Reproducible genomic DNA preparation from diverse crop species for molecular genetic applications

Kelvin T Chiong,Shankar R Pant,Carlos A Avila,Kranthi K Mandadi,Mona B Damaj,Ninfa R Ramos,T Erik Mirkov,Denise V Carvalho,Carmen S Padilla

doi:10.1186/s13007-017-0255-6

Kelvin T Chiong, Shankar R Pant + Show 7 more

Open Access

https://doi.org/10.1186/s13007-017-0255-6

Copy DOI

Abstract

BackgroundSeveral high-throughput molecular genetic analyses rely on high-quality genomic DNA. Copurification of other molecules can negatively impact the functionality of plant DNA preparations employed in these procedures. Isolating DNA from agronomically important crops, such as sugarcane, rice, citrus, potato and tomato is a challenge due to the presence of high fiber, polysaccharides, or secondary metabolites. We present a simplified, rapid and reproducible SDS-based method that provides high-quality and -quantity of DNA from small amounts of leaf tissue, as required by the emerging biotechnology and molecular genetic applications.ResultsWe developed the TENS-CO method as a simplified SDS-based isolation procedure with sequential steps of purification to remove polysaccharides and polyphenols using 2-mercaptoethanol and potassium acetate, chloroform partitioning, and sodium acetate/ethanol precipitation to yield high-quantity and -quality DNA consistently from small amounts of tissue (0.15 g) for different plant species. The method is simplified and rapid in terms of requiring minimal manipulation, smaller extraction volume, reduced homogenization time (20 s) and DNA precipitation (one precipitation for 1 h). The method has been demonstrated to accelerate screening of large amounts of plant tissues from species that are rich in polysaccharides and secondary metabolites for Southern blot analysis of reporter gene overexpressing lines, pathogen detection by quantitative PCR, and genotyping of disease-resistant plants using marker-assisted selection.ConclusionTo facilitate molecular genetic studies in major agronomical crops, we have developed the TENS-CO method as a simple, rapid, reproducible and scalable protocol enabling efficient and robust isolation of high-quality and -quantity DNA from small amounts of tissue from sugarcane, rice, citrus, potato, and tomato, thereby reducing significantly the time and resources used for DNA isolation.

Highlights

Several high-throughput molecular genetic analyses rely on high-quality genomic DNA
The presence of high amounts of sodium chloride (NaCl) (0.5 M) in the TENS-CO extraction buffer was efficient in increasing the solubility of polysaccharides, reducing their coprecipitation with the DNA in subsequent steps [48]
The incorporation of high concentrations of potassium acetate (KOAc) (5 M) aided in neutralizing the lysis produced by the TENS-CO buffer reaction and precipitating proteins that bind to sodium dodecyl sulfate (SDS), thereby eliminating excess of proteins that form in the pellet after centrifugation

Summary

Introduction

Several high-throughput molecular genetic analyses rely on high-quality genomic DNA. Copurification of other molecules can negatively impact the functionality of plant DNA preparations employed in these procedures. Isolation of DNA from agronomically important crops, such as sugarcane, rice, citrus, potato and tomato remains a limiting step due to the presence of high polysaccharides, polyphenols, and other secondary metabolites [1,2,3,4,5]. Levels of these compounds accumulate in plants under abiotic and biotic stresses, such as drought or pathogen infection [6, 7]. Most methods use phenol/ chloroform for purification of the extracted DNA; while others add a purification step, by binding the extracted DNA to glass fiber coated plate wells [33] or to an anion exchange column [34]

Methods

Results

Conclusion