Locked nucleic acid pentamers as universal PCR primers for genomic DNA amplification.
Locked nucleic acid pentamers as universal PCR primers for genomic DNA amplification.
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DOI：
10.1371/journal.pone.0003701
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发表时间：
2008
期刊：
影响因子：
3.7
通讯作者：
Liu W
中科院分区：
文献类型：
--
作者：
Sun Z;Chen Z;Hou X;Li S;Zhu H;Qian J;Lu D;Liu W
多重技术可以在一次反应中同时检测多个核酸序列，与传统的单反应检测方法相比，可以节省大量时间、成本和劳动力。然而，目前使用的多重方法需要精确的手工操作和许多复杂的步骤，因此需要一种新的、更简单的技术。含有锁定核酸残基的寡核苷酸是一种有吸引力的工具，因为它们对其互补靶标具有很强的亲和力，它们已被用来避免二聚体形成和错配杂交并增强有效的引发。在本研究中，我们旨在研究锁定核酸五聚体在基因组 DNA 扩增和多重基因分型中的用途。我们设计了锁定核酸五聚体作为基因组 DNA 扩增的通用 PCR 引物。锁定核酸五聚体能够引发所研究基因组内选定序列的扩增，并且所得产物的长度与通过限制性消化获得的产物相似。在来自三种细菌物种的基因组 DNA 的实时 PCR 中，锁定核酸五聚体显示出高引发效率。偏倚测试的数据表明，锁定核酸五聚体对肺炎克雷伯菌基因组中测试的六个基因中的每一个都具有相同的亲和力。结合悬浮阵列基因分型，基于锁定核酸五聚体的 PCR 扩增能够通过基因和物种特异性探针鉴定出总共 15 个菌株，包括 3 种细菌。在测定中使用的 32 个物种中，使用该方法清楚地鉴定了 28 个物种和 50 个不同的基因。作为一种新型基因组 DNA 扩增，使用锁定核酸五聚体作为通用引物对，结合悬浮阵列基因分型，可以通过单一扩增程序鉴定多个不同的基因或物种。这表明基于锁核酸五聚体的PCR可广泛用于病原体鉴定。
Multiplexing technologies, which allow for simultaneous detection of multiple nucleic acid sequences in a single reaction, can save a lot of time, cost and labor compared to traditional single reaction detection methods. However, the multiplexing method currently used requires precise handiwork and many complicated steps, making a new, simpler technique desirable. Oligonucleotides containing locked nucleic acid residues are an attractive tool because they have strong affinities for their complementary targets, they have been used to avoid dimer formation and mismatch hybridization and to enhance efficient priming. In this study, we aimed to investigate the use of locked nucleic acid pentamers for genomic DNA amplification and multiplex genotyping. We designed locked nucleic acid pentamers as universal PCR primers for genomic DNA amplification. The locked nucleic acid pentamers were able to prime amplification of the selected sequences within the investigated genomes, and the resulting products were similar in length to those obtained by restriction digest. In Real Time PCR of genomic DNA from three bacterial species, locked nucleic acid pentamers showed high priming efficiencies. Data from bias tests demonstrated that locked nucleic acid pentamers have equal affinities for each of the six genes tested from the Klebsiella pneumoniae genome. Combined with suspension array genotyping, locked nucleic acid pentamer-based PCR amplification was able to identify a total of 15 strains, including 3 species of bacteria, by gene- and species-specific probes. Among the 32 species used in the assay, 28 species and 50 different genes were clearly identified using this method. As a novel genomic DNA amplification, the use of locked nucleic acid pentamers as universal primer pairs in conjunction with suspension array genotyping, allows for the identification of multiple distinct genes or species with a single amplification procedure. This demonstrates that locked nucleic acid pentamer-based PCR can be utilized extensively in pathogen identification.
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