Positional Cloning: Single-Gene Cardiovascular Disorders

Positional cloning is a comprehensive genetic strategy used to identify a disease-causing gene without any prior knowledge of the pathogenesis or protein defects involved in the disease process. The basic process involves collection of accurately diagnosed patients and their family members, genotyping DNAs with polymorphic DNA markers mapped to specific regions on chromosomes, genetic linkage analysis to determine markers that are in close proximity to the chromosome location of the disease gene and to define the critical region by haplotype analysis, identification and selection of candidate genes residing in the critical region, and, eventually, identification of the disease-causing DNA sequence variants by various methods. Many molecular techniques are utilized in positional cloning. Bioinformatics and computation analysis are significant and indispensable components of such a study, and are detailed elsewhere in this volume. This chapter presents a few basic laboratory protocols for conducting positional cloning: genomic DNA preparation, genotyping polymorphic markers, DNA sequencing, and related procedures that serve on fluorescent-labeled and capillary electrophoresis-based semi-automated genetic analysis systems.