DNA RECOVERY FROM SOILS OF DIVERSE COMPOSITION

A simple, rapid method for bacterial lysis and direct extraction of DN A from soils with minimal shearing was developed to address the risk o f chimera formation from small template DNA during subsequent PCR, The method was based on lysis with a high-salt extraction buffer (1.5 M N aCl) and extended heating (2 to 3 h) of the soil suspension in the pre sence of sodium dodecyl sulfate (SDS), hexadecyltrimethylammonium brom ide, and proteinase K. The extraction method required 6 h and was test ed on eight soils differing in organic carbon, clay content, and pH, i ncluding 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 b acteria was 92 to 99%. When the method was tested on all eight unseede d soils, microscopic examination of indigenous bacteria in soil pellet s before and after extraction showed variable cell lysis efficiency (2 6 to 92%). Crude DNA yields from the eight soils ranged from 2.5 to 26 .9 mu g of DNA g(-1), and these were positively correlated with the or ganic carbon content in the soil (r = 0.73), DNA yields from gram-posi tive bacteria from pure cultures were two to six times higher when the high-salt-SDS-heat method was combined with mortar-and-pestle grindin g and freeze-thawing, and most DNA recovered was of high molecular wei ght, Four methods for purifying crude DNA were also evaluated for perc ent recovery, fragment size, speed, enzyme restriction, PCR amplificat ion, and DNA-DNA hybridization. In general, all methods produced DNA p ure enough for PCR amplification, Since soil type and microbial commun ity characteristics will influence DNA recovery, this study provides g uidance for choosing appropriate extraction and purification methods o n the basis of experimental goals.