A method for construction of long randomized open reading frames and polypeptides.
A method is presented for construction of randomized open reading frame sequences (ORFs) and gene libraries containing them. The building blocks for the ORFs were 75 bp long DNA fragments generated by cloning sequences from a single synthetic oligonucleotide preparation by bridge mutagenesis. The fragments had the property that, regardless of their orientation in the ligated product, the ORF of the construct was maintained. The heterogeneity of the ORFs resulted from the random ligation of 2000 different DNA fragments. The randomized ORFs were cloned downstream from the lac promoter in a multicopy plasmid in Escherichia coli. To test the method, a library of 10(6) clones was constructed.
Mandecki
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Corporate Molecular Biology, Abbott Laboratories, Abbott Park, IL 60064.
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics*; D004262:DNA Restriction Enzymes; D019003:Deoxyribonuclease HpaII; D015252:Deoxyribonucleases, Type II Site-Specific; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics*; D010957:Plasmids; D011401:Promoter Regions, Genetic
D010455:Peptides; D004247:DNA; D004262:DNA Restriction Enzymes; D019003:Deoxyribonuclease HpaII; D015252:Deoxyribonucleases, Type II Site-Specific