PCR-Free Site-Directed Mutagenesis on Repetitive Sequences Using Single-Stranded DNA-Assisted Double-Stranded DNA Nicking by DNAzymes.

ABSTRACT: Site-directed mutagenesis (SDM) is a key driver for many biochemical investigations and biotechnological applications. Despite decades of development, it is still difficult to perform SDM on some sequences, including highly repetitive ones, because polymerase chain reaction (PCR) is often used as the most effective method for SDM, but results in complicated products with these sequences. To overcome this limitation, we report herein a PCR-free SDM method that uses DNA-cleaving DNAzymes. Since these DNAzymes lack the capability of cleaving double-stranded DNA, we employed single-stranded DNAs as assisting DNAs to open superhelical plasmids, allowing the DNA-cleaving DNAzymes to cleave the plasmid. Such a system, named single-stranded DNA-assisted double-stranded DNA nicking by DNAzymes (DAN

SUBMITTER: Lyu M 

PROVIDER: S-EPMC12416466 | biostudies-literature | 2025 Sep

REPOSITORIES: biostudies-literature