Linker tailing: unphosphorylated linker oligonucleotides for joining DNA termini

Abstract

Current procedures for inserting linker oligonucleotides between DNA termini involve terminal addition of linker duplexes followed by restriction enzyme cleavage and religation . Such methods have the disadvantage that target DNA molecules containing internal recognition sites are cut during the linker cleavage step. We describe a method for adding linker oligonucleotides to DNA termini which eliminates the requirement for subsequent restriction enzyme treatment. Nonphosphorylated linker duplexes are ligated to target DNA termini on one strand alone. Removal of the unligated strand generates a single-stranded protrusion at each terminus. Two such tailed termini may now be linked by annealing their single-stranded complementary tails, resulting in a hybrid DNA molecule containing a single linker insertion. Each step of the procedure has been studied to optimize the recovery of recombinant molecules, and we present examples of how the method may be applied.