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RNase H-dependent PCR (rhPCR): improved specificity and single nucleotide polymorphism detection using blocked cleavable primers

机译：RNase H依赖性PCR（rhPCR）：使用封闭的可裂解引物提高特异性和检测单核苷酸多态性

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Background The polymerase chain reaction (PCR) is commonly used to detect the presence of nucleic acid sequences both in research and diagnostic settings. While high specificity is often achieved, biological requirements sometimes necessitate that primers are placed in suboptimal locations which lead to problems with the formation of primer dimers and/or misamplification of homologous sequences. Results Pyrococcus abyssi ( P.a .) RNase H2 was used to enable PCR to be performed using blocked primers containing a single ribonucleotide residue which are activated via cleavage by the enzyme (rhPCR). Cleavage occurs 5'-to the RNA base following primer hybridization to the target DNA. The requirement of the primer to first hybridize with the target sequence to gain activity eliminates the formation of primer-dimers and greatly reduces misamplification of closely related sequences. Mismatches near the scissile linkage decrease the efficiency of cleavage by RNase H2, further increasing the specificity of the assay. When applied to the detection of single nucleotide polymorphisms ( SNPs ), rhPCR was found to be far more sensitive than standard allele-specific PCR. In general, the best discrimination occurs when the mismatch is placed at the RNA:DNA base pair. Conclusion rhPCR eliminates the formation of primer dimers and markedly improves the specificity of PCR with respect to off-target amplification. These advantages of the assay should find utility in challenging qPCR applications such as genotyping, high level multiplex assays and rare allele detection.
机译：背景技术聚合酶链反应（PCR）通常用于在研究和诊断环境中检测核酸序列的存在。尽管通常可以实现高特异性，但是生物学要求有时需要将引物置于次优位置，这会导致引物二聚体的形成和/或同源序列的错误扩增。结果使用深渊热球菌（P.a.）RNase H2使PCR能够使用含有单个核糖核苷酸残基的封闭引物进行，该引物通过酶的裂解而被激活（rhPCR）。引物与靶DNA杂交后，RNA碱基发生5'裂解。引物首先与靶序列杂交以获得活性的需要消除了引物二聚体的形成，并大大减少了紧密相关的序列的错误扩增。易断裂键附近的错配降低了RNase H2切割的效率，从而进一步提高了测定的特异性。当用于检测单核苷酸多态性（SNP）时，rhPCR被发现比标准等位基因特异性PCR灵敏得多。通常，将错配置于RNA：DNA碱基对时会产生最佳区分。结论rhPCR消除了引物二聚体的形成，并显着提高了脱靶扩增PCR的特异性。该测定法的这些优点应可用于挑战性qPCR应用中，例如基因分型，高水平多重测定法和罕见的等位基因检测。

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