Reverse genetics: Identification of Arabidopsis lines with T-DNA insertions in a gene of interest
Brenda G. Leicht & Chi-Lien Cheng
Tested Studies in Laboratory Teaching, 2009, Volume 30
Abstract
We have made use of the excellent resources of the Arabidopsis research community to demonstrate the use of reverse genetics to students taking a course in Genetics and Biotechnology. Students begin with a gene of interest (e.g., our students have previously searched for a gene encoding a co-chaperone protein and another that encodes an exportin protein) and use the TAIR and SIGnAL websites to find T-DNA insertions in that gene. They next use a primer design tool, also at the SIGnAL website, to retrieve the sequences of PCR primers that will give PCR products that distinguish the three possible genotypes (homozygotes for the T-DNA insertion, heterozygotes for the T-DNA insertion, and homozygotes for the wild type allele). The wet lab portion of the exercise involves growing the T-DNA insertion mutation lines (which are a genotypically mixed population of T3 seeds), isolating genomic DNA from individual T3 plants, and using PCR and agarose gel electrophoresis to determine the genotype (specifically those that are hetero- or homozygous for the T-DNA insertion) of the individual plants.
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