Method for the amplification of unknown flanking dna sequence

Abstract

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.