A METHOD FOR THE AMPLIFICATION OF UNKNOWN FLANKING DNA - TARGETED INVERTED REPEAT AMPLIFICATION

A new method has been developed that permits the rapid amplification o f unknown DNA flanking a known site so that one can walk into an uncha racterized region of DNA. This method eliminates the steps and sequenc e artifacts associated with cloning and permits genome walking into un clonable regions of DNA. In this method, human genomic DNA is restrict ion enzyme digested and then ligated to the 3' end of a 5-phosphorylat ed oligonucleotide using a short bridging oligonucleotide as a splint. The phosphorylated oligonucleotide is designed to create 5'-end exten sions that are complementary to the known sequence. Following denatura tion and reannealing under dilute conditions that promote intra-strand annealing and under high stringency, only those DNA strands that cont ain the known sequence will form a stem-loop structure with a recessed and phosphorylated 5' end. This stem-loop renders a substrate for a s ubsequent heat-stable ligation reaction to another oligonucleotide tha t anneals to the known sequence immediately adjacent to the phosphoryl ated oligonucleotide high-stringency annealing site. The oligonucleoti de appended to the phosphorylated oligonucleotide by the heat-stable l igase can, when present in its free, non-ligated form, prime DNA polym erase-mediated amplification of those strands modified by site-specifi c ligation to this same oligonucleotide. This is followed by one or tw o nested DNA amplifications, with the final amplification primed by th e phosphorylated oligonucleotide in its free, non-ligated form. We suc cessfully applied this method to the specific amplification of 2.2 kb of DNA flanking the 5' end of the cystic fibrosis transmembrane conduc tance regulator cDNA using primers that anneal to the cDNA sequence an d to the specific amplification of 2.2 kb of human genomic beta-globin DNA flanking the primer annealing sites.