DNA Extraction Protocol for Plants with High Levels of Secondary Metabolites and Polysaccharides without Using Liquid Nitrogen and Phenol.

Sunil Kumar Sahu,Muthusamy Thangaraj,Kandasamy Kathiresan

doi:10.5402/2012/205049

Abstract

Mangroves and salt marsh species are known to synthesize a wide spectrum of polysaccharides and polyphenols including flavonoids and other secondary metabolites which interfere with the extraction of pure genomic DNA. Although a plethora of plant DNA isolation protocols exist, extracting DNA from mangroves and salt marsh species is a challenging task. This study describes a rapid and reliable cetyl trimethylammonium bromide (CTAB) protocol suited specifically for extracting DNA from plants which are rich in polysaccharides and secondary metabolites, and the protocol also excludes the use of expensive liquid nitrogen and toxic phenols. Purity of extracted DNA was excellent as evident by A260/A280 ratio ranging from 1.78 to 1.84 and A260/A230 ratio was >2, which also suggested that the preparations were sufficiently free of proteins and polyphenolics/polysaccharide compounds. DNA concentration ranged from 8.8 to 9.9 μg μL−1. The extracted DNA was amenable to RAPD, restriction digestion, and PCR amplification of plant barcode genes (matK and rbcl). The optimized method is suitable for both dry and fresh leaves. The success of this method in obtaining high-quality genomic DNA demonstrated the broad applicability of this method.

Highlights

The isolation of pure, intact, and high-quality DNA is very crucial for any molecular studies [1]
DNA isolation from plants is usually compromised by excessive contamination by secondary metabolites
The DNA isolation methods need to be adjusted to each plant species and even to each plant tissue because of the presence of these metabolites, unlike animals and microbes [2]

Summary

Introduction

The isolation of pure, intact, and high-quality DNA is very crucial for any molecular studies [1]. The search for a more efficient means of extracting DNA of both higher quality and yield has led to the development of several protocols for isolating DNA from plants containing high levels of secondary metabolites [3,4,5,6,7]. The mangroves and salt marsh are specially adapted to harsh environment such as marshy anoxic anaerobic soil and fluctuating salinity of the water bodies [8]. To avoid these stress conditions mangroves and salt marsh plants synthesize high amounts of polysaccharides, polyphenols, and other secondary metabolites such as alkaloids and flavanoids which impede DNA extraction [9, 10]

Objectives

Methods

Results

Conclusion