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Nucleic Acids Research, Vol 26, Issue 7 1812-1818, Copyright © 1998 by Oxford University Press

ARTICLES

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, M Rosenberg, M Przybylska, E Ananiev and D Straus
Biology Department, Brandeis University, Waltham, MA 02254-9110, USA.

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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J. Li, F. Wang, V. Zabarovska, C. Wahlestedt, and E. R. Zabarovsky
Cloning of polymorphisms (COP): enrichment of polymorphic sequences from complex genomes
Nucleic Acids Res., January 15, 2000; 28(2): e1 - e1.
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