Barnes W N, Reznikoff W S
J Biol Chem. 1975 Oct 25;250(20):8184-92.
A sequential DNA-RNA hybridization procedure is described whereby RNA homologous to a target DNA region 35 to 140 base pairs in length can be purified up to 6700-fold from a complex in vitro transcript to a homogeneity sufficient for sequence analysis. Requirements for the procedure include: (a) uniform transcription over the target DNA region in vitro; (b) specialized transducing phages which carry genetic deletions defining the target region on either side; and (c) specialized transducing phages which carry the target DNA in opposite orientations. These requirements have been met for the genetic control region (promoter, operator) of the lactose operon of Escherichia coli, to which the method was applied. The procedure is independent of the activity of the genetic control signals under study and can therefore be applied without modification to the study of point mutations introduced into the template.
本文描述了一种连续的DNA-RNA杂交方法，通过该方法，长度为35至140个碱基对的与目标DNA区域同源的RNA可从复杂的体外转录物中纯化出来，纯化倍数高达6700倍，达到足以进行序列分析的纯度。该方法的要求包括：（a）在体外目标DNA区域上进行均匀转录；（b）携带在两侧定义目标区域的基因缺失的特殊转导噬菌体；（c）携带目标DNA反向排列的特殊转导噬菌体。已满足对大肠杆菌乳糖操纵子的遗传控制区域（启动子、操纵基因）的这些要求，并将该方法应用于此。该方法与所研究的遗传控制信号的活性无关，因此无需修改即可应用于对引入模板中的点突变的研究。
