DNA recovery from soils of diverse composition

TLDR

Soil type and microbial community characteristics influence DNA recovery, underscoring the need for tailored extraction and purification strategies. The study aimed to develop a simple, rapid bacterial lysis and DNA extraction method that minimizes shearing and reduces chimera formation, while offering guidance on method selection based on soil properties. The method uses high‑salt (1.5 M NaCl) extraction buffer with SDS, hexadecyltrimethylammonium bromide, proteinase K, and extended heating (2–3 h) to lyse cells, and was tested on eight diverse soils, with four purification protocols evaluated for recovery and purity. The protocol yielded high‑molecular‑weight DNA (>23 kb) with 92–99 % recovery from seeded soils, 26–92 % lysis efficiency across unseeded soils, and crude yields of 2.5–26.9 µg g⁻¹ positively correlated with organic carbon (r = 0.73); purification methods produced PCR‑ready.

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 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.