Screening DNA Pools for T-DNA Insertions in Arabidopsis Genes
This protocol was adapted from “Obtaining Mutants,” Chapter 2 in Arabidopsis by Detlef Weigel and Jane Glazebrook. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2002.INTRODUCTION
Many investigators are interested in studying the functions of genes whose sequences or expression patterns suggest roles in particular biological processes. A powerful way to identify a mutation in the gene of interest and to test mutant plants for phenotypes that are predicted to result from loss of function of that gene is by PCR screening. Pools of insertion lines are screened using one primer corresponding to the gene of interest and one primer corresponding to the end of the insertion element. The synthesis of a product indicates the presence of an insertion in the gene of interest. The pool is then repeatedly subdivided until a single plant carrying the desired insertion is identified.
