Reverse genetics: Identification of Arabidopsis lines with T-DNA insertions in a gene of interest

 

Tested Studies in Laboratory Teaching, 2009, Volume 30

Brenda G. Leicht & Chi-Lien Cheng

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.

Keywords:  Arabidopsis, reverse genetics, chaperone gene, biotechnology

University of Toronto, Mississauga (2008)