Comparison of methods to preserve Rheum palmatum (Polygonaceae) for efficient DNA extraction and PCR amplification

Abstract

ABSTRACT. In this study, we compared the quality of DNA extracted using the modified CTAB method, from Rheum palmatum leaves preserved using fourteen different methods, including ones used commonly in other species: under ultra-cold (-80°C) temperatures, after drying with an absorbent paper, desiccating using a silica gel, drying at 60°C, in 70% ethanol, absolute ethanol, 70% ethanol supplemented with 50 mM EDTA, SDS-DNA extracting solution, nuclear separation buffer, improved NaCl-CTAB solution, TE-buffer, I-solution, or IIsolution. DNA extracted from fresh leaves was used as the control. The quality of extracted DNA was evaluated based on the success of PCR amplification of the ITS2 region and a microsatellite marker. DNA was not extracted from samples preserved in the nuclear separation buffer and II-solution. The purities of DNA extracted from leaves preserved in ultra-cold temperatures, 70% ethanol, and 70% ethanol with 50 mM EDTA, and after desiccating using a silica gel and drying were higher, and comparable to the purity of DNA extracted from fresh leaves, than 2 M. Huang et al. Genetics and Molecular Research 15 (3): gmr.15038019 those of leaves preserved using other methods. In the present study, combined with the PCR amplifications, the preservation using ultra-cold temperatures, silica gel desiccation, or drying, and PCR amplification of the extracted DNA can be used for further molecular studies in R. palmatum.

Research Article Improved methods for storing and extracting DNA from <i>Ilex</i> <i>paraguariensis</i> (Aquifoliaceae) tissue samples

“…precipitation ), which apparently did not occur in the samples, id fragments. The same was not observed for R. , for which conservation methods influenced DNA quality CTAB were not amplified, ded high quality fragments (Huang et al, 2016b). In , storage with silica gel made it impossible to amplify the 20 ºC favored the preservation of the samples (Feres et al, study, it should be noted that oxidation of phenolic compounds present in the leaf samples of mate stored in CTAB, at room temperature for 90 days, did not impede DNA amplification.…”

“…Methods for storing and extracting DNA from DNA extraction in CTAB buffer was not successful from leaves of Huang et al, 2016 From the analysis of the amplification products of the plastid fragment we observed that with all of the storage methods, or with fresh leaves, we obtained DNA without the presence of residues that impede the acti Inappropriate storage methods may result in DNA degradation, as well as co of PCR inhibitors (Huang et al, 2016b), which apparently did not occur in the samples, since there was amplification of the plastid fragments. The same was not observed for K. lathrophyton, for which conservation methods influenced DNA quality and PCR reactions.…”

“…In addition, CTAB may be a viable alternative for the collection of desiccation-sensitive samples that are difficult to preserve using other methodologies. These results obtained with the CTAB buffer should be associated with the presence of chemicals that help prevent DNA degradation, such as Tris-HCl, which provides good buffering capacity, and EDTA, which chelates the cations Mg 2+ and Ca 2+ and inhibits DNAse activity (Huang et al, 2016b). In addition, we used PVP, which prevents oxidation of phenolic compounds due to its antioxidant action.…”

“…The method of preserving plant tissue with CTAB buffer was successfully originated in a study with high quality DNA extracted successfully from leaf samples that were preserved for 30 days (Rogstad, 1992), and samples from the stems of Plinia cauliflora, in which it avoided the oxidation of phenolic compounds (Danner et al, 2011). This method was also used for the conservation of fungal spores, maintained at room temperature or at 4ºC for two weeks (Huang et al, 2016b). Contrary to these results, DNA extraction in CTAB buffer was not successful from leaves of result of its deg we observed that with all of the storage methods, or with fresh leaves, we obtained DNA without the presence of residues that impede the acti Inappropriate storage methods may result in DNA degradation, as well as co of PCR inhibitors (Huang et al, 2016 since there was amplification of the plast palmatum and PCR reactions.…”

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“…Preserving DNA through deactivation of nucleases, removal of cations or lowering temperature becomes crucial to inhibit enzymes that degrade DNA. Most studies have utilized freezing conditions (Carroll et al, 2012;Wu et al, 2012) or the use of preservatives such as ethanol (Murphy et al, 2002;Bressan et al, 2014;Huang et al, 2016), RNAlater (Nechvatal et al, 2008;Sorensen et al, 2016), and glycerol:PBS (McKain et al, 2013;Fliegerova et al, 2014) to preserve bacterial DNA before extraction. However, few studies have examined the combination of chemical buffers and temperature reduction to preserve DNA and optimize bacterial analysis.…”