Extraction of DNA from plant and fungus tissues in situ

Abstract

BackgroundWhen samples are collected in the field and transported to the lab, degradation of the nucleic acids contained in the samples is frequently observed. Immediate extraction and precipitation of the nucleic acids reduces degradation to a minimum, thus preserving accurate sequence information. An extraction method to obtain high quality DNA in field studies is described.FindingsDNA extracted immediately after sampling was compared to DNA extracted after allowing the sampled tissues to air dry at 21°C for 48 or 72 hours. While DNA extracted from fresh tissues exhibited little degradation, DNA extracted from all tissues exposed to 21°C air for 48 or 72 hours exhibited varying degrees of degradation. Yield was higher for extractions from fresh tissues in most cases. Four microcentrifuges were compared for DNA yield: one standard electric laboratory microcentrifuge (max rcf = 16,000×g), two battery-operated microcentrifuges (max rcf = 5,000 and 3,000 ×g), and one manually-operated microcentrifuge (max rcf = 120×g). Yields for all centrifuges were similar. DNA extracted under simulated field conditions was similar in yield and quality to DNA extracted in the laboratory using the same equipment.ConclusionsThis CTAB (cetyltrimethylammonium bromide) DNA extraction method employs battery-operated and manually-operated equipment to isolate high quality DNA in the field. The method was tested on plant and fungus tissues, and may be adapted for other types of organisms. The method produced high quality DNA in laboratory tests and under simulated field conditions. The field extraction method should prove useful for working in remote sites, where ice, dry ice, and liquid nitrogen are unavailable; where degradation is likely to occur due to the long distances between the sample site and the laboratory; and in instances where other DNA preservation and transportation methods have been unsuccessful. It may be possible to adapt this method for genomic, metagenomic, transcriptomic and metabolomic projects using samples collected in situ.

Evolutionary insights into Bean common mosaic necrosis virus and Cowpea aphid borne mosaic virus using global isolates and thirteen new near complete genomes from Kenya

“…(from Busia). Leaf samples collected from symptomatic plants were stored using two methods: silica gel and the paper press method (Abu Almakarem et al , 2012). Samples were then transported to the Bioscience eastern central Africa (BecA-ILRI) laboratories in Nairobi, Kenya for processing.…”

Validation of loop‐mediated isothermal amplification for fast and portable sex determination across the phylogeny of birds

“…It must be noted, though, that crucial parameters such as incubation time or temperature might change depending on the amount of template/inhibitors added. Because of this, future work to deploy LAMP under field conditions must focus on developing easy, fast, clean and yield‐controlled DNA extraction protocols for biological tissues (see e.g., Abu Almakarem, Heilman, Conger, Shtarkman, & Rogers, for DNA extraction of plant and fungus tissues).…”

Optimization of CTAB-based RNA Extraction for in planta Fusarium oxysporum f. sp. cubense Gene Expression Study

“…Lithium chloride does not effectively precipitate DNA and proteins (Barlow et al 1963;Sambrook & Russel 2001) and hence any potential degradation caused by Foc endogenous RNases can be minimised. Moreover, phenol acts through the disruption of protein structures and hence helps in denaturing RNases (Abu Almakarem et al 2012).…”