Random oligonucleotide mutagenesis: Application to a large protein coding sequence of a major histocompatibility complex class I gene, H-2DP

We have used random oligonucleotide mutagenesis (or saturation mutagenesis) to create a library of point mutations in the α1 protein domain of a Major Histocompatibility Complex (MHC) molecule. This protein domain is critical for T cell and B cell recognition. We altered the MHC class I H-2DP gene sequence such that synthetic mutant α1 exons (270 bp of coding sequence), which contain mutations identified by sequence analysis, can replace the wild type α1 exon. The synthetic exons were constructed from twelve overlapping oligonucleotides which contained an average of 1.3 random point mutations per intact exon. DNA sequence analysis of mutant α1 exons has shown a point mutant distribution that fits a Poisson distribution, and thus emphasizes the utility of this mutagenesis technique to "scan" a large protein sequence for important mutations. We report our use of saturation mutagenesis to scan an entire exon of the H-2DP gene, a cassette strategy to replace the wild type α1 exon with individual mutant α1 exons, and analysis of mutant molecules expressed on the surface of trans-fected mouse L cells.