Evaluation and Optimization of DNA Extraction and Purification Procedures for Soil and Sediment Samples

Abstract

We compared and statistically evaluated the effectiveness of nine DNA extraction procedures by using frozen and dried samples of two silt loam soils and a silt loam wetland sediment with different organic matter contents. The effects of different chemical extractants (sodium dodecyl sulfate [SDS], chloroform, phenol, Chelex 100, and guanadinium isothiocyanate), different physical disruption methods (bead mill homogenization and freeze-thaw lysis), and lysozyme digestion were evaluated based on the yield and molecular size of the recovered DNA. Pairwise comparisons of the nine extraction procedures revealed that bead mill homogenization with SDS combined with either chloroform or phenol optimized both the amount of DNA extracted and the molecular size of the DNA (maximum size, 16 to 20 kb). Neither lysozyme digestion before SDS treatment nor guanidine isothiocyanate treatment nor addition of Chelex 100 resin improved the DNA yields. Bead mill homogenization in a lysis mixture containing chloroform, SDS, NaCl, and phosphate-Tris buffer (pH 8) was found to be the best physical lysis technique when DNA yield and cell lysis efficiency were used as criteria. The bead mill homogenization conditions were also optimized for speed and duration with two different homogenizers. Recovery of high-molecular-weight DNA was greatest when we used lower speeds and shorter times (30 to 120 s). We evaluated four different DNA purification methods (silica-based DNA binding, agarose gel electrophoresis, ammonium acetate precipitation, and Sephadex G-200 gel filtration) for DNA recovery and removal of PCR inhibitors from crude extracts. Sephadex G-200 spin column purification was found to be the best method for removing PCR-inhibiting substances while minimizing DNA loss during purification. Our results indicate that for these types of samples, optimum DNA recovery requires brief, low-speed bead mill homogenization in the presence of a phosphate-buffered SDS-chloroform mixture, followed by Sephadex G-200 column purification.

Incorporating polymerase chain reaction-based identification, population characterization, and quantification of microorganisms into aerosol science: A review

“…For most environmental bacteria and fungi, an additional step such as highenergy agitation with micron-sized beads (bead beating) or rapid freeze-thaw cycling is necessary for complete cell wall disruption. Indeed, the use of these physical disruption steps has been associated with 99% cell disruption in environmental samples and large increases in DNA yields from soils and aerosol samples (Haugland et al, 2002, Zhou et al, 1996, Miller et al, 1999. Other researchers, however, have cautioned that methods such as bead beating and sonication can shear genomic DNA into fragments (Miller et al, 1999;Picard et al, 1992) and that these fragments may lead to either incomplete PCR amplification or chimera (PCR amplicon composed of DNA from different, multiple microorganisms) production.…”

“…Indeed, the use of these physical disruption steps has been associated with 99% cell disruption in environmental samples and large increases in DNA yields from soils and aerosol samples (Haugland et al, 2002, Zhou et al, 1996, Miller et al, 1999. Other researchers, however, have cautioned that methods such as bead beating and sonication can shear genomic DNA into fragments (Miller et al, 1999;Picard et al, 1992) and that these fragments may lead to either incomplete PCR amplification or chimera (PCR amplicon composed of DNA from different, multiple microorganisms) production. Miller and coworkers describe speeds and times for bead beating that reduce this fragmentation (Miller et al, 1999), and extraction by sonication is not recommended.…”

“…Other researchers, however, have cautioned that methods such as bead beating and sonication can shear genomic DNA into fragments (Miller et al, 1999;Picard et al, 1992) and that these fragments may lead to either incomplete PCR amplification or chimera (PCR amplicon composed of DNA from different, multiple microorganisms) production. Miller and coworkers describe speeds and times for bead beating that reduce this fragmentation (Miller et al, 1999), and extraction by sonication is not recommended. Clearly described protocols exist for thorough cell wall lysis for small quantities of fungi (Haugland et al, 2002;Schabereiter-Gurtner et al, 2001) and all bacteria (Frank et al, 2003), and both have been employed successfully in aerosol studies.…”

“…Recent aerosol studies independently concluded that inhibitory compounds produced by either fungal cell lysates or by the environmental matrix (dust or particulate matter) can significantly inhibit PCR, and that these compounds and their associated inhibition can be removed by nucleic acid purification techniques such as phenol:chloroform extraction with ethanol precipitation (Dean et al, 2005;Williams et al, 2001) or by using spin columns with cleanup and elution (Haugland et al, 2002). The literature contains a limited amount of nucleic acid extraction optimization studies that provide very useful information for PCR-based aerosol studies (Dean et al, 2005;Haugland et al, 2002;Miller et al, 1999;Porteous et al, 1997;Zhou et al, 1996). Details of DNA extraction protocols used in previous aerosol studies are presented in Table 2.…”

Rapid Detection of Mycobacterium tuberculosis by Recombinase Polymerase Amplification

“…For samples that were smear negative but culture positive 8/14 (57.1%) and 2/5 (40%) were identified as positives via the IS6110 and IS1081 assays, respectively. It should be noted a crude DNA extraction procedure was used and improved sample handling and DNA extraction may improve detection from complex sample matrixes such as sputum [42]. Similarly whereas 0.5 ml inoculums were used for culture the volume taken for DNA extraction was only 0.25 ml, with just 5 mL being used for each RPA assay and there may be opportunity to improve sensitivity by testing larger sample volumes or by incorporating a sample concentration step.…”