文献类型： 外文期刊

作者： Yan, YX ¹ ; An, CC ² ; Li, L ² ; Gu, JY ² ; Tan, GH ² ; Chen, ZL ² ;

作者机构： 1.Peking Univ, Coll Life Sci, Natl Lab Prot Engn & Plant Genet Engn, Beijing 100871, Peoples R China

2.Peking Univ, Coll Life Sci, Natl Lab Prot Engn & Plant Genet Engn, Beijing 100871, Peoples R China; Yunnan Acad Agr Sci, Oilcrop Inst, Kunming 650205, Peoples R China

期刊名称：NUCLEIC ACIDS RESEARCH （ 影响因子：16.971； 五年影响因子：15.542 ）

ISSN： 0305-1048

年卷期： 2003 年 31 卷 12 期

页码：

收录情况： SCI

摘要： Dozens of PCR-based methods are available for chromosome walking from a known sequence to an unknown region. These methods are of three types: inverse PCR, ligation-mediated PCR and randomly primed PCR. However, none of them has been generally applied for this purpose, because they are either difficult or inefficient. Here we describe a simple and efficient PCR strategy-T-linker-specific ligation PCR (T-linker PCR) for gene or chromosome walking. The strategy amplifies the template molecules in three steps. First, genomic DNA is digested with 3' overhang enzymes. Secondly, primed by a specific primer, a strand of the target molecule is replicated by Taq DNA polymerase and a single A tail is generated on the 3' unknown end of the target molecule, and then a 3' overhang-T linker (named T-linker) is specifically ligated onto the target. Thirdly, the target is amplified by two rounds of nested PCR with specific primers and T-linker primers. T-linker PCR significantly improves the existing PCR methods for walking because it uses specific T/A ligation instead of arbitrary ligation or random annealing. To show the feasibility and efficiency of T-linker PCR, we have exploited this method to identify vector DNA or T-DNA insertions in transgenic plants.