SIZE OF DNA DETERMINED BY VISCOELASTIC MEASUREMENTS - RESULTS ON BACTERIOPHAGES, BACILLUS-SUBTILIS AND ESCHERICHIA-COLI
SIZE OF DNA DETERMINED BY VISCOELASTIC MEASUREMENTS - RESULTS ON BACTERIOPHAGES, BACILLUS-SUBTILIS AND ESCHERICHIA-COLI
复制标题
DOI：
10.1016/0022-2836(72)90191-x
复制
发表时间：
1972-01-01
影响因子：
5.6
通讯作者：
ZIMM, BH
中科院分区：
文献类型：
--
作者：
KLOTZ, LC;ZIMM, BH
描述了一种粘弹性方法，该方法对于确定非常大的DNA分子的尺寸特别有用。所用的仪器是以前描述过的笛卡尔潜水员圆柱仪器的发展（查普曼，克洛茨，汤普森和齐姆，1969年）。在这种仪器中，溶液的粘度可以像往常一样测量，但另外，可以观察到在驱动应力被移除后发生的弹性反冲。对于大DNA分子的溶液，这种反冲在很长一段时间内呈指数衰减;时间常数称为延迟时间，是DNA分子量的敏感函数。同时测定特性粘度和延迟时间，可以得到三种计算分子量的方法：仅从特性粘度计算、仅从延迟时间计算和从延迟时间与特性粘度的比值计算。本文给出了T2噬菌体DNA、T7噬菌体DNA和枯草芽孢杆菌W23和大肠杆菌B稳定期细胞裂解液中DNA的测定结果。对于噬菌体DNA，假设DNA分子是无规卷曲，通过所有三种方法计算的分子量与公认的值吻合良好。对于细胞裂解物DNA，最可靠的计算分子量的方法（仅从阻滞时间）给出了合理的值（2.0 × 109，对于B.枯草芽孢杆菌为2.7 × 109;大肠杆菌）。其他两种方法的结果存在一定的差异，这可能是由于部分DNA的降解，将特性粘度与分子量相关的经验公式中的错误，或两者的组合。在任何情况下，清楚的是，这些裂解物中至少一半的DNA对应于整个染色体。延迟时间对细胞裂解物中的许多可能的人为因素不敏感，例如部分DNA的降解、剪切应力的影响和剪切依赖性聚集效应（流变固定），因此它是特别有用的量。粘度对上述三种都很敏感。结果表明，在该仪器上测得的粘度和延迟时间对研究分子量至少为10 ~（10）道尔顿的DNA是有用的。
A viscoelastic method is described which is particularly useful for determining the sizes of very large DNA molecules. The instrument used is a development of a Cartesian-diver rotating-cylinder instrument previously described (Chapman, Klotz, Thompson & Zimm, 1969). In this instrument the viscosity of the solution can be measured as usual, but in addition the elastic recoil, occurring after the driving stress is removed, can be observed. With solutions of large DNA molecules this recoil decays exponentially at long times; the time constant, called the retardation time, is a sensitive function of the molecular weight of the DNA. Measurement of both intrinsic viscosity and retardation time gives three methods of calculating molecular weight: from intrinsic viscosity alone, from retardation time alone and from the ratio of retardation time to intrinsic viscosity. Results of measurements on solutions of T2 bacteriophage DNA, T7 bacteriophage DNA, and the DNA in stationary-phase cell lysates ofBacillus subtilisW23 andEscherichia coliB are presented. For the bacteriophage DNA's, the molecular weights calculated by all three methods, assuming that the DNA molecules are random coils, agree well with the accepted values. For the cell-lysate DNA's, the most reliable method of calculating molecular weight (from the retardation time alone) gives reasonable values (2.0 × 109forB. subtilisand 2.7 × 109forE. coli). The results from the other two methods are in some disagreement, which may be due to degradation of part of the DNA, an error in the empirical formula relating intrinsic viscosity to molecular weight, or a combination of both. In any case it is clear that at least half of the DNA in these lysates corresponds to the whole chromosome. The retardation time is insensitive to many possible artifacts in the cell lysates, such as degradation of part of the DNA, effects of shear stress and shear-dependent aggregation effects (rheopexy), so it is a particularly useful quantity. Viscosity is sensitive to all three of the above. The results presented here indicate that viscosity and retardation time measurements obtained on this instrument are useful for studying DNA of molecular weight up to at least 1010daltons.