Novel MHC Variants Spliced by Overlap Extension

Abstract

We have developed a new technique, based on the polymerase chain reaction (Ho 1989), called “splicing by overlap extension” (SOE), which simplifies site-directed mutagenesis and allows the introduction of larger systematic changes in gene structure in a manner independent of the distribution of restriction endonuclease sites in the targeted area of the gene (Ho 1989). An added feature of SOE is that it provides a straight-forward strategy for the generation of chimeric genes from unrelated sources, such that our ability to make genetic constructs is limited by knowledge of how protein domains will interact and not by the structure of the genes that encode them (Horton 1989). We have used the SOE strategy to generate novel MHC class I variants designed to investigate the antigen presenting properties of the encoded glycoproteins. Here we present examples of how SOE has been applied in the generation of these constructs and the characterization of their gene products using mAbs.