Abstract

BackgroundThe world’s top three cereals, based on their monetary value, are rice, wheat, and corn. In cereal crops, DNA extraction is difficult owing to rigid non-cellulose components in the cell wall of leaves and high starch and protein content in grains. The advanced techniques in molecular biology require pure and quick extraction of DNA. The majority of existing DNA extraction methods rely on long incubation and multiple precipitations or commercially available kits to produce contaminant-free high molecular weight DNA.ResultsIn this study, we compared three different methods used for the isolation of high-quality genomic DNA from the grains of cereal crop, Zea mays, with minor modifications. The DNA from the grains of two maize hybrids, M10 and M321, was extracted using extraction methods DNeasy Qiagen Plant Mini Kit, CTAB-method (with/without 1% PVP) and modified Mericon extraction. Genes coding for 45S ribosomal RNA are organized in tandem arrays of up to several thousand copies and contain codes for 18S, 5.8S and 26S rRNA units separated by internal transcribed spacers ITS1 and ITS2. While the rRNA units are evolutionary conserved, ITS regions show high level of interspecific divergence and have been used frequently in genetic diversity and phylogenetic studies. In this study, the genomic DNA was then amplified with PCR using primers specific for ITS gene. PCR products were then visualized on agarose gel.ConclusionThe modified Mericon extraction method was found to be the most efficient DNA extraction method, capable to provide high DNA yields with better quality, affordable cost and less time.

Highlights

  • The world’s top three cereals, based on their monetary value, are rice, wheat, and corn

  • Since the first use of cetyltrimethyl ammonium bromide (CTAB)-based method for extraction of DNA from plant leaves [14, 15], it has been modified several times to reduce contaminants such as polyphenols and polysaccharides that are present in the plant tissues [16,17,18]

  • All currently published methods of DNA extraction have demonstrated their effectiveness in isolating DNA that is suitable for polymerase chain reaction (PCR) amplification or restriction digestion, they require long incubations, multiple precipitation steps, and ethanol washes to produce RNA-free genomic DNA of high purity

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Summary

Introduction

The world’s top three cereals, based on their monetary value, are rice, wheat, and corn. The majority of existing DNA extraction methods rely on long incubation and multiple precipitations or commercially available kits to produce contaminant-free high molecular weight DNA. The extraction and purification of high-quality DNA from cereals is generally difficult due to the presence of polysaccharides, proteins, and DNA polymerase inhibitors such as tannins, Abdel‐Latif and Osman Plant Methods (2017) 13:1 the high cost of liquid nitrogen. Commercially available column-based extraction kits are effective in isolating contaminant-free DNA from recalcitrant plant species, there is still loss of significant amounts of DNA on the column. DNA quality required for PCR and sequencing is often very high with DNA of high molecular weight and with less shearing, free of contamination from protein, RNA or polysaccharides, and 260/280 nm absorbance ratio of approximately 1.8–2.0. The aim of this study was to compare quality and quantity of DNA isolated using three different extraction methods

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