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Updated: Mar 27, 2026

In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity
Published on: March 25, 2020
Complementary annealing mediated by exonuclease: a method for seamless cloning and conditioning site-directed
Shuhui Sun1, Hao Huang1, Yingchuan Billy Qi1
1Institute of Developmental and Regenerative Biology, College of Life and Environmental Sciences, Hangzhou Normal University , #16 Xuelin Street, Hangzhou , Zhejiang 310036 , China.
Abstract:
Traditional cut-paste DNA cloning is often limited by the availability of restriction enzyme sites. Here, we described the complementary annealing mediated by exonuclease (CAME), in which the insert or vector fragment is amplified to carry sequences complementary to the other, and both fragments are modified by exonuleases to create directional single-stranded overhangs. The two recessed DNA fragments are joined through complementary strand annealing. The CAME is highly efficient for cloning the DNA of at least 12 kb and single DNA fragment out of a complex DNA sample. Moreover, the application of CAME greatly improved the efficiency of site-directed mutagenesis.
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