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Title: CKC: isolation of necleic acids from a diversity of plants using CTAB and slica columns

item Henderson, David
item Hammond, John

Submitted to: Molecular Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/3/2012
Publication Date: 1/24/2012
Citation: Henderson, D.C., Hammond, J. 2012. CKC: isolation of necleic acids from a diversity of plants using CTAB and slica columns. Molecular Biotechnology. DOI: 10.1007/s12033-012-9494-y.

Interpretive Summary: Nucleic acid extraction from plants forms the first step of many molecular biology procedures, including methods for plant disease diagnosis. Methods applicable to almost any plant species are desirable in order to allow routine analyses for a variety of purposes, including microarray analysis to determine gene expression levels or to assay for plant pathogens. However, chemicals present in many plants, including phenolic compounds, polysaccharides, latex, and other secondary metabolites, may interfere with either the extraction of pure nucleic acids, or adversely affect the quality such that the extracted nucleic acids are not suitable for use in molecular biology procedures such as enzymatic modification. Extraction methods that are suitable for some plants are often not applicable to other plants with different levels or combinations of secondary metabolites. A method has been developed by which high quality total nucleic acids can be obtained simply and reproducibly in less than two hours from a diverse range of plant materials containing different combinations of secondary metabolites, including plant species known to be problematic using previously available methods. This method will allow the use of a single protocol for extraction of high quality nucleic acids, suitable for a variety of molecular biology applications, from almost any commonly encountered plant species. One application will be for the detection and identification of plant virus diseases using a diagnostic plant virus microarray, allowing quarantine officials and diagnosticians to more rapidly identify viruses from any source plant material.

Technical Abstract: To assay for viruses in plant samples, we required a method for isolation of nucleic acid that is rapid, simple, and applicable to the widest variety of plants. A protocol for isolation of total nucleic acid (TNA) was developed by combining common CTAB methods with silica spin columns. We report data on TNA purity and RNA quality from over 30 plant genera representing 25 families; RNA quality was measured and determined to be suitable to generate reliable real-time PCR results. One-step RT-PCR was successfully performed on all samples. The protocol can be completed in less than two hours.