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A simple and rapid method for DNA isolation from xylophagous insects.

Calderón-Cortés N, Quesada M, Cano-Camacho H, Zavala-Páramo G - Int J Mol Sci (2010)

Bottom Line: Published methods to isolate DNA from insects are not always effective in xylophagous insects because they have high concentrations of phenolics and other secondary plant compounds in their digestive tracts.A simple, reliable and labor-effective cetyltrimethylammonium bromide-polyvinylpyrrolidone (CTAB-PVP) method for isolation of high quality DNA from xylophagous insects is described.DNA isolated by the CTAB-PVP method could be used in most molecular analyses.

View Article: PubMed Central - PubMed

Affiliation: Center for Ecosystems Research (CIEco), National Autonomous University of Mexico (UNAM), Morelia, Mexico; E-Mail: mquesada@oikos.unam.mx (M.Q.).

ABSTRACT
Published methods to isolate DNA from insects are not always effective in xylophagous insects because they have high concentrations of phenolics and other secondary plant compounds in their digestive tracts. A simple, reliable and labor-effective cetyltrimethylammonium bromide-polyvinylpyrrolidone (CTAB-PVP) method for isolation of high quality DNA from xylophagous insects is described. This method was successfully applied to PCR and restriction analysis, indicating removal of common inhibitors. DNA isolated by the CTAB-PVP method could be used in most molecular analyses.

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Analysis of Oncideres albomarginata chamela-DNA digested with different restriction enzymes and separated by 1% agarose gel electrophoresis. (a) Genomic DNA isolated with the CTAB-method, and (b) Genomic DNA isolated with the CTAB-PVP method. For both (a) and (b), Lane M, DNA size marker (1 Kb plus DNA ladder; Invitrogen, Carlsbad, CA, USA); Lane 1, restriction digestion with XbaI; Lane 2, restriction digestion with NotI; Lane 3, restriction digestion with EcoRI.
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f3-ijms-11-05056: Analysis of Oncideres albomarginata chamela-DNA digested with different restriction enzymes and separated by 1% agarose gel electrophoresis. (a) Genomic DNA isolated with the CTAB-method, and (b) Genomic DNA isolated with the CTAB-PVP method. For both (a) and (b), Lane M, DNA size marker (1 Kb plus DNA ladder; Invitrogen, Carlsbad, CA, USA); Lane 1, restriction digestion with XbaI; Lane 2, restriction digestion with NotI; Lane 3, restriction digestion with EcoRI.

Mentions: The isolated DNA using both methods was tested for PCR amplification. Amplifications of a mitochondrial cytochrome oxidase I (COI) gene fragment using fresh DNA obtained with both methods were successfully achieved. However, amplification of the COI gene fragment was observed only for the CTAB-PVP isolated-DNA after the DNA samples had been stored for three months (Figure 2). These results indicate that DNA isolated by the traditional CTAB method is not suitable for longer storage periods. Similar results have been previously reported [18,22]. DNA preparations containing contaminants have a shorter storage lifespan [18]. The most common contaminants are polysaccharides, RNA and phenolics [10–12,18–22,25,30,31]. Polysaccharides and phenolics usually produce highly viscous and brown-colored solutions, respectively [10,20,30]. Given that RNA contamination is normally removed by treatment with RNase [30], and the isolated DNA was not viscous, it is likely that phenolics are the contaminants present in the CTAB isolated-DNA. In addition, the inclusion of PVP and β-mercaptoethanol cleared the DNA solutions. This suggests that DNA isolated by the CTAB-PVP method had lower concentrations of phenolics compared with the traditionally used CTAB method. The purity and quality of the isolated DNA was also validated by digestion with different restriction endonucleases. The results showed a complete digestion of CTAB-PVP isolated-DNA (Figure 3a), while CTAB isolated-DNA showed only partial digestion (Figure 3a), indicating the presence of contaminants in this DNA preparation. The CTAB-PVP method demonstrated to be applicable to other xylophagous insects, since isolated DNA from three additional species of xylophagous beetles proved amenable for PCR amplification (Figure 4a) and restriction digestion (Figure 5), whereas DNA isolated with the CTAB-method was not suitable for PCR amplification (Figure 4a) and showed only partial digestion (Figure 5a).


A simple and rapid method for DNA isolation from xylophagous insects.

Calderón-Cortés N, Quesada M, Cano-Camacho H, Zavala-Páramo G - Int J Mol Sci (2010)

Analysis of Oncideres albomarginata chamela-DNA digested with different restriction enzymes and separated by 1% agarose gel electrophoresis. (a) Genomic DNA isolated with the CTAB-method, and (b) Genomic DNA isolated with the CTAB-PVP method. For both (a) and (b), Lane M, DNA size marker (1 Kb plus DNA ladder; Invitrogen, Carlsbad, CA, USA); Lane 1, restriction digestion with XbaI; Lane 2, restriction digestion with NotI; Lane 3, restriction digestion with EcoRI.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3100841&req=5

f3-ijms-11-05056: Analysis of Oncideres albomarginata chamela-DNA digested with different restriction enzymes and separated by 1% agarose gel electrophoresis. (a) Genomic DNA isolated with the CTAB-method, and (b) Genomic DNA isolated with the CTAB-PVP method. For both (a) and (b), Lane M, DNA size marker (1 Kb plus DNA ladder; Invitrogen, Carlsbad, CA, USA); Lane 1, restriction digestion with XbaI; Lane 2, restriction digestion with NotI; Lane 3, restriction digestion with EcoRI.
Mentions: The isolated DNA using both methods was tested for PCR amplification. Amplifications of a mitochondrial cytochrome oxidase I (COI) gene fragment using fresh DNA obtained with both methods were successfully achieved. However, amplification of the COI gene fragment was observed only for the CTAB-PVP isolated-DNA after the DNA samples had been stored for three months (Figure 2). These results indicate that DNA isolated by the traditional CTAB method is not suitable for longer storage periods. Similar results have been previously reported [18,22]. DNA preparations containing contaminants have a shorter storage lifespan [18]. The most common contaminants are polysaccharides, RNA and phenolics [10–12,18–22,25,30,31]. Polysaccharides and phenolics usually produce highly viscous and brown-colored solutions, respectively [10,20,30]. Given that RNA contamination is normally removed by treatment with RNase [30], and the isolated DNA was not viscous, it is likely that phenolics are the contaminants present in the CTAB isolated-DNA. In addition, the inclusion of PVP and β-mercaptoethanol cleared the DNA solutions. This suggests that DNA isolated by the CTAB-PVP method had lower concentrations of phenolics compared with the traditionally used CTAB method. The purity and quality of the isolated DNA was also validated by digestion with different restriction endonucleases. The results showed a complete digestion of CTAB-PVP isolated-DNA (Figure 3a), while CTAB isolated-DNA showed only partial digestion (Figure 3a), indicating the presence of contaminants in this DNA preparation. The CTAB-PVP method demonstrated to be applicable to other xylophagous insects, since isolated DNA from three additional species of xylophagous beetles proved amenable for PCR amplification (Figure 4a) and restriction digestion (Figure 5), whereas DNA isolated with the CTAB-method was not suitable for PCR amplification (Figure 4a) and showed only partial digestion (Figure 5a).

Bottom Line: Published methods to isolate DNA from insects are not always effective in xylophagous insects because they have high concentrations of phenolics and other secondary plant compounds in their digestive tracts.A simple, reliable and labor-effective cetyltrimethylammonium bromide-polyvinylpyrrolidone (CTAB-PVP) method for isolation of high quality DNA from xylophagous insects is described.DNA isolated by the CTAB-PVP method could be used in most molecular analyses.

View Article: PubMed Central - PubMed

Affiliation: Center for Ecosystems Research (CIEco), National Autonomous University of Mexico (UNAM), Morelia, Mexico; E-Mail: mquesada@oikos.unam.mx (M.Q.).

ABSTRACT
Published methods to isolate DNA from insects are not always effective in xylophagous insects because they have high concentrations of phenolics and other secondary plant compounds in their digestive tracts. A simple, reliable and labor-effective cetyltrimethylammonium bromide-polyvinylpyrrolidone (CTAB-PVP) method for isolation of high quality DNA from xylophagous insects is described. This method was successfully applied to PCR and restriction analysis, indicating removal of common inhibitors. DNA isolated by the CTAB-PVP method could be used in most molecular analyses.

Show MeSH