Froehlich Thomas, Geulen Oliver
Roche Diagnostics GmbH, Roche Applied Science, Penzberg, Germany.
Methods Mol Biol. 2008;429:117-33. doi: 10.1007/978-1-60327-040-3_9.
Real-time polymerase chain reaction (PCR) has become a standard tool in both quantitative gene expression and genetic variation analysis. Data collection is performed throughout the PCR process, thus combining amplification and detection into a single step. This can be achieved by combining a variety of different fluorescent chemistries that correlate the concentration of an amplified PCR product to changes in fluorescence intensity. Hybridization probe pairs and single-labeled probes are sequence-specific, dye-labeled oligonucleotides, used in real-time PCR approaches, in particular for genotyping of single nucleotide polymorphisms (SNPs). In that case, a detector probe is designed to cover the polymorphism. Allelic variants are identified and differentiated via post-PCR melting curve analysis. A single melting curve can distinguish different T (m)s, and differently labeled probes may be used, theoretically allowing multiplexed genotyping of several SNPs.
实时聚合酶链反应（PCR）已成为定量基因表达和遗传变异分析的标准工具。在整个PCR过程中进行数据收集，从而将扩增和检测合并为一个步骤。这可以通过结合多种不同的荧光化学方法来实现，这些方法将扩增的PCR产物浓度与荧光强度变化相关联。杂交探针组和单标记探针是序列特异性的、染料标记的寡核苷酸，用于实时PCR方法，特别是用于单核苷酸多态性（SNP）的基因分型。在这种情况下，设计一个检测探针来覆盖多态性。通过PCR后熔解曲线分析来鉴定和区分等位基因变体。单一熔解曲线可以区分不同的解链温度（Tm），并且可以使用不同标记的探针，理论上允许对多个SNP进行多重基因分型。
