Comparison of five DNA extraction methods in three medicinal plants: Peganum harmala L., Tamarix ramosissima Ledeb., and Potentilla reptans L

Abstract

Extracting high-yield, high-quality DNA from plant samples is challenging due to the presence of the cell wall, pigments, and some secondary metabolites. The main CTAB method, two of its modified protocols (beta-mercaptoethanol or ammonium acetate were eliminated), the modified Murray and Thompson method, and the Gene All kit were statistically compared based on the quantity and quality of the total DNA (tDNA) extracted from fresh and dried leaves of three medicinal herbs P. harmala, T. ramosissima, and P. reptans. The suitability of the tDNAs for molecular studies was evaluated by polymerase chain reaction (PCR) of the fragments of the internal transcribed spacer (ITS) in nuclear DNA and the trnL-F region in chloroplast DNA. Some significant differences were found between the tDNAs extracted by five extraction methods. With the exception of P. harmala, where the PCR of both the ITS fragments and the trnL-F region worked successfully in all DNA samples, but only the ITS fragments, not the chloroplast trnL-F region, were amplified in the DNA samples of T. ramosissima and P. reptans. The chloroplast trnL-F region was amplified only in DNA samples extracted from fresh and dried leaves of the three studied herbs using the commercial kit. Gene All kit, the main CTAB method, and its modified protocols were the less time-consuming protocols that yielded DNA suitable for downstream PCR vis-a-vis the modified Murray and Thompson method.

Keywords: CTAB; DNA extraction; Murray and Thompson method; Peganum harmala L.; Potentilla reptans L.; Tamarix ramosissima Ledeb..

Figure 1

The quality assessment of the extracted total DNA from (a) Peganum harmala L., (b) Tamarix. ramosissima Ledeb., and (c) Potentilla reptans L. using the studied extraction methods. About 500 µg of DNA was analyzed by electrophoresis on a 0.8% agarose gel. 1) the main CTAB method - fresh leaves, 2) the main CTAB method - dried leaves, 3) the first CTAB method - fresh leaves, 4) the first CTAB method - dried leaves, 5) the second CTAB method - fresh leaves, 6) the second CTAB method - dried leaves, 7) the modified Murray and Thompson method - fresh leaves, 8) the modified Murray and Thompson method - dried leaves, 9) the Gene All kit- fresh leaves, and 10) the Gene All kit- dried leaves

Figure 2

The results of the PCR of the ITS fragments in the nuclear ribosomal DNA of Peganum harmala L., Tamarix ramosissima Ledeb., and Potentilla reptans L. using the universal primers of ITS4 and ITS5m. M: DNA molecular ladder (DM2300), 1) the main CTAB method - fresh leaves, 2) the first CTAB method - fresh leaves, 3) the second CTAB method - fresh leaves, 4) the modified Murray and Thompson method - fresh leaves, 5) Gene All kit - fresh leaves, 6) negative control, 7) the main CTAB method - dried leaves, 8) the first CTAB method - dried leaves, 9) the second CTAB method - dried leaves, 10) the modified Murray and Thompson method - dried leaves, 11) the Gene All kit - dried leaves, and 12) negative control. (d, e, and f) The results of the PCR of the trnL-F region in the chloroplast genome of P. harmala, T. ramosissima, and P. reptans, respectively, using the universal primers of trnL(f) and trnL(c). M: DNA molecular ladder (DM2300); (d): 1) the Gene All kit - fresh leaves, 2) Gene All kit - dried leaves, 3) the second CTAB method - fresh leaves, 4) the second CTAB method - dried leaves, 5) the main CTAB method - fresh leaves, 6) the main CTAB method - dried leaves, 7) negative control, 8) the first CTAB method - fresh leaves, 9) the first CTAB method - dried leaves, 10) the modified Murray and Thompson method - fresh leaves, and 11) the modified Murray and Thompson method - dried leaves. (e): 1) Gene All - fresh leaves, 2) Gene All Kit - dried leaves, 3) the second CTAB method - fresh leaves, 4) the second CTAB method - dried leaves, 5) negative control. (f): 1) Gene All Kit - fresh leaves, 2) Gene All Kit - dried leaves, 3) the second CTAB method - dried leaves, 4) the first CTAB method - dried leaves, 5) the main CTAB method - dried leaves, 6) negative control