Revie D, Smith D W, Yee T W
Department of Biology, University of California, San Diego, La Jolla 92093.
Nucleic Acids Res. 1988 Nov 11;16(21):10301-21. doi: 10.1093/nar/16.21.10301.
Kinetic equations describing ligation of DNA to circular recombinant forms were developed and solved for four types of reactions: (a) a homogeneous population of singly restricted DNA fragments, (b) insertion of singly restricted insert into vector, (c) forced directional insertion of doubly restricted insert into vector, and (d) insertion of singly restricted insert into phosphatased vector. The effects of varying vector and insert sizes, starting concentrations, and phosphatase treatment on the yield of circular 1:1 recombinants were analyzed. Selected theoretical predictions were experimentally tested and verified. Our suggestions on optimizing ligation reactions in several cases are at variance with common practice. For example, optimum conditions in case (b) and (d) ligations are best specified as individual insert and vector concentrations rather than as insert/vector molar ratios, except in case (d) ligations involving very small insert size. In case (c) ligations, highest efficiencies are obtained when both vector and insert are at relatively low concentration.
我们推导了描述DNA连接成环状重组形式的动力学方程，并针对四种反应类型进行了求解：(a) 单酶切DNA片段的均匀群体；(b) 将单酶切的插入片段插入载体；(c) 将双酶切的插入片段强制定向插入载体；(d) 将单酶切的插入片段插入经磷酸酶处理的载体。分析了载体和插入片段大小、起始浓度以及磷酸酶处理对环状1:1重组体产量的影响。对选定的理论预测进行了实验测试和验证。我们在几种情况下优化连接反应的建议与常规做法不同。例如，在(b)和(d)类连接反应中，最佳条件最好指定为插入片段和载体的个体浓度，而不是插入片段/载体摩尔比，但(d)类连接反应涉及非常小的插入片段大小的情况除外。在(c)类连接反应中，当载体和插入片段浓度都相对较低时，可获得最高效率。
