Limits...
Optimization of DNA extraction and PCR protocols for phylogenetic analysis in Schinopsis spp. and related Anacardiaceae.

Mogni VY, Kahan MA, de Queiroz LP, Vesprini JL, Ortiz JP, Prado DE - Springerplus (2016)

Bottom Line: The modifications proposed allowed the extraction of 70-120 µg of non-degraded genomic DNA per gram of dry tissue that resulted useful for PCR amplification.Sequence analyses of amplicons showed similarity with the corresponding Schinopsis accessions available at GenBank.The methodology presented here can be routinely applied for molecular studies of the group aimed to clarify not only aspects on the molecular biology but also the taxonomy and phylogeny of this fascinating group of vascular plants.

View Article: PubMed Central - PubMed

Affiliation: Facultad de Ciencias Agrarias, Universidad Nacional de Rosario, Campo Experimental Villarino, S2125ZAA Zavalla, Santa Fe Argentina ; IICAR, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET-UNR), Zavalla, Argentina.

ABSTRACT
The Anacardiaceae is an important and worldwide distributed family of ecological and socio-economic relevance. Notwithstanding that, molecular studies in this family are scarce and problematic because of the particularly high concentration of secondary metabolites-i.e. tannins and oleoresins-that are present in almost all tissues of the many members of the group, which complicate the purification and amplification of the DNA. The objective of this work was to improve an available DNA isolation method for Schinopsis spp. and other related Anacardiaceae, as well as the PCR protocols for DNA amplification of the chloroplast trnL-F, rps16 and ndhF and nuclear ITS-ETS fragments. The modifications proposed allowed the extraction of 70-120 µg of non-degraded genomic DNA per gram of dry tissue that resulted useful for PCR amplification. PCR reactions produced the expected fragments that could be directly sequenced. Sequence analyses of amplicons showed similarity with the corresponding Schinopsis accessions available at GenBank. The methodology presented here can be routinely applied for molecular studies of the group aimed to clarify not only aspects on the molecular biology but also the taxonomy and phylogeny of this fascinating group of vascular plants.

No MeSH data available.


0.8 % Agarose gel electrophoresis depicting results of DNA extraction of some accessions of Schinopsis spp. and related species. a DNA extracted with the optimized protocol. Lanes1, 2, 8, 14 and 15 molecular marker (Lambda EcoRI/HindIII), 3 and 4S. lorentzii, 5 and 6S. marginata, 7Lithraea molleoides, 9–13S. brasiliensis. b DNA extracted with the DNAEasy Plant Mini Kit (Quiagen Inc. Valencia, CA) used as control. 1 Molecular weight marker (100 bp DNA Ladder), 2–11 different specimens of S. brasiliensis (asterisk reference bands of the marker)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4835408&req=5

Fig1: 0.8 % Agarose gel electrophoresis depicting results of DNA extraction of some accessions of Schinopsis spp. and related species. a DNA extracted with the optimized protocol. Lanes1, 2, 8, 14 and 15 molecular marker (Lambda EcoRI/HindIII), 3 and 4S. lorentzii, 5 and 6S. marginata, 7Lithraea molleoides, 9–13S. brasiliensis. b DNA extracted with the DNAEasy Plant Mini Kit (Quiagen Inc. Valencia, CA) used as control. 1 Molecular weight marker (100 bp DNA Ladder), 2–11 different specimens of S. brasiliensis (asterisk reference bands of the marker)

Mentions: Briefly, the modifications introduced to the protocol described by Permingeat et al. (1998), that allowed the isolation of total DNA of all species tested were the following: the decrease of the initial quantity of plant material (20–25 vs. 500–1000 mg); the addition of sterile sand (or liquid nitrogen in the Eppendorf tubes) for disrupting leaf tissue and create the lysate; the extension in the incubation time and the temperature increment (150 min at 75 °C vs. 60 min at 60 °C) of the Extraction Buffer; the duplication of the chloroform step for protein removal and the final precipitation with Ethanol in presence of 5 % V/V NaCl 5 M (instead of NaAc 3 M pH 5.2). The result of the extraction methods are shown in Fig. 1. The modified protocol produced clear bands of high molecular weight corresponding to the total DNA in most of the accessions (38/41), although some samples showed smearing consistent with partially degraded DNA (Fig. 1a). The assays performed with the DNeasy Plant Mini Kit (control) showed similar results and allowed the extraction of the 12 samples tested as well, and less smearing was observed (Fig. 1b). Comparison by eye of ethidium bromide fluorescence produced by the samples to the Lambda DNA and spectrophotometric quantification showed values ranging from 70 to 120 µg of DNA per gram of tissue (vs. 75–130 µg obtained with the control, see Fig. 1b), indicating a relative good yield and the presence of high molecular weight DNA, and that the samples can be used directly for PCR reactions.Fig. 1


Optimization of DNA extraction and PCR protocols for phylogenetic analysis in Schinopsis spp. and related Anacardiaceae.

Mogni VY, Kahan MA, de Queiroz LP, Vesprini JL, Ortiz JP, Prado DE - Springerplus (2016)

0.8 % Agarose gel electrophoresis depicting results of DNA extraction of some accessions of Schinopsis spp. and related species. a DNA extracted with the optimized protocol. Lanes1, 2, 8, 14 and 15 molecular marker (Lambda EcoRI/HindIII), 3 and 4S. lorentzii, 5 and 6S. marginata, 7Lithraea molleoides, 9–13S. brasiliensis. b DNA extracted with the DNAEasy Plant Mini Kit (Quiagen Inc. Valencia, CA) used as control. 1 Molecular weight marker (100 bp DNA Ladder), 2–11 different specimens of S. brasiliensis (asterisk reference bands of the marker)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4835408&req=5

Fig1: 0.8 % Agarose gel electrophoresis depicting results of DNA extraction of some accessions of Schinopsis spp. and related species. a DNA extracted with the optimized protocol. Lanes1, 2, 8, 14 and 15 molecular marker (Lambda EcoRI/HindIII), 3 and 4S. lorentzii, 5 and 6S. marginata, 7Lithraea molleoides, 9–13S. brasiliensis. b DNA extracted with the DNAEasy Plant Mini Kit (Quiagen Inc. Valencia, CA) used as control. 1 Molecular weight marker (100 bp DNA Ladder), 2–11 different specimens of S. brasiliensis (asterisk reference bands of the marker)
Mentions: Briefly, the modifications introduced to the protocol described by Permingeat et al. (1998), that allowed the isolation of total DNA of all species tested were the following: the decrease of the initial quantity of plant material (20–25 vs. 500–1000 mg); the addition of sterile sand (or liquid nitrogen in the Eppendorf tubes) for disrupting leaf tissue and create the lysate; the extension in the incubation time and the temperature increment (150 min at 75 °C vs. 60 min at 60 °C) of the Extraction Buffer; the duplication of the chloroform step for protein removal and the final precipitation with Ethanol in presence of 5 % V/V NaCl 5 M (instead of NaAc 3 M pH 5.2). The result of the extraction methods are shown in Fig. 1. The modified protocol produced clear bands of high molecular weight corresponding to the total DNA in most of the accessions (38/41), although some samples showed smearing consistent with partially degraded DNA (Fig. 1a). The assays performed with the DNeasy Plant Mini Kit (control) showed similar results and allowed the extraction of the 12 samples tested as well, and less smearing was observed (Fig. 1b). Comparison by eye of ethidium bromide fluorescence produced by the samples to the Lambda DNA and spectrophotometric quantification showed values ranging from 70 to 120 µg of DNA per gram of tissue (vs. 75–130 µg obtained with the control, see Fig. 1b), indicating a relative good yield and the presence of high molecular weight DNA, and that the samples can be used directly for PCR reactions.Fig. 1

Bottom Line: The modifications proposed allowed the extraction of 70-120 µg of non-degraded genomic DNA per gram of dry tissue that resulted useful for PCR amplification.Sequence analyses of amplicons showed similarity with the corresponding Schinopsis accessions available at GenBank.The methodology presented here can be routinely applied for molecular studies of the group aimed to clarify not only aspects on the molecular biology but also the taxonomy and phylogeny of this fascinating group of vascular plants.

View Article: PubMed Central - PubMed

Affiliation: Facultad de Ciencias Agrarias, Universidad Nacional de Rosario, Campo Experimental Villarino, S2125ZAA Zavalla, Santa Fe Argentina ; IICAR, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET-UNR), Zavalla, Argentina.

ABSTRACT
The Anacardiaceae is an important and worldwide distributed family of ecological and socio-economic relevance. Notwithstanding that, molecular studies in this family are scarce and problematic because of the particularly high concentration of secondary metabolites-i.e. tannins and oleoresins-that are present in almost all tissues of the many members of the group, which complicate the purification and amplification of the DNA. The objective of this work was to improve an available DNA isolation method for Schinopsis spp. and other related Anacardiaceae, as well as the PCR protocols for DNA amplification of the chloroplast trnL-F, rps16 and ndhF and nuclear ITS-ETS fragments. The modifications proposed allowed the extraction of 70-120 µg of non-degraded genomic DNA per gram of dry tissue that resulted useful for PCR amplification. PCR reactions produced the expected fragments that could be directly sequenced. Sequence analyses of amplicons showed similarity with the corresponding Schinopsis accessions available at GenBank. The methodology presented here can be routinely applied for molecular studies of the group aimed to clarify not only aspects on the molecular biology but also the taxonomy and phylogeny of this fascinating group of vascular plants.

No MeSH data available.