Research Article An optimized protocol for DNA extraction in plants with a high content of secondary metabolites, based on leaves of Mimosa tenuiflora (Willd.) Poir. (Leguminosae).

Abstract

Some species are characterized by a high content of tannins, alkaloids, and phenols in their leaves. These secondary metabolites are released during DNA extraction and might hinder molecular studies based on PCR (polymerase chain reaction). To provide an efficient method to extract DNA, Mimosa tenuiflora, an important leguminous plant from Brazilian semiarid region used in popular medicine and as a source of fuelwood or forage, was used. Eight procedures previously reported for plants were tested and adapted from leaf tissues of M. tenuiflora stored at -20°C. The optimized procedure in this study encompassed the utilization of phenol during deproteinization, increased concentrations of cetyltrimethylammonium bromide and sodium chloride, and a shorter period and lower temperature of incubation concerning other methods. The extracted DNA did not present degradation, and amplification via PCR was successful using ISSR, trnL, ITS, and ETS primers. Besides M. tenuiflora, this procedure was also tested and proved to be efficient in genetic studies of other plant species.

A modified protocol for rapid DNA isolation from cotton (Gossypium spp.)

“…However, high endogenous levels of polysaccharides and polyphenols interfere with the isolation of good quality DNA, thereby rendering it unsuitable for downstream analyses [[1], [2], [3]]. During cell disruption, phenolic compounds come out of the vacuoles, become readily oxidized and irreversibly bind with nucleic acids and proteins, thus resulting in a dark DNA pellet unsuitable for most enzymatic manipulations [4]. On the other hand, the viscous nature of polysaccharides makes extracted DNA fractious to pipetting; and, it also interferes with various biological enzymes, and especially hinders the PCR reaction by inhibiting the Thermus aquaticus DNA polymerase ( Taq.…”

Optimizing Genomic DNA Isolation and PCR Amplification For Pasak Bumi (Eurycoma longifolia)

“…CTAB able to produce high quality genomic DNA amenable to RAPD (Random amplified Polymorphic DNA), restriction digestion, and amplification of plant barcode genes (matK and rbcL) with reduced cost [24]. The same result also found in M. tenuiflora, tanin producing trees [8].…”

“…Different with herbaceous plant, many part of trees containing of some DNA inhibitor such as polysaccharides, tannin, phenol and other secondary metabolites substances. These substances will inhabit DNA polymerase activity during PCR process [8]. other inhibitory substances and has been used successfully for some higher plant and forest trees [9].…”

Comparison of DNA Yield from Different Plant Materials of Plumeria sp. (Apocynaceae)

“…The phenolic compound that present in DNA solution can inhibit enzymatic reaction such as polymerase chain reaction (PCR). Modification of extraction protocols and DNA purification need to be done to proceed the enzymatic reaction [9].…”