Yokota H, Johnson F, Lu H, Robinson R M, Belu A M, Garrison M D, Ratner B D, Trask B J, Miller D L
Department of Molecular Biotechnology, University of Washington, Seattle, WA 98195, USA.
Nucleic Acids Res. 1997 Mar 1;25(5):1064-70. doi: 10.1093/nar/25.5.1064.
We have developed an improved method of straightening DNA molecules for use in optical restriction mapping. The DNA was straightened on 3-aminopropyltriethoxysilane-coated glass slides using surface tension generated by a moving meniscus. In our method the meniscus motion was controlled mechanically, which provides advantages of speed and uniformity of the straightened molecules. Variation in the affinity of the silanized surfaces for DNA was compensated by precoating the slide with single-stranded non-target blocking DNA. A small amount of MgCl2 added to the DNA suspension increased the DNA-surface affinity and was necessary for efficient restriction enzyme digestion of the straightened surface-bound DNA. By adjusting the amounts of blocking DNA and MgCl2, we prepared slides that contained many straight parallel DNA molecules. Straightened lambda phage DNA (48 kb) bound to a slide surface was digested by EcoRI restriction endonuclease, and the resulting restriction fragments were imaged by fluorescence microscopy using a CCD camera. The observed fragment lengths showed excellent agreement with their predicted lengths.
我们开发了一种改进的拉直DNA分子的方法，用于光学限制性图谱分析。利用移动弯月面产生的表面张力，将DNA在3-氨丙基三乙氧基硅烷包被的载玻片上拉直。在我们的方法中，弯月面运动是通过机械方式控制的，这为拉直分子的速度和均匀性提供了优势。通过用单链非靶标阻断DNA预包被载玻片，补偿了硅烷化表面对DNA亲和力的变化。向DNA悬浮液中添加少量MgCl2可增加DNA与表面的亲和力，这对于有效酶切拉直的表面结合DNA是必需的。通过调整阻断DNA和MgCl2的量，我们制备了包含许多笔直平行DNA分子的载玻片。与载玻片表面结合的拉直的λ噬菌体DNA（48 kb）用EcoRI限制性内切酶消化，然后使用CCD相机通过荧光显微镜对产生的限制性片段进行成像。观察到的片段长度与预测长度高度一致。
