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United States Department of Agriculture

Agricultural Research Service

Title: Use of Class Iis Restriction Enzymes for Site-Directed Mutagenesis: Variations on Phoenix Mutagenesis

item Hirschi, Kendal
item Shigaki, Toshiro - BAYLOR COLLEGE MED

Submitted to: Analytical Biochemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: April 4, 2001
Publication Date: September 28, 2001

Interpretive Summary: In this laboratory report, we briefly describe an innovative improvement on a technique that researchers use to make so-called designer genes. This improved approach is faster and more convenient than the standard protocols, and it is also inexpensive and highly accurate. In coming up with our discovery, we utilized a technique to amplify DNA, and produced a variation of this method to introduce changes in the DNA. Our benchmark report will facilitate scientists' work in many respects with regard to expediting future genetic work oriented toward specific enhancements in human health and the alleviation of human suffering due to disease states or adverse conditions.

Technical Abstract: We have developed a new, easier-to-use variation of PCR-based site- directed mutagenesis. This highly accurate approach takes advantage of the property of Class IIS restriction enzymes, whose cleavage sites are often separated from the recognition sites. When a recognition site of a Class IIS restriction enzyme is included at the 5' end of a mutagenic PCR primer, ,the recognition sequence is cleaved off, leaving a cohesive end. The othe primer can be designed so that 1) it creates a compatible end when digested with the same enzyme, and 2) it recreates the coding sequence adjacent to the altered bases. This strategy offers a more reliable and easier mode of site-directed mutagenesis than conventional protocols, which involve blunt- end ligation or circular amplification of a plasmid.

Last Modified: 4/22/2015
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