The study of regions involved in the regulation of gene expression typically involves analysis of nested sets of genetic deletions that enter the region of interest from both upstream and downstream directions. In many cases, however, the region to be studied is too large to be readily analyzed by substituting individual bases. In such cases, it may be preferable to use "linker scanning" mutagenesis to search within the regulatory region and locate sequences that are particularly important. This method was originally developed by McKnight and Kingsbury (1), in order to study the transcriptional control signals of the thymidine kinase (tk) gene of Herpes simplex virus . In preference to mutating separately each of the 50-100 nucleotides that composed the control sequence of interest, they developed the "linker scanning" method, to introduce clustered sets of point mutations at desired locations. In this specific example, a set of point mutations was constructed to scan systematically across a region of DNA known to harbor the control elements of the tk gene. McKnight and Kingsbury then tested the retention of transcriptional competence using a microinjection assay in individual cells and were able to identify three distinct regions upstream of the tk gene that are required for in vivo gene expression.
More recent refinements of this technique have been described in some detail (2-4). The most commonly used method requires a unique restriction site adjacent to the region being mutagenized, and it uses complementary oligonucleotides. The sequence of interest is initially cloned into a plasmid vector. The plasmid is linearized, and a nested series of 5′ and 3′ deletion mutations is created using restriction enzymes. The fragments generated are ligated together with complementary oligonucleotides, filling in the gap between the sequence of interest and the nearby restriction site. A series of these mutants is created in order to scan the site of interest. An alternative procedure utilizes site-directed mutagenesis procedures to introduce smaller clusters of point mutations throughout the region being studied (2).
