Walter N G
Max-Planck-Institute for Biophysical Chemistry, Department of Biochemical Kinetics, Göttingen, Germany.
J Mol Biol. 1995 Dec 15;254(5):856-68. doi: 10.1006/jmbi.1995.0661.
Evolution comprises cycles of amplification, mutagenesis and selection. To study evolutionary phenomena, isothermal strand displacement amplification (SDA) of double-stranded DNA as an in vitro model for rolling-circle replication of viruses has been coupled to a positive selection procedure. First, two subsequent amplification reactions utilizing exo- Klenow polymerase were performed under direct observation using the fluorescent dye thiazole orange. Under the chosen conditions, the mutation rate was 1.5 x 10(-3) and 0.4 x 10(-3) for base substitutions and deletions, respectively. Then, a 16mer oligodeoxynucleotide with an acridine moiety coupled to its 5' end was used to select for double strands that retained their ability to form a triple-helix with the oligodeoxynucleotide. Conditions for triple-helix formation were chosen such that only 10 to 40% of the SDA products were allowed to bind the third strand. Non-denaturing polyacrylamide gel electrophoresis was used to separate triple-helices from unmodified double strands, and only triplex strands were used to initiate a new round of error-prone amplification and selection. Nine such rounds with about 270 molecular generations were performed. The final mutant spectrum was characterized and compared with those of amplification reactions without additional selection pressure. While without selection pressure base substitutions and deletions throughout the initial wild-type rapidly produce a diverse mutant distribution, the consensus after nine selection rounds clearly shows two mutational hotspot positions. Using gel shift assays and a newly developed non-radioactive DNase I footprinting technique, it could be shown that both the initial wild-type and the final consensus do not differ significantly in their triplex formation ability. As opposed to this, they do show different amplification efficiencies. The final consensus sequence is amplified with the highest rate in the exponential reaction phase, while the most abundant clone, which is characterized by two additional point deletions, is the sequence with the highest amplification rate in the linear growth phase.
进化包括扩增、诱变和选择的循环。为了研究进化现象，双链DNA的等温链置换扩增（SDA）作为病毒滚环复制的体外模型已与正向选择程序相结合。首先，使用荧光染料噻唑橙在直接观察下利用外切酶Klenow聚合酶进行两个连续的扩增反应。在选定的条件下，碱基替换和缺失的突变率分别为1.5×10⁻³和0.4×10⁻³。然后，使用一个5'端连接有吖啶部分的16聚体寡脱氧核苷酸来选择保留与该寡脱氧核苷酸形成三链体能力的双链。选择三链体形成的条件，使得仅允许10%至40%的SDA产物与第三条链结合。非变性聚丙烯酰胺凝胶电泳用于从未修饰的双链中分离三链体，并且仅三链体链用于启动新一轮的易错扩增和选择。进行了九轮这样的反应，约有270个分子世代。对最终的突变谱进行了表征，并与没有额外选择压力的扩增反应的突变谱进行了比较。在没有选择压力的情况下，整个初始野生型中的碱基替换和缺失会迅速产生多样化的突变分布，而经过九轮选择后的共有序列清楚地显示出两个突变热点位置。使用凝胶迁移分析和新开发的非放射性DNase I足迹技术，可以表明初始野生型和最终共有序列在其三链体形成能力上没有显著差异。与此相反，它们确实显示出不同的扩增效率。最终的共有序列在指数反应阶段扩增速率最高，而以另外两个点缺失为特征的最丰富的克隆是线性生长阶段扩增速率最高的序列。
