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An efficient one-step site-directed and site-saturation mutagenesis protocol
Open accessAbstract
We have developed a new primer design method based on the QuickChange site-directed mutagenesis protocol, which significantly improves the PCR amplification efficiency. This design method minimizes primer dimerization and ensures the priority of primer-template annealing over primer self-pairing during the PCR. Several different multiple mutations (up to 7 bases) were successfully performed with this partial overlapping primer design in a variety of vectors ranging from 4 to 12 kb in length. In comparison, all attempts failed when using complete-overlapping primer pairs as recommended in the standard QuickChange protocol. Our protocol was further extended to site-saturation mutagenesis by introducing randomized codons. Our data indicated no specific sequence selection during library construction, with the randomized positions resulting in average occurrence of each base in each position. This method should be useful to facilitate the preparation of high-quality site saturation libraries.
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Cited by
Zhang, G., & Tandon, A. (2012) · Scientific Reports · doi:10.1038/srep00415 · Cited by 1,012
Betz, C., Stracka, D., Prescianotto-Baschong, C., Frieden, M., Demaurex, N., & Hall, M. N. (2013) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1302455110 · Cited by 538
Reetz, M. T. (2011) · Angewandte Chemie International Edition · doi:10.1002/anie.201000826 · Cited by 538
A cyclic oligonucleotide signaling pathway in type III CRISPR-Cas systems
Kazlauskiene, M., Kostiuk, G., Venclovas, Č., Tamulaitis, G., & Siksnys, V. (2017) · Science · doi:10.1126/science.aao0100 · Cited by 521
The Crucial Role of Methodology Development in Directed Evolution of Selective Enzymes
Qu, G., Li, A., Acevedo‐Rocha, C. G., Sun, Z., & Reetz, M. T. (2020) · Angewandte Chemie International Edition · doi:10.1002/anie.201901491 · Cited by 473
Metadata
- Authors
- Zheng, L.
- Venue
- Nucleic Acids Research
- Publisher
- Oxford University Press (OUP)
- Published
- 2004-08-03
- Volume
- 32
- Issue
- 14
- Pages
- e115-e115
- ISSN
- 0305-1048 (ISSN-L)
- 1362-4962
- OpenAlex
- W2144175569
- Cited by
- 1,178
- References
- 0
- FWCI
- 4.37
- Type
- journal article
- Language
- en
- Open access
- green
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