High-efficiency thermal asymmetric interlaced PCR for amplification of unknown flanking sequences
High-efficiency thermal asymmetric interlaced PCR for amplification of unknown flanking sequences
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DOI：
10.2144/000112601
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发表时间：
2007-11-01
期刊：
影响因子：
2.7
通讯作者：
Chen, Yuanling
中科院分区：
文献类型：
--
作者：
Liu, Yao-Guang;Chen, Yuanling
分离已知序列两侧的未知DNA序列是分子生物学研究中的一项重要任务。热不对称交错聚合酶链式反应(Tail-PCR)是一种有效的方法。但原始Tail-PCR的成功率还有待提高，更希望获得更大尺寸的目的产物。在这里，我们提出了一种显著改进的尾部聚合酶链式反应程序，具有特殊的引物设计和优化的加热条件。这种高效的尾部聚合酶链式反应(hiTAIL-PCR)结合了尾部循环法和抑制性-聚合酶链式反应的优点，可以阻断非靶产物的扩增，抑制小靶序列的扩增，但可以有效扩增大的靶序列。利用这种方法，我们从转基因水稻品系中分离出了T-DNA插入片段的基因组侧翼序列。在我们的测试中，反应的成功率高于90%，并且在大多数情况下获得的主要产物的大小为1-3kb。
Isolation of unknown DNA sequences flanked by known sequences is an important task in molecular biology research. Thermal asymmetric interlaced PCR (TAIL-PCR) is an effective method for this purpose. However the success rate of the original TAIL-PCR needs to be increased, and it is more desirable to obtain target products with larger sizes. Here we present a substantially improved TAIL-PCR procedure with special primer design and optimized thermal conditions. This high-efficiency TAIL-PCR (hiTAIL-PCR) combines the advantages of the TAIL-cycling and suppression-PCR, thus it can block the amplification of nontarget products and suppress small target ones, but allow efficient amplification of large target sequences. Using this method, we isolated genomic flanking sequences of T-DNA insertions from transgenic rice lines. In our tests, the success rates of the reactions were higher than 90%, and in most cases the obtained major products had sizes of 1-3 kb.