Padlock Probes to Detect Single Nucleotide Polymorphisms
Padlock Probes to Detect Single Nucleotide Polymorphisms
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DOI：
10.1007/978-1-4939-7213-5_14
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发表时间：
2018-01-01
期刊：
影响因子：
--
通讯作者：
Nilsson, Mats
中科院分区：
文献类型：
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作者：
Krzywkowski, Tomasz;Nilsson, Mats
高通量DNA分析方法的快速发展，使研究对象的遗传景观和畸变的全球表征在时间和成本有效的方式。然而，在大多数方法中，由于样品制备需要将核酸从其原生环境中分离出来，因此丢失了空间组织背景。我们在此提出最新的方案，用于多路复用，在原位检测mrna和单核苷酸多态性使用挂锁探针和滚动圈扩增。我们利用保守的ACTB mRNA中的单核苷酸变异成功地分化了人和小鼠共培养细胞，并将所提出的方案应用于人脑中PCDH X和Y同源基因型。我们提供了一种在单个细胞或选定组织区域中自动表征和定量目标mRNA的方法。具有多态性的mRNA首先被反向转录为cDNA。等位基因特异性挂锁探针与cDNA靶杂交，并通过酶环化与亲本mRNA分子保持物理联系。最后，利用滚动圆放大机制原位复制圆形探针，以方便检测。
Rapid development of high-throughput DNA analyzation methods has enabled global characterization of genetic landscapes and aberrations in study subjects in a time and cost effective fashion. In most methods, however, spatial tissue context is lost since sample preparation requires isolation of nucleic acids out of their native environment. We hereby present the most recent protocol for multiplexed, in situ detection of mRNAs and single nucleotide polymorphisms using padlock probes and rolling circle amplification. We take advantage of a single nucleotide variant within conserved ACTB mRNA to successfully differentiate human and mice cocultured cells and apply presented protocol to genotype PCDH X and Y homologs in human brain. We provide a method for automated characterization and quantitation of target mRNA in single cells or chosen tissue area. mRNA of interest, harboring a polymorphism, is first reverse-transcribed to cDNA. Allele specific padlock probes are hybridized to the cDNA target and enzymatically circularized maintaining a physical link with the parent mRNA molecule. Lastly, circularized probes are replicated in situ, using rolling circle amplification mechanism to facilitate detection.