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 alpha 1 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 alpha 1 exons (270 bp of coding sequence), which contain mutations identified by sequence analysis, can replace the wild type alpha 1 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 alpha 1 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 alpha 1 exon with individual mutant alpha 1 exons, and analysis of mutant molecules expressed on the surface of transfected mouse L cells.
D000595:Amino Acid Sequence; D000818:Animals; D000954:Antigens, Surface / Q000235:genetics / Q000302:isolation & purification; D001483:Base Sequence; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D004274:DNA, Recombinant / Q000138:chemical synthesis / Q000302:isolation & purification*; D006183:H-2 Antigens / Q000235:genetics* / Q000302:isolation & purification; D007739:L Cells; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000235:genetics*; D012150:Polymorphism, Restriction Fragment Length
D000954:Antigens, Surface; D004274:DNA, Recombinant; D006183:H-2 Antigens; D009841:Oligonucleotides