Mechanism of DNA multimerization caused by strand-displacement DNA polymerases

Abstract

It has been recently shown that for Bst DNA polymerase, the side isothermal amplification reaction named multimerization (MM) proceeds under certain conditions. MM hinders interpretation of amplification results and reduces the accuracy and reliability of DNA/RNA diagnostics. Here, the mechanism of MM caused by strand-displacement DNA polymerases is reported. The mechanism includes the following key stages: 1) envelopment of the enzyme globule by the synthesized DNA strand, facilitated by DNA breathing, 2) convergence of the 3′-ends of the DNA strands and pseudo-cyclic trigger DNA structure formation, 3) synthesis of the products with repeated motifs resulting in their expansion due to DNA slippage. Initiation of MM reaction occurs with extremely low probability, however, the resulting few trigger DNA structures are efficiently amplified and ultimately lead to the accumulation of nonspecific amplicons (multimers). Molecular models with certain steric and thermodynamic characteristics were used to confirm the proposed mechanism. The highest MM efficiency was observed for DNA templates and reaction conditions that facilitated DNA breathing, complete envelopment of the enzyme globule with DNA strands and convergence of their 3′-ends.