Abstract
A quick in vitro mutagenesis method for the construction of nested deletion libraries was developed. Many deletions can be obtained in a single inverse PCR (IPCR) by replacing one of the two primers with a mixture of 5'-truncated oligodeoxynucleotides. Since chemical DNA synthesis proceeds from the 3'to the 5'end, such a mixture of 5'-truncated oligodeoxynucleotides can easily be obtained in a single automated DNA synthesis under reduced coupling efficiency. This deletion mutagenesis method yields many different deletions in a defined short DNA segment and is, therefore, best suited for a deletion analysis at base pair level. Applications might include functional analysis of regulatory DNA segments and protein engineering work that requires libraries for the expression of N-terminal, C-terminal or internal truncated proteins as well as fusion proteins having different splice sites.
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Selected References
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- Barnes W. M. PCR amplification of up to 35-kb DNA with high fidelity and high yield from lambda bacteriophage templates. Proc Natl Acad Sci U S A. 1994 Mar 15;91(6):2216–2220. doi: 10.1073/pnas.91.6.2216. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Imai Y., Matsushima Y., Sugimura T., Terada M. A simple and rapid method for generating a deletion by PCR. Nucleic Acids Res. 1991 May 25;19(10):2785–2785. doi: 10.1093/nar/19.10.2785. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Langridge J., Langridge P., Bergquist P. L. Extraction of nucleic acids from agarose gels. Anal Biochem. 1980 Apr;103(2):264–271. doi: 10.1016/0003-2697(80)90266-3. [DOI] [PubMed] [Google Scholar]
- Ogel Z. B., McPherson M. J. Efficient deletion mutagenesis by PCR. Protein Eng. 1992 Jul;5(5):467–468. doi: 10.1093/protein/5.5.467. [DOI] [PubMed] [Google Scholar]
- Wang K., Koop B. F., Hood L. A simple method using T4 DNA polymerase to clone polymerase chain reaction products. Biotechniques. 1994 Aug;17(2):236–238. [PubMed] [Google Scholar]