Complementary annealing mediated by exonuclease: a method for seamless cloning and conditioning site-directed mutagenesis

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...

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Detalles Bibliográficos
Autores principales: Sun, Shuhui, Huang, Hao, Qi, Yingchuan Billy, Qiu, Mengsheng, Dai, Zhong-Min
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Taylor & Francis 2015
Materias:
Article; Medical Biotechnology
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4686905/
https://www.ncbi.nlm.nih.gov/pubmed/26740790
http://dx.doi.org/10.1080/13102818.2014.988094
Descripción
Sumario: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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