A comparative study of extraction and purification methods for environmental DNA from soil and sludge samples

Abstract

An important prerequisite for a successful metagenome library construction is an efficient extraction procedure for DNA out of environmental samples. In this study we compared three indirect and four direct extraction methods, including a commercial kit, in terms of DNA yield, purity and time requirement. A special focus was set on methods which are appropriate for the extraction of environmental DNA (eDNA) from very limited sample sizes (0.1 g) to enable a highly parallel approach. Direct extraction procedures yielded on average 100-fold higher DNA amounts than indirect ones. A drawback of direct extraction was the small fragment size of appr. 12 kb. The quality of the extracted DNA was evaluated by the ability of different restriction enzymes to digest the environmental DNA. Only the commercial kit and a direct extraction method using freeze-thaw cell lysis in combination with an in gel patch electrophoresis with hydroxyapatite to remove humic acid substances yielded DNA, which was completely digested by all restriction enzymes. Moreover, only DNA extracted by these two procedures could be used as template for the amplification of fragments of several 16S rDNA, 18S rDNA groups under standard PCR conditions.