Volume 21, Issue 5 (2019)                   JAST 2019, 21(5): 1215-1226 | Back to browse issues page

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Spadoni A, Sion S, Gadaleta S, Savoia M A, Piarulli L, Fanelli V, et al . A Simple and Rapid Method for Genomic DNA Extraction and Microsatellite Analysis in Tree Plants. JAST 2019; 21 (5) :1215-1226
URL: http://jast.modares.ac.ir/article-23-13213-en.html
1- SINAGRI S. r. l. - Spin off University of Bari, Via Amendola 165/A, 70126, Bari, Italy.
2- Department of Soil, Plant and Food Sciences, University of Bari, Via Amendola 165/A, 70126, Bari, Italy.
3- DISSPA, University of Bari, BARI, Italy
Abstract:   (4369 Views)
A new and optimized protocol, here called 6hDNA (i.e. a genomic DNA obtained by a six-hour extraction method), has been developed based on the traditional Cetyl-TrimethylAmmonium Bromide (CTAB) method. It allows a fast and easy isolation of genomic DNA from plant species, especially from those with high polyphenol and polysaccharide contents. Co-precipitation of polysaccharides was avoided by adding higher concentrations of selective precipitants of nucleic acid, CTAB 3% (w/v) and sodium chloride (NaCl) (1.42M). PolyVinylPyrrolidone (PVP) 1% (w/v) was applied to remove polyphenols as PCR inhibitors. Proteins were degraded by treatments of chloroform:isoamyl alchol (24:1) and phenol:chloroform:isoamyl alchol (25:24:1) and removed by centrifugation from plant extracts. The yield of total DNA from leaves of Vitis vinifera, Citrus sinensis and Olea europaea ranged from 42 to 980 ng µL-1 with A260/A280 ratio values between 1.6 and 2.06. The purity and integrity of the obtained DNA guarantees successful downstream applications including PCR and microsatellite markers. The use of lyophilized plant material and the reduced time of the total procedure make this new 6hDNA protocol more convenient when compared to the most common DNA isolation protocols, such as: “Doyle and Doyle”, “Lodhi”, “Li”, or those using the DNAzol reagent and the Nucleospin Plant Minikit.
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Article Type: Research Paper | Subject: Agricultural Economics/Agriculture Marketing and Supply Chains
Received: 2018/01/10 | Accepted: 2018/09/15 | Published: 2019/09/15

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