Single-step Isolation of DNA from the Soil Samples for PCR-analysis Using Two-Component System Containing Polyaniline-Modified Silica and Alginate Microspheres

The method providing one-step isolation of DNA from soil extracts was developed. Isolation of nucleic acids from the biological objects presenting in a soil usually is carried out by in situ lysis of the sample followed by DNA purification. The purification step is complicated by the fact that about 60% of all soil organic substances are humic substances (HSs), which are powerful inhibitors of the polymerase. The currently known methods of DNA isolation from soil samples are usually laborious and multistage. In our opinion, a promising approach to solve this problem could be based on the use of a two-component bioseparating element. The first component of such element is a special sorbent demonstrating the effect of "negative selection" in relation to DNA (when DNA is not retained by the sorbent while the admixtures are effectively held), and the second polymeric component exhibits high selectivity in binding of HSs. The effectiveness of the use of such two-component system for one-step isolation of DNA from soil extracts with simultaneous purification of DNA from HSs was confirmed by spectroscopic, electrophoretic, and chromatographic methods. The developed element was represented as a compact spin-cartridge containing two layers of different sorbents. In particular, polyaniline-modified silica effectively retained proteins, and polymer alginate particles provided the retention of hydrophobic (similar to 98%) and hydrophilic (>75%) fractions of HSs. The both used components did not retain DNA. Achieved purity of the isolated DNA provided the possibility of its direct use in PCR analysis. The yield of purified DNA estimated by the model experiments was more than 90% .