A High-Throughput Arabidopsis Reverse Genetics System

A collection of Arabidopsis lines with T-DNA insertions in known sites was generated to increase the efficiency of functional genomics. A high-throughput modified thermal asymetric interlaced (TAIL)-PCR protocol was developed and used to amplify DNA fragments flanking the T-DNA left borders from ∼10...

Full description

Saved in:
Bibliographic Details
Published in:The Plant cell 2002-12, Vol.14 (12), p.2985-2994
Main Authors: Sessions, Allen, Burke, Ellen, Presting, Gernot, Aux, George, McElver, John, Patton, David, Dietrich, Bob, Ho, Patrick, Bacwaden, Johana, Ko, Cynthia, Clarke, Joseph D., Cotton, David, Bullis, David, Snell, Jennifer, Miguel, Trini, Hutchison, Don, Kimmerly, Bill, Mitzel, Theresa, Katagiri, Fumiaki, Glazebrook, Jane, Law, Marc, Goff, Stephen A.
Format: Article
Language:English
Subjects:
Agrobacterium tumefaciens - genetics
Arabidopsis - genetics
Binding Sites - genetics
Blasts
Chromosomes, Plant - genetics
Databases, Genetic
DNA, Bacterial - chemistry
DNA, Bacterial - genetics
DNA, Plant - chemistry
DNA, Plant - genetics
Genes
Genome, Plant
Genomes
Genomics
Genomics Article
Internet
Mutagenesis, Insertional
Plant cells
Plants
Plants, Genetically Modified
Polymerase chain reaction
Polymerase Chain Reaction - methods
Promoter regions
Reverse genetics
Seeds - genetics
Sequence Analysis, DNA
Sequencing
Citations: Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:A collection of Arabidopsis lines with T-DNA insertions in known sites was generated to increase the efficiency of functional genomics. A high-throughput modified thermal asymetric interlaced (TAIL)-PCR protocol was developed and used to amplify DNA fragments flanking the T-DNA left borders from ∼100,000 transformed lines. A total of 85,108 TAIL-PCR products from 52,964 T-DNA lines were sequenced and compared with the Arabidopsis genome to determine the positions of T-DNAs in each line. Predicted T-DNA insertion sites, when mapped, showed a bias against predicted coding sequences. Predicted insertion mutations in genes of interest can be identified using Arabidopsis Gene Index name searches or by BLAST (Basic Local Alignment Search Tool) search. Insertions can be confirmed by simple PCR assays on individual lines. Predicted insertions were confirmed in 257 of 340 lines tested (76%). This resource has been named SAIL (Syngenta Arabidopsis Insertion Library) and is available to the scientific community at www.tmri.org.
ISSN:1040-4651
1532-298X
DOI:10.1105/tpc.004630