Ustar Biotechnologies (Hangzhou), Ltd., Hangzhou, Zhejiang, 310012, China.
Sci Rep. 2012;2:246. doi: 10.1038/srep00246. Epub 2012 Feb 3.
CPA is a class of isothermal amplification reactions that is carried out by a strand displacement DNA polymerase and does not require an initial denaturation step or the addition of a nicking enzyme. At the assay temperature of 63°C, the formation of a primer-template hybrid at transient, spontaneous denaturation bubbles in the DNA template is favored over re-annealing of the template strands by the high concentration of primer relative to template DNA. Strand displacement is encouraged by the annealing of cross primers with 5' ends that are not complementary to the template strand and the binding of a displacement primer upstream of the crossing primer. The resulting exponential amplification of target DNA is highly specific and highly sensitive, producing amplicons from as few as four bacterial cells. Here we report on the basic CPA mechanism - single crossing CPA - and provide details on alternative mechanisms.
CPA 是一类等温扩增反应，由链置换 DNA 聚合酶进行，不需要初始变性步骤或添加切口酶。在 63°C 的检测温度下，与模板链的重新退火相比，在 DNA 模板中短暂的、自发的变性泡中形成引物-模板杂交体更有利于高浓度的引物相对于模板 DNA。通过退火与模板链不互补的 5' 端的交叉引物以及在交叉引物上游结合置换引物，鼓励链置换。目标 DNA 的指数扩增具有高度特异性和高度灵敏性，可从少至四个细菌细胞中产生扩增子。在这里，我们报告了基本的 CPA 机制——单交叉 CPA，并提供了替代机制的详细信息。
