Restriction Cutting Independent Method for Cloning Genomic DNA Segments Outside the Boundaries of Known Sequences

Abstract

We present a simple method for cloning genomic DNA segments outside the boundaries of known sequences, which is not dependent on restriction cutting or mapping. In the first step of the method, a library of single-stranded flanking sequences is generated by linear amplification with one primer in the known region. A homooligomeric cytosine tail is added to each of the single-stranded fragments by a terminal transferase catalyzed reaction. The tailed fragments are then amplified by PCR with a nested primer in the known region and a poly-guanine primer complementary to the cytosine tail in the unknown 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 tRNALeu(UAA) gene from three cyanobacteria belonging to the genus Microcystis.