Abstract
Here we show that a commercial blocking reagent (G2) based on modified eukaryotic DNA significantly improved DNA extraction efficiency. We subjected G2 to an inter-laboratory testing, where DNA was extracted from the same clay subsoil using the same batch of kits. The inter-laboratory extraction campaign revealed large variation among the participating laboratories, but the reagent increased the number of PCR-amplified16S rRNA genes recovered from biomass naturally present in the soils by one log unit. An extensive sequencing approach demonstrated that the blocking reagent was free of contaminating DNA, and may therefore also be used in metagenomics studies that require direct sequencing.
Highlights
Modern microbial ecology studies are often based on extraction of community nucleic acids, followed by molecular analyses of the recovered DNA or RNA
Nucleic acids released from lysed cells can be immobilized on particulate adsorption sites before the extraction procedure is completed[5]
Since adsorption is rarely taken into consideration during protocol development, this potential confounder may have a significant influence on the efficiency of DNA extractions from complex matrices, such as soil
Summary
Modern microbial ecology studies are often based on extraction of community nucleic acids, followed by molecular analyses of the recovered DNA or RNA. In protocols for direct DNA and RNA extraction from environmental samples, one parameter of success has been extraction yield, which could be problematic if the aim is to study viable present day organisms. To minimize such contamination by soil-bound nucleic acid, one solution would be to extract and recover cells from soil prior to lysis and DNA extraction other biases like differential cell lysis may be introduced[11,12]. Used in connection with a custom phenol/ chloroform-based protocol, the G2 blocking reagent can increase extraction of DNA and mRNA from clay rich groundwater sediments more than 10,000-fold[16]
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