Influence of Nucleotide Context on Non-Specific Amplification of DNA with Bst exo – DNA Polymerase

In this study, the effect of nucleotide sequence on the Bst exo– binding with DNA and on the efficiency of multimerization initiation, are reported. Preference for binding of the “closed” form of Bst exo– to the purine-rich DNA sequences, especially those containing dG at the 3 ′-end of the growing chain was revealed using molecular docking of the single-stranded trinucleotides (sst) and trinucleotide duplexes (dst). The data obtainedin  silico were confirmed in the experiments using oligonucleotide templates that differ in the structure of the 3 ′- and 5′-terminal motifs. It has been shown that templates with the oligopurine 3′-terminal fragment and oligopyrimidine 5′-terminal part contribute to the earlier start of multimerization. The results can be used for design of nucleotide sequences suitable for reliable isothermal amplifica tion. To avoid multimerization, DNA templates and primers containing terminal dA and/or dG nucleotides should be excluded.

Source: Biochemistry (Moscow) - January 1, 2024 Category: Biochemistry Source Type: research