Once one or more loci have been identified through a genomic screen as possibly containing a gene of interest, additional techniques are needed to locate the exact gene responsible. Positional cloning is the process of identifying a disease gene based on its location on a chromosome. Genetic screening is the inital step of positional cloning. The usual steps are: (1) linkage (locates a chromosome area); (2) fine mapping (narrows down the initial genomic area to a smaller region); (3) candidate gene analysis (looks for mutations in genes lying in that small area). By using positional cloning, researchers can identify or "clone" a gene knowing only its location on a chromosome, as determined through linkage analysis. Once the location is identified, a physical representation of the genes and DNA in the linked region is constructed.
Before the Human Genome Project was completed, such a physical representation was constructed by using a "contig," a group of overlapping DNA fragments that together cover the linked region. The contig is a scaffold or platform on which to place genes and other sequences in the correct position. The geneticist continues to collect families looking for new recombinations that reduce the piece of DNA that all the affected family members share but that has not been inherited by any of the unaffected individuals.
Positional cloning is a laborious process if the region is large and if the genes and polymorphisms making up the contig are not known. This portion of the process has been greatly helped by the Human Genome Project, as it provides the contig all filled out and correctly mapped.
In most cases, the smallest inherited piece of DNA is still quite large molecularly, and can contain many genes. Only one gene causes the disease, though, so each gene must be tested for mutations that segregate with the trait. This can be very time consuming as well. If no mutations are found, the process is repeated with the next gene in the smallest shared region of DNA.
The third gene identified through positional cloning was the cystic fibrosis transmembrane conductance regulator gene. The gene, identified in 1989, regulates chloride ion transport across the plasma membrane and consists of twenty-seven exons spread over a 230-kilobase-pair region on chromosome seven. This gene was difficult to clone because of the lack of chromosome abnormalities that would have helped locate it. No human genome sequence was available at that time. Numerous technical problems also arose in constructing a physical map of the linkage region, creating the need to screen numerous libraries to obtain a full-length clone of the gene. More than 550 mutations were identified by late 2001.