Site Directed Mutagenesis and other Recombinant Manipulations

Gene Synthesis

CBI has applied sophisticated technologies to construct completely synthetic “designer genes” expressing the exact desired protein. Genes are synthesized by combining overlapping complementary oligonucleotides, filling in the gaps with a thermostable nucleotide polymerase, and amplifying the product by PCR. These amplified products are cloned into the desired plasmid vector and overexpressed. Genes up to ~2.0 kbp have been synthesized, but there is no theoretical limit to the size.

Synthetic “designer genes” normally have unique DNA sequences that have been modified to optimize the codon usage for the target host. In addition, restriction sites, leader sequences, homing signals, or other modified sequences can be incorporated for special applications.

Site Directed Mutagenesis

CBI normally performs site-directed mutagenesis using modifications of a mega-primer PCR protocol. Using this technology, substitutions of 1-10 bp can easily be generated. These substitutions are useful for any site-directed mutagenesis project, and have been often applied for alanine-scanning type of protein functional probing or “linker-scanning” analyses. Clones can be verified by sequence analysis.

Please call 800-735-9224 or e-mail for additional information or a quotation.