ABSTRACT
Figure 8-1 TILLING procedure. From Till et al (2003) with small modifi cation. Seeds are mutagenized with EMS, which causes G/C-to-A/T point mutations. To avoid ambiguities caused by chimerism of mutant plants in the fi rst (M1) generation, they are self-fertilized; and M2 progeny from single seed descent are used for screening. A Yyoung leafve is collected from each M2 plant, and then DNA is extracted. Plants are self-fertilized, and the M3 seed is collected and shipped to the Arabidopsis Biological Resource Center for distribution. For screening, DNAs are pooled eightfold to maximize the effi ciency of mutation detection. PCR is performed using 5′-end-labeled gene-specifi c primers to target the desired locus, and heteroduplexes are formed by heating and cooling the PCR products. CEL I nuclease is used to cleave at base mismatches, and products representing induced mutations are visualized with denaturing polyacrylamide gel electrophoresis.
