Target-selected mutant screen by TILLING in Drosophila

Abstract

The availability of the full Drosophila genomic DNA sequence prompts the development of a method to efficiently obtain mutations in genes of interest identified by their sequence homologies or biochemically. To date, molecularly characterized mutations have been generated in around 6000 of the approximately 15,000 annotated fly genes, of which around one-third are essential for viability. To obtain mutations in essential and nonessential genes of interest, we took a reverse genetics approach, based on the large-scale detection of point mutations by Cel-I-mediated heteroduplex cleavage. A library of genomic DNA from 2086 EMS-mutagenized lines was established. The library was screened for mutations in three genes. A total of 6.1 Mb were screened, and 44 hits were found in two different mutagenesis conditions. Optimal conditions yielded an average of one mutation every 156 kb. For an essential gene tested, five of 25 mutations turned out to cause lethality, confirming that EMS mutagenesis leads to high frequency of gene inactivation. We thereby established that Cel-I-mediated TILLING can be used to efficiently obtain mutations in genes of interest in Drosophila.

Figure 1.

Cel-I-based Tilling strategy. We prepared a genomic DNA library from the mutagenized b pr cn sca/CyO, hb-lacZ fly lines and performed a three-step (1,2,3) nested PCR reaction (A) for a particular gene of interest. The third PCR reactions included the IRDye700 (red) and IRDye800 (blue) fluorophores bound to the forward and reverse primers, thereby differentially labeling the two ends of the amplicons (B). Then we denatured and reannealed the fragments to generate balancer/mutant heteroduplexes (C) that were digested by Cel-I (D). When a SNP is present, cleavage of the heteroduplexes generates two fragments labeled by IRDye700 and IRDye800, respectively, which can be detected upon denaturing polyacrylamide gel electrophoresis in a LI-COR setup. A typical LI-COR signal shows two fragments with complementary sizes that must sum up the original amplicon size. Subsequently, we sequenced the PCR products to confirm the molecular lesions (see Methods).

Figure 2.

Tilling screen. Example of a gel used for mutation detection within the Sara-4 amplicon in 96 balanced mutant lines; 700 (left) and 800 nm (right) emission images showing the fragments generated by Cel-I-based heteroduplex cleavage. (Arrowheads) Fragments generated by cleavage at 14 different silent background polymorphisms in the balancer that could be reidentified by sequencing; (circles) two novel mutations. Notice that both the polymorphisms and the novel mutations appear in both channels with complementary sizes. In the case of the second mutation, the longer fragment (800-nm channel) is less distinguishable (asterisk). After sequencing, we could, however, reconfirm that this signal corresponds to a bona fide mutation. Note also that the silent polymorphisms are present in all 96 lines, reflecting the fact that the balancer is itself isogenic. New mutations appear only in individual lines. Full-length product, 747 bp (arrow). Cleavage of the two new mutants yields 440 bp (700 nm) + 315 bp (800 nm) (lane 4) and 90 bp (700 nm) and 660 bp (800 nm) (lane 80).

Figure 3.

Mutation spectrum in three genes. Genomic organization of Sara, Alp23B, and Arf6. White boxes represent the ORFs. Black boxes below (Sara-1 to 5, Alp23B-1 to 3, and Arf6-1,2) correspond to the position of the amplicons considered in this study. Total DNA screened, number of SNPs, missense, nonsense, and lethal mutations are listed. Lines within the ORF and introns represent the position of the balancer polymorphisms.