Farrand S K, Eden F C, Chilton M D
Biochim Biophys Acta. 1975 May 16;390(3):264-75. doi: 10.1016/0005-2787(75)90347-0.
A systematic study of the DNA-DNA-filter reaction is presented which measures its ability to detect small amounts of simple DNA (bacterial or bacteriophage) in model mixtures of DNA immobilized on filters. Saturation curves show qualitatively that significant binding occurs when there is 10% Agrobacterium tumefaciens DNA on the filter but not 1%. PS8 bacteriophage DNA is detectable at a level of 0.1%. True saturation is not attained in the bacterial DNA reaction : radioactivity bound represents only 3% of the theoretical saturation value. The bacteriophage DNA reactions attain 15-30% of the expected saturation value. When crown gall tumor DNA filters were tested for the presence of A. tumefaciens or PS8 bacteriophage DNA by saturation reactions, an apparently significant amount of binding was observed compared with usual background levels for heterologous DNA filters. However thermal dissociation profiles revealed that no well-matched duplexes were formed. Normal tobacco callus DNA filters exhibited the same type of binding of labeled DNA to a similar extent (50-100% as much as tumor DNA filters). Both types of DNA-filters bound Bacillus subtilis and bacteriophage T4 DNA as efficiently as A. tumefaciens and PS8 DNA. The high non-specific background binding of labeled DNA by filters containing DNA isolated from plant tissue culture materials is ascribed to low single strand molecular weight of the filterbound DNA. This study provides no evidence for foreign DNA in crown gall tumors, and raises objections to the interpretation of the data of earlier investigators (Quetier, F., Huguet, T. and Guille, E. (1969) Biochem, Biophys. Res, Commun. 34, 128-133 and Srivastava, B.I.S. (1970) Life Sci. 9, 889-892) who claimed to detect A. tumefaciens DNA in crown gall tumors by DNA-DNA-filter hybridization.
本文介绍了一项关于DNA - DNA - 滤膜反应的系统研究，该研究测量了其在固定于滤膜上的DNA模型混合物中检测少量简单DNA（细菌或噬菌体）的能力。饱和曲线定性地表明，当滤膜上有10%的根癌土壤杆菌DNA时会发生显著结合，但1%时则不会。PS8噬菌体DNA在0.1%的水平可被检测到。细菌DNA反应未达到真正的饱和：结合的放射性仅占理论饱和值的3%。噬菌体DNA反应达到预期饱和值的15 - 30%。当通过饱和反应检测冠瘿瘤DNA滤膜中根癌土壤杆菌或PS8噬菌体DNA的存在时，与异源DNA滤膜的通常背景水平相比，观察到明显大量的结合。然而，热解离图谱显示未形成良好匹配的双链体。正常烟草愈伤组织DNA滤膜在相似程度上表现出相同类型的标记DNA结合（为肿瘤DNA滤膜的50 - 100%）。两种类型的DNA滤膜结合枯草芽孢杆菌和噬菌体T4 DNA的效率与根癌土壤杆菌和PS8 DNA相同。含有从植物组织培养材料中分离的DNA的滤膜对标记DNA的高非特异性背景结合归因于滤膜结合DNA的低单链分子量。本研究没有提供冠瘿瘤中存在外源DNA的证据，并对早期研究者（Quetier, F., Huguet, T.和Guille, E. (1969) Biochem, Biophys. Res, Commun. 34, 128 - 133以及Srivastava, B.I.S. (1970) Life Sci. 9, 889 - 892）的数据解释提出了异议，这些研究者声称通过DNA - DNA - 滤膜杂交在冠瘿瘤中检测到了根癌土壤杆菌DNA。
