[DNA assembly by multi-fragment digestion/ligation and homologous recombination].
Sheng Wu Gong Cheng Xue Bao
; 40(5): 1559-1570, 2024 May 25.
Article
en Zh
| MEDLINE
| ID: mdl-38783816
ABSTRACT
To develop an accurate and efficient protocol for multi-fragment assembly and multi-site mutagenesis, we integrated and optimized the common multi-fragment assembly methods and validated the established method by using fructose-1,6-diphosphatase 1 (FBP1) with 4 mutant sites. The fragments containing mutations were assembled by introducing mutant sites and Bsa I recognition sequences. After digestion/ligation, the ligated fragment was amplified with the primers containing overlap region to the linearized vector. The amplified fragment was ligated to the linearized vector and the ligation product was transformed into Escherichia coli. After screening and sequencing, the recombinant plasmid with 4 mutant sites was obtained. This protocol overcame the major defects of Gibson assembly and Golden Gate assembly, serving as an efficient solution for multi-fragment assembly and multi-site mutagenesis.
Palabras clave
Texto completo:
1
Base de datos:
MEDLINE
Asunto principal:
Fructosa-Bifosfatasa
/
Escherichia coli
/
Recombinación Homóloga
Idioma:
Zh
Revista:
Sheng Wu Gong Cheng Xue Bao
Asunto de la revista:
BIOTECNOLOGIA
Año:
2024
Tipo del documento:
Article
País de afiliación:
China