Journal Article

An adapter ligation-mediated PCR method for high-throughput mapping of T-DNA inserts in the Arabidopsis genome

Kim, Christopher J, Ecker, Joseph R, Leisse, Thomas J, O'Malley, Ronan C, Alonso, Jose M

2007

Overview

Agrobacterium transfer DNA (T-DNA) is an effective plant mutagen that has been used to create sequence-indexed T-DNA insertion lines in Arabidopsis thaliana as a tool to study gene function. Creating T-DNA insertion lines requires a dependable method for locating the site of insertion in the genome. In this protocol, we describe an adapter ligation-mediated PCR method that we have used to screen a mutant library and identify over 150,000 T-DNA insertional mutants; the method can also be applied to map individual mutants. The procedure consists of three steps: a restriction enzyme-mediated ligation of an adapter to the genomic DNA; a PCR amplification of the T-DNA/genomic DNA junction with primers specific to the adapter and T-DNA; and sequencing of the T-DNA/genomic junction to enable mapping to the reference genome. In most cases, the sequenced genomic region extends to the T-DNA border, enabling the exact location of the insert to be identified. The entire process takes 2 weeks to complete.

• Publisher: Nature Publishing Group UK,Nature Publishing Group
• Subject: Agrobacterium tumefaciens / Analytical Chemistry / Arabidopsis - genetics / Binding sites / Biological Techniques / Biomedical and Life Sciences / Chromosome Mapping - methods / Computational Biology/Bioinformatics / Deoxyribonucleic acid / DNA / DNA Primers / DNA, Bacterial - chemistry / DNA-ligand interactions / Enzymes / Gene mutations / Genes / Genome, Plant / Genomes / Genomics / Life Sciences / Methods / Microarrays / Mutagenesis, Insertional / Mutants / Organic Chemistry / Plasmids / Polymerase Chain Reaction - methods / Protocol / Sequence Analysis, DNA / Transformation, Genetic