Creating random mutagenesis libraries using megaprimer PCR of whole plasmid.
The conventional method for cloning a DNA fragment is to insert it into a vector and ligate it. Although this method is commonly used, it is labor intensive because the ratio and concentrations of the DNA insert and the vector need optimizing. Even then, the resultant library is often plagued with unwanted plasmids that have no inserts or multiple inserts. These species have to be eradicated to avoid tedious screening, especially when producing a mutant gene library. To overcome these problems, we modified the QuikChange protocol so that each plasmid carries a single insert. Although the QuikChange was originally developed for site-directed mutagenesis using complementary mutagenic oligonucleotide primers in whole plasmid PCR, we found that the protocol also worked for megaprimers consisting of hundreds of nucleotides. Based on this discovery, we used insert fragments, which we wanted to clone, as the primers in the QuikChange reaction. The resultant libraries were virtually free from species with no inserts or multiple inserts. The present method, which we designated MEGAWHOP (megaprimer PCR of whole plasmid), is thus ideal for creating random mutagenesis megalibraries.
Miyazaki
Kentaro
K
Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki, Japan. miyazaki-kentaro@aist.go.jp
Takenouchi
Misa
M
D003001:Cloning, Molecular / Q000379:methods*; D017931:DNA Primers; D004274:DNA, Recombinant / Q000235:genetics* / Q000302:isolation & purification; D004259:DNA-Directed DNA Polymerase; D015723:Gene Library; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics; D016296:Mutagenesis; D016133:Polymerase Chain Reaction / Q000379:methods*; D011897:Random Allocation; D019914:Taq Polymerase; D013698:Templates, Genetic
D017931:DNA Primers; D004274:DNA, Recombinant; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; C072146:Pfu DNA polymerase; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase