Muecke Merlind, Samuels Martin, Davey Megan, Jeruzalmi David
Department of Molecular and Cellular Biology, Harvard University, 7 Divinity Avenue, Cambridge, MA 02138, USA.
Structure. 2008 Jun;16(6):837-41. doi: 10.1016/j.str.2008.04.008.
We describe a method for preparing large, linear DNA molecules in amounts that are suitable for structural studies. The procedure employs self-primed DNA amplification on a starting molecule that consists of the sequence of interest flanked by the cohesive end sequences from bacteriophage lambda as well as endonuclease recognition sites. Amplification produces long polymers of DNA, tens of kilobases in length, which harbor many copies of the sequence of interest. Endonuclease digestion of these polymers, followed by chromatographic purification, yields high-quality preparations of the DNA molecule of interest. Reliance on the cohesive end sequences to initiate self-primed amplification effectively enables the synthesis of DNA molecules of interest with minimal restriction on length and sequence.
我们描述了一种制备大量线性DNA分子的方法，其数量适用于结构研究。该程序在起始分子上采用自引发DNA扩增，起始分子由感兴趣的序列组成，两侧是来自噬菌体λ的粘性末端序列以及内切核酸酶识别位点。扩增产生长度达数十千碱基的长DNA聚合物，其中含有许多感兴趣序列的拷贝。对这些聚合物进行内切核酸酶消化，然后进行色谱纯化，可得到高质量的感兴趣DNA分子制剂。依靠粘性末端序列启动自引发扩增，能够有效地合成对长度和序列限制最小的感兴趣DNA分子。
