DNA recovery from soils of diverse composition

• Published in: Applied and Environmental Microbiology Vol. 62; no. 2; pp. 316 - 322
• Main Authors: Zhou, J. (Michigan State University, East Lansing, MI.), Bruns, M.A, Tiedje, J.M
• Format: Journal Article
• Language: English
• Published: Washington, DC American Society for Microbiology 01.02.1996
• Subjects: ADN; AMPLIFICATION CHAINE POLYMERASE; Animal, plant and microbial ecology; BACTERIA; BACTERIA GRAM NEGATIVA; BACTERIA GRAM POSITIVA; BACTERIE GRAM NEGATIF; BACTERIE GRAM POSITIF; Biological and medical sciences; CALOR; Cetrimonium Compounds; CHALEUR; CHLORURE DE SODIUM; CLORURO SODICO; Deoxyribonucleic acid; Detergents; DNA; DNA, Bacterial - genetics; DNA, Bacterial - isolation & purification; Evaluation Studies as Topic; FLORA DEL SUELO; FLORE DU SOL; Fundamental and applied biological sciences. Psychology; General aspects. Techniques; Gram-Negative Bacteria - chemistry; Gram-Negative Bacteria - genetics; Gram-Positive Bacteria - chemistry; Gram-Positive Bacteria - genetics; Methods and techniques (sampling, tagging, trapping, modelling...); Polymerase Chain Reaction; Povidone - analogs & derivatives; PURIFICACION; PURIFICATION; REACCION DE CADENAS DE POLIMERASA; Sodium Dodecyl Sulfate; Soil Microbiology; Soils; TIPOS QUIMICOS DE SUELOS; TIPOS TEXTURALES DE SUELOS; TYPE DE SOL CHIMIQUE; TYPE DE SOL TEXTURAL; Performance evaluation; Clay; Organic carbon; Purification; Method; Optimization; Soils; DNA; Content; Lysis; Bacteria; Extraction; Comparative study; Physicochemical properties
• ISSN: 0099-2240; 1098-5336
• DOI: 10.1128/aem.62.2.316-322.1996

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 micrograms 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