Positional Cloning

A genetic disease gene can be identified by three approaches: (1) Functional cloning in which a disease gene is identified based on biological background of a disease and the gene function without knowledge of chromosomal position of the gene, for example, identification of the globin gene mutations responsible for certain forms of anemia. This cloning strategy relies heavily on detailed information of the disease and the gene. However, such information is not available for the majority of single-gene disorders in human. (2) Positional candidate gene cloning is another approach which can be used to identify a disease gene. This technique combines information of chromosomal location of a disease locus and a candidate gene locus. Once the disease and a candidate gene loci are colocalized on the chromosomal region, the gene will then be cloned and sequenced. Recently, we have mapped two genetic mouse disorders, chondrodysplasia (cho ) to α1 of type XI collagen (Col11a1 ) locus on chromosome 3 and disproportionate micromelia (Dmm ) to α1 type II collagen (Col2a1 ) locus on chromosome 15. We have identified the mutation in cho , which is a deletion of a cytosine residue in the coding region of Col11a1 mRNA (1 ). In Dmm , a deletion of three nucleotides in the coding region of Col2a1 mRNA was found (2 ). (3) Positional cloning is an approach by which a disease gene can be cloned by sole information of its physical location on a chromosome. Because high-density chromosome maps of polymorphic markers of human and mouse have been developed and yeast, bacterial, and phase artificial chromosome libraries of human and mouse are available, positional cloning of a disease gene can be accomplished in a reasonable short time span. In 1986, the first human genetic disease gene, chronic granulomatous disease, was identified by positional cloning (3 ). Since then, the number of disease genes cloned by this technique has been dramatically increased. The experimental strategy of the positional cloning includes three basic steps (4 ). The first step is to establish a fine genetic linkage map of a disease gene locus by identifying markers that are close to the gene. Currently, 6580 mouse and 5264 human short tandem repeat polymorphic markers and 797 mouse restriction fragment length polymorphic markers have been assigned to chromosomes and are available (5 ,6 ). This makes it possible to map a gene of interest within 1 cM. The second step is to construct a physical map of the gene locus. Yeast artificial chromosome (YAC) (7 ,8 ), bacterial artificial chromosome (BAC) (9 ,10 ), 9and phage P1-derived artificial chromosome (PAC) (11 -13 ) libraries have been utilized by many laboratories to define a genomic region that contains genes of interest. The third step is to search for the gene in the defined genomic region by the techniques of cDNA selection (14 -16 ), exon trapping (17 -19 ), or GpC island searching (20 ). Although the protocols described in this chapter have been developed for the mouse, they could also be adapted to other species, such as rat, zebrafish, and even humans with the exception of the breeding plan.