Quaite F E, Sutherland J C, Sutherland B M
Biology Department, Brookhaven National Laboratory, Upton, NY 11973.
Plant Mol Biol. 1994 Feb;24(3):475-83. doi: 10.1007/BF00024115.
Quantitation of UV-induced DNA damages in nanogram quantities of non-radioactive DNA from irradiated plants by gel electrophoresis requires a prompt, efficient, high-yield method of isolating DNA yielding high-molecular-weight, enzymatically digestible DNA. To meet these criteria we devised a high-yield method for isolating from plant tissue, DNA whose single-strand molecular length is greater than about 170 kb. Leaf tissue is embedded in agarose plugs, digested with Proteinase K in the presence of detergent, and treated with phenylmethylsulfonyl fluoride (PMSF). The agarose plugs are then soaked with buffer appropriate to the desired enzyme treatment. Evaluation of the DNA on neutral and alkaline gels indicates its high molecular length and low frequency of single-strand breaks. The DNA can be digested with damage-specific and other endonucleases. The method is especially suitable for DNA damage quantitation, as tissue processing is carried out immediately after harvesting (allowing DNA lesion measurement at precisely known times after irradiation), and many samples can be easily handled at once. It should also be useful for molecular analysis of large numbers of plant samples available only in small quantities. We here use this method to quantitate DNA damage induced by 297 and 365 nm radiation, and calculate the relative damaging effects of these wavebands in today's solar spectrum.
通过凝胶电泳对辐照植物中纳克量的非放射性DNA进行紫外线诱导的DNA损伤定量分析，需要一种快速、高效、高产的方法来分离出高分子量、可酶切的DNA。为满足这些标准，我们设计了一种高产方法，用于从植物组织中分离单链分子长度大于约170 kb的DNA。将叶片组织包埋在琼脂糖块中，在去污剂存在的情况下用蛋白酶K消化，并用苯甲基磺酰氟（PMSF）处理。然后用适合所需酶处理的缓冲液浸泡琼脂糖块。在中性和碱性凝胶上对DNA的评估表明其高分子长度和低单链断裂频率。该DNA可用损伤特异性内切酶和其他内切酶进行消化。该方法特别适用于DNA损伤定量分析，因为组织处理在收获后立即进行（允许在辐照后精确已知的时间测量DNA损伤），并且可以一次轻松处理许多样品。它对于仅以少量形式存在的大量植物样品的分子分析也应该是有用的。我们在此使用该方法对297和365 nm辐射诱导的DNA损伤进行定量分析，并计算这些波段在当今太阳光谱中的相对损伤效应。
