Restriction cutting independent method for cloning genomic DNA segments outside the boundaries of known sequences

We present a simple method for cloning genomic DNA segments outside the bou ndaries of known sequences, which is not dependent on restriction cutting o r mapping. In the first step of the method a library of single-stranded fla nking sequences is generated by linear amplification with one primer in the known region. A homooligomeric cytosine tail is added to each of the singl e-stranded fragments by a terminal transferase catalyzed reaction. The tail ed fragments are then amplified by PCR with a nested primer in the known re gion and a poly-guanine primer complementary to the cytosine tail in the un known region. Finally the different fragments are separated by cloning and characterized by sequencing. The method was used to clone both the upstream (5') and the downstream (3') genomic regions of an intron-interrupted tRNA (Leu)(UAA) gene from three cyanobacteria belonging to the genus Microcystis .