T-linker-specific ligation PCR (T-linker PCR): an advanced PCR technique for chromosome walking or for isolation of tagged DNA ends
T-linker-specific ligation PCR (T-linker PCR): an advanced PCR technique for chromosome walking or for isolation of tagged DNA ends
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DOI：
10.1093/nar/gng068
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发表时间：
2003-06-15
影响因子：
14.9
通讯作者：
Chen, ZL
中科院分区：
文献类型：
--
作者：
Yan, YX;An, CC;Chen, ZL
有数十种基于 PCR 的方法可用于从已知序列到未知区域的染色体行走。这些方法分为三种类型：反向 PCR、连接介导的 PCR 和随机引物 PCR。然而，它们都没有被普遍应用于此目的，因为它们要么困难要么效率低下。在这里，我们描述了一种简单高效的PCR策略——用于基因或染色体步移的T-linker特异性连接PCR（T-linker PCR）。该策略分三步扩增模板分子。首先，用 3' 突出端酶消化基因组 DNA。其次，在特异性引物的引导下，Taq DNA聚合酶复制目标分子的一条链，并在目标分子的3'未知端产生单个A尾，然后将3'突出端-T接头（称为T-linker）特异性连接到目标分子上。第三，使用特异性引物和T-接头引物通过两轮巢式PCR扩增靶标。 T-linker PCR 显着改进了现有的步行 PCR 方法，因为它使用特异性 T/A 连接而不是任意连接或随机退火。为了证明 T-linker PCR 的可行性和效率，我们利用这种方法来鉴定转基因植物中的载体 DNA 或 T-DNA 插入。
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.