Positional cloning without a genome map: using 'Targeted RFLP Subtraction' to isolate dense markers tightly linked to the regA locus of Volvox carteri

The ability to isolate genes defined by mutant phenotypes has fueled the rapid progress in understanding basic biological mechanisms and the causes of inherited diseases. Positional cloning, a commonly used method for isolating genes corresponding to mutations, is most efficiently applied to the small number of model organisms for which high resolution genetic maps exist. We demonstrate a new and generally applicable positional cloning method that obviates the need for a genetic map. The technique is based on Restriction Fragment Length Polymorphism (RFLP) Subtraction, a method that isolates RFLP markers spanning an entire genome. The new method, Targeted RFLP Subtraction (TRS), isolates markers from a specific region by combining RFLP Subtraction with a phenotypic pooling strategy. We used TRS to directly isolate dense markers tightly linked to the regA gene of the eukaryotic green alga Volvox. As a generally applicable method for saturating a small targeted region with DNA markers, TRS should facilitate gene isolation from diverse organisms and accelerate the process of physically mapping specific regions in preparation for sequence analysis.