Large-scale discovery of induced point mutations with high-throughput TILLING.

TILLING (Targeting Induced Local Lesions in Genomes) is a general reverse-genetic strategy that provides an allelic series of induced point mutations in genes of interest. High-throughput TILLING allows the rapid and low-cost discovery of induced point mutations in populations of chemically mutagenized individuals. As chemical mutagenesis is widely applicable and mutation detection for TILLING is dependent only on sufficient yield of PCR products, TILLING can be applied to most organisms. We have developed TILLING as a service to the Arabidopsis community known as the Arabidopsis TILLING Project (ATP). Our goal is to rapidly deliver allelic series of ethylmethanesulfonate-induced mutations in target 1-kb loci requested by the international research community. In the first year of public operation, ATP has discovered, sequenced, and delivered >1000 mutations in >100 genes ordered by Arabidopsis researchers. The tools and methodologies described here can be adapted to create similar facilities for other organisms.

[1]  S. Henikoff,et al.  Predicting deleterious amino acid substitutions. , 2001, Genome research.

[2]  C Cruz,et al.  Genetic studies of the lac repressor. XIV. Analysis of 4000 altered Escherichia coli lac repressors reveals essential and non-essential residues, as well as "spacers" which do not require a specific sequence. , 1994, Journal of molecular biology.

[3]  L. Stein,et al.  RNAi analysis of genes expressed in the ovary of Caenorhabditis elegans , 2000, Current Biology.

[4]  P. Zipperlen,et al.  Functional genomic analysis of C. elegans chromosome I by systematic RNA interference , 2000, Nature.

[5]  Steven Henikoff,et al.  Targeted screening for induced mutations , 2000, Nature Biotechnology.

[6]  S Rozen,et al.  Primer3 on the WWW for general users and for biologist programmers. , 2000, Methods in molecular biology.

[7]  N. Mitsukawa,et al.  The Arabidopsis ERECTA gene encodes a putative receptor protein kinase with extracellular leucine-rich repeats. , 1996, The Plant cell.

[8]  E. Myers,et al.  Basic local alignment search tool. , 1990, Journal of molecular biology.

[9]  S. Henikoff,et al.  High-throughput screening for induced point mutations. , 2001, Plant physiology.

[10]  Ronald W. Davis,et al.  Functional profiling of the Saccharomyces cerevisiae genome , 2002, Nature.

[11]  Kei-Hoi Cheung,et al.  Large-scale analysis of the yeast genome by transposon tagging and gene disruption , 1999, Nature.

[12]  S. Henikoff,et al.  Genome-Wide Profiling of DNA Methylation Reveals Transposon Targets of CHROMOMETHYLASE3 , 2002, Current Biology.

[13]  Shmuel Pietrokovski,et al.  Increased coverage of protein families with the Blocks Database servers , 2000, Nucleic Acids Res..

[14]  S. Henikoff,et al.  Targeting induced local lesions IN genomes (TILLING) for plant functional genomics. , 2000, Plant physiology.

[15]  S. Henikoff,et al.  High-throughput TILLING for functional genomics. , 2003, Methods in molecular biology.

[16]  Ronald W. Davis,et al.  Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis. , 1999, Science.

[17]  B Reardon,et al.  High-throughput isolation of Caenorhabditis elegans deletion mutants. , 1999, Genome research.

[18]  J H Miller,et al.  Genetic studies of the lac repressor. XIII. Extensive amino acid replacements generated by the use of natural and synthetic nonsense suppressors. , 1990, Journal of molecular biology.

[19]  Christophe Person,et al.  Disruption and sequence identification of 2,000 genes in mouse embryonic stem cells , 1998, Nature.