, SDS, NaCl, and phosphate-Tris buffer(pH 8) was found to be the best physical lysis technique when DNA yield and cell lysis efficiency were used as criteria. The bead mill homogenizationconditions were also optimized for speed and duration with two different homogenizers. Recovery of high-molecular-weight DNA was greatest when we used lower speeds and shorter times (30 to 120 s). We evaluated four differentDNA purification methods (silica-based DNA binding, agarose gel electrophoresis, ammonium acetate precipitation, and Sephadex G-200 gel filtration) for DNA recovery and removal of PCR inhibitors from crude extracts. SephadexG-200 spin column purification was found to be the best method for removing PCR-inhibiting substances while minimizing DNA loss during purification. Our results indicate that for these types of samples, optimum DNA recovery requires brief, low-speed bead mill homogenization in the presence of a phosphate-buffered SDS-chloroform mixture, followed by Sephadex G-200 column purification.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 24/09/20 alle ore 02:06:11