Experimental Techniques.docxVIP

Appendix D:Experimental Techniques? ? ? ? ? ? ? ? 附录D:实验技术? ? ? ? Elucidating the molecular basis of life is no easy task.??Our current understanding of complex processes like replication, transcription or translation has required thousands of scientists laboring for decades.??Yet, considering the tiny scale on which molecular biologists work, the amount known is admirable.??This understanding is due to a powerful set of techniques, tools that allow our immense hands to cut and past molecules, and our blurry eyes to peer inside cells.? ?In this chapter we review the most important of these techniques used today.? ? ? ? ? ? ? ? 要弄清楚生命的分子基础并不是一件容易的事。我们现在了解的复杂过程，如复制、转录或转译，是几千位科学家通过几十年的辛勤研究得来的。不过，考虑到分子生物学家工作的对象是如此微小，已获得的知识还是值得称道的。这样的理解得益于一套有效的技术，这是一些使我们的大手能够去切割并粘贴分子和使我们模糊的眼睛能够窥探细胞内部的工具。本章我们回顾现今使用的一些最重要的技术。? ? ? ? D.1??DNA Manipulation ? ? ? ? Techniques? ? ? ? ? ? ? ? D.1??DNA操作技术? ? ? ? D.1.1??Gel Electrophoresis and ? ? ? ? Southern Blotting? ? ? ? ? ? ? ? D.1.1??凝胶电泳与? ? ? ? Southern印迹法? ? ? ? Gel electrophoresis is a technique used to separate a mixture of DNA molecules according to length (Figure D.1). DNA molecules are pulled through a gel by a voltage that attracts the negative charges on phosphodiester bonds.??The gel is a tangle of polymers that is quite dense, but contains holes through which DNA molecules can pass.??Shorter molecules fit more easily through these holes than larger molecules; as a result, the speed at which a DNA molecule moves down the gel is directly related to its size.? ? ? ? ? ? ? ? 凝胶电泳是用来将DNA分子混合物根据它们的长度分开的技术（图D.1）。DNA分子的磷酸二酯键上带有负电荷，在电场作用下会在凝胶中发生移动。凝胶是一团密度较大但有孔的聚合物，DNA分子可以从孔中穿过。较小的分子比大分子更容易进入这些孔；结果，一个DNA分子在凝胶中的移动速度直接与它的大小有关。? ? ? ? After the gel has been ‘run’ for some time, DNA molecules of different lengths will each occupy different places on the gel, appearing as individual bands.??Very fine gels can be made that can allow DNA molecules to be resolved even if they differ in length by only one base pair.??Note that gel electrophoresi