Over the last two decades, isothermal amplification of nucleic acids has gained more attention due to a number of advantages over the widely used polymerase chain reaction. For isothermal amplification, DNA polymerases with strand-displacement activity are needed, and Bst exo- polymerase is one of the most commonly used. Unfortunately, Bst exo- causes nonspecific DNA amplification (so-called multimerization) under isothermal conditions that results in undesirable products (multimers) consisting of tandem nucleotide repeats. Multimerization occurs only for short ssDNA or primer dimers, and the efficiency of multimerization depends significantly on the reaction conditions, but slightly depends on the sequence of DNA templates. In this study we report the prevention of DNA multimerization using a new type of modified oligonucleotide primers with internucleosidic phosphates containing 1,3-dimethyl-2-imino-imidazolidine moieties (phosphoryl guanidine (PG) groups). Primers with one, two or three PG groups located at the 3'- or 5'-ends or in the middle of the primers were designed. It turned out, such bulky groups interfere with the moving of Bst exo- polymerase along DNA chains. However, one modified phosphate does not notably affect the efficiency of polymerization, and the elongation is completely inhibited only when three contiguous modifications occur. Multimerization of the linear ssDNA templates is blocked by three modifications in the middle of both primers whereas specific amplification of the circular ssDNA by rolling circle amplification is not inhibited. Thus, incorporation of three PG groups is sufficient to prevent multimerization and allows to create improved primers for reliable isothermal amplification with Bst exo- DNA polymerase.

中文翻译：

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在用Bst exo-DNA聚合酶等温扩增过程中，使用磷酰胍引物防止DNA多聚。

在过去的二十年中，核酸的等温扩增由于与广泛使用的聚合酶链反应相比具有许多优势而受到更多关注。对于等温扩增，需要具有链置换活性的DNA聚合酶，而Bst外切聚合酶是最常用的酶之一。不幸的是，Bst外显子在等温条件下引起非特异性DNA扩增（所谓的多聚），从而导致由串联核苷酸重复序列组成的不良产物（多聚体）。多聚仅发生在短的ssDNA或引物二聚体上，多聚的效率在很大程度上取决于反应条件，但在一定程度上取决于DNA模板的序列。在这项研究中，我们报告了一种新型修饰寡核苷酸引物与含有1,3-二甲基-2-亚氨基亚氨基咪唑烷部分（磷酰基胍（PG）基团）的核苷间磷酸酯的作用，以防止DNA多聚化。设计具有在3'或5'末端或在引物中间的一个，两个或三个PG基团的引物。事实证明，这种庞大的基团干扰了Bst外聚合酶沿DNA链的移动。但是，一种改性的磷酸盐并不显着影响聚合效率，并且仅当发生三个连续的改性时，才完全抑制了伸长率。线性ssDNA模板的多聚化被两个引物中间的三个修饰所阻断，而环状ssDNA通过滚环扩增的特异性扩增则不受抑制。