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 fr...

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Bibliographic Details
Published in:Protein engineering 1990-01, Vol.3 (3), p.221-226
Main Author: MANDECKI, W
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Base Sequence
Biological and medical sciences
Cloning, Molecular
Deoxyribonuclease HpaII
Deoxyribonucleases, Type II Site-Specific
Diverse techniques
DNA - genetics
DNA Restriction Enzymes
Escherichia coli - genetics
evolution
Fundamental and applied biological sciences. Psychology
Gene Library
gene synthesis
Genetic Vectors
Molecular and cellular biology
Molecular Sequence Data
mutagenesis
oligonucleotide
Peptides - genetics
Plasmids
Promoter Regions, Genetic
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Description
Summary: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 106 clones was constructed.
ISSN:1741-0126
0269-2139
1741-0134
1460-213X
DOI:10.1093/protein/3.3.221