DNA recovery from soils of diverse composition

Jizhong Zhou, Maryann Victoria Bruns, James M. Tiedje

Research output: Contribution to journalArticle

2137 Citations (Scopus)

Abstract

A simple, rapid method for bacterial lysis and direct extraction of DNA from soils with minimal shearing was developed to address the risk of chimera formation from small template DNA during subsequent PCR. The method was based on lysis with a high-salt extraction buffer (1.5 M NaCl) and extended heating (2 to 3 h) of the soil suspension in the presence of sodium dodecyl sulfate (SDS), hexadecyltrimethylammonium bromide, and proteinase K. The extraction method required 6 h and was tested on eight soils differing in organic carbon, clay content, and pH, including ones from which DNA extraction is difficult. The DNA fragment size in crude extracts from all soils was >23 kb. Preliminary trials indicated that DNA recovery from two soils seeded with gram-negative bacteria was 92 to 99%. When the method was tested on all eight unseeded soils, microscopic examination of indigenous bacteria in soil pellets before and after extraction showed variable cell lysis efficiency (26 to 92%). Crude DNA yields from the eight soils ranged from 2.5 to 26.9 μg of DNA g-1, and these were positively correlated with the organic carbon content in the soil (r = 0.73). DNA yields from gram-positive bacteria from pure cultures were two to six times higher when the high-salt-SDS-heat method was combined with mortar-and-pestle grinding and freeze-thawing, and most DNA recovered was of high molecular weight. Four methods for purifying crude DNA were also evaluated for percent recovery, fragment size, speed, enzyme restriction, PCR amplification, and DNA-DNA hybridization. In general, all methods produced DNA pure enough for PCR amplification. Since soil type and microbial community characteristics will influence DNA recovery, this study provides guidance for choosing appropriate extraction and purification methods on the basis of experimental goals.

Original languageEnglish (US)
Pages (from-to)316-322
Number of pages7
JournalApplied and Environmental Microbiology
Volume62
Issue number2
StatePublished - Feb 1996

Fingerprint

Soil
DNA
soil
lysis
sodium dodecyl sulfate
Sodium Dodecyl Sulfate
Polymerase Chain Reaction
bacterium
amplification
methodology
Carbon
Salts
organic carbon
sodium
salts
heat
sulfate
salt
purification methods
chimera

All Science Journal Classification (ASJC) codes

  • Environmental Science(all)
  • Biotechnology
  • Microbiology

Cite this

Zhou, Jizhong ; Bruns, Maryann Victoria ; Tiedje, James M. / DNA recovery from soils of diverse composition. In: Applied and Environmental Microbiology. 1996 ; Vol. 62, No. 2. pp. 316-322.
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DNA recovery from soils of diverse composition. / Zhou, Jizhong; Bruns, Maryann Victoria; Tiedje, James M.

In: Applied and Environmental Microbiology, Vol. 62, No. 2, 02.1996, p. 316-322.

Research output: Contribution to journalArticle

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