Improved plasmid multi-site directed mutagenesis method based on polymerase chain reaction (PCR)
By performing site-directed mutagenesis and PCR primer design on plasmid DNA, simultaneous recombination of functional switching and mutation is achieved, solving the problem of low DpnI enzyme digestion efficiency, improving the efficiency and accuracy of site-directed mutagenesis, and simplifying the screening of mutant plasmids.
Patent Information
- Application Number
- CN202480056288.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Priority Date
- 2024-02-14
- Filing Date
- 2024-08-12
- Publication Date
- 2026-04-10
AI Technical Summary
In existing technologies, DpnI enzymes cleave hemimethylated DNA slowly and with reduced activity, resulting in an increase in wild-type DNA content in the final mixture and affecting the efficiency of site-directed mutagenesis.
By performing site-directed mutagenesis on plasmid DNA and using PCR with primer pairs targeting functional switches and adding mutations, PCR products were isolated and functional switches and mutations were achieved during the recombination stage, avoiding the use of DpnI enzyme digestion. Target cells were directly transformed and wild-type DNA was removed through functional screening.
It effectively reduced the content of wild-type DNA in the final mixture, improved the efficiency and accuracy of site-directed mutagenesis, and simplified the screening process for mutant plasmids.