[Immobilization of DNA on microporous membranes using UV-irradiation].
[Immobilization of DNA on microporous membranes using UV-irradiation].
复制标题
[利用紫外线照射将 DNA 固定在微孔膜上]。
DOI：
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发表时间：
1992
期刊：
影响因子：
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通讯作者：
G. Dymshits
中科院分区：
文献类型：
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作者：
S. Kalachikov;B. A. Adarichev;G. Dymshits
比较了硝酸纤维素和尼龙微孔膜固定DNA的各种技术。尽管在印迹过程中DNA在这些膜上有很强的初级吸附，但在杂交和洗涤后，靶DNA的保留差，杂交信号低。UV照射后DNA的共价交联导致靶DNA的定量固定。对DNA中单个碱基的光固定量子产率估计约为10（-4）。足以固定已知长度的DNA片段的UV照射剂量可以通过公式Ilc =（22.3 +/-4.8）c/l计算，其中l是DNA片段长度（以碱基对计），c是待固定的DNA部分（%）。对于大多数杂交实验，约0.6- 0.8kJ/m2的UV照射剂量是最佳的。高于0.8-1 kJ/m2的剂量可能导致杂交效率的损失。在最佳的固定条件下，杂交信号增加5倍的硝酸纤维素膜和50倍的不带电的尼龙膜相比，这些膜在真空中烘烤。
Various techniques of DNA immobilization onto nitrocellulose and nylon microporous membranes have been compared. Despite a strong primary adsorption of DNA onto these membranes during blotting procedures, poor retention of the target DNA and low hybridization signals are obtained after hybridization and washings. Covalent cross-linking of DNA upon UV irradiation leads to a quantitative immobilization of target DNA. Quantum yield of DNA photoimmobilization estimated for a single base in DNA is about 10(-4). UV irradiation dose sufficient for immobilization of DNA fragment of a known length can be calculated by the formula Ilc = (22.3 +/- 4.8) c/l, where l is the DNA fragment length (in base pairs), c is the DNA part (%) to be immobilized. The UV irradiation dose about 0.6-0.8 kJ/m2 is optimal for most hybridization experiments. Doses higher than 0.8-1 kJ/m2 may cause a loss in the hybridization efficiency. Under optimal immobilization conditions, hybridization signals increasing five-fold for nitrocellulose membranes and fifty-fold for uncharged nylon membranes as compared with baking these membranes in vacuum.