Mapping of the 150 kb spoIIIC-pheA region of the Bacillus subtilis chromosome using Long Accurate PCR and three yeast artificial chromosomes.

We constructed a PCR map of the 150 kb spoIIIC-pheA region of the Bacillus subtilis chromosome. It was established using known sequences of the spoIIIC, blt, aadK, sacC, spoVB and pheA loci and eight random sequence tags. The tags were generated using PFGE-purified DNA of yeast artificial chromosome (YAC) 11-17 from the yeast clone which carries the major part of this region. The ends of two other YACs were positioned on the map using total DNA extracted from yeast cells carrying them. The procedure allowed the placement of precisely known and new (putative) genes on the physical chromosome map and the generation of sufficient amounts of DNA for sequencing this region. Apart from allowing correction of the genetic map in this region, these results demonstrate how a collection of long segments of bacterial chromosome and Long Accurate PCR can be used for reliable high-resolution physical mapping of an extended chromosome area.