Sequence Independent Base-excision Method using Engineered Nucleotide for Synthetic Biology

Abstract

Restriction endonuclease(RE) is an enzyme to make cleavage site within DNA according to its recognition site. It is used to separate wanted gene fragment. However, it is difficult to cut nucleotide on demand. As RE can recognize 4 to 6 bases length of recognition site, there might be redundancy of recognition site in target DNA strand. Moreover, most of RE treatment requires additional enzyme treatment before applying to DNA ligation or molecular cloning. It is burdensome for users to be aware of enzyme recognition site and having to have to avoid repetitive recognition sites in target DNA fragment. Here, I showed that one-base precision restriction mechanism by incorporating i unnatural base pair as a recognition site and endonuclease V which could preferentially cut unnatural base pair as a restriction enzyme. Deaminated adenosine, inosine was inserted into primer site. Amplified DNA strands, using designed primer, were targeted by specific endonuclease. It works well as a universal cleavage method. This mechanism reduces cost and time-consuming. This novel method can be used in the field of molecular and synthetic biology. This sequence independent cleavage method requires primers containing deaminated adenosine during Polymerase chain reaction(PCR) and endonuclease V recognizing deaminated adenosine and nicking around it. This approach can facilitate removal of designated site. We can obtain DNA that consists of extra treatment of enzyme with a blunt end, which would be assembled into long gene structure or applied to diverse DNA fragments. Therefore, this method could improve oligo retrieval system at array-based synthetic technology which used Type IIs restriction endonuclease, sequence dependent cleavage system. This universal invented cleavage method can reduce labor and cost such in high-throughput scale experiment.