Frequent problems and their resolutions by using thermal asymmetric interlaced PCR (TAIL-PCR) to clone genes in Arabidopsis T-DNA tagged mutants

• Published in: Biotechnology, biotechnological equipment Vol. 29; no. 2; pp. 260 - 267
• Main Authors: Wu, Lei, Di, Dong-Wei, Zhang, Dan, Song, Bin, Luo, Pan, Guo, Guang-Qin
• Format: Journal Article
• Language: English
• Published: England Taylor & Francis 04.03.2015; Taylor & Francis Ltd
• Subjects: Agriculture and Environmental Biotechnology; Arabidopsis; Asymmetry; Cloning; complex T-DNA insertion; Deoxyribonucleic acid; DNA; Gene sequencing; high-throughput sequencing; Insertional mutagenesis; Mutagenesis; Mutants; Mutation; Next-generation sequencing; Nucleotide sequence; Polymerase chain reaction; T-DNA; T-DNA tagged mutants; TAIL-PCR; Tails; TAIL-PCR; high-throughput sequencing; T-DNA tagged mutants; complex T-DNA insertion
• ISSN: 1310-2818; 1314-3530
• DOI: 10.1080/13102818.2014.998161

T-DNA insertional mutagenesis is a powerful tool in Arabidopsis functional 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.