Environ Health Toxicol.  2017 ;32(1):e2017013. 10.5620/eht.e2017013.

Necessity of purification during bacterial DNA extraction with environmental soils

• Lim HJ
• Choi JH
• Son A

• KMID: 2411245
• DOI: http://doi.org/10.5620/eht.e2017013

Abstract

Complexity and heterogeneity of soil samples have often implied the inclusion of purification steps in conventional DNA extraction for polymerase chain reaction (PCR) assays. Unfortunately the purification steps are also time and labor intensive. Therefore the necessity of DNA purification was re-visited and investigated for a variety of environmental soil samples that contained various amounts of PCR inhibitors. Bead beating and centrifugation was used as the baseline (without purification) method for DNA extraction. Its performance was compared with that of conventional DNA extraction kit (with purification). The necessity criteria for DNA purification were established with environmental soil samples. Using lysis conditions at 3000 rpm for 3 minutes with 0.1 mm glass beads, centrifugation time of 10 minutes and 1:10 dilution ratio, the baseline method outperformed conventional DNA extraction on cell seeded sand samples. Further investigation with PCR inhibitors (i.e., humic acids, clay, and magnesium [Mg]) showed that sand samples containing less than 10 μg/g humic acids and 70% clay may not require purifications. Interestingly, the inhibition pattern of Mg ion was different from other inhibitors due to the complexation interaction of Mg ion with DNA fragments. It was concluded that DNA extraction method without purification is suitable for soil samples that have less than 10 μg/g of humic acids, less than 70% clay content and less than 0.01% Mg ion content.

Keyword

DNA purification; DNA extraction; Cell lysis; Quantitative polymerase chain reaction; Polymerase chain reaction inhibitors; Humic acids

MeSH Terms

Centrifugation
DNA
DNA, Bacterial*
Glass
Humic Substances
Magnesium
Methods
Polymerase Chain Reaction
Population Characteristics
Soil*
DNA
DNA, Bacterial
Humic Substances
Magnesium
Soil