Simple biological assay for the error rate upon cloning of synthetic oligodeoxyribonucleotides.
A method was developed for determination of the rate of undesired point mutations upon cloning of synthetic DNA. The method relies on cloning of an oligonucleotide(s) into the E. coli alkaline phosphatase gene inactivated due to a small deletion within the active site. The oligonucleotide adds back the deleted sequence, but simultaneously introduces a missense mutation at a critical position. The activity of the enzyme is restored only if there is a predefined sequence change within the codon specifying an essential residue of the active site. The clones carrying the reactivated gene are detected by colony color screening on plates. The method is fast and simple, does not require specialized equipment nor enzymatic reactions, although a separate oligonucleotide needs to be provided for each sequence change to be evaluated. The procedure allows for the use of crude extracts of oligonucleotides and distinguishes between different types of sequence changes.
Mandecki
W
W
Abbott Laboratories, Corporate Molecular Biology, Abbott Park, IL 60064-3500.
Shallcross
M A
MA
Kavanaugh
T J
TJ
D000469:Alkaline Phosphatase / Q000235:genetics; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D001681:Biological Assay; D003001:Cloning, Molecular; D005813:Genes, Synthetic; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D009154:Mutation; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics
D001426:Bacterial Proteins; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D000469:Alkaline Phosphatase