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. 1998 Apr 1;26(7):1812–1818. doi: 10.1093/nar/26.7.1812

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

J Corrette-Bennett 1, M Rosenberg 1, M Przybylska 1, E Ananiev 1, D Straus 1
PMCID: PMC147462  PMID: 9512557

Abstract

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.

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Selected References

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