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Asian Journal of Plant Sciences

Year: 2007 | Volume: 6 | Issue: 6 | Page No.: 977-981
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Authors

A. Dehestani

S.K. Kazemi Tabar

Keywords

Research Article

A Rapid Efficient Method for DNA Isolation from Plants with High Levels of Secondary Metabolites

A. Dehestani and S.K. Kazemi Tabar
This research was designated to optimize a rapid, inexpensive method to isolate desirable DNA from plants with high concentrations of secondary products. DNA extraction procedures of plants with high levels of secondary metabolites e.g., Tea (Camellia sinensis) Pokeweed (Phytolacca dodecandra), Broad bean (Vicia faba L.) and most of medicinal plants is so complicated due to polyphenolics, tanins, alkaloids and other metabolites that makes it difficult to obtain high quality DNA with good maintenance time. To overcome this problem, we designed a simple but efficient method. In this method, an extraction buffer is primarily used to reduce the secondary metabolite levels followed by a separate lysis buffer comprising of a sarcosyl detergent (N-lauryl sarcosine sodium salt), 2 different reducing agents and higher concentrations of chelating agents. CTAB/NaCl precipitation of polysaccharides and elimination of proteins through chloroform:isoamyl alcohol extraction resulted a more pure DNA. Presented method consists of few steps. Furthermore, protein and polysaccharide contamination was noticeably reduced. This method does not require expensive reagents and modern laboratory equipments and it is possible to isolate several DNA samples per day in a general biology lab. DNA derived using this method was tested electrophoretically and then was examined spectrophotometrically: A260/280 (protein contamination) and A260/230 (Polyphenol and polysaccharide residuals). Polymerase Chain Reaction (PCR) amplification was performed using different RAPD primers and EcoRI, BamHI and HindIII were used for restriction enzyme reactions at different digestion conditions. Results indicated that DNA produced in this method is desirable for must of genetic assays and because polyphenolic compounds were removed or neutralized, maintenance time of DNA samples increased.
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samir abisb Reply

this paper is very important for me because I am phd student working on faba bean dna

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