Construction of a cDNA expression library in a binary vector using a nicking enzyme

摘要

Ligation-independent cloning (LIC), such as Gibson Assembly, tends to produce clones without an insert, depending on the sequences present at the ends of linearized vectors. We used a nicking enzyme-mediated LIC (NE-LIC) method to construct a cDNA library in a binary vector pER8. Prior to constructing the cDNA library, pilot experiments were carried out, in which the GUS coding sequence was cloned into pER8 using NE-LIC. Approximately 12% of input vector DNAs were converted to plasmids carrying a GUS insert, and no plasmids without an insert were detected, indicating that this strategy is highly effective for cloning with the binary vector pER8. Therefore, NE-LIC was adopted to construct a cDNA library in pER8, by using cDNA that was PCR-amplified from a library constructed in another vector. As a result, a cDNA library in pER8 was successfully constructed. During library construction, it is important to exclude plasmids without an insert, since contamination from plasmids without inserts decreases the efficiency of screening. Therefore, NE-LIC is useful for the construction of cDNA libraries.