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CELL BIOLOGY & MOLECULAR GENETICS

Isolation of High Molecular Length DNA

Alfalfa, Pea, Rice, Sorghum, Soybean, and Spinach

^a B.M. Sutherland, Biology Dep., Brookhaven National Laboratory, Upton, NY 11973-5000
^b Kyoto Univ., Gokanoshi, Uji, Kyoto 611-0011, Japan
^c Institute of Genetic Ecology, Tohoku Univ., Sendai, Japan
^d Dep. of Pathology, Biological and Biomedical Sciences Program, Harvard Medical School, Boston, MA 02115
^e Dep. of Natural Resource Science and Landscape Architecture, Univ. of Maryland, College Park MD 20742
^f Argonne National Laboratory, Argonne, IL 60439
^g Dep. of Biology, Toho Univ. School of Medicine, Tokyo, Japan

Corresponding author (bms{at}bnl.gov)

Measuring DNA damage in higher plants is important in assessing the impacts of environmental conditions, e.g., increased UV resulting from ozone depletion, and in testing the relationship of productivity to DNA damage and repair. Sunlight exposure of plants produces UV-induced DNA damages measurable by treating DNA with damage-specific enzymes and dispersion of DNA molecules in denaturing media. Such DNA must be enzyme-digestible, with few single strand breaks. DNA isolation must preclude repair, providing a "snapshot" of DNA damage. We developed a method for isolating DNA from several crop plants, both monocots and dicots—alfalfa (Medicago sativa L.), pea (Pisum sativum L.), rice (Oryza sativa L.), soybean [Glycine max (L.) Merr.], sorghum [Sorghum bicolor (L.) Moench], and spinach (Spinacia oleraceae L.). This method is simple, readily deals with multiple samples, and avoids organic solvents. We show that pyrimidine dimers can readily be quantified in DNA prepared by this method. This method should also be useful for other experiments requiring high molecular length, enzymatically digestible plant DNA.

Abbreviations: CPD, cyclobutyl pyrimidine dimers • kb, kilobase • LN₂, liquid nitrogen • Mb, megabase • UV, ultraviolet radiation • UVA, 320–400 nm • UVB, 290–320 nm