(see following sections). For example, if a mutation defining a gene of interest has been mapped to a region of the genome for which the entire DNA sequence is known, the “candidate gene” approach can be used to identify it. Computer analysis of the genomic sequence can predict which sequences in the region represent coding sequences and open-reading frames (ORFs) of genes and what proteins these DNA sequences encode. It is then often possible to guess one or a few most likely candidate genes and confirm that one of these is correct by sequencing one (or preferably more) mutant allele and finding the responsible sequence alteration(s) or by expressing the candidate gene to see if its encoded product reverts the mutant phenotype back to wild type.
A new method called genomic mismatch scanning (GMS), using DNA microchip technology, will allow more rapid identification of the candidate gene and the mutational lesion in one step. Oligonucleotides representing the entire sequences of all candidate genes in the region to be tested, as well as all possible single base-change mutational variants of each sequence, are synthesized and fixed in an indexed array on a microchip (see description of the method in Chapter 5). The chip is then annealed to differently labeled probes from nonmutant and mutant forms of the cloned gene. By comparing these patterns, both the correct candidate gene and the nature of the mutational lesion can be determined.
With the increasing availability of genomic sequence information, the following somewhat different approach is becoming more useful for studying biological processes, especially in organisms such as mammals, for which the forward genetic approach is difficult. It is called reverse genetics, because it starts with a cloned gene of potential interest. The cloned gene is then used to obtain animals with defects in the gene or its expression for functional analysis. The steps in this approach are as follows:
Identify a gene of interest from its sequence (e.g., the mouse homolog of a developmentally important gene in Drosophila) and obtain a clone of the gene by standard methods based on sequence similarity (such as screening a mouse library (collection) of genomic DNA clones with the cloned Drosophila gene).
Determine its expression pattern (as described above) for clues to its function.
Inactivate the gene (often referred to as “knocking out,” “targeted inactivation,” or “homologous recombination” of the gene) and observe the phenotypic consequences for more definitive information on function. This can be done either transiently, by injection of an antisense or double-stranded mRNA that specifically prevents gene expression, or permanently (preferable, but requiring considerably more effort), by generating animals that carry a null mutation in the gene. In the nematode C. elegans, the double-stranded mRNA method works