US Patent 5,470,722
United States Patent and Trademark Office
University of Iowa Research Foundation (Oakdale, IA, US)
A method that permits the rapid amplification of unknown DNA that flanks a known site, such that one can walk into an uncharacterized region of DNA. In this method, human genomic DNA is restriction enzyme digested and then ligated to a 5' phosphorylated-oligonucleotide so that the 5' end of each strand of genomic DNA is extended and phosphorylated. The phosphorylated-oligonucleotide is constructed to render 5' end extensions that are complementary to the known sequence. Following denaturation and re-annealing under dilute conditions that promote intrastrand annealing and under high stringency, only those DNA strands containing the known sequence will form a stem-loop structure with a recessed and phosphorylated 5' end, rendering a substrate for a subsequent heat-stable ligation reaction to another oligonucleotide. This second oligonucleotide is complementary to the sequence immediately adjacent to the phosphorylated-oligonucleotide high stringency annealing site. The heat-stable ligation reaction appends a known sequence to the DNA segments containing the two known contiguous DNA sequences used for oligonucleotide annealing. This heat-stable ligation of known sequence permits the subsequent highly specific amplification of the unknown flanking DNA.
Jones, Douglas H. Method for the amplification of unknown flanking DNA sequence. US Patent 5,470,722 filed May 06, 1993 and issued November 28, 1995. https://ir.uiowa.edu/patents/143