Targeted screening for induced mutations.
With the accumulation of large-scale sequence data, emphasis in genomics has shifted from determining gene structure to testing gene function, and this relies on reverse genetic methodology. Here we explore the feasibility of screening for chemically induced mutations in target sequences in Arabidopsis thaliana. Our TILLING (Targeting Induced Local Lesions IN Genomes) method combines the efficiency of ethyl methanesulfonate (EMS)-induced mutagenesis with the ability of denaturing high-performance liquid chromatography (DHPLC) to detect base pair changes by heteroduplex analysis. Importantly, this method generates a wide range of mutant alleles, is fast and automatable, and is applicable to any organism that can be chemically mutagenized.
McCallum
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CM
Basic Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109-1024, USA.
Comai
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Greene
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EA
Henikoff
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D000483:Alleles; D019943:Amino Acid Substitution; D017360:Arabidopsis / Q000187:drug effects / Q000235:genetics*; D020137:Base Pair Mismatch; D020029:Base Pairing; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D018388:Codon, Terminator; D017124:Conserved Sequence; D004248:DNA (Cytosine-5-)-Methyltransferases; D015254:DNA Modification Methylases / Q000235:genetics; D017931:DNA Primers; D005020:Ethyl Methanesulfonate / Q000494:pharmacology; D005818:Genetic Engineering / Q000379:methods; D007438:Introns; D016296:Mutagenesis; D017354:Point Mutation; D016133:Polymerase Chain Reaction
D018388:Codon, Terminator; D017931:DNA Primers; D005020:Ethyl Methanesulfonate; D015254:DNA Modification Methylases; D004248:DNA (Cytosine-5-)-Methyltransferases; C112810:chromomethylase