A Simple and Convenient Sticky/Blunt-End Ligation Method for Fusion Gene Construction

作者：Gao, Song; Zhang, Jiannan; Miao, Tianjin; Ma, Di; Su, Ying; An, Yingfeng^*; Zhang, Qingrui

来源：Biochemical Genetics, 2015, 53(1-3): 42-48.

DOI：10.1007/s10528-015-9669-x

• Overlap extension PCR
• Chimeric gene library
• Fusion gene library
• T4 polynucleotide kinase
• Directed evolution

Here we present a simple and convenient sticky/blunt-end ligation method for fusion gene construction. The fusion gene is constructed by seamless ligation of 5'-end phosphorylated blunt ends instead of by overlap extension PCR (OE-PCR). Therefore, the challenge of amplifying large DNA fragments (e.g., the large bifunctional enzyme gene constructed by fusion of two monofunctional enzyme genes) by PCR can be avoided. In addition, synthesis of the inner primers for OE-PCR is not necessary, indicating that this method should be especially convenient for construction of fusion genes with various combinations of multiple fragments (e.g., chimeric gene libraries, fusion gene libraries). As a modification of the commonly used fusion gene construction technique, this method may find a wide range of applications in bioscience and biotechnology.

• 出版日期2015-4
• 单位青岛科技大学; 沈阳农业大学