title: Frequent problems and their resolutions by using thermal asymmetric interlaced PCR (TAIL-PCR) to clone genes inArabidopsisT-DNA tagged mutants.
abstract: T-DNA insertional mutagenesis is a powerful tool inArabidopsisfunctional genomics research. Previous studies have developed thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) as an efficient strategy in isolation of DNA sequences adjacent to known sequences in T-DNA tagged mutants. However, a number of problems are encountered when attempts are made to clone flanking sequences in T-DNA tagged mutants. Therefore, it is necessary to improve the efficiency of cloning mutagenesis. Here, we present the most frequent problems and provide an improved method to increase TAIL-PCR efficiency. Even then, it is not always possible to successfully obtain flanking sequences; in such cases, we recommend using high-throughput sequencing to determine the mutations.
authors.affiliation: Department of MOE Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences, Lanzhou University, Lanzhou , Gansu , P.R. China.
authors.forename: Lei
authors.initials: L
authors.lastname: Wu
authors.affiliation: Department of MOE Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences, Lanzhou University, Lanzhou , Gansu , P.R. China.
authors.forename: Dong-Wei
authors.initials: DW
authors.lastname: Di
authors.affiliation: Department of MOE Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences, Lanzhou University, Lanzhou , Gansu , P.R. China.
authors.forename: Dan
authors.initials: D
authors.lastname: Zhang
authors.affiliation: Department of MOE Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences, Lanzhou University, Lanzhou , Gansu , P.R. China ; Department of Life Science College, Xiamen University, Xiamen , Fujian , P.R. China.
authors.forename: Bin
authors.initials: B
authors.lastname: Song
authors.affiliation: Department of MOE Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences, Lanzhou University, Lanzhou , Gansu , P.R. China.
authors.forename: Pan
authors.initials: P
authors.lastname: Luo
authors.affiliation: Department of MOE Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences, Lanzhou University, Lanzhou , Gansu , P.R. China.
authors.forename: Guang-Qin
authors.initials: GQ
authors.lastname: Guo
keywords: T-DNA tagged mutants; TAIL-PCR; complex T-DNA insertion; high-throughput sequencing