Beskin A D, Zevin-Sonkin D, Sobolev I A, Ulanovsky L E
Department of Structural Biology, Weizmann Institute of Science, Rehovot, Israel.
Nucleic Acids Res. 1995 Aug 11;23(15):2881-5. doi: 10.1093/nar/23.15.2881.
Modular primers are strings of three contiguously annealed unligated oligonucleotides (modules) as short as 5- or 6-mers, selected from a presynthesized library. It was previously found that such strings can prime DNA sequencing reactions specifically, thus eliminating the need for the primer synthesis step in DNA sequencing by primer walking. It has remained largely a mystery why modular primers prime uniquely, while a single module, used alone in the same conditions, often shows alternative priming of comparable strength. In a puzzling way, the single module, even in a large excess over the template, no longer primes at the alternative sites, when modules with which it can form a contiguous string are also present. Here we describe experiments indicating that this phenomenon cannot be explained by cooperative annealing of the modules to the template. Instead, the mechanism seems to involve competition between different primers for the available polymerase. In this competition, the polymerase is preferentially engaged by longer primers, whether modular or conventional, at the expense of shorter primers, even though the latter can otherwise prime with similar or occasionally higher efficiency.
模块化引物是由三个连续退火的未连接寡核苷酸（模块）组成的序列，短至5或6聚体，从预先合成的文库中选择。先前发现，这样的序列可以特异性地引发DNA测序反应，从而消除了通过引物步移进行DNA测序时引物合成步骤的需要。模块化引物为何能独特地引发反应，而单个模块在相同条件下单独使用时却常常表现出强度相当的替代引发，这在很大程度上仍是个谜。令人费解的是，即使单个模块相对于模板有大量过剩，当存在能与之形成连续序列的模块时，它也不再在替代位点引发反应。在这里，我们描述的实验表明，这种现象不能用模块与模板的协同退火来解释。相反，其机制似乎涉及不同引物对可用聚合酶的竞争。在这种竞争中，聚合酶优先与较长的引物结合，无论是模块化引物还是传统引物，而牺牲较短的引物，尽管后者在其他情况下可以以相似或偶尔更高的效率引发反应。
