DNA EXTRACTION TECHNIQUES FOR SOME WILD SPECIES FROM UZBEKISTAN

Abstract

Although DNA extraction is already a routine procedure and protocols have been developed for a large number of plant species, various plant tissues and organs, solving the problem of extracting pure undegraded DNA from plant is the main step in molecular approaches for study plant organisms. The extraction of high-quality DNA from plant materials is an important factor for DNA barcoding. The effective DNA extraction protocol for samples with high content of admixture, which prevents the restriction reaction and the PCR, or degraded DNA should be established for each case. This work presents the comparison of DNA isolation techniques from silica gel dried and herbarium samples of some wild species collected in Uzbekistan. In this work, we optimized the technique for isolating the total DNA, on the examples of 16 representatives of Salvia, Dracocephalum, Phlomoides, Tulipa, Iris, Allium genera from Uzbekistan. The results of a comparative analysis of three methods of DNA extraction from leaf tissues of wild plants are represented in this article. DNA amount and purity degrees were measured using spectrophotometer. The efficiency of the DNA extraction method was assessed according to two criteria: the quantitative parameters -DNA concentration (ng/μl), and the qualitative parameters - DNA purity (nm). The DNA concentration variate from 758.1 ng/μl obtained with the CTAB method to 13.89 ng/μl obtained with commercial column extraction kits. λ = 260/280 ratio is range from 1.7 to 1.98 nm. Additionally, PCR amplification was realized to reflect the validation of the DNA isolation protocol, using specific primers of four chloroplast (rbcL, matK, psbA, trnL-F) and one nuclear (ITS) plant DNA barcode regions. The sequence length was 300–800 bp appropriately.