Linker scanning mutagenesis

Abstract

Abstract Techniques for in vitro mutagenesis of DNA play a major role in molecular biology. They are powerful tools in dissecting the relationship between structure and function of a given gene. A typical analysis of gene function proceeds in several steps with an increasing degree of resolution. Deletion mutations are usually analysed first; once a functionally important region has been delimited by deletion mapping, this site can be characterized in more detail by linker scanning mutagenesis and by single point mutations introduced by methods described elsewhere in this book. In linker scanning (LS) mutations a short segment of the DNA of interest is replaced by a new synthetic sequence without inserting or deleting base-pairs, thereby introducing a cluster of point mutations at the replacement site. Generation of a series of mutations in which the replacement site is systematically moved, allows scanning of the DNA of interest to identify regions which are critical for the activity of a gene product or in regulating gene expression. LS mutations give a relatively high degree of resolution, while the number of mutations required to perform a systematic analysis of a region of interest is reduced, compared to single base substitutions. In contrast to deletions, LS mutations do not alter the original distances between different DNA sequences. This makes the unequivocal interpretation of experimental results much easier.