| 摘要 |
<p>The present invention provides a simple and rapid method for site-directed mutagenesis of more than, for example, 10 sites simultaneously with up to 100% efficiency. The method uses two terminal tailed primers, specific for each end of the gene (or DNA sequence) to be mutated, with a unique nucleotide tail each that are simultaneously annealed to template DNA together with a set of mutagenic primers in between. Following synthesis of the mutant strand by primer extension and ligation with, for example, T4 DNA polymerase and ligase, the unique mutant strand-specific tails of the terminal primers are used as anchors to specifically amplify the mutant strand by high-fidelity polymerase chain reaction.</p> |
