title: Positional cloning without a genome map: using 'Targeted RFLP Subtraction' to isolate dense markers tightly linked to the regA locus of Volvox carteri.
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.
authors.affiliation: Biology Department, Brandeis University, Waltham, MA 02254-9110, USA.
authors.forename: J
authors.initials: J
authors.lastname: Corrette-Bennett
authors.forename: M
authors.initials: M
authors.lastname: Rosenberg
authors.forename: M
authors.initials: M
authors.lastname: Przybylska
authors.forename: E
authors.initials: E
authors.lastname: Ananiev
authors.forename: D
authors.initials: D
authors.lastname: Straus
mesh_terms: D020418:Algal Proteins; D001483:Base Sequence; D015139:Blotting, Southern; D000460:Chlorophyta / Q000235:genetics*; D002874:Chromosome Mapping / Q000379:methods*; D003001:Cloning, Molecular / Q000379:methods; D003433:Crosses, Genetic; D015723:Gene Library; D017343:Genes, Plant; D005819:Genetic Markers; D018745:Genome, Plant; D008957:Models, Genetic; D009838:Oligodeoxyribonucleotides; D012150:Polymorphism, Restriction Fragment Length; D011506:Proteins / Q000235:genetics*
chemical_list: D020418:Algal Proteins; D005819:Genetic Markers; D009838:Oligodeoxyribonucleotides; D011506:Proteins