Institute of Biochemistry and Genetics, Ufa Federal Research Center, Russian Academy of Sciences, 450054, Prosp. Oktyabrya, 71, Ufa, Bashkortostan, Russia.
Institute of Fundamental Medicine and Biology, Kazan Federal University, 420021, Parizhskoy Kommuny Str., 9, Kazan, Tatarstan, Russia.
Anal Biochem. 2021 Sep 1;628:114267. doi: 10.1016/j.ab.2021.114267. Epub 2021 Jun 3.
DNA polymerases with strand-displacement activity allow to amplify nucleic acids under isothermal conditions but often lead to undesirable by-products. Here, we report the increase of specificity of isothermal amplification in the presence of poly (aspartic) acids (pAsp). We hypothesized that side reactions occur due to the binding of the phosphate backbone of synthesized DNA strands with surface amino groups of the polymerase, and weakly acidic polyelectrolytes could shield polymerase molecules from DNA and thereby inhibit nonspecific amplification. Suppression of nonspecific polymerase activity by pAsp was studied on multimerization as a model side reaction. It was found that a low concentration of pAsp (0.01%) provides successful amplification of specific DNA targets. The inhibitory effect of pAsp is due to its polymeric structure since aspartic acid did affect neither specific nor nonspecific amplification. Strongly acidic polyelectrolyte heparin does not possess the same selectivity since it suppresses any DNA synthesis. The applicability of pAsp to prevent nonspecific reactions and reliable detection of the specific target has been demonstrated on the genetic material of SARS-CoV-2 coronavirus using Loop-mediated isothermal amplification.
具有链置换活性的 DNA 聚合酶可以在等温条件下扩增核酸，但通常会导致不理想的副产物。在这里，我们报告了在聚（天冬氨酸）（pAsp）存在下等温扩增特异性的提高。我们假设，由于合成 DNA 链的磷酸骨架与聚合酶表面的氨基结合，导致副反应发生，而弱酸性聚电解质可以使聚合酶分子免受 DNA 的影响，从而抑制非特异性扩增。我们研究了 pAsp 对多聚化的抑制作用，作为一种模型副反应。结果发现，低浓度的 pAsp（0.01%）可成功扩增特定的 DNA 靶标。pAsp 的抑制作用归因于其聚合结构，因为天冬氨酸既不影响特异性扩增，也不影响非特异性扩增。强酸性聚电解质肝素不具有相同的选择性，因为它抑制任何 DNA 合成。使用环介导等温扩增，已经证明了 pAsp 可用于防止非特异性反应并可靠检测特定靶标，该方法已经应用于 SARS-CoV-2 冠状病毒的遗传物质中。
