title: A method for amplification of unknown flanking sequences based on touchdown PCR and suppression-PCR.
abstract: Thermal asymmetric staggered PCR is the most widely used technique to obtain the flanking sequences. However, it has some limitations, including a low rate of positivity, and complex operation. In this study, a improved method of it was made based on suppression-PCR and touchdown PCR. The PCR fragment obtained by the amplification was used directly for sequencing after gel purification. Using this improved method, the positive rate of amplified flanking sequences of the ATMT mutants reached 99%. In addition, the time from DNA extraction to flanking sequence analysis was shortened to 2 days with about 6 dollars each sample.
authors.affiliation: Department of Pathogenobiology, Jilin University Mycology Research Center, Key Laboratory of Pathobiology, Ministry of Education, College of Basic Medical Sciences, Jilin University, Changchun, 130021, China. Electronic address: gaosong850118@me.com.
authors.forename: Song
authors.initials: S
authors.lastname: Gao
authors.affiliation: Department of Pathogenobiology, Jilin University Mycology Research Center, Key Laboratory of Pathobiology, Ministry of Education, College of Basic Medical Sciences, Jilin University, Changchun, 130021, China. Electronic address: hedan@jlu.edu.cn.
authors.forename: Dan
authors.initials: D
authors.lastname: He
authors.affiliation: Department of Pathogenobiology, Jilin University Mycology Research Center, Key Laboratory of Pathobiology, Ministry of Education, College of Basic Medical Sciences, Jilin University, Changchun, 130021, China; Jilin Provincial Key Laboratory on Molecular and Chemical Genetic, Second Hospital of Jilin University, Changchun, 130021, China. Electronic address: liguangquan@jlu.edu.cn.
authors.forename: Guangquan
authors.initials: G
authors.lastname: Li
authors.affiliation: Department of Pathogenobiology, Jilin University Mycology Research Center, Key Laboratory of Pathobiology, Ministry of Education, College of Basic Medical Sciences, Jilin University, Changchun, 130021, China; Department of Biology, Plant Science College of Jilin University, Changchun, 130062, China. Electronic address: yh_zhang@jlu.edu.cn.
authors.forename: Yanhua
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Department of Pathogenobiology, Jilin University Mycology Research Center, Key Laboratory of Pathobiology, Ministry of Education, College of Basic Medical Sciences, Jilin University, Changchun, 130021, China; Science Research Certer, China-Japan Union Hospital, Jilin University, Changchun, 130021, China. Electronic address: lvhuiying52@126.com.
authors.forename: Huiying
authors.initials: H
authors.lastname: Lv
authors.affiliation: Department of Pathogenobiology, Jilin University Mycology Research Center, Key Laboratory of Pathobiology, Ministry of Education, College of Basic Medical Sciences, Jilin University, Changchun, 130021, China. Electronic address: wli99@jlu.edu.cn.
authors.forename: Li
authors.initials: L
authors.lastname: Wang
mesh_terms: D016960:Agrobacterium tumefaciens / Q000737:chemistry* / Q000235:genetics; D001232:Aspergillus fumigatus / Q000737:chemistry* / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry* / Q000235:genetics; D004271:DNA, Fungal / Q000737:chemistry* / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D013175:Sporothrix / Q000737:chemistry* / Q000235:genetics
chemical_list: D004269:DNA, Bacterial; D004271:DNA, Fungal
keywords: Agrobacterium tumefaciens-Mediated transformation; Flanking sequences; Suppression-PCR; Thermal asymmetric staggered PCR (TAIL-PCR); Touchdown TAIL-PCR (TD-TAIL PCR)