Open AccessRandom Mutagenesis by Whole-Plasmid PCR Amplification
Author(s) -
Asit Parikh,
F. Peter Guengerich
Publication year - 1998
Publication title -
biotechniques/biotechniques
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.617
H-Index - 131
eISSN - 1940-9818
pISSN - 0736-6205
DOI - 10.2144/98243st01
Subject(s) - mutagenesis , plasmid , biology , polymerase chain reaction , genetics , multiple displacement amplification , microbiology and biotechnology , computational biology , dna , mutation , gene , dna extraction
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 DpnI 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.