The elucidation of DNA and protein sequences in recent years has led to the identification of many genes, using sequence patterns in genomic DNA and the sequence similarity of the encoded proteins with proteins of known function. As discussed in Chapter 8, the general functions of proteins identified by sequence searches can be predicted by analogy with known proteins. However, the precise in vivo roles of such “new” proteins may be unclear in the absence of mutant forms of the corresponding genes. In this section, we describe several ways of disrupting the normal function of a specific gene in the genome of an organism. Analysis of the resulting mutant phenotype often helps reveal the in vivo function of the normal gene and its encoded protein.
Three basic approaches underlie these gene inactivation techniques: (1) replacing a normal gene with other sequences, (2) introducing an allele whose encoded protein inhibits the functioning of the expressed normal protein, and (3) promoting destruction of the mRNA transcribed from a gene. The normal endogenous gene is modified in techniques based on the first approach, but is not modified in the other approaches.
Often researchers desire to study the effect of a particular allele of a gene rather than observing the effect of complete inactivation of the gene. For example, testing the effect of a dominant allele of an oncogene on cell division might require replacing one of the normal copies of the gene with that dominant allele. Until recently, this type of precise genome editing was nearly impossible to achieve, but now a variation on a bacterial system for making sequence-