Plant & Animal Genomes XIV Conference

January 14-18, 2006
Town & Country Convention Center
San Diego, CA

High Through-Put Targeting Induced Local Lesions In Genomes (TILLING) Using Applied Biosystems’ 3730 Series Capillary Electrophoresis System.

David Baker¹ , Lee MacPherson¹ , Kilian Hann¹ , Jonathan H. Clark¹

¹  John Innes Centre Genome Laboratory, Norwich, NR4 7UH, UK

²  Applera Deutschland GmbH, Frankfurter Strasse 129B, D-64293, Darmstadt, Germany

TILLING is a strategy used in reverse genetic studies to identify series of chemically induced point mutations in specific genes. The detection of point mutations exploits the ability of the CEL1 endonuclease to cleave genomic DNA at mis-matched hetero-duplexes. Poly-acrylamide slab-gel electrophoresis systems are traditionally used to detect the cleavage products. These methods are labour intensive and not easily automated. We have developed the use of Applied Biosystems’ 3730 series capillary electrophoresis system for cleavage product detection. Following optimization of the CEL1 cleavage reaction, clean-up and capillary run conditions point mutations could be identified in populations with up to 12 fold pooling. For high through-put fragment analysis the AFLP Analysis Method in Applied Biosystems’ GeneMapper® Software v3.7 was optimized for effective peak detection and enables accurate cleavage product sizing from 1Kb target regions. We present our progress in TILLING a range of organisms (Medicago, Drosphila and Zebrafish, Rat & C.elegans) and discuss strategies for the development of an automated TILLING system.