Plant Genotyping - Methods and Protocols
Plant Genotyping - Methods and Protocols
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植物基因分型 - 方法和方案
DOI：
10.1007/978-1-0716-3024-2_20
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发表时间：
2023
期刊：
影响因子：
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通讯作者：
Hardinge P
中科院分区：
文献类型：
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作者：
Hardinge P
在聚合酶链反应（PCR）中，已经证明了使用分子信标（MB）来检测含有突变或单核苷酸多态性（SNP）的扩增序列的高特异性。MB已经通过在优化的条件下靶向单链环序列而适于与等温核酸扩增技术环介导扩增（LAMP）一起使用，以实现诸如植物基因分型的应用。与PCR相比，LAMP具有几个优点，例如快速扩增、能够实现低成本设备的单一温度反应条件以及对某些PCR抑制剂的鲁棒性。然而，尽管所需的引物数量增加，但LAMP的特异性是有限的，并且假阳性结果可能是有问题的。在本章中，分子信标在LAMP检测的设计考虑，以及MB-LAMP扩增和检测的方法，在转基因（GM）玉米样品的基因序列的例子进行了描述。
High specificity has been demonstrated in polymerase chain reaction (PCR) with the use of molecular beacons (MBs) to detect amplified sequences containing mutations or single-nucleotide polymorphisms (SNPs). MBs have been adapted for use with the isothermal nucleic acid amplification technology loop-mediated amplification (LAMP) by targeting single-stranded loop sequences under optimized conditions to enable applications such as plant genotyping. LAMP has several benefits over PCR, such as rapid amplification, single-temperature reaction conditions enabling low-cost equipment, and robustness to certain PCR inhibitors. However, and despite the increased number of primers required, the specificity of LAMP is limited, and false positive results can be problematic. In this chapter, design considerations for molecular beacons in LAMP assays are described, as well as a method for MB-LAMP amplification and detection, with an example of gene sequences in genetically modified (GM) maize samples.