A high-throughput arabidopsis reverse genetics system

Allen Sessions, Ellen Burke, Gernot Presting, George Aux, John McElver, David Patton, Bob Dietrich, Patrick Ho, Johana Bacwaden, Cynthia Ko, Joseph D. Clarke, David Cotton, David Bullis, Jennifer Snell, Trini Miguel, Don Hutchison, Bill Kimmerly, Theresa Mitzel, Fumiaki Katagiri, Jane GlazebrookMarc Law, Stephen A. Goff

Research output: Contribution to journalArticlepeer-review

709 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)2985-2994
Number of pages10
JournalPlant Cell
Volume14
Issue number12
DOIs
StatePublished - Dec 1 2002

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