Disease genes can sometimes be isolated with the aid of positional cloning, a process also known as reverse genetics (Collins 1995). Positional cloning is the process used to identify a disease gene, based only on knowledge of its chromosomal location, without any knowledge of the biological function of the gene. Positional cloning requires a genetic map of unique DNA segments or genes (genetic markers), with known chromosomal location, that exist in several alternative forms (alleles). These allelic variations (polymorphisms) allow comparisons of the wild type as the "diseased" genotype.
Linkage analysis is a method of localizing one or more genes influencing a trait to specific chromosomal regions. This is performed by examining the cosegregation of the phenotype of interest with genetic markers. Relatives who are phenotypically alike will share common alleles at markers surrounding the genes influencing the phenotype, whereas other relatives (i.e., those who are phenotypically dissimilar) will not carry these alleles. To carry out linkage analyses, investigators need, minimally, a set of families in which phenotyped individuals have known relationships to one another and the genotypes of these individuals, including one or more genetic markers.
Once the chromosomal location of the disease gene has been ascertained, the area of chromosomal DNA can be cloned. Until recently, the process of positional cloning involved laborious efforts to build a physical map and to sequence the region. (The sequencing of the human genome has obviated this step.) Physical maps can be produced by isolating and linking together yeast and/or bacterial artificial chromosomes containing segments of human DNA from the region. These fragments are then sequenced and ordered, and from these data, the genomic DNA sequence for the region of the candidate gene region is determined.
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