Using JelMarker TM Image Reader Software

Targeting Induced Local Lesions In Genomes or TILLING® was first introduced in 2000, through the Arabidopsis genome project (1). The TILLING technique uses chemical mutagenesis with ethylmethanesulfonate (EMS) to induce point mutations throughout an entire genome. The mutagenized founder population is crossbred and the resultant population is analyzed for gene specific point mutations. EcoTILLING takes regular TILLING one step further in that it allows for haplotyping groups or species based on natural allele variation identified through SNP discovery. To start the TILLING procedure, the genes of interest are identified with gene specific primers and PCR amplified. The primers are labeled with two dye colors – the forward primer is labeled with a shorter wavelength wavelength fluorescent dye (LI-COR’s IRDye® 700 blue) and the reverse primer is labeled with a longer wavelength fluorescent dye (LI-COR’s IRDye 800 green). Fragments are then run through a thermal-cycler and heteroduplexes are formed at the point of the polymorphism between two hybridized DNA fragments. The hybridized fragments are then cleaved at the heteroduplex site by CEL I nuclease or SurveyorTM Nuclease. The DNA is denatured and the resulting shorter fragments are run through gel electrophoresis and a scanned image of the gel is produced (2, 3).