Efficient One-Step Fusion PCR Based on Dual-Asymmetric Primers and Two-Step Annealing.
Mol Biotechnol
; 60(2): 92-99, 2018 Feb.
Article
em En
| MEDLINE
| ID: mdl-29230626
ABSTRACT
Gene splicing by fusion PCR is a versatile and widely used methodology, especially in synthetic biology. We here describe a rapid method for splicing two fragments by one-round fusion PCR with a dual-asymmetric primers and two-step annealing (ODT) method. During the process, the asymmetric intermediate fragments were generated in the early stage. Thereafter, they were hybridized in the subsequent cycles to serve as template for the target full-length product. The process parameters such as primer ratio, elongation temperature and cycle numbers were optimized. In addition, the fusion products produced with this method were successfully applied in seamless genome editing. The fusion of two fragments by this method takes less than 0.5 day. The method is expected to facilitate various kinds of complex genetic engineering projects with enhanced efficiency.
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Base de dados:
MEDLINE
Assunto principal:
Proteínas Recombinantes de Fusão
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Reação em Cadeia da Polimerase
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Primers do DNA
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Escherichia coli
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Hibridização de Ácido Nucleico
Idioma:
En
Ano de publicação:
2018
Tipo de documento:
Article