Structured oligonucleotides for target indexing to allow single-vessel PCR amplification and solid support microarray hybridization.
Structured oligonucleotides for target indexing to allow single-vessel PCR amplification and solid support microarray hybridization.
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用于靶标索引的结构化寡核苷酸，可实现单容器 PCR 扩增和固相支持微阵列杂交。
DOI：
10.1039/c4an01352b
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发表时间：
2015
期刊：
影响因子：
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通讯作者：
Bergeron,MichelG
中科院分区：
文献类型：
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作者：
Girard,LaurieD;Boissinot,Karel;Peytavi,Régis;Boissinot,Maurice;Bergeron,MichelG
分子诊断技术的组合越来越多地被用来克服灵敏度、特异性或多路复用能力的限制，并提供高效的芯片实验室设备。两种这样的技术，即PCR扩增和微阵列杂交被串联使用，以利用前者的高灵敏度和特异度，以及后者的高多重能力。这些方法通常在不同的缓冲液和反应室中进行。然而，这些复杂的方法具有很高的复杂性和成本，涉及试剂要求、液体存储和集成到自动化设备中的反应室数量。此外，微阵列杂交具有序列依赖的效率，但并不总是可预测的。在这项工作中，我们发展了一种结构化寡核苷酸探针的概念，它是通过从聚合酶核酸外切酶活性中切割来激活的。这项技术被称为SCISSOHR，用于结构切割诱导的单链寡核苷酸杂交反应。SCISSOHR探针能够将目标序列索引到标签序列。SCISSOHR技术还允许在仅存在聚合酶链式反应缓冲液的情况下，在单个血管中结合核酸扩增和微阵列杂交。SCISSOHR技术使用在靶标存在时不可逆修改的扩增探针，释放单链DNA标签用于微阵列杂交。每个标签由一个依赖于3个核苷酸序列的片段和一个独特的“不依赖于靶序列”的14个核苷酸片段组成，从而以最小的交叉杂交实现最佳杂交。我们评估了支持微阵列杂交的五(5)个PCR缓冲器的性能，并与传统的杂交缓冲器进行了比较。最后，作为概念验证，我们开发了一种用于扩增、检测和鉴定三(3)个DNA靶标的多重检测方法。这项新技术将促进芯片上实验室微流控设备的设计，同时还可以降低消耗品成本。到目前为止，它将使用于检测和识别遗传目标的高度多元化的分析实现具有成本效益的自动化。
The combination of molecular diagnostic technologies is increasingly used to overcome limitations on sensitivity, specificity or multiplexing capabilities, and provide efficient lab-on-chip devices. Two such techniques, PCR amplification and microarray hybridization are used serially to take advantage of the high sensitivity and specificity of the former combined with high multiplexing capacities of the latter. These methods are usually performed in different buffers and reaction chambers. However, these elaborate methods have high complexity and cost related to reagent requirements, liquid storage and the number of reaction chambers to integrate into automated devices. Furthermore, microarray hybridizations have a sequence dependent efficiency not always predictable. In this work, we have developed the concept of a structured oligonucleotide probe which is activated by cleavage from polymerase exonuclease activity. This technology is called SCISSOHR for Structured Cleavage Induced Single-Stranded Oligonucleotide Hybridization Reaction. The SCISSOHR probes enable indexing the target sequence to a tag sequence. The SCISSOHR technology also allows the combination of nucleic acid amplification and microarray hybridization in a single vessel in presence of the PCR buffer only. The SCISSOHR technology uses an amplification probe that is irreversibly modified in presence of the target, releasing a single-stranded DNA tag for microarray hybridization. Each tag is composed of a 3-nucleotide sequence-dependent segment and a unique “target sequence-independent” 14-nucleotide segment allowing for optimal hybridization with minimal cross-hybridization. We evaluated the performance of five (5) PCR buffers to support microarray hybridization, compared to a conventional hybridization buffer. Finally, as a proof of concept, we developed a multiplexed assay for the amplification, detection, and identification of three (3) DNA targets. This new technology will facilitate the design of lab-on-chip microfluidic devices, while also reducing consumable costs. At term, it will allow the cost-effective automation of highly multiplexed assays for detection and identification of genetic targets.