Prevention of DNA multimerization using phosphoryl guanidine primers during isothermal amplification with Bst exo- DNA polymerase

Abstract

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.

Keywords: Blocking modifications; Bst exo- DNA polymerase; Isothermal amplification; Multimerization; Phosphoryl guanidine oligonucleotides (PGO); Rolling Circle Amplification.