7 Gene Isolation and Analysis of Multiple Mutant Alleles

There are many ways to isolate a DNA fragment containing a specific gene. The methods vary depending on the organism and the genetic and recombinant DNA technology available for working with that particular organism. Moreover, the approach chosen will vary depending on several other factors. These include the phenotype of strains carrying the mutant allele, whether the mutation is dominant or recessive, and whether one has developed a selection or a screen for identifying strains with the wild-type or mutant phenotype. Additionally, cloned genes homologous to the gene of interest or sequence information might be available. Therefore, the particular circumstances will be different for different genes, different mutant alleles, and different organisms. Broadly speaking, there are three basic approaches to cloning a gene. Cloning by complementation uses in vivo functional activity (phenotype) to isolate the dominant allele of a gene. Positional cloning locates a gene to a particular chromosomal region based on information on its map position. Conservation of the sequence of a protein or a particular protein domain, and the implied conservation of gene sequence, can be used to isolate a gene by a method we will refer to as cloning by sequence homology. All three methods require the construction of libraries. The type of library, its size, and the vectors chosen will vary with the organism under investigation and the intended cloning approach.