title: Complementary annealing mediated by exonuclease: a method for seamless cloning and conditioning site-directed mutagenesis.
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.
authors.affiliation: Institute of Developmental and Regenerative Biology, College of Life and Environmental Sciences, Hangzhou Normal University , #16 Xuelin Street, Hangzhou , Zhejiang 310036 , China.
authors.forename: Shuhui
authors.initials: S
authors.lastname: Sun
authors.affiliation: Institute of Developmental and Regenerative Biology, College of Life and Environmental Sciences, Hangzhou Normal University , #16 Xuelin Street, Hangzhou , Zhejiang 310036 , China.
authors.forename: Hao
authors.initials: H
authors.lastname: Huang
authors.affiliation: Institute of Developmental and Regenerative Biology, College of Life and Environmental Sciences, Hangzhou Normal University , #16 Xuelin Street, Hangzhou , Zhejiang 310036 , China.
authors.forename: Yingchuan Billy
authors.initials: YB
authors.lastname: Qi
authors.affiliation: Institute of Developmental and Regenerative Biology, College of Life and Environmental Sciences, Hangzhou Normal University, #16 Xuelin Street, Hangzhou, Zhejiang310036, China; Department of Anatomical Sciences and Neurobiology, School of Medicine, University of Louisville, Louisville, Kentucky, KY40292, USA.
authors.forename: Mengsheng
authors.initials: M
authors.lastname: Qiu
authors.affiliation: Institute of Developmental and Regenerative Biology, College of Life and Environmental Sciences, Hangzhou Normal University , #16 Xuelin Street, Hangzhou , Zhejiang 310036 , China.
authors.forename: Zhong-Min
authors.initials: ZM
authors.lastname: Dai
keywords: complementary annealing mediated by exonuclease; ligation-independent cloning; seamless cloning; site-directed mutagenesis