Lvovsky L, Ioshikhes I, Raja M C, Zevin-Sonkin D, Sobolev I A, Liberzon A, Shwartzburd J, Ulanovsky L E
Department of Structural Biology, Weizmann Institute of Science, Rehovot 76100, Israel.
Nucleic Acids Res. 1998 Dec 1;26(23):5525-32. doi: 10.1093/nar/26.23.5525.
Here we analyze the effect of DNA folding on the performance of short primers and describe a simple technique for assessing hitherto uncertain values of thermodynamic parameters that determine the folding of single-stranded DNA into secondary structure. An 8mer with two degenerate positions is extended simultaneously at several complementary sites on a known template (M13mp18) using one, two or three (but never all four) of the possible dNTPs. The length of the extension is site specific because it is limited by the first occurrence in the downstream template sequence of a base whose complementary dNTP is not present. The relative priming efficiencies of different sites are then ranked by comparing their band brightnesses on a gel. The priming efficiency of a short primer (unlike conventional long primers) depends dramatically on the secondary structure of the template at and around the priming site. We calculated the secondary structure and its effect on priming using a simple model with relatively few parameters which were then optimized to achieve the best match between the predictions and the actual rankings of the sites in terms of priming efficiency. This work introduces an efficient and conceptually novel approach that in the future can make use of more data to optimize a larger set of DNA folding parameters in a more refined model. The model we used, however crude it may be, significantly improved the prediction of priming efficiencies of 8mer primers and appreciably raised the success rate of our DNA sequencing technique (from 67 to 91% with a significance of P < 7 x 10(-5)), which uses such primers.
在此，我们分析了DNA折叠对短引物性能的影响，并描述了一种简单技术，用于评估那些决定单链DNA折叠成二级结构的热力学参数的迄今尚未确定的值。使用一种、两种或三种（但绝不是全部四种）可能的脱氧核糖核苷三磷酸（dNTP），在已知模板（M13mp18）上的几个互补位点同时延伸一个具有两个简并位点的8聚体。延伸的长度是位点特异性的，因为它受到下游模板序列中第一个出现的碱基（其互补dNTP不存在）的限制。然后通过比较不同位点在凝胶上的条带亮度来对其相对引发效率进行排序。短引物（与传统的长引物不同）的引发效率极大地取决于引物位点及其周围模板的二级结构。我们使用一个参数相对较少的简单模型计算二级结构及其对引发的影响，然后对这些参数进行优化，以使预测结果与位点在引发效率方面的实际排序达到最佳匹配。这项工作引入了一种高效且概念新颖的方法，未来可以利用更多数据，在更精细的模型中优化更大组的DNA折叠参数。然而，我们使用的模型尽管可能很粗糙，但显著改善了对8聚体引物引发效率的预测，并明显提高了我们使用此类引物的DNA测序技术的成功率（从67%提高到91%，P值小于7×10⁻⁵，具有显著性）。
