A variation of subtractive DNA cloning in which the restriction-nuclease-cleaved tester DNA is biotinylated before annealing with an excess of randomly sheared driver DNA. Annealing is halted at about 90% completion and the single-stranded DNA is separated from double-stranded DNA. As the unique tester fragments are present at lowest concentration, they will be slowest to find complementary strands and anneal and will, therefore, be enriched in the remaining single-stranded DNA fraction. They are isolated on the basis of biotin's affinity for immobilised avidin and subjected to subsequent cycles of purification.