Optimising the method of isolating nucleic acids from kefir using a silica-based sorbent
Abstract
The most common methods of nucleic acid isolation are those using sorption carriers. The general principle of the methods for solid-phase extraction is based on the use of silica whose unique properties provide for selective DNA and RNA binding. The purpose of the work was to analyse the quality of sorption-based extraction systems using guanidine thiocyanate of various concentrations and 4 types of detergents in the lysis solution when extracting nucleic acids from kefir as a fermented dairy food product containing bacterial and fungal cells.
For the study, we used a commercially available fermented dairy food product “Kefir” with the bacteria content of at least 107 CFU/g and yeast content of at least 104 CFU/g. 99% silica (Sigma-Aldrich, USA) was used to isolate DNA. The experiment considered 3 variants of guanidine thiocyanate concentrations: 3.5 M, 5 M, and 6.5 M, as well as 4 variants of detergents: 1% Triton X-100, 1% Tween 20, 1% Tween 80, and 1% CTAB. Thus, DNA extraction was carried out in 12 different variants.
DNA obtained after extraction was visualised by electrophoresis in 2% agarose gel. The DNA concentration was measured using a Qubit 4 fluorometer (Thermo Fisher Scientific, USA). The polymerase chain reaction was performed using Taq polymerase on the CFX96 Real-Time System (Bio-Rad, USA). Statistical data processing was carried out using ANOVA dispersion analysis in the Statistica software.
Electrophoresis in agarose gel showed that using 5 M and 6.5 M concentrations of guanidine thiocyanate contributed to obtaining more pronounced rRNA bands on the electropherogram when using Tween 80, whereas when using Tween 20, the optimal concentration of guanidine thiocyanate for DNA extraction was 5 M.
The analysis of the effect of detergents on the quality of DNA isolation showed that the minimum concentration of extracted DNA was obtained when using CTAB and it was 1.03 ng/μl. Triton X-100 used as a detergent allowed obtaining a DNA concentration 5.9 times higher than with the CTAB detergent (p < 0.001). The values obtained from the extraction using Tween 80 and Tween 20 were 3.1 and 3.9 times higher, respectively, than the concentrations when using CTAB (p<0.05). Real-Time PCR conducted to assess the threshold cycle parameter (Ct) showed that the obtained Ct values with ITS1 and ITS4 primers in all variants of DNA extraction do not allow assessing the quality of isolation of nucleic acids from fungi. The analysis of Ct with primers 337F and 1100R suggests that the optimal concentration value for guanidine thionation was 5 M (the average value of Ct for all detergents was 25.0). According to the results of the experiment, it was found that when isolating nucleic acids from kefir, it is necessary to use guanidine thionation at a concentration of 5 M or 6.5 M. The concentration of isolated DNA was higher when using Triton X-100 and Tween 20 as a detergent. The use of Tween 20 or Tween 80 as detergents significantly increased the RNA fraction in the nucleic acid preparation.
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References
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