The Applicability of Three DNA Isolation Methods in SSR Analysis of Hexaploid Plum (Prunus domestica L.) Cultivars

Abstract

The main goal of any DNA extraction procedure is to ensure reliable and reproducible results in a simple, fast and inexpensive manner. When it comes to plant tissues, this goal is challenging to achieve due to the presence of a variety of metabolites that interfere with DNA during isolation and downstream analysis. In this study, we compared the efficiency of three methods for DNA extraction from plum kernels: 1) the standard CTAB Soltis method which is the most common protocol for DNA extraction from various plant tissues (seeds, young leafs, mature leafs, root); 2) CTAB-based method originally described for DNA isolation from medicinal plants with high levels of secondary metabolites; 3) and one of various commercially available kits. The usefulness of the obtained DNA was evaluated by SSR analysis with seven microsatellite markers. Although the latter two extraction protocols retrieved genomic DNA that gave positive PCR results, only DNA isolated by kit produced full SSR profile

Assessment of pollution at the former uranium waste dumpsite near kaji-Say Village/Kyrgyzstan: a genetic and physiological investigation

“…Frozen leaf tissue (0.5 g) was grounded using a mortar. Following modified CTAB, DNA extraction protocol (Aboul-Ftooh Aboul-Maaty & Abdel-Sadek Oraby, 2019; Fu et al, 2017;Hanjalic et al, 2018) was employed for genomic DNA extraction using the grounded leaf tissue. At final step, the genomic DNA obtained was air-dried for 4 h for removing ethanol and 100 μL RNase-free water was added for dissolving the genomic DNA in it.…”

SUITABILITY OF VARIOUS BUFFERS FOR GENOMIC DNA EXTRACTION OF PINEAPPLE (Ananas comosus) VAR. MD2

“…Furthermore, the usage of liquid nitrogen facilitates the grinding process as the latter abruptly converts tissues into brittle solid (Kasajima, 2018) and also keeps the cellular enzymes deactivated to prevent shearing and damage to DNA structure (Aboul-Maaty & Oraby, 2019). The addition of pre-warmed isolation buffers further break sample tissue as the high temperature at 60-70°C provides a favourable environment for components in the buffer to work better hence enhances the recovery of high-quality DNA (Hanjalić et al, 2018;Sudan et al, 2017). The vigorous vortexing prior to the incubation in the water bath is significant to allow further mixing of tissue with isolation buffer (Sudan et al, 2017).…”

“…In contrast to our work, phenol is excluded due to its hazardous property and replaced with chloroform: isoamyl alcohol (24:1) and of b-me. The presence of b-me antioxidant properties allow chloroform: isoamyl alcohol to further eliminate proteins from DNA (Hanjalić et al, 2018) and weaken the disulfide bonding between cysteine residues to remove tannins and polyphenols (Yu et al, 2017). Furthermore, phenol-using protocols are typically go to denaturing step twice with phenol, then chloroform: isoamyl alcohol.…”