Henry D. Hunt

Gene splicing by overlap extension

Publisher Summary Conventional methods of engineering recombinant DNA make use of restriction enzymes to cut molecules apart at specific nucleotide sequences and ligases to rejoin the parts. A significant limitation of this technology is that restriction enzymes are sequence dependent and these recognition sequences appear more or less randomly in DNA. That is, restriction enzymes cut where recognition sites are located and not necessarily at optimal positions along the gene for purposes of genetic engineering. The polymerase chain reaction (PCR) has made possible a sequence-independent engineering method that is referred to as “gene splicing by overlap extension” or “SOE.” This technology is especially useful in complicated constructions that require precise recombination points—such as joining two coding sequences in frame—and it also provides a straightforward way of performing site-directed mutagenesis.

Novel MHC Variants Spliced by Overlap Extension

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.

The Functional Significance of Amino Acid Polymorphisms in Class I MHC Molecules

The x-ray crystal model of the HLA-A2 molecule depicts an antigen presenting domain consisting of two α-helices under which are found eight antiparallel β-strands. Furthermore, the position, orientation, and functional contribution of many amino acid side chains has been proposed (Bjorkman 1987a,b). In general, amino acid residues that point inward/upward from the α-helices or point upward from the β-strand floor are thought to be involved with either peptide binding or T-cell receptor (TcR) interaction. Amino acid side chains that point outward from the α-helices, project downward from the β-strand floor, or are located on loops outside of the antigen recognition site (ARS) are thought to be silent with regard to TcR interaction or peptide binding (Bjorkman 1987b).