Molecular nature of RAPD markers from Haemophilus influenzae Rd genome

Despite the widespread application of the random amplified polymorphic DNA (RAPD) technique, there is no experimental evidence of the molecular mechan ism of random amplification starting from a complex template. To investigat e this mechanism, we cloned and sequenced 23 selected RAPD bands amplified from Haemophilus influenzae Rd genomic DNA using eight decamer primers diff erent in GC content and/or nucleotide sequence. As the whole genome sequenc e of H. influenzae Rd has been reported, the exact nucleotide sequence of e ach primer-template annealing site was identified. Results showed that, on an average, a homology of eight base pairs was involved in priming events a nd that the number of nonhomologous base pairings declined exponentially fr om the 5' end of the primer to its 3' end. The interaction between the prim er and the template DNA was stabilized by the formation of secondary struct ures, and a perfect match of the 3' terminal region of the primer was not n ecessary for successful amplification. The complexity of the annealing proc ess suggested that, in the studied reaction conditions, many primer-templat e annealing sites were extended in the first cycles and that differences in the efficiency of priming and replication processes led to amplification o f RAPD fragments. Moreover, the distribution of the amplified regions on th e H, influenzae chromosome was analyzed. (C) Elsevier, Paris.