Research Article

Comparison of small scale methods for the rapid and efficient extraction of mitochondrial DNA from wheat crop suitable for down-stream processes

Published: December 04, 2014
Genet. Mol. Res. 13 (4) : 10320-10331 DOI: https://doi.org/10.4238/2014.December.4.27
Cite this Article:
(2014). Comparison of small scale methods for the rapid and efficient extraction of mitochondrial DNA from wheat crop suitable for down-stream processes. Genet. Mol. Res. 13(4): gmr4510. https://doi.org/10.4238/2014.December.4.27
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Abstract

We evaluated and compared 2 mitochondrial DNA (mtDNA) extraction methods in terms of DNA quality and success of subsequent polymerase chain reaction (PCR) amplifications from yel­low etiolated shoots of wheat crop (Triticum aestivum). mtDNA ex­traction is difficult because the presence of metabolites interfere with DNA isolation procedures and downstream applications such as DNA restriction, amplification, and cloning. The method (with modification) involved inactivation of genomic DNA by DNase I enzyme, RNA by RNase enzyme, contaminant proteins by using proteinase K, and pre­cipitation of polysaccharides in the presence of a high salt concentra­tion. The DNase I and RNA enzyme ratio was adjusted to 10:8 mL. The purity of mtDNA was confirmed by PCR amplification of genomic, mitochondrial, and chloroplast (rbcL) gene. The mitochondrial COXIII gene of 400 bp was amplified; the b-actin and chloroplast genes were not amplified. A260/A280 (1.89) and A260/A230 (2.07) ratios were calcu­lated using a spectrophotometer. The isolated mtDNA was amenable to amplification and restriction digestion. The technique is fast, reproduc­ible, and suitable for PCR-based markers.

We evaluated and compared 2 mitochondrial DNA (mtDNA) extraction methods in terms of DNA quality and success of subsequent polymerase chain reaction (PCR) amplifications from yel­low etiolated shoots of wheat crop (Triticum aestivum). mtDNA ex­traction is difficult because the presence of metabolites interfere with DNA isolation procedures and downstream applications such as DNA restriction, amplification, and cloning. The method (with modification) involved inactivation of genomic DNA by DNase I enzyme, RNA by RNase enzyme, contaminant proteins by using proteinase K, and pre­cipitation of polysaccharides in the presence of a high salt concentra­tion. The DNase I and RNA enzyme ratio was adjusted to 10:8 mL. The purity of mtDNA was confirmed by PCR amplification of genomic, mitochondrial, and chloroplast (rbcL) gene. The mitochondrial COXIII gene of 400 bp was amplified; the b-actin and chloroplast genes were not amplified. A260/A280 (1.89) and A260/A230 (2.07) ratios were calcu­lated using a spectrophotometer. The isolated mtDNA was amenable to amplification and restriction digestion. The technique is fast, reproduc­ible, and suitable for PCR-based markers.