Improved DNA purification with quality assurance for evaluation of the microbial genetic content of constructed wetlands

Efficient isolation of target DNA is a crucial first step of DNA-based metagenomic analyses of environmental samples. Insufficient quantity and purity of DNA isolated using commercial kits result in missing genetic information, especially for large-diameter substrates in constructed wetlands (CWs). Here, we addressed this problem by devising a cost-effective calcium chloride lysozyme-sodium dodecyl sulfate (SDS) method (CCLS), with key improvements in the steps of humic acid removal and cell lysis. The buffer comprising Tris, EDTA, Na2O2P7 and PVPP (TENP), and skim milk, could reduce adsorption between microorganisms and substrates, and calcium chloride precipitated and removed over 94% of humic acid. This humic acid removal step, when compared to the PowerSoil DNA kit (MO BIO Laboratories Inc.) (MBKIT), significantly enhanced the DNA purity (A260/230) from 0.68 to 1.63 (p < 0.01). When gentle and extended cell lysis in CCLS replaced the short but violent bead-beating in the MBKIT, DNA yield and the amount of lysed bacteria detected by quantitative real-time polymerase chain reaction (qPCR) on average increased by 2 and 4 folds, respectively, compared to that obtained using the MBKIT (p < 0.01). Furthermore, the full-length bacterial 16S rRNA gene and nirK gene from denitrifying microorganisms were successfully amplified from CCLS-generated DNA. Additionally, bacterial diversity indices of richness, Shannon, and evenness examined by denaturing gradient gel electrophoresis (DGGE) increased by 75, 30, and 7%, respectively, by CCLS compared to that using the MBKIT. Hence, the CCLS method enables improved evaluation of microbial density and diversity in CW systems.