Raja M C, Zevin-Sonkin D, Shwartzburd J, Rozovskaya T A, Sobolev I A, Chertkov O, Ramanathan V, Lvovsky L, Ulanovsky L E
Center for Mechanistic Biology and Biotechnology, Argonne National Laboratory IL 60439-4833, USA.
Nucleic Acids Res. 1997 Feb 15;25(4):800-5. doi: 10.1093/nar/25.4.800.
Here we describe template directed enzymatic synthesis of unique primers, avoiding the chemical synthesis step in primer walking. We have termed this conceptually new technique DENS (differential extension with nucleotide subsets). DENS works by selectively extending a short primer, making it a long one at the intended site only. The procedure starts with a limited initial extension of the primer (at 20-30 degrees C) in the presence of only two out of the four possible dNTPs. The primer is extended by 6-9 bases or longer at the intended priming site, which is deliberately selected, (as is the two-dNTP set), to maximize the extension length. The subsequent termination reaction at 60-65 degrees C then accepts the extended primer at the intended site, but not at alternative sites, where the initial extension (if any) is generally much shorter. DENS allows the use of primers as long as 8mers (degenerate in two positions) which prime much more strongly than modular primers involving 5-7mers and which (unlike the latter) can be used with thermostable polymerases, thus allowing cycle-sequencing with dye-terminators compatible with Taq DNA polymerase, as well as making double-stranded DNA sequencing more robust.
在此，我们描述了独特引物的模板导向酶促合成方法，避免了引物步移中的化学合成步骤。我们将这种概念上全新的技术称为DENS（核苷酸亚群差异延伸）。DENS的工作原理是选择性地延伸短引物，使其仅在预期位点成为长引物。该过程首先在仅存在四种可能的dNTP中的两种的情况下，在20 - 30摄氏度对引物进行有限的初始延伸。引物在特意选择的预期引物位点（与二dNTP组一样）延伸6 - 9个碱基或更长，以最大化延伸长度。随后在60 - 65摄氏度的终止反应接受预期位点的延伸引物，但不接受其他位点的引物，在其他位点初始延伸（如果有的话）通常要短得多。DENS允许使用长达8聚体（在两个位置简并）的引物，其引发能力比涉及5 - 7聚体的模块化引物强得多，并且（与后者不同）可与热稳定聚合酶一起使用，从而允许使用与Taq DNA聚合酶兼容的数据终止剂进行循环测序，以及使双链DNA测序更稳健。
