Critical evaluation of random mutagenesis by error-prone polymerase chain reaction protocols, Escherichia coli mutator strain, and hydroxylamine treatment.
Random mutagenesis methods constitute a valuable protein modification toolbox with applications ranging from protein engineering to directed protein evolution studies. Although a variety of techniques are currently available, the field is lacking studies that would directly compare the performance parameters and operational range of different methods. In this study, we have scrutinized several of the most commonly used random mutagenesis techniques by critically evaluating popular error-prone polymerase chain reaction (PCR) protocols as well as hydroxylamine and a mutator Escherichia coli strain mutagenesis methods. Relative mutation frequencies were analyzed using a reporter plasmid that allowed direct comparison of the methods. Error-prone PCR methods yielded the highest mutation rates and the widest operational ranges, whereas the chemical and biological methods generated a low level of mutations and exhibited a narrow range of operation. The repertoire of transitions versus transversions varied among the methods, suggesting the use of a combination of methods for high-diversity full-scale mutagenesis. Using the parameters defined in this study, the evaluated mutagenesis methods can be used for controlled mutagenesis, where the intended average frequency of induced mutations can be adjusted to a desirable level.
Rasila
Tiina S
TS
Program in Cellular Biotechnology, Institute of Biotechnology, Viikki Biocenter, University of Helsinki, FIN-00014 Helsinki, Finland.
Pajunen
Maria I
MI
Savilahti
Harri
H
D019943:Amino Acid Substitution; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000201:enzymology*; D005787:Gene Frequency; D019811:Hydroxylamine / Q000737:chemistry*; D016296:Mutagenesis; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods*; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D019914:Taq Polymerase / Q000235:genetics / Q000378:metabolism
D004269:DNA, Bacterial; D011994:Recombinant Proteins; D019811:Hydroxylamine; D019914:Taq Polymerase