Comparative study of methods for extraction and purification of environmental DNA from soil and sludge samples.
An important prerequisite for successful construction of a metagenome library is an efficient procedure for extracting DNA from environmental samples. 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 on methods that 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 approx 12 kb. The quality of the extracted DNA was evaluated by the ability of different restriction enzymes to digest the eDNA. 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 polymerase chain reaction conditions.
Roh
Changhyun
C
School of Chemical Engineering, Institute of Molecular Biology and Genetics, Seoul National University, Seoul, South Korea.
Villatte
Francois
F
Kim
Byung-Gee
BG
Schmid
Rolf D
RD
D004275:DNA, Ribosomal / Q000737:chemistry / Q000302:isolation & purification*; D004784:Environmental Monitoring / Q000379:methods; D006812:Humic Substances; D012722:Sewage / Q000737:chemistry*; D012988:Soil Microbiology
D004275:DNA, Ribosomal; D006812:Humic Substances; D012722:Sewage