Evaluation of purification procedures for DNA extracted from rich organic samples: interference with humic substances

Abstract

The polymerase chain reaction (PCR) has only recently become a powerful tool for the detection of microbial organisms from environmental samples. The application of this molecular technology has so far been hampered by the presence of various impurities co-extracted with soil DNA, such as humic acids and metal ions. These interfering substances can inhibit PCR most likely due either to chelation of humic acids with magnesium ions required by Taq polymerase, or to the binding of primers that reduce the sensitivity of detection [1]. A number of works devoted to methodological approaches outline the difficulty to recover pure DNA extracted from soil or sediment samples. Removing humic substances from the DNA samples represents actually a methodological challenge. Time consuming methods like CsCl gradients [2,3] or extensive and repetitive precipitation steps [4] have been developed. Methods such as diluting crude extract samples have been used but this latter reduces the detection limit [1]. In order to simplify the purification steps, the use of various minicolumns has been recently tested by some authors [1,[5][6][7][8].Our laboratory has focused attention for several years on the impact of tropical termites on physical and chemical soil properties and more specifically on the distribution of humic substances [9,10]. Our aim was to design molecular tools in order to study bacterial changes induced by termite activities. However, the samples of soil-feeding termite nests typically impede the purification of DNA because such nests are mostly made of termite dejections which are rich in organic matter and humic substances. Here we compare electrophoretic and chromatographic methods to purify DNA samples. The quality of DNA was then tested by performing amplification by Polymerase Chain Reaction (PCR). Considering physical and chemical properties of both DNA and humic substances, we also sought to explain how these molecules compete and interact during purification procedures. MaterialThe samples originated from the rainforest of the NsimiZoetele region (South Cameroun). Termite mounds samples belong to the soil-feeding species Cubitermes subarquatus (Termitinae). Soils without termite activity were defined as controls. Termite mounds of C. subarquatus contain around 60% of clay, 45 mg/g of Total Organic Carbon (TOC) including 15% of humic acids. Control soils A2 (2 -5 cm) and A6 (100 -140 cm) have 44.4 and 67.4% of clay, 18.8 and 5.76 mg/g of TOC with 9% and 7% of humic acids, respectively. DNA extraction and purification were made in duplicate on termite mound and A2 samples. No DNA was noticeable in the A6 deeper strata. To test the efficiency of the procedures A2 and A6 sterilised subsamples were inoculated with a bacterial strain of Escherichia coli (Pharmacia, strain NM 522). DNA extractionThe detailed procedure is described elsewhere [11]. Briefly, 0.4 g samples are submitted to a lysis solution containing lysozyme, proteinase K, and sodium dodecyl sulfate. Protein and cell debris were eliminated by a salting ...

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From bacterial genomics to metagenomics: concept, tools and recent advances

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