TILLING Workflow

(based on Arabidopsis as an example organism)

	Seeds are mutagenized to induce point mutations throughout the genome.
	A founder population is grown from mutagenized seeds.
	Founder population is self-fertilized to produce a crossed population.
	Seeds from the crossed population are stored and DNA samples are collected in 96-well plates.
	Up to eight 96-well plates are pooled into one and the samples (768) subjected to PCR with two gene-specific primers labeled with different IRDye® infrared dyes.
	Resulting amplicons are heated and cooled, resulting in heteroduplexes between wild type and mutant samples.

	CEL I nuclease is used to cleave at base mismatches.
	Samples are denatured and electrophoresed on a LI-COR 4300 DNA Analysis System.
	In lanes that have a mutation in the pool, a band will be visible below the wild type band on the IRDye® 700 infrared dye image. A counterpart band will be visible in the same lane on the IRDye® 800 infrared dye image. This band is the cleavage product labeled with IRDye® 800 infrared dye from the complementary DNA strand. The sum of the length of the two counterpart bands is equal to the size of the amplicon, which makes it easy to distinguish mutations from amplification artifacts.
	After detection of a mutation in a pool (lane), the individual DNA samples in the pool are screened again to find out which of the eight pooled samples from the crossed population has the mutation.

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