Random mutagenesis by whole-plasmid PCR amplification.
Random mutagenesis has become a powerful means of studying the effects of a large number of permutations of a particular DNA sequence. As a prime example, libraries of randomized cDNA clones, when translated into their corresponding proteins, can be useful in investigating the functional contributions of a mutagenized region to the overall properties of a protein. Existing molecular cloning techniques for random mutagenesis are tedious and frequently plagued with high levels of background from wild-type (nonmutagenized) template. We report a PCR-based method involving amplification of an entire plasmid containing a gene sequence of interest with partially complementary degenerate oligonucleotides for randomization of up to 12 consecutive nucleotide residues. Sequential treatment of the PCR product with Dpn/and a second specific restriction endonuclease and T4 DNA ligase followed by high-efficiency electroporation permits the generation of libraries with very low background. This technique should prove useful for studies on enzyme structure-function relationships as well as for other diverse applications.
Parikh
A
A
Vanderbilt University School of Medicine, Nashville, TN, USA.
Guengerich
F P
FP
D003062:Codon; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics; D017931:DNA Primers / Q000138:chemical synthesis; D015723:Gene Library; D006801:Humans; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA
D003062:Codon; D017931:DNA Primers; D003577:Cytochrome P-450 Enzyme System