An adapter ligation-mediated PCR method for high-throughput mapping of T-DNA inserts in the Arabidopsis genome.
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.
O'Malley
Ronan C
RC
Genomic Analysis Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037, USA.
Alonso
Jose M
JM
Kim
Christopher J
CJ
Leisse
Thomas J
TJ
Ecker
Joseph R
JR
D017360:Arabidopsis / Q000235:genetics*; D002874:Chromosome Mapping / Q000379:methods*; D017931:DNA Primers; D004269:DNA, Bacterial / Q000737:chemistry*; D018745:Genome, Plant; D016254:Mutagenesis, Insertional; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA; D014170:Transformation, Genetic
D017931:DNA Primers; D004269:DNA, Bacterial; C036483:T-DNA