Cassette hybridization for vector assembly application in antibody chain shuffling

Abstract

Gene assembly methods are an integral part of molecular cloning experiments. The majority of existing vector assembly methods stipulate a need for exonucleases, endonucleases and/or the use of single-stranded DNA as starting materials. Here, we introduced a vector assembly method that employs conventional PCR to amplify stable double-stranded DNA fragments and assembles them into functional vectors specifically for antibody chain shuffling. We successfully formed vectors using cassettes amplified from different templates and assembled an array of single chain fragment variable clones of fixed variable heavy chain, with different variable light chains – a chain shuffling process for antibody maturation. The method provides an easy alternative to the conventional cloning process.

Keywords

• antibody chain shuffling
• DNA assembly
• homologous assembly
• PCR-based cloning
• scFv