A method for the amplification of unknown flanking DNA: targeted inverted repeat amplification

Abstract

A new method has been developed that permits the rapid amplification of unknown DNA flanking a known site so that one can walk into an uncharacterized region of DNA. This method eliminates the steps and sequence artifacts associated with cloning and permits genome walking into unclonable regions of DNA. In this method, human genomic DNA is restriction enzyme digested and then ligated to the 3' end of a 5'-phosphorylated oligonucleotide using a short bridging oligonucleotide using a short bridging oligonucleotide as a splint. The phosphorylated oligonucleotide is designed to create 5'-end extensions that are complementary to the known sequence. Following denaturation and reannealing under dilute conditions that promote intra-strand annealing and under high stringency, only those DNA strands that contain the known sequence will form a stem-loop structure with a recessed and phosphorylated 5' end. This stem-loop renders a substrate for a subsequent heat-stable ligation reaction to another oligonucleotide that anneals to the known sequence immediately adjacent to the phosphorylated oligonucleotide high-stringency annealing site. The oligonucleotide appended to the phosphorylated oligonucleotide by the heat-stable ligase can, when present in its free, non-ligated form, prime DNA polymerase-mediated amplification of those strands modified by site-specific ligation to this same oligonucleotide. This is followed by one or two nested DNA amplifications, with the final amplification primed by the phosphorylated oligonucleotide in its free, non-ligated form. We successfully applied this method to the specific amplification of 2.2 kb of DNA flanking the 5' end of the cystic fibrosis transmembrane conductance regulator cDNA using primers that anneal to the cDNA sequence and to the specific amplification of 2.2 kb of human genomic beta-globin DNA flanking the primer annealing sites.