The extraction of DNA from plant seeds is an essential step for satisfactory results in molecular studies particularly those involving plant genetics (Junior et al. 2016). Different seeds belonging to related genera or different orders contain many components with variable complexities that badly interfere with purity of the extracted DNA and molecular investigations following isolation procedures (Porebski et al. 1997; Ribeiro and Lovato 2007).
To insure isolation of DNA with better yield and quality from seeds of diverse plant orders, we implemented several steps in the present modified protocol. Liquid nitrogen was used to break the cell wall and disrupt the cell membrane (Clark 1997) while keeping cellular enzymes and other undesired chemicals deactivated, thus reducing shearing and damaging of the DNA. Other methods used for disrupting plant tissues, such as digestion with pectinase and cellulose (Manen et al. 2005), are not as reproducible or accurate as the use of liquid nitrogen.
High concentration of the 3× CTAB was also used to disrupt the cells and nuclear membranes in order to expose the genetic components (Amani et al. 2011). In the present modified method, the 3× CTAB buffer also contains the highest recommended concentration level (0.3%) of 2-β-mercaptoethanol which successfully removed polyphenols (Horne et al. 2004; Li et al. 2007) giving rise a clear translucent DNA pellet. The CTAB extraction buffer also includes 1.4 M of NaCl which improved the quality of the extracted DNA (Sahu et al. 2012).
To remove the remaining polysaccharides during DNA extraction from all plant samples included in the present work, a modification for the precipitation of DNA was also performed by increasing the concentration of sodium chloride and potassium acetate. The concentration of NaCl varied with plant species in a range between 0.7 M (Clark 1997) and 6 M (Aljanabi et al. 1999; Moreira and Oliveira 2011). In the present standardized protocol, we used 6 M NaCl (Moreira and Oliveira 2011) and 3 M potassium acetate (Paterson et al. 1993). These modifications successfully removed polysaccharides impurities from DNA extracted by this modified protocol from all plant samples and produced pure and high-quality DNA suitable for further molecular analysis. Proteins, most lipids, and cellular debris were removed by binding with non-aqueous compounds and precipitated during the chloroform-isoamyl alcohol step.
Longer incubation of the extracted DNA at − 20 °C also enhanced precipitation of DNA. In general, the quantity and quality of isolated DNA depend on precipitation temperature and duration (Michiels et al. 2003). Low-temperature precipitation employed in the present modified protocol increased DNA yield. Extracted DNA were re-suspended in minimum amount of 1× TE buffer since the presence of chelating agents in TE buffer can affect the PCR and other molecular analysis of the extracted DNA.
The method employed in the present work proved to be successful and applicable for extraction of DNA with high yield and purity from 19 different plant species that belong to seven different plant orders. The matrix variation effects on the purity and quality of the isolated genomic DNA were minimized by using the same plant samples as starting materials for both protocols employed in the present investigation.
Electrophoresis separation of DNA extracted by the present protocol showed intense bands very close to the gel wells (Fig. 1, upper lane) signifying high degree of purity and intact DNA. It is known that the presence of smear could be a sign of degradation of the extracted DNA which easily affects the quality of the subsequent molecular application results (Devi et al. 2013).
DNA samples extracted by the present protocol were assessed for successful PCR amplification with RAPD primer (OPZ-09). The presence of clear and well-differentiated band patterns (Fig. 4) reflects the efficiency of the protocol to produce genomic DNA with high purity suitable for molecular studies that based on PCR techniques (Devi et al. 2013).
Purification of DNA is also an important step for analyzing and measuring genetically modified (GM) food products (Ateş Sönmezoğlu and Keskin 2015). The DNA extracted by our standardized protocol yielded detectable and reproducible bands for NPTII (173 bp target) proving its suitability for PCR amplification as well as for the identification of GM crops using the PCR assay.
The A260/A280 purity ratio is an important measure for estimating the polyphenol contamination levels of the extracted DNA. Ratios of A260/A280 below 1.8 render the extracted DNA inappropriate for molecular investigations (Sambrook and Russell 2001). Therefore, higher level of 2-β-mercaptoethanol (0.3%) used in the present standardized method successfully removed polyphenols giving rise to translucent final DNA pellets (Suman et al. 1999).
In the present modified CTAB-based protocol, although the RNase A enzyme was not used during isolation and purification of DNA, the ratios of absorption A260/A280 of the extracted DNA (Table 2) were higher than the recommended optimal limit of DNA purity (Sambrook and Russell 2001). Similar results were also observed by Sambrook and Russell (2001) which were taken to be associated with RNA contamination. In our case, the resulted intact DNA bands, very close to the wells (Fig. 1, upper lane), indicated high purity of the extracted DNA with no RNA contamination, particularly that the recommended and the most accurate way to determine RNA contamination is to run the sample on an agarose gel where another band of the RNA, if present, will be visible in the gel (Wang et al. 2012). Therefore, the higher ratios of absorption A260/A280 in our case may be attributed to slight changes in the pH of the extracted samples (Wilfinger et al. 1997).
Polysaccharide contamination was also assessed (Table 2) by estimating the absorbance ratio A260/A230 as a secondary measure of nucleic acid purity (Wilson and Walker 2005). This ratio is important to evaluate the level of salt residues in the purified DNA. It is recommended to be greater than 1.5 and preferably close to 1.8. The reported values of A260/A230 ratio in most of the DNA plant samples extracted by the present modified protocol are higher than those of the DNA samples extracted by the other classical method.