High-Throughput Screening for Induced Point Mutations

Adaptations of the targeting induced local lesions in genomes are reported, which help to understand gene function and effective reverse genetic strategies are increasingly in demand.

Abstract

With the completion of genome sequencing projects, emphasis in genomics has shifted from analyzing sequences to understanding gene function, and effective reverse genetic strategies are increasingly in demand. Here we report adaptations of the targeting induced local lesions in genomes (TILLING) reverse genetic strategy (McCallum et al., 2000a) to make it suitable for large-scale screening of chemically induced mutations in Arabidopsis and other plants. TILLING has several advantages over other reverse genetic strategies. Unlike methods that provide only knockout mutations (e.g. Altmann et al., 1995), TILLING yields a traditional allelic series of point mutations. This will be especially valuable for essential genes, where sublethal alleles are required for phenotypic analysis. Because chemical mutagenesis causes a high density of mutations (Koornneef et al., 1982), virtually all genes can be targeted by screening relatively few individuals. Furthermore, the generality of chemical mutagenesis means that TILLING can be applied to plants without requiring transgenic or sophisticated tissue culture methodology. However, point mutations are relatively subtle changes, so their detection can be challenging. This problem has received much current attention because of the importance of discovering single nucleotide polymorphisms in humans for genotyping, and numerous strategies have been introduced (Kristensen et al., 2001). In the original TILLING method, we described the use of denaturing HPLC (dHPLC) for sensitive mutation discovery in pools (McCallum et al., 2000a). The availability of an automated column injector that accepts a 96-well microtiter plate (Underhill et al., 1997) meant that TILLING could be routinely performed in a central facility at the rate of about one gene per week. To obtain high throughput for genomic applications, we desired a reliable and inexpensive point mutation discovery method that could be performed more rapidly than dHPLC and in a robust manner.